DNA marker assisted breeding in interspecific crosses to improve canola

(Brassica napus L.)

Christopher James Schelfhout BSc.Agric.(Hons)

‘This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia’

Faculty of Natural and Agricultural Sciences School of Plant Biology

2007

i

Statement of candidate contribution

I declare that this thesis is my own composition and the result of my own

research work. All the contributions made by other individuals have been duly

acknowledged. This thesis contains no material which has been accepted for

the award of any other degree in this university or another institution and to the

best of my knowledge and belief, contains no material previously published or

written by another person, except where due reference has been made in the

text.

Several papers have been published from this thesis (page iv). Chris

Schelfhout contributed 90% of the work for all experimental research,

preliminary drafts and literature research. The co-authors contributed 10% for

research guidance, editing and funding. All co-authors agree to the use of

these published works as chapters in this thesis.

………………………………….

Christopher James Schelfhout

................................................

Associate Professor Wallace Cowling

………………………………….

Dr. Janet Wroth

ii

Abstract

In order to expand the gene pool of canola-quality rapeseed (Brassica napus)

reciprocal interspecific crosses were made between B. napus cv. Mystic and

near canola-quality B. juncea breeding line JN29. F1 progeny from these

crosses were used to make backcrosses to both parents in all possible

combinations and directions, and were selfed to form F2-derived lines. The

highest frequencies of viable F2 and BC1 progeny were obtained when B. napus

was the maternal parent of the interspecific hybrid. BC1 and F2 progeny (and

subsequent generations) were grown under field conditions to identify

agronomic improvements over the parents. Transgressive segregation was

observed in F2 and BC1 and in subsequent generations for agronomic traits

(seed yield under high or low rainfall conditions, plant biomass, harvest index,

height, branching and days to anthesis) and seed quality traits (oil, protein,

glucosinolates, oleic acid). The majority of progeny conformed to B. napus

morphology, and a minority segregated to B. juncea morphology in subsequent

generations. Some of the B. juncea morphotypes had lower glucosinolates and

higher oleic acid than the parent JN29, with no detectable erucic acid, and

thereby conformed to canola quality.

Methods were developed for tracing B-genome in interspecific progeny.

A repetitive DNA sequence pBNBH35 from B. nigra (genome BB, 2n = 16) was

used to identify B-genome chromosomes and introgressions in interspecific

progeny. Specific primers were designed for pBNBH35 in order to amplify the

repetitive sequence by PCR. A cloned sub-fragment of 329 bp was confirmed

by sequencing as part of pBNBH35. PCR and hybridisation techniques were

used on an array of Brassica species to confirm that the pBNBH35 sub-

fragment was Brassica B-genome specific. Fluorescence in situ hybridisation

(FISH) in B nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38)

showed that the pBNBH35 sub-fragment was present on all eight Brassica B-

genome chromosomes and absent from A- and C-genome chromosomes. The

pBNBH35 repeat was localised to the centromeric region of each B-genome

chromosome. FISH clearly distinguished the B-genome chromosomes from the

A-genome chromosomes in the amphidiploid species B. juncea. This is the first

known report of a B-genome repetitive marker that is present on all Brassica B-

genome chromosomes.

iii

The pBNBH35 sub-fragment was used as a PCR-based marker and

FISH probe to trace and identify Brassica B-genome chromosomes and

chromosomal introgressions in the interspecific progeny B. napus x B. juncea.

B-genome positive PCR products were identified in 67% of fertile F2 progeny,

which was more than double the proportion found in fertile BC1 progeny. The

majority of these progeny were indistinguishable from B. napus in morphology.

Four B-genome positive F2-derived families and one BC1-derived family were

fixed or segregating for B. juncea morphology. Based on FISH assays, the B.

juncea morphotypes could be grouped into two types in the F4 and BC1S2

generation: (i) those with a B. juncea complement of 36 chromosomes, 16 of

which gave B-genome positive signals, and (ii) those with a B. napus

complement of 38 chromosomes and no (or few) B-genome signals. One of

these B. juncea morphotypes with 38 chromosomes exhibited isolated and

weak B-genome FISH signals on 11 chromosomes and typical A-genome FISH

signals. FISH signals were not detected in several B-genome PCR positive

progeny. The results suggest that novel B. napus genotypes have been

generated containing introgressions of B-genome chromatin from B. juncea

chromosomes. B. juncea morphology occurred in interspecific progeny with a

chromosome complement similar to B. napus (2n = 38) and without the entire B-

genome present. It also is highly likely that recombination has occurred

between the A-genome of the two Brassica species.

This research has demonstrated that the secondary gene pool of B.

napus may be accessed by selfing interspecific hybrids, and without sacrificing

canola quality, if the B. juncea parent is near canola-quality. Interspecific

progeny may be screened to enhance the proportion with B-genome positive

signals. Some progeny with B. juncea–type morphology had improved seed

quality over the JN29 parent.

iv

Acknowledgements

This thesis was completed under the supervision of Associate Professor

Wallace Cowling and Dr. Janet Wroth. I am very grateful and fortunate to have

had such committed supervisors who lead by example and strive for excellence

in the work they do. Their encouragement and enthusiasm has enabled me to

achieve so much during the course of this study. Wallace provided a seemingly

endless supply of innovative ideas and he has shown me that persistence and

commitment is to be rewarded. Janet is a lateral thinker who can always be

relied upon to give an alternative perspective on things. Thanks also to Janet

for all the little pieces of maternal advice you gave along the way. I thankfully

acknowledge the financial support of the Australian Research Council and

industry partners Council of Grain Grower Organisations Ltd and Export Grains

Centre Ltd, who funded my scholarship and operating expenses.

There are many others who supported my research in various ways.

These include Milton Sanders and Kylie Edwards who tirelessly helped with

field trials and Jeremy English and Catherine Borger who also assisted with

harvest. Thanks to Michael Blair for your assistance with trials at the Shenton

Park field station. Thank you to Graham Walton, Tim Trent and staff at the

Merredin Research Station for their assistance with the trials at Merredin. I

would also like to thank Matthew Nelson, Anouska Cousin and Michael Francki

for the numerous discussions and advice given during our shared time in the

molecular genetics laboratory. Thanks also to Nick Larkin for your friendship

and advice during our time shared in the office.

Many thanks to Dr. Rod Snowdon and colleagues at Justus Liebig

Universitat, Germany. Rod’s generous donation of time, advice and resources

proved to be a productive partnership. It was a very rewarding and memorable

experience.

I sincerely thank my parents for their support and assistance along the

way. Their continued encouragement has been invaluable. I must thank my

three very own F1 progeny Louis, Mary and George who all arrived during the

course of this study and kept life interesting. I apologise for the late nights and

missed opportunities. Finally, to my beautiful wife Anni, thank you so much for

your patience and support, it has been a long time coming but now I’m finally at

the end and I would like to dedicate this thesis to you.

v

Journal publications arising from this thesis

Schelfhout CJ, Snowdon R, Cowling WA, Wroth JM (2004) A PCR based B-

genome-specific marker in Brassica species. Theoretical and Applied

Genetics 109, 917-921

Schelfhout CJ, Snowdon R, Cowling WA, Wroth JM (2006) Tracing B-genome

chromatin in Brassica napus × B. juncea interspecific progeny. Genome

49, 1490-1497

Schelfhout CJ, Wroth JM, Yan G, Cowling WA (2008) Enhancement of genetic

diversity in canola-quality Brassica napus and B. juncea by interspecific

hybridisation. Australian Journal of Agricultural Research (in press).

Conference publications arising from this thesis

Schelfhout, C., Cowling, W. and Wroth, J. (2002) Tracing the B genome in B.

napus x B. juncea interspecific hybrids. Proceedings of the 12th

Australasian Plant Breeding Conference, Perth Western Australia.

Cowling, W., Wroth, J. and Schelfhout, C. (2002). Interspecific crossing to

widen the gene pool of canola. Proceedings of the 12th Australasian

Plant Breeding Conference, Perth, Western Australia.

Schelfhout, C., Cowling, W. and Wroth J. (2003) B-genome detection and

influence in interspecific crosses of Brassica napus and B. juncea.

Proceedings of the 11th International Rapeseed Congress. Copenhagen,

Denmark.

vi

Table of contents

Statement of candidate contribution ………………………………...……….………i

Abstract ……………………………………………………..……..….…….…….……ii

Acknowledgments …..………….…………………………………………...………..iv

Publications arising from this thesis....……………………………….….……….….v

Table of contents ..………………………………………………………….….……..vi

Chapter 1. General Introduction ..……………………………………………….…..1

Chapter 2. Literature Review .……………………………………….……………….6

Chapter 3. Published paper “A PCR based B-genome specific marker in

Brassica species” ….…………………………………………………………...……28

Chapter 4. Published paper “Tracing B-genome chromatin in B. napus × B.

juncea interspecific progeny” ………………………………………………...……34

Chapter 5. In press paper “Enhancement of genetic diversity in canola-quality

Brassica napus and B. juncea via reciprocal gene exchange” ………...……….43

Chapter 6. General Discussion …………………………………………….…....…65

References ………………….……………………………………………………..…72

1

Chapter 1 - General Introduction

Rotation with canola (Brassica napus L.) has improved weed, insect and

disease control in subsequent cereal crops in Australia, and has improved

profitability in Australian farming systems. The area sown to canola increased

markedly during the early 1990s (Norton et al. 1999). Expansion into rainfall

zones as low as 280 mm per annum was made possible with the introduction of

shorter season varieties (Salisbury and Wratten 1999) however these options

do not always extend to growers the same relative gain as in higher rainfall

cropping regions. In drier areas canola crops are usually only sown when

opportunistically favourable conditions prevail early in the cropping season.

Australian canola growers are also faced with threats from blackleg

(Leptosphaeria maculans) disease as a result of peak ascospore releases in

June, July and August coinciding with crop sowing and establishment (Bokor

1975, Salisbury et al. 1995, Khangura and Barbetti 2004).

In 30 years of oilseed rape breeding in Australia since 1970, B. napus

was converted into canola quality, based on Canadian sources of low erucic

acid in oil and European sources of low glucosinolates in meal, and selected for

adaptation to the Australian winter-spring growing environment with high levels

of blackleg resistance (Salisbury and Wratten 1999). By default, the Australian

breeding programs became one large “closed recurrent selection” program with

an effective population size of 11 and a population inbreeding coefficient of 0.21

after 5 cycles of selection over 30 years (Cowling 2007). This narrowing of the

gene pool in Australia over 30 years is of concern, and new sources of genetic

variation are vital to the future of Australian canola breeding.

The scope for genetic improvement of B. napus from within its primary

gene pool is limited due to the narrowness of the gene pool. Becker et al.

(1995) discuss probable causes for such a narrow gene pool, such as its

relatively recent origins from a few natural interspecific hybridizations between

B. rapa and B. oleracea, and selection for adaptation to winter and spring

cropping environments in Europe, followed by further selection into canola

quality germplasm. New genetic diversity in B. napus will aid the adaptation of

this species to new environments, as suggested by Lewis and Thurling (1994)

in Australia.

2

The secondary gene pool of B. napus, including all the diploid and

amphidiploid species in U’s triangle (U 1935), has long been considered as a

source of new alleles for B. napus breeding. B. juncea (genome AABB) is

grown widely in drought-prone areas and has been the subject of selection for

drought tolerance (Chauhan et al. 2007). This species may provide alleles to

improve adaptation to low-rainfall environments in B. napus (genome AACC).

Previous crosses between B. napus (canola quality) and B. juncea (mustard

quality) resulted in B. napus progeny with potentially useful blackleg resistance,

but lower seed quality (Barret et al. 1998, Roy 1980a, 1980b, 1984, Sacristan

and Gerdemann 1986), earliness (Rao et al. 1993a) or shatter resistance

(Prakash and Chopra 1988). Potentially beneficial alleles from B. juncea or

other donor species are mostly lost during backcrossing to restore canola

quality, unless they are major alleles that are readily tracked by phenotype or

with molecular markers. A potential solution is to use near canola-quality B.

juncea as the donor of beneficial alleles. B. juncea shares a common genome

(AA) with B. napus and may also contribute useful B-genome alleles through

introgression with the A or C genomes of B. napus. This research aimed to

determine if progeny of such crosses maintained their canola quality upon

selfing, with possible reciprocal benefits to both species through a single

interspecific cross.

With this in mind, experiments were set up with reciprocal crossing and

backcrossing in all possible combinations to examine the potential for

introducing valuable alleles from B. juncea into B. napus and also, in the

reverse direction, useful quality and agronomic traits into B. juncea from B.

napus. The first backcross was used to test the value of increasing the

proportion of alleles from the recurrent parent.

The progeny and parental lines were observed in the field in the F2 and

BC1 generation, and fertile selfed selections were tested in contrasting

agricultural environments (low and high rainfall) in the F3 and BC1S1.

Measurement of agronomic, disease and quality traits allowed a comparison of

performance of various cross combinations (reciprocals and backcross versus

self-pollination).

Molecular and cytogenetic techniques were developed to assist in the

detection of B-genome within B. napus-type progeny. An RFLP type B-genome

marker has been reported (Gupta et al. 1992). If converted to a PCR-based B-

3

genome specific marker, this would be useful to detect B-genome in

interspecific progeny and parents. With such a test, individuals carrying B-

genome could be selected in the F2 and BC1, and those with putative B-genome

introgressions could be detected by FISH reactions in F2-derived and BC1-

derived lines.

If canola quality is maintained in interspecific progeny through the

approach outlined here, then interspecific progeny may be selected for superior

performance in target agricultural environments with the knowledge that the

selections are of immediate value in commercial canola breeding programs.

Further backcrossing to the parent species to improve canola quality should not

be necessary. Minor alleles contributing to valuable economic traits, such as

yield, quality or disease resistance, from interspecific crossing will be available

for further genetic improvement in elite canola breeding programs.

The main hypothesis of this thesis is that progeny from crossing between

canola-quality B. napus (AACC) and near canola-quality B. juncea (AABB) will

retain their canola quality and demonstrate transgressive segregation for key

agronomic traits in the fertile F2 and BC1 progeny. Further, meiosis and

recombination in the F1 (AABC) will result in few fertile F2 and BC1 progeny. B-

genome chromatin will be present in some of these progeny. It is expected,

based on past results (Roy 1980a), that cross direction will greatly influence the

fertility of progeny. It is hypothesized that both B. napus (AACC) and B. juncea

(AABB) progeny will be detected in F2-derived and BC1-derived progeny, and

some of these will be canola quality with superior performance in for relevant

agricultural traits in target environments.

A secondary aim of the thesis was to develop PCR-based molecular

assays to identify individuals with B-genome introgressions from B. juncea early

in the selfing and backcrossing process. It is hypothesised that many of these

B-genome positive progeny will have B. napus morphology. FISH cytogenetic

studies will help to localise the site of introgression of B-genome in B. napus

type progeny. The association of B-genome with new agronomic variation

within the progeny will be examined. It may also be possible to identify specific

influences of B-genome on traits observed in B. napus type progeny, such as

blackleg resistance or adaptation to low rainfall environments.

Finally, the research aimed to identify unique interspecific progeny with

superior agronomic, disease or quality traits. Superior lines may be of either B.

4

juncea or B. napus morphology. It is hypothesised that BC1 progeny

backcrossed to B. juncea will have higher proportions of B-genome and more B.

juncea type progeny, than those backcrossed to the B. napus parent; likewise,

progeny backcrossed to B. napus will have less B-genome than selfed progeny.

On the basis of previous results (Roy 1980a), it was expected that the majority

of progeny derived from selfing of the F1 would be B. napus morphology.

The use of near canola-quality B. juncea in crossing with canola quality

B. napus should reduce the so-called deleterious “linkage drag” which is often

observed in interspecific crosses, where extensive backcrossing is necessary to

restore seed quality or agronomic traits. Selection of high quality progeny with

agronomic improvements from B. juncea may be possible in target

environments, such as the low rainfall cropping regions of Australia. By

minimizing backcrossing, minor alleles for complex agronomic traits should be

available for selection in interspecific progeny with canola quality.

This thesis is divided into a number of chapters as listed below. Apart

from the published and submitted papers all of the references are listed at the

end of the thesis:

Chapter 1. General introduction: provides the background to this research

project.

Chapter 2. Literature review: reviews the literature on;

• The history of B. napus as a crop species in Australia

• Agronomic factors that limit expansion of canola in Australian farming

systems

• B. napus, its evolution and relatives and,

• Sources of new genetic variation for the improvement of B. napus

Chapter 3. Published paper:

Schelfhout CJ, Snowdon R, Cowling WA, Wroth JM (2004) A PCR based B-

genome-specific marker in Brassica species. Theoretical and Applied

Genetics 109, 917-921

5

This chapter outlines the isolation and development of a B-genome specific

marker that is believed to be the first B-genome marker present on all Brassica

B-genome chromosomes.

Chapter 4. Published paper:

Schelfhout CJ, Snowdon R, Cowling WA, Wroth JM (2006) Tracing B-genome

chromatin in Brassica napus × B. juncea interspecific progeny. Genome

49, 1490-1497

This chapter outlines the further development of the B-genome specific probe

outlined in Chapter 3. Here the marker is developed into a probe for

fluorescence in-situ hybridisation (FISH) and used to identify putative B-genome

introgressions in B. napus × B. juncea interspecific progeny. Genomic

relationships are discussed.

Chapter 5. “in press” (June 2008):

Schelfhout CJ, Wroth JM, Yan G, Cowling WA (2008) Enhancement of genetic

diversity in canola-quality Brassica napus and B. juncea by interspecific

hybridisation. Australian Journal of Agricultural Research (in press).

This chapter outlines the extensive field testing of the Brassica interspecific

progeny. It also discusses the transgressive segregation observed and the

reciprocal nature of genetic improvement in both B. napus and B. juncea. It

also discusses the effects of the interspecific cross on seed quality.

Chapter 6. General discussion;

This chapter critically discusses the key information gained from this research

and proposes some opportunities for further investigation.

6

Chapter 2 - Review of Literature

Brassica napus as a crop species in Australia

History

Brassica napus first appeared as a commercial crop (rapeseed) in Australia in

1969 during a period when cereal growers were faced with the introduction of

wheat quotas (Colton and Potter 1999). Lack of resistance to the fungal

disease ‘blackleg’ (causal agent Leptosphaeria maculans) in these early

varieties from Canadian germplasm caused widespread failure of rapeseed

crops within a few years. In Western Australia blackleg disease was so severe

that the area sown to rapeseed was reduced from 49,000 hectares in 1973 to

3,000 hectares in 1974 (Salisbury et al. 1995).

The major focus of research and breeding during the 1970s was to

improve blackleg resistance and to reduce anti-nutritional components, namely

erucic acid in oil and a range of glucosinolates in the seed, as discussed in

several reviews (Downey 1990, Downey and Rimmer 1993, Scarth 1995). The

term ‘double-low’ rapeseed was used to denote varieties of rapeseed with less

than 2% erucic acid and less than 30 µmoles of total glucosinolates per gram of

meal, and in 1979 the term ‘canola’ was introduced in Canada to describe

varieties with these improved seed quality traits (Anon. 2007, Colton and Potter

1999). The current quality requirements for Australian canola being traded in

the open bulk commodity market are listed in Table 1.

Canola breeding in Australia in the 1980s continued to focus on the

improvement of blackleg resistance as well as the introduction of hybrid and

herbicide resistant varieties. Development of Australian cultivars is based on a

very limited germplasm pool of about 11 ancestral parents introduced in 1970

(Cowling 2007). There has been little introduction of new germplasm over the

past 30 years and recent Australian varieties are the product of crossing within

this gene pool over 5 generations of closed recurrent selection (Cowling 2007).

7

Table 1. Quality standards for Australian canola (modified from Australian

Oilseeds Federation, Salisbury and Potter 1999).

Commodity Quality trading standard Price adjustments and comments

Oil – 40% (Western Australia 42%)

1.5% premium or deduction for each 1% above or below 40% respectively

Impurity – max. 3% (Rejected above )

Gross weight will be adjusted by 1% for each 1% up to 3% max. 2 for 1 penalty over 4%

Moisture – max. 8% (Reject above)

If accepted over max, 2% deduction for each 1% over the allowed level

Broken seed – max. 7% (Rejected above)

If accepted over max, 0.5% deduction for each 1% over the allowed level

Canola (Brassica napus or Brassica rapa) Glucosinolates as specified: maximum 30 micromoles (any one or any mixture of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, and 2-hydroxy-4-pentenyl glucosinolate) per gram of oil-free air-dry solids Erucic acid in oil maximum 2%

Damaged seed - 3% Sprouted – max. 5% Green - nil (2%) Total – max. 10% (Rejected above)

0.5% deduction for each 1% over the allowed level - except for green seeds which shall incur a penalty of 1% for each 1% over zero (nil) to a maximum of 2% over which green seed level, the seed is rejectable

Canola production in Australia: the current status

During the 1990s, improved varieties were released and Australian canola

production increased from approximately 0.1 Mt in 1990 to 1.7 Mt in 1999

(Colton and Potter 1999). While production was limited to the higher rainfall

areas, by the late 1990s herbicide tolerant and shorter season varieties became

available leading to expansion of the industry into rainfall zones as low as 280

mm per annum (Salisbury and Wratten 1999). Australian production peaked in

1999 and has declined in recent years due to several unfavourable seasons.

Table 2 shows the area sown to canola and the corresponding production for

the last nine seasons (Australian Oilseeds Federation, 2007).

8

Table 2. Figures for the area sown to canola in Australian and the production in

years 1998/99 to 2007/08 est. Source:

http://www.australianoilseeds.com/australian_oilseeds_industry/industry_facts_

and_figures.

Season 99/00 00/01 01/02 02/03 03/04 04/05 05/06 06/07 07/08 est.

Area sown (‘000 ha) 1728 1334 1130 972 1005 1119 940 943 1060

Production (‘000 t) 2400 1680 1608 790 1622 1531 1438 512 953

The benefits of growing canola

In addition to direct benefits in seed oil and meal, canola provides grain growers

with several useful rotational benefits. Canola, a broadleaf species, permits

greater control of grass weeds for subsequent cereal crops and allows chemical

group rotation to avoid herbicide resistance in weeds (Oilseeds WA 2006).

Canola also acts as a break crop for cereal diseases (Norton et al. 1999).

Kirkegaard et al. (1994, 1997, 2000) have suggested that canola may exhibit

soil fumigation properties by releasing glucosinolates that are toxic to soil borne

plant pathogens. While the mechanism by which canola provides benefits to

subsequent cereal crops is debated, the boost in wheat yield is significant –

from a survey of 226 wheat crops in 1995 across Victoria, wheat yield following

canola was 3.9 t/ha where wheat following wheat was 2.8 t/ha (Norton et al.

1999).

Rising oil prices and increasing interest in biofuels has bolstered farmers’

confidence in canola as a profitable crop. Recent drought and tight supply for

oilseeds has kept global prices for canola buoyant with an outlook for possible

future rises (Gorey 2007).

9

Agronomic factors that limit expansion of canola in Australian farming

systems

Climate

Traditionally canola has been restricted to higher rainfall regions of southern

Australia (>450 mm). With the advent of shorter season varieties canola,

production has expanded into low rainfall environments with annual averages of

280 - 350 mm (Salisbury and Wratten et al. 1999). Unlike many other canola

regions of the world, canola crops in southern Australia mature under increasing

temperatures and ensuing terminal drought stress. Soil moisture stress at pod

fill can cause considerable yield loss through poor seed set, pod abortion and

quality problems such as reduced oil (Mailer and Pratley 1990) and increased

seed glucosinolate content (Champolivier and Merrien 1996). Lewis and

Thurling (1994) observed higher yields in low rainfall environments from B.

juncea compared with B. napus cultivars and concluded that the higher yields

were achieved through higher rates of post-anthesis growth in the B. juncea

lines.

In tandem with decreasing soil moisture, temperatures rise during the

maturity of Australian canola crops. High temperatures and low rainfall during

pod fill are associated with low oil content of canola seed (Pritchard et al. 2000).

There is a strong inverse relationship of seed oil and protein content under

Australian conditions (Si et al. 2003). Si and Walton (2004) reported limits to oil

content in short season, low rainfall environments. High temperatures at crop

maturity contributed to an increase in the production of erucic acid in the seed

oil (Wilmer et al. 1997). There have been suggestions that high temperatures

may reduce unsaturated fatty acid composition, however experiments utilising

short periods of high temperature have failed to prove this (Pritchard et al.

2000).

Crop establishment

Good crop establishment can be hampered by inconsistent rainfall events and

patchy soil moisture. By selecting larger seed, Kant and Tomar (1995)

demonstrated in B. juncea that germination, emergence seedling length and

early vigour are improved. This may help establishment under low moisture

conditions.

10

Soils and crop nutrition

Some areas in South-Eastern Australia are young, fertile soils, whilst those of

Western Australia are ancient, infertile soils and require the regular application

of fertilisers to be productive (Hunt and Gilkes 1992). Canola crops require

approximately 25% more nitrogen, phosphorous and potassium and five times

more sulphur than Australian wheat crops (Hocking et al. 1999). The higher

input costs of canola compared to wheat increases the financial risk of growing

canola, especially in low rainfall regions.

Extensive use of nitrogen fertilisers has led to the development of soil

acidity over large areas of Australia’s agricultural regions (Hunt and Gilkes

1992). Low soil pH values can result in aluminium toxicity that causes stunted

root growth (Kochian 1995, Huang et al. 2002) and manganese toxicity that

induces chlorosis and distorted leaves (Hocking et al. 1999). Application of lime

is a common approach to combating low soil pH. However, introgression of

aluminium tolerance from wild Brassica relatives has been proposed to improve

oilseed rape production on acid soils (Huang et al. 2001), and there have been

reports of aluminium tolerance in Brassica species (Huang et al. 2002).

Diseases

The fungal pathogen that causes blackleg, Leptosphaeria maculans (asexual

form Phoma lignam) is by far the most serious disease of canola and commonly

causes crop lodging during the seed fill stage (Salisbury et al. 1995). The

Mediterranean climate of southern Australia is particularly favourable for the

retention of the blackleg fungus on crop residues over the summer months. Hot

dry summers inhibit fungal growth and development, which resumes in the

cooler autumn and winter months with the release of ascospores that are

usually dispersed several hours after rain (Hall 1992) or following heavy dew

(McGee 1977). In Australia, peak ascospore showers occur in June, July and

August (winter) and coincide with the time of crop sowing and establishment

(Bokor 1975, Salisbury et al. 1995, Khangura and Barbetti 2004).

Resistance to blackleg has increased in Australian breeding programs

since the 1970s (Cowling 2007). Cargeeg and Thurling (1980) proposed that

11

‘adult plant resistance’ to blackleg was polygenic due to the range in the level of

resistance among breeding populations.

Early work by Roy (1978) and Sacristan and Gerdemann (1986)

putatively transferred adult plant resistance to blackleg from the A genome of

Brassica juncea (Genome = AABB) to the A genome of B. napus (Genome =

AACC). By 1984 a low erucic acid line (OnapJR) with complete (seedling and

adult) resistance to blackleg had been found in progeny from B. napus – B.

juncea interspecific hybridisation (Roy 1984). OnapJR was later crossed with

partially resistant B. napus lines to produce progeny adapted to cold, wet and

highly diseased conditions (Roy 1984). Roy (1984) did not conduct cytological

studies in an attempt to identify introgressed donor germplasm and was unable

to establish homozygous or stable lines with the B. juncea type resistance.

Attempts to incorporate the ‘B genome resistance’ in B. napus were

made by producing B. napus – B. nigra somatic hybrids (Gerdemann-Knorck et

al. 1995). High levels of resistance to blackleg were achieved using this

method, although further breeding work is required to stabilise the resistance in

individual plants. B. napus – B. juncea hybrids generated by Barret et al.

(1998) were claimed to have high levels of blackleg resistance (including

cotyledon resistance) introgressed from B. juncea. Two theories were proposed

for the introgression of this fragment. The first theory suggested that the

resistance was carried on a chromosome that had substituted for an entire B.

napus chromosome. The second theory suggested the loci carrying resistance

were located on a fragment homologous to that in B. napus, thus permitting

homologous recombination. Earlier work by Roy (1978, 1984) indicated that the

B. juncea ‘complete’ resistance was carried on the B genome thus making it

unlikely to be transferred via homologous recombination. Results published by

Barret et al. (1998) indicates possible incorporation of improved A genome type

resistance from B. juncea.

In the study of a cross between the susceptible B. napus cultivar ‘Tower’

and a B. juncea-type resistant B. napus line, Pang and Halloran (1996)

suggested that adult plant resistance to blackleg is governed by three major

genes with complex interactive effects. Mayerhofer et al. (1997) mapped

blackleg resistance to a single locus in the Australian cultivars Shiralee and

Maluka and subsequently used these cultivars to generate Canadian cultivars

with high levels of blackleg resistance.

12

The B. napus cultivar Surpass 400, released in 2000 in Australia,

contained a powerful new resistance gene that was monogenically dominant (Li

and Cowling 2003). Cultivars with this gene, introduced from a wild B. sylvestris

plant (Crouch et al. 1994), had excellent resistance to blackleg until a break

down of the B. sylvestris resistance was observed (Li et al. 2003). Breakdown

of this resistance was subsequently confirmed by surveys conducted in 2003 on

cultivars with this monogenic resistance (Canola Association of Australia 2004).

In the same year canola crops on the Eyre Peninsula and several other regions

of Australia suffered serve blackleg related losses. L. maculans can rapidly

overcome major gene resistance to blackleg in Australia (Li et al. 2005) and in

Europe (Brun et al. 2001).

Pests

Key invertebrate pests of canola attack crops at the establishment stage or at

the flowering and pod fill phase (Miles and McDonald 1999). During crop

establishment canola is attacked by a number of insect pests including the red-

legged earth mite (Halotydeus destructor), blue oat mite (Penthaleus spp.),

lucerne flea (Sminthurus viridis) and false wireworm (Isopteron punctatissimus,

Adelium spp.). Pests that specifically attack canola crops during the flowering

and pod fill phase include aphids (turnip aphid, Lipaphis erysimi, cabbage

aphid, Brevicoryne brassicae, and green peach aphid, Myzus persicae), native

budworm (Helicopvera puntigera) and Rutherglen bug (Nysius vinitor).

Recently diamondback moth (Plutella xylostella) has emerged as a serious

threat to canola crops. This pest became prominent in the Northern agricultural

region of Western Australia in 2000 and the major outbreak was attributed to

unseasonal weather conditions (Cook et al. 2000).

Yield

Yield improvement in canola remains the primary breeding objective as for most

other crop species. Yield is a complex trait, the end result of many

physiological and morphological traits that are quantitatively inherited as well as

environmental factors that affect a crop over the entire growing season.

Thurling (1974a, 1974b) found that a reduction of yield occurred in both B.

napus and B. rapa with delays in sowing date and that this was most likely a

13

result of a reduced duration of pre-anthesis growth. Thurling and Vijendra Das

(1979) suggested that higher yield is determined by pre-anthesis vegetative

growth. In low rainfall zones of Southern Australia, particularly at lower

latitudes, drought stress and high spring temperatures have potential to hamper

crop yield and quality. One solution is the breeding of early flowering varieties

that can form siliqua and start filling seed prior to the onset of heat and drought

stress. However, early flowering itself may not increase yield because this trait

shortens the period of vegetative growth prior to anthesis (Thurling 1983). In

order to maintain viable yields whilst reducing the time to flowering it is

important to increase the relative growth rate of the plant during this pre-

anthesis period. In addition to this, Lewis and Thurling (1994) demonstrated

that yield may be improved in crops facing terminal drought through improved

water use efficiency during the post-anthesis period. In that study B. juncea

had better post-anthesis water use efficiency than B. napus and B. rapa, but

similar total water use during the growing season. The efficient post-anthesis

water use of B. juncea enabled this species to generate a higher yield (Lewis

and Thurling 1994). Niknam et al (2003) found that osmotic adjustment

occurred in water stressed Brassica and varied between species and cultivars.

It was also observed that those accessions with a high osmotic adjustment

suffered less yield reduction in water limiting conditions.

Weeds

Canola is regularly challenged by a number of grass and broadleaf weeds

including many weeds from the Brassicaceae family such as wild turnip (B.

tuornefortii) and wild radish (Raphanus raphanastrum) (Sutherland 1999).

Minimum or no-till cropping relies on herbicide application at both pre- and post-

emergent stages of crop development. Post emergent weed control is also

dependent on resistance in the crop species to the herbicide being applied.

Herbicide resistance is a valuable trait carried in a number of Australian

canola varieties. The most common resistance is to the Group C triazine

herbicides. Triazine tolerance was discovered in B. rapa (Maltais and Bouchard

1978) and was later found to be carried in cytoplasmic DNA and inherited

maternally (Souza-Machado et al 1978). This trait has since been introduced in

B. napus populations (Beversdorf et al 1980). This expands the control options

14

for related (and other) broadleaf leave weeds such as wild radish as well as

annual ryegrass that are serious competitors and contaminators of canola crops

(Littlewood and Garlinge 2001). A yield penalty is observed with the triazine

tolerant (TT) cultivars (Röbbelen 1987, Robertson et al 2002). Despite this yield

suppression, canola production is dominated by triazine tolerant canola in

Australia due to major benefits from weed control in minimum till cropping

systems. Recently varieties with resistance to the Group B imidazolinone

herbicides have been introduced to Australia, which provide farmers with

greater flexibility in weed control (Littlewood and Garlinge 2001). However,

resistance to Group B herbicides develops rapidly in weed species and

consequently producers must plan carefully to integrate these herbicide

resistant canola varieties into their crop rotations (Sutherland 1999).

Silique shatter

Most Australian canola crops are swathed to regulate seed maturity and to limit

pod shattering. Swathing adds $20/ha to input costs (Carmody 2001) and has a

significant bearing on the gross margin of canola production, particularly in

marginal regions where yields are low. While breeding has reduced the degree

of pod shatter, almost all varieties will shatter to some degree and yield losses

increase proportionally as swathing is delayed beyond maturity. Optimal

swathing time is when half or more of the seed has lost its green colour

(Carmody 2001). A number of Brassica relatives with higher levels of

resistance to shatter have been identified as a potential source of shatter

resistance in B. napus, such as Sinapis alba (Brown et al. 1997) and B. juncea

(Prakash and Chopra 1988; Prakash and Chopra 1990). Shatter resistance has

also been introduced into B. napus through the resynthesis of B. napus from its

diploid progenitor species B. rapa and B. oleracea (Morgan et al. 1998), and is

believed to be inherited polygenically and independently of other agronomic

traits (Morgan et al. 2000).

Oil content and quality

Seed oil content is inversely related to seed protein content (Singh et al. 2001,

Si et al. 2003) and this is particularly prominent in low rainfall, high temperature

regions. The oil content of canola in these areas often falls below the current

15

standard (Table 1) and in unfavourable years large areas of the Australian

canola crop are affected by low seed oil. Further breeding effort is needed to

stabilize oil content and quality under drought and heat stress conditions.

Nevertheless, Australian canola meets the global standards for oil content and

is widely recognised for its low levels of moisture, admixture and chlorophyll, all

of which assure a major advantage for buyers and processors of Australian

canola (Australian Oilseeds Federation 2007).

The frying vegetable oil market may demand a reduction in saturated fats

and increase in the mono-unsaturated oleic acid content of Australian canola in

the future (Mailer 1999).

In 1999 large areas in Western Australia were planted to a single cultivar,

Karoo (Nelson 2000). Unfortunately, this variety had a low proportion of

individual plants with high levels of erucic acid that affected the overall oil

quality and caused problems for the Australian export market (Nelson 2000,

Cowling et al. 2001). Canola breeding is necessary to develop high quality

cultivars with stable seed quality and greater adaptation to season-end

stresses.

Brassica napus, its evolution and relatives

Over 95% of oilseed rape grown in Australia is Brassica napus var. oleifera

(West et al. 2001). B. napus is a member of the Brassicaceae family of plants

that have a multitude of uses and have benefited mankind for thousands of

years. This family includes species that are cultivated as oilseeds, vegetables

and condiments. The vegetable Brassicas (B. oleracea and B. rapa) have a

range of morphotypes including leafy rosettes, heading forms, swollen stems

and enlarged roots (Labana and Gupta 1993), whereas the other species B.

nigra, B. napus, B. juncea and B. carinata are predominantly oleiferous forms

with some morphotypes suitable for use as vegetable crops.

It is believed that B. napus first appeared near Flanders around the year

1600 where it quickly replaced the winter turnip rape throughout north-western

Europe (Labana and Gupta 1993). Its origins, despite being relatively recent,

parallel other amphidiploid Brassica species that appeared many years earlier.

It is assumed that B. napus originated in South-Eastern Europe around the

16

Mediterranean where populations of its progenitor species B. rapa and B.

oleracea overlap (Labana and Gupta 1993).

U’s triangle and the Brassica genomes

A Korean botanist working in Japan (U 1935) proposed the theory of a

triangular relationship amongst six oilseed and vegetable Brassicas. This

triangle (Fig. 2), traditionally known as U’s triangle, allocates alphabetic genome

descriptors and illustrates the relationship between the three main diploid

species B. rapa (AA), B. nigra (BB) and B. oleracea (CC) and their amphidiploid

progeny B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC).

Figure 2. The ‘Triangle of U’, showing genome descriptors and progenitor

genomes of the amphidiploid species (from U 1935).

The triangle of U has been well supported by a number of research groups

using different techniques. These included studies on amphidiploid production

and chromosome pairing (Prakash and Hinata 1980), nuclear DNA content

(Verma and Rees 1974), DNA analysis (Erickson et al. 1983) and the use of

genome specific markers (Hosaka et al. 1990). Cytogenetics and genome

relationships among Brassica crops and relationships to the model plant

Arabidopsis are reviewed recently by Snowdon (2007).

B. rapa

AA

2n = 2x = 20

B. oleracea

CC

2n = 2x = 18

B. nigra

BB

2n = 2x = 16

B. juncea

AABB

2n = 4x = 36

B. carinata

BBCC

2n = 4x = 34

B. napus

AACC

2n = 4x = 38

17

Studies on pachytene chromosome analysis provide evidence to suggest

there are six Brassica genomes observed within the diploid Brassica species

(Röbbelen 1960, Venkateswarlu and Kamala 1971) and are classified as the A,

B, C, D, E, and F genomes. Other reports suggest there may have been as few

as five or as many as seven (Truco et al. 1996). Chromosomes of these five to

seven original types within the present three diploid genomes have lost

homology due to duplication and rearrangements (Prakash and Chopra 1993,

Truco et al. 1996). Cytogenetic reports indicate the three diploid species

evolved from a common progenitor species (Prakash and Chopra 1993),

however more recent molecular studies (Song et al. 1988, Yanagino et al. 1987,

Truco et al. 1996), suggest a biphyletic origin of the diploid Brassicas where B.

rapa and B. oleracea evolved from one lineage and B. nigra evolved separately

along another lineage.

The amphidiploid oilseed B. napus appears to have arisen

spontaneously through natural hybridisation of B. oleracea and B. rapa (Chen

and Heenan 1989). The progenitor species of B. napus (B. rapa and B.

oleracea) have many sub-species or varieties (Prakash and Hinata 1980).

Chen and Heenan (1989) deduced that the narrow range of genetic variation

found within B. napus resulted from the hybridisation of very few of these

parental genomes. Other investigators (Hosaka et al. 1990, Olsson 1960 and

Song and Osborn 1992) suggest that B. napus arose from polyphyletic origins

rather than a single hybridisation event between the two parent genomes.

Despite these theories of polyphylogeny in B. napus it is accepted that the

genetic diversity of oilseed rape (Brassica napus) is small. Becker et al. (1995)

identifies two probable causes for this: (1) rapeseed is of relatively recent origin

and extensive cultivation and breeding of this crop species did not occur until

little over 50 years ago; and (2) the species has a narrow genetic base. The

present breeding material of oilseed rape is derived from very few interspecific

hybrid plants that occurred spontaneously some centuries ago (Becker et al.

1995).

Many studies have attempted to re-create the amphidiploid Brassica

species using a number of hybridisation techniques. B. carinata and B. juncea

were easier to resynthesise than B. napus (Prakash and Chopra 1993), possibly

as a result of the more recent evolution of B. napus compared with its

amphidiploid relatives. Approaches taken to generate synthetic B. napus from

18

B. rapa and B. oleracea have included grafting (Hosoda et al. 1963 and Namai

1971), mixed pollination (Feng 1955 and Sarashima 1964), style excision

(Hososda et al. 1963), embryo culture (Nishi et al. 1961, Nishi et al. 1970 and

Matsuzawa 1984) and ovary culture (Inomata 1978). More recently resynthesis

of B. napus has become a common practice for the introduction of desirable

variation from its diploid parents (Chen and Heenan 1989, Becker et al. 1995)

and has practical implications for crop improvement.

The Brassica genomes have been the subject of many studies on

chromosome interaction, homology and wide hybridisation. The basis of these

studies has paved the way for future research into germplasm improvement of

B. napus and its oilseed relatives.

Introducing genetic variation for the improvement of Brassica napus

Kumar et al. (1988) state that the genus Brassica, with its large number of wild

species, had the potential to donate many new nuclear/organelle genes to the

different cultivated species for improvement in the range of edible oils and

vegetables. However, because of pre- and post fertilisation barriers,

hybridisation between wild and cultivated species has not been very successful.

Sources of genetic variation for Brassica napus

Spontaneous mutations may often go unnoticed although occasionally they

provide useful variation for crop improvement (Muangprom and Osborn 2004).

Induced variation has been used extensively with Brassicas to provide genetic

variation for disease resistance (Yadav et al. 2001), altered oil profile (Velasco

et al. 1998), shatter resistance (Kadkol 1987) and yield (Rahman and Das

1991). Despite valuable results that have been obtained by a number of

groups, the use of induced mutagenesis is limited due to diplontic selection,

adverse linkages and the undesirable pleiotropic effects normal associated with

mutants (Dhillon et al. 1993).

Genetic transformation is another method of creating genetic variation in

B. napus where gene sequences may be taken from any organism. Genetic

modification by plant transformation has advanced more rapidly in Brassica

19

species than many others because of the rapid advance of tissue culture

methods (Moloney and Holbrook 1993). By using genetic transformation,

interspecies and wider genetic barriers can be by-passed. Genetic

transformation is an expensive and time consuming approach but does provide

a direct approach for the introduction or improvement of a desired trait.

New variation has been observed for a number of agronomic traits in

progeny derived from B. juncea somaclones (Katiyar 1997, Jain et al. 1989).

Despite promising variation arising from somaclonal variation, direct application

of these techniques to generate variation are uncommon in contemporary

breeding programs where breeders are seeking specific trait improvements.

In most crop species, crossing and recombination of genes and alleles is

still the most widely used and basic method of introducing new genetic

variation. However, its application becomes limited when the target alleles lie

across species barriers. This problem is compounded by the restricted genetic

diversity within the primary gene pool of B. napus oilseed rape germplasm

(Becker et al. 1995).

The secondary and wider gene pools

Centres of origin as first described by Vavilov are regions of high genetic

diversity for domesticated species (Vavilov 1951). These centres of origin may

provide a source of new genes and alleles for particular traits in breeding

programs. This is valuable in some crop species where local varieties have

become extinct or where domestication has reduced the genetic base to a

limited number of varieties (Borojevic 1990). Wild populations of B. napus have

not been found (Prakash and Hinata 1980) around its proposed centre of origin,

thus the only ancestral sources of variation for B. napus may lie with

domesticated and wild relatives of the progenitor genomes in B. rapa and B.

oleracea.

Using interspecific hybridisation to introduce genetic variation in B. napus

Hermsen (1992) outlined the importance of selecting new alleles for crop

improvement from within the species where possible. This approach avoids the

introduction of large quantities of alien DNA and the subsequent associated

genetic disruption. As previously outlined, the gene pool of B. napus is limited

20

(Becker et al. 1995). Breeders have little choice but to search the secondary

gene pool to find sources of improvement for desired traits.

Two populations constitute different species when there is no gene flow

between them because of genetic barriers (Lacadena 1978). With manipulation

these interspecies barriers may be overcome to make use of beneficial

germplasm of closely related species. Interspecific hybridisation within the

Brassicas has enabled improvement of disease resistance (Roy 1984, Somers

et al. 2002), herbicide resistance (Ayotte et al. 1987), pod shatter resistance

(Prakash and Chopra 1990), oil profile (Getinet at al. 1994) and flowering time

(Rao et al. 1993a, Rao et al. 1993b).

Interspecific barriers to wide hybridisations

Barriers such as spatial isolation, non-synchronous flowering and cleistogamy

may be easily controlled in a breeding program (Hermsen 1992). For the

complete process of interspecific hybridisation to take place, there must be no

restriction imposed on the processes that take place after pollination and there

must be homology in the genetic processes that take place between the pollen

and the host plant. Hogenboom (1973) uses several terms when referring to

interspecific crosses such as ‘incongruity’ to describe non-homology in the

genetic process, ‘penetration capacity’ to describe mechanisms controlled by

the pollen genes to circumvent barriers against hybridising alien females, and

‘barrier capacity’ to note the genetic regulation of barriers against hybridisation

within the female plant by male pollen.

Prezygotic barriers may occur on the stigma, style or ovary of the host

plant and are well documented for many interspecies crosses. Hybridisation

may not occur when pollen fails to germinate due to a mismatch of stigma

fluids. However, Heizmann et al. (2000) found few problems with the adhesion

of pollen to stigmas in both intra- and interspecific crosses of Brassicaceae

species. Wider crosses in this family were shown to exhibit reduced pollen

adhesion to the stigma (Luu et al. 1998). Barriers to pollen tube growth occur in

either the stigma or style of the host plant. Yadav et al. (2002) observed

variation in the rate of pollen tube growth amongst several Brassica interspecific

hybrids. Rate and frequency of pollen tube growth was seen to be affected by

the direction of the cross, where reciprocal crosses favoured one direction. Luu

21

et al. (1998) suggested most interspecific pre-zygotic barriers occur within the

style after the pollen grains have germinated.

Post zygotic impedance of sexual hybridisation in remotely related

species may result in fully sterile F1 plants due to lack of homologous pairing of

chromosomes (Hermsen 1992). Chromosome elimination, sub-lethality, or

other abnormalities of plant growth, male sterility, poor flowering of F1 plants,

disharmonious karyotypes in segregating generations, all causing hybrid

breakdown are typical post-zygotic barriers to hybridisation. Subsequently

hybrids with these faults rarely produce viable seed and their genetic variation is

lost.

Overcoming barriers to interspecific hybridisation

Pre-fertilisation barriers can be overcome by grafting, mixed pollination, style

excision, treatment of the style with chemicals and embryo rescue (Prakash and

Chopra 1993), whilst post-fertilisation barriers are overcome by in-vitro

techniques such as ovary, ovule and embryo rescue. Techniques used to

overcome Brassica interspecific hybridisation barriers are listed in Table 2

(modified from Prakash and Chopra 1993).

Table 2. Techniques for overcoming barriers to interspecific hybridisation in

Brassica species (modified from Prakash and Chopra 1993).

Technique Reference Grafting Hosoda et al. (1963), Namai (1971) Mixed pollination Feng (1955), Sarashima (1964) Style excision Hosoda et al. (1963) Embryo culture Nishi et al. (1961), Ayotte et al.

(1987), Chrungu et al. (1999), Kumar et al. (2001)

Ovary culture Inomata (1978), Inomata (2002)

Despite wild B. napus populations never being found there is an immense

amount of genetic diversity within its progenitor genomes. Research has shown

that interspecific barriers in the Brassica family can be overcome.

22

Analysis of Brassica interspecific hybrid progeny

Lacadena (1977) stated that the main objective of interspecific hybridisation in

plant breeding was to expand the gene pool and to introduce alien genes

carried by wild species into cultivated varieties. This statement may be

extended to include not only wild species but also closely related species in all

stages of domestication. Location and identification of genetic sequences

responsible for new variation in recipient Brassica genomes assist in the

stabilisation of these traits in future generations. Interspecific progeny have

been analysed both phenotypically and/or genotypically to characterise the

range of new genetic variation.

Phenotypic analyses

The progeny of Brassica interspecific hybrids are screened for morphological

and physiological traits such as yield, disease and/or quality traits that differ

from the parental material. Environmental influences are known to significantly

affect phenotypic analyses. Richards (1978) found maximum response to

selection for drought stress in B. rapa and B. napus in the target (low rainfall)

environment.

Genotypic analyses

The ability to detect, locate and characterise foreign DNA from a donor genome

in progeny of an interspecific cross will assist selection of desirable genotypes

and phenotypes (Basunanda et al. 2007). Selection may occur for alien DNA in

the early generations of selfing or backcrossing, thus increasing the probability

of securing useful new traits to the host species.

Identification of alien chromatin in wide crosses has been achieved by

using cytogenetic techniques to identify chromosome insertions (Snowdon

1997), flow cytometry to quantify chromosome content (Sabharwal and Dolezel

1993) and molecular markers to trace chromosome and DNA transfer (Quiros

1991, Basunanda et al. 2007).

Cytogenetics

Direct observation of chromosomes can be a useful tool in the analysis of

interspecific hybrids. Chromosomal rearrangements may be observed and

23

correlations drawn between these and phenotypic observations. The in situ

detection of nucleic acid sequences, whether of genes on chromosomes or

viruses or of mRNA in tissues, provides a direct visualisation of the spatial

location of sequences that is crucial for elucidation of the organisation and

function of genes (Wilkinson 1998). In situ hybridisation involves using a

labelled probe (radioactive or fluorescent) to detect complementary sequences

in target DNA. The probe may be either digested genomic DNA (genomic in

situ hybridisation, or GISH) or any gene or DNA sequence used as a probe

(fluorescence in situ hybridisation, or FISH).

GISH has been used as tool to identify Brassica genomes in interspecific

and intergenomic hybrids. By using genomic DNA probes from the three diploid

species the A and B genome chromosomes could be distinguished in B. juncea,

as well as the B and C genome chromosomes in B. carinata (Itoh et al. 1991,

Snowdon et al. 1997). Hosaka et al. (1990) were not able to distinguish

between the chromosomes of these two genomes, possibly due to the relative

homology of the A and C genomes. On the basis of this report, along with the

small size of Brassica chromosomes, it appears GISH may not be a suitable

method for identifying C genome introgressions in the AA (B. rapa) or AABB (B.

juncea) background.

Using a sequence originally reported by Harrison and Heslop-Harrison

(1995), Armstrong et al. (1998) used FISH with a highly repeated sequence

(pBcKB4) from B. campestris (B. rapa) to demonstrate the presence of C-

genome in wide Brassica crosses. This probe was observed to co-localise with

pericentomeric heterochromatin on the nine chromosomes of B. oleracea (CC).

The probe was not totally genome specific, but may prove useful in the

detection of whole C genome chromosomes in the B. juncea (AABB)

background. This marker may however fail to identify translocation of distal or

telomeric C genome chromosome fragments in an A or B genome background.

FISH markers, based on reverse transcriptase domains in retroelements of B.

oleracea, showed characteristic distributions on the C genome (Alix et al. 2005).

Localisation of alien introgressions

There has been much discussion on the ability of transgenes in B. napus

to recombine with other species in interspecific and other wide crosses

24

(Jorgensen and Andersen 1994). This has prompted investigations of ‘safe

integration’ sites within the B. napus genome which minimise transgene

recombination in wide hybrids (Mikkelsen et al. 1996, Tomiuk et al. 2000). This

concept has implications for the introgression of alien DNA during the creation

of Brassica interspecific hybrids. It would be virtually impossible to target

specific sites in B. napus for introgression through interspecific hybridisation.

However, it would be noteworthy to determine if there are certain chromosomal

regions that are consistently recombining in these wide crosses.

The stability of B genome additions or introgressions in the A or C

genome of B. napus needs further investigation. Non-homologous

chromosome pairing has been reported in the A and B genomes of Brassicas

(Prakash 1973) but putative introgressions have not always been stable (Roy

1984). There is little research to date that shows preferential introgression or

recombination in the progeny of Brassica interspecific hybrids.

Molecular genetics

Koornneef et al (1997) state that genetics becomes more powerful when

combined with molecular genetics, which links DNA and phenotype. An array of

molecular techniques has emerged over the past 25 years, many of which have

benefited plant breeding. Perez de la Vega (1993) outlines three marker

systems that supplement phenotypic analysis in plant populations. These are

(1) biological compounds or non-protein low molecular weight plant compounds,

(2) proteins, including enzymes and storage proteins and (3) DNA markers

obtained by restriction enzymes, by PCR and base sequences. The third group

mentioned here have become particularly prominent in modern plant breeding.

Some examples of molecular genetic uses for a range of breeding and genome

mapping exercises in the Brassica species include DNA markers based on

restriction fragment length polymorphisms (RFLP) by Lagercrantz and Lydiate

(1996), randomly amplified polymorphic DNA (RAPD) (Thormann et al. 1994),

and PCR based microsatellite DNA sequences (Uzunova and Ecke 1999) and

have been comprehensively reviewed by Snowdon and Friedt (2004). These

techniques are often used with other genotypic analyses to study Brassica

interspecific hybrids and have all proven to be useful tools in the advancement

of Brassica germplasm.

25

Genome specific sequences

Genome and species specific marker studies in Brassica have been reported a

number of times. Chen and Heenan (1990) have used genome specific

isozyme markers to study chromosome behaviour in interspecific hybrids of B.

napus and B. campestris (B. rapa). Chèvre et al. (1991) used isozyme, RFLP

and fatty acid markers that expressed genome specificity in the analysis of B.

napus B-genome addition lines.

Hosaka et al. (1990) generated genome specific markers based on RFLP

probes that were useful in identifying A and C genome DNA (Table 3). The C

genome specific markers were specific to the C genome when probed to

genomic DNA in B. oleracea, B. napus and a synthetic B. napus line. Two

probes were identified that bound exclusively to C genome DNA (pB547 and

pB870) in both diploid B. oleracea and amphidiploid B. napus. Not all of the

probes studied bound exclusively to species containing C genome. Hosaka et

al. (1990) stated that these two probes were dispersed and provided evidence

to suggest that these probes hybridise to five of the C genome chromosomes.

It is not stated whether these probes hybridise to locations on all of the nine C

genome chromosomes. Some of the probes that initially appeared to be C

genome specific also hybridised to fragments from alternative genomes (pB845)

suggesting a degree of genome homology.

Iwabuchi et al. (1991) isolated and cloned an 88 base pair repetitive

sequence present in the A genome of B. campestris (syn. rapa) (pCS1). This

sequence showed no homology to the C-genome found in B. oleracea. This

probe may also hybridise to A-genome chromosomes of B. napus. The

sequence was reported to be a middle repetitive element with an approximate

copy number of 1,680 (Iwabuchi et al. 1991). In situ hybridisation analysis

exhibited strong signals on three of the ten A-genome chromosomes indicating

that this sequence is not distributed across the entire A-genome. Itoh et al.

(1991) successfully used the probe CS1 in the detection of A-genome

chromosomes in somatic hybrids of B. campestris and B. oleracea. The

genome specific markers mentioned here are based on digested genomic

clones.

26

Table 3. Genome specific markers generated by Hosaka et al. (1990)

Clone Presence Absence

pB177 A C B

pB185 C A B

pB320 C A B

pB370 A C B

pB485 A B C

pB488 A B C

pB547 C A B

pB845 C A B

pB850 C A B

pB870 C A B

Quiros et al. (1991) reported the application of RAPD based genome specific

markers. Sixty-five genome specific markers were identified found in both the

diploid and derived amphidiploid species. Sixteen of these markers were A-

genome specific, 37 were B-genome specific and 12 were C-genome specific.

Out of the 37 B-genome specific markers, 11 were mapped to four independent

B-genome chromosomes in B. napus-nigra addition lines. RAPD markers have

also been used to identify seven of the eight B-genome chromosomes (Struss

et al. 1992).

To date, there have been no publications that identify A or C genome

specific markers that are dispersed across the entire genome of the respective

diploid species. Quiros et al. (1991) could not confirm whether their markers

were dispersed across every chromosome for each of the three genomes. The

difficulty involved in identifying unique A and C genome specific markers

supports theories on the relative homology of these two genomes (Song et al.

1988). Snowdon et al. (2002) was able to distinguish between A and C-genome

chromosomes in B. napus using cytogenetic techniques however molecular

markers would be even more useful for plant breeding applications wherever

wide crosses are made through interspecific hybridisation of Brassica species.

Gupta et al. (1992) reported the isolation of Brassica B genome specific

sequences. Two sequences were used as hybridisation probes with one

proving to be highly B genome specific. This 496 base pair sequence

(pBNBH35) was observed to hybridise to species carrying Brassica B-genome

chromosomes. Gupta et al. (1992) identified another B genome specific repeat

27

sequence (pBN-4), that is believed to be highly dispersed across all B-genome

chromosomes (Kapila et al. 1996). With respect to their repetitive and broad

distribution, coupled with the low homology of the B genome with the A and C

genomes (Song et al. 1988, Yanagino et al. 1987), these markers may prove

efficient in identifying B genome introgressions into A and or C genome

backgrounds subject to further investigation and development.

Conclusions

Rapid adoption of canola in Australia during the 1990s demonstrates the need

for this crop in Australian farming systems, although recent reductions in area

indicate that genetic improvements are needed. Yield, quality and disease

resistance remain major breeding objectives coupled with improved agronomic

practices. It is unlikely that the yield limits of this crop are fully realised.

Improved cultivars will come from breeding programs that select new genetic

variation and useful allelic combinations for the benefit of the Australian canola.

Canola in Australia has similar restrictions in genetic diversity to canola in other

countries. Given the limited genetic base of B. napus, genetic resources in the

secondary gene pool become important for future genetic improvements. There

is a useful array of genetic tools and analyses that can be used to assist in the

improvement of crop species today.

28

Chapter 3 – Published paper

Schelfhout CJ, Snowdon R, Cowling WA, Wroth JM (2004) A PCR based B-

genome-specific marker in Brassica species. Theoretical and Applied Genetics

109, 917-921.

Theor Appl Genet (2004) 109: 917–921DOI 10.1007/s00122-004-1713-x

ORIGINAL PAPER

C. J. Schelfhout . R. Snowdon . W. A. Cowling .J. M. Wroth

A PCR based B-genome-specific marker in Brassica species

Received: 19 January 2004 / Accepted: 27 April 2004 / Published online: 26 June 2004# Springer-Verlag 2004

Abstract Previous hybridisation studies showed that therepetitive DNA sequence pBNBH35 from Brassica nigra(genome BB, 2n=16) bound specifically to the B-genomeand not to the A- or C-genomes of Brassica species. Weamplified a sub-fragment of pBNBH35 from B. nigra byPCR, cloned and sequenced this sub-fragment, andconfirmed that it was a 329-bp sub-fragment ofpBNBH35. PCR and hybridisation techniques were usedto confirm that the pBNBH35 sub-fragment was BrassicaB-genome-specific. Fluorescence in situ hybridisation(FISH) in B. nigra, B. juncea (AABB, 2n=36) and B.napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica B-genomechromosomes and absent from the A- and C-genomechromosomes. The pBNBH35 repeat was localised to thecentromeric region of each B-genome chromosome. FISHclearly distinguished the B-genome chromosomes fromthe A-genome chromosomes in the amphidiploid speciesB. juncea. This is the first known report of a B-genomerepetitive marker that is present on all B-genomechromosomes. It will be a useful tool for the detectionof B chromosomes in interspecific hybrids and may proveuseful for phylogenetic studies in Brassica species.

Introduction

Large regions of eukaryote genomes are characterised byrepetitive DNA sequences (Flavell 1980; Jelinek andSchmid 1982) which generally appear in one of severaldifferent forms—tandemly repeated sequences, retro-elements and other unique classes such as telomericsequences or rDNA units (Heslop-Harrison 2000). Theserepetitive DNA sequences have been useful as markers inphylogenetic studies (Halldén et al. 1987) as repetitivesequence motifs tend to be highly conserved withinspecies but vary across species (Heslop-Harrison 2000).Species-specific sequences have been identified in Bras-sica species, and homology often exists between A- andC-genome repetitive sequences while there are fewersimilarities between B-genome and A- or C-genomesequences (Hosaka et al. 1990; Chèvre et al. 1991). Thisis consistent with the results of genomic studies ofBrassica species that have identified close relationshipsbetween the A- and C-genomes and more distantassociations with the B-genome (Song et al. 1988; Quiroset al. 1991; Truco et al. 1996).

Prior to this communication no Brassica B-genomespecific sequences had been shown to be present on alleight B-genome chromosomes. Gupta et al. (1992) clonedtwo B. nigra genome-specific sequences, one of which—pBNBH35—proved to be highly B-genome specific.Southern hybridisation analysis showed that this 496-bpsequence hybridised to all species carrying the Brassica B-genome (Gupta et al. 1992). Southern hybridisationanalysis suggested that pBNBH35 is a highly dispersedsequence. Kapila et al. (1996) showed that pBNBH35 waspresent on five monosomic addition lines of B. nigra in aB. napus background (Chèvre et al. 1991), however theywere unable to identify this sequence on all eight B. nigrachromosomes, and there have been no further studiesreported.

The objective of the investigation reported here was toconvert pBNBH35 into a PCR-based marker and confirmits specificity to all eight chromosomes of the Brassica B-genome using PCR, DNA hybridisation and in situ

Communicated by H. C. Becker

C. J. Schelfhout (*) . W. A. Cowling . J. M. WrothSchool of Plant Biology, Faculty of Natural and AgriculturalSciences, The University of Western Australia,35 Stirling Hwy,Crawley, WA, 6009, Australiae-mail: cschelfh@agric.uwa.edu.auTel.: +61-8-64882560Fax: +61-8-64881108

R. SnowdonInstitut für Pflanzenbau und Pflanzenzüchtung I, Justus LiebigUniversitat,Heinrich-Buff-Ring 26-32,35392 Giessen, Germany

W. A. CowlingCanola Breeders Western Australia Proprietary Limited,15/219 Canning Highway,South Perth, WA, 6151, Australia

hybridisation techniques. The potential use of this se-quence as a B-genome-specific marker is discussed.

Materials and methods

Plant material

The plant material used in this study is shown in Table 1. Seeds weresown in pots containing a standard potting mix and slow-releasefertiliser and were propagated in an air-conditioned glasshouse at20–25°C. At the six-leaf stage, the youngest two leaves from eachplant were collected, snap frozen in liquid nitrogen and stored at−80°C.

Total genomic DNA extraction and quantification

Sufficient leaf tissue to half-fill a 1.5-ml tube was removed from−80°C storage and macerated in 600 μl of DNA extraction buffer(10 g l−1 N-lauroyl-sarcosine, 3.2 g l−1 EDTA, 12.1 g l−1 Trizmabase, 12.6 g l−1 sodium sulfite, 5.8 g l−1 sodium chloride, pH 8.5).The tubes were kept on ice for 10 min, after which 600 μl of phenol:chloroform:iso-amyl alcohol (25:24:1) was added. The contentswere mixed for 1 min and centrifuged at 13,000 rpm for 10 min. Thesupernatant was transferred to a fresh tube along with 40 μl of 3 Msodium acetate and 250 μl isopropanol, and the tubes were invertedto encourage precipitation. After 10 min at room temperature thetubes were centrifuged at 13,000 rpm for 10 min. The supernatantwas discarded and the DNA pellet washed with 500 μl 70% ethanol,then centrifuged at 13,000 rpm for 5 min. Excess ethanol wasdecanted and the DNA pellet gently vacuum-dried for 10 min. DNAwas resuspended in 100 μl of R40 (2 μl RNase A in 1 ml TE buffer).Total genomic DNA concentration was estimated against a DNAmass ladder (Gibco, Gaithersburg, Md.) by electrophoresis on a1.5% agarose gel followed by ethidium bromide staining and UVlight visualisation.

PCR assay

Primers were designed to amplify a sub-fragment of the pBNBH35sequence published by Gupta et al. (1992) (Fig. 1). A PCR protocolwas optimised to amplify this sequence in species containing theBrassica B-genome. Template DNA (1 μl of 100 ng/μl) from each ofthe respective Brassica species (Table 1) was added to 24 μl ofmaster mix [2.5 μl 10× reaction buffer (Sigma, St. Louis, Mo.),4.0 μl 1.25 mM dNTPs (Promega, Madison, Wis.), 1 μl 10 mMPrimer 1 (Geneworks), 1 μl 10 mM Primer 2 (Geneworks), 1 μlREDTaq polymerase (Sigma) and 15.3 μl MilliQ water]. The PCRanalyses were run on the following program in a Hybaidthermocycler: one cycle of 4 min at 94°C; 30 cycles of 1 min at94°C, 2 min at 60°C, 2 min at 72°C; a final hold at 4°C.

Sequence analysis

The size of the PCR products was determined by electrophoresis ona 1.5% agarose gel, ethidium bromide staining and visualisationunder UV light. The PCR products were isolated from the gel andcleaned on PCR cleanup columns (Roche, Indianapolis, Ind.).Cleaned samples were again checked for size, then ligated into apGEM T-Easy (Promega) plasmid vector and transformed intoEscherichia coli-competent cells (JB 109, Promega) before over-night cultivation in LB media at 32°C. A sample of the clonecontaining the pBNBH35 sub-fragment was stored at −80°C inglycerol stocks. Sequence analysis was conducted on twoindependently cloned PCR products isolated from B. nigra.Sequencing was performed using the BigDye terminator protocol(Applied Biosystems, Foster City, Calif.) and sequence comparisonsmade using Vector NTI software. Homology of the amplified sub-fragment of pBNBH35 to published DNA sequences was examinedusing a BLASTN database query (Web Angis-BioManager website:http://biomanager.angis.org.au/) on the Genebank main and ESTdatabases.

Specificity of PCR product

To check the B-genome specificity of pBNBH35, DNA wasextracted from each Brassica species in U’s triangle (U 1935)(Table 1) and used as template DNA in the PCR-based assaydescribed above. The cleaned PCR products (10 μl) were screenedon a 1.5% agarose gel as described above. Fragment size wasestimated against a 100-bp ladder (Promega).

Slot blot hybridisation assay

Genomic DNA (20 ng) from B. juncea and B. napus was denaturedin an equal volume of 0.4 N sodium hydroxide and blotted ontoHybond N+ nylon membrane that had been pre-soaked in 2× SSC

Table 1 Brassica species usedin this study

Brassica species Variety name Genome description

B. rapa Pak Choy (Chinese cabbage) 2n=20 (AA)B. nigra Black mustard (ATC 90745) 2n=16 (BB)B. oleracea Cabbage 2n=18 (CC)B. napus Canola cv. Mystic 2n=38 (AACC)B. juncea Indian mustard (line JN29) 2n=36 (AABB)B. carinata Ethiopian mustard 2n=34 (BBCC)

Fig. 1 pBNBH35, a 496-bp B-genome-specific sequence publishedby Gupta et al. (1992) with primers designed (bold) to amplify a329-bp sub-fragment

918

(0.3 M NaCl, 0.03 M Na Citrate) using a Bio-Rad (Hercules, Calif.)slot blot apparatus. Following blotting, the membrane was rinsed in2× SSC, blotted again and dried at 60°C for 30 min. Membraneswere pre-hybridised for 3–4 h at 65°C in the pre-hybridisationsolution [per 30-ml tube: 0.3 g dextran sulphate, 17.55 ml sterilewater, 6 ml of 20× SETS (3M NaCl, 20 mM EDTA, 0.6M Tris-HCl,11 mM tetra-sodium pyrophosphate, pH 8), 6 ml 50× Denhardt’ssolution and 3 ml of 10% sodium dodecyl sulphate (SDS)]. ThepBNBH35 sub-fragment (100 ng) was used as a probe in thehybridisation. It was prepared by denaturation for 10 min at 100°Cwith 1.2 μl random primers (50 μg ml−1) and immediate quenchingon ice. This solution was then combined with 3.2 μl sterile water,2.0 μl oligo buffer (Promega), 0.6 μl Klenow (Promega) at 5 U μl−1

and 3 μl [32 P]-dCTP (20 MBq) and incubated at 37°C for 2 h.Following the incubation period 30 μl 0.8 M EDTA was added tostop the labelling reaction, and the contents of the tube were brieflycentrifuged. The probe solution was then centrifuged for 10 min at10,000 rpm in a sephadex spin column to remove unincorporatednucleotides. The pre-hybridisation solution was replaced with thehybridisation solution (per tube: 0.5 g dextran sulphate, 2.7 mlsterile water, 1 ml SETS, 1 ml 50× Denhardt’s and 50 μl 10% SDS).The probe solution was added and the membrane hybridisedovernight at 65°C. The following day the hybridisation solution wasdiscarded, and the membrane was given two 15-min washes in 2×SSC. Membranes were exposed to film (Fuji medical X-ray) forapproximately 4 days at −80°C and developed in an auto-developer(All-pro imaging). The slot blot matrix was crossed-checked for thepresence of bands.

Fluorescence in situ hybridisation (FISH) assay

Binding of the amplified sub-fragment (pBNBH35-sub) to chromo-somes within the Brassica complex was examined by FISH in orderto confirm its B-genome specificity and distribution within B-genome chromosomes of B. juncea.Four-day-old root tips were obtained from Brassica species

(Table 1) for observation of mitotic chromosomes. Seeds weregerminated on canola germination media [MS salts + B5 vitamins,3% (w/v) sucrose, 0.8% purified agar and myo-inositol (100 mg l−1]at 25°C for 3 days and moved to 4°C for 24 h prior to the excision of1-cm-long root tips. To accumulate metaphases we placed theexcised root tips in 2 mM 8-hydroxyquinoline for 2 h at roomtemperature on a shaker and then transferred them to 4°C for 2 hwithout shaking. The root tips were blotted dry on filter paper andtransferred to Farmer’s fixative (3:1, ethanol:acetic acid). After 24 h,the root tips were transferred to fresh Farmer’s fixative and stored at−20°C. Root tips were transferred to 100% ethanol for long-termstorage at ambient temperatures.To make chromosome preparations we removed the root tips from

the ethanol and washed them briefly in sterile distilled water. Fifteenroot tips were selected from each Brassica species (Table 1). One-millimeter segments of the root tip were excised and transferred to50 μl enzyme solution consisting of 2% (w/v) cellulase (Calbio-chem) and 20% (v/v) pectinase (Sigma) dissolved in enzyme buffer(40 mM citric acid, 60 mM tri-sodium citrate, pH 4.8; filter-sterilisedthrough a 0.2-μm filter). The material was digested for 2 h at 37°C.The enzyme solution was then replaced with 50 μl hypotonicsolution (75 mM KCl), renewed once and left at room temperaturefor 40 min. The hypotonic solution was replaced with 50 μl 60%acetic acid, renewed once and left for 25 min at room temperature,with gentle mixing every few minutes. The second acetic acidsolution was replaced with 50 μl Farmer’s fixative and renewedonce. The root tips were gently sheared with a pipette to separatecells, and 15–20 μl of the cell suspension was loaded onto pre-cleaned cold microscope slides. We immediately added 30 μl ofFarmer’s fixative, and the slides were allowed to air dry slowly. Theslides were viewed by phase-contrast microscopy to identify suitablemetaphase stage cells for hybridisation.For preparation of the FISH probe the pBNBH35 sub-sequence

was directly labelled with the fluorochrome Cy3-11-dUTP (Amers-

ham Life Science, UK) via PCR amplification. Unincorporatedfluorochrome was removed using a PCR cleanup column (Qiagen,Valencia, Calif.), and the labelled probe was eluted at a concentra-tion of approximately 10 ng μl−1 in a hybridisation solutioncontaining 50% formamide, 2× SSC and 10% dextran sulphate.Approximately 15 μl of probe solution was applied to the area of

the slide to be hybridised, covered with a 24×24-mm cover slip andsealed with rubber cement. Denaturation was achieved by incuba-tion at 80°C for 4 min on a heated metal plate. The slides wereimmediately transferred to 37°C for overnight hybridisation. Thefollowing day the rubber seal and cover slip were carefully removedand the slides washed at 42°C for 5 min in 2× SSC followed by twowashes for 5 min in 0.2× SSC and a final wash of 5 min in 2× SSC.The slides were stored in 4× SSC containing 0.5% Tween prior tostaining.DAPI-Antifade (20 μl) (Appligene-Oncor) was added to each

slide, covered with a cover slip, left for 2 min, then squashed under apaper towel to remove excess stain. Slides were stored in the darkuntil viewing. They were viewed under a Leica DM-R fluorescencemicroscope with a single-bypass and photographed with aCohu 4912 uncooled CCD camera. Individual images were mergedusing the Leica Q-FISH software. No image manipulation wasnecessary.

Results

Sequence analysis of the two independently cloned sub-fragments of pBNBH35, amplified by PCR from B. nigra,confirmed their homology with the sequence published byGupta et al. (1992) (Fig. 1). When aligned usingVECTOR NTI software both of the analysed sequencesshowed 92% homology with pBNBH35. The BLASTN

programme indicated no homology greater than 10% withany other sequence in the Genebank main and ESTdatabases. The PCR product showed B-genome specificity

Fig. 2 PCR based assay of six Brassica species with differentgenome compositions. L 100-bp ladder, AA Brassica rapa, BB B.nigra, CC B. oleracea, AB B. juncea, AC B. napus, BC B. carinata,N negative control

919

when amplified from several Brassica species from U’striangle (U 1935) (Fig. 2).

The pBNBH35 sub-fragment was amplified only fromthose samples where the template DNA contained theBrassica B-genome (B. nigra, B. juncea and B. carinata)(Fig. 2). A single band, equivalent to the expected size of329 bp from pBNBH35 (Fig. 1), was generated in eachBrassica species with the B-genome (Fig. 2).

Slot blot hybridisation of the pBNBH35 sub-fragmentgave positive signals with B. juncea (AABB) but not withB. napus (AACC) (data not shown).

Results from in situ hybridisation with the pBNBH35-sub-fragment are shown in Figs. 3 and 4. The molecularcytogenetic results supported the results of the PCR assayand the Southern hybridisations. Strong FISH signals fromthe pBNBH35 sub-fragment were observed on B. nigrachromosomes, whereas no signals were seen in B. napus.Strong hybridisation signals were also observed on 16chromosomes in B. juncea (2n=36), presumably corre-sponding to the 16 B-genome chromosomes. NopBNBH35 sub-fragment signals were observed in theremaining 20 chromosomes of B. juncea.

The pBNBH35 repeat was localised in large blockssurrounding the centromeres of all B-genome chromo-somes, with a gap often visible at the centromere (Fig. 3).Signals did not extend to the telomeric regions.

Discussion

FISH has become the method of choice for analysis of thechromosomal distribution of repetitive DNA sequenceelements in plant genomes (Heslop-Harrison 2000). In theinvestigation described here, FISH was used to investigatethe previously unknown distribution of a Brassica B-genome repeat sequence on B-genome chromosomes. Weconfirmed that pBNBH35 is a repetitive sequence with B-genome specificity, as indicated by Gupta et al. (1992),and we converted the sequence into a PCR product that isamplified only in species containing the Brassica B-genome. The FISH assay showed that this sequence islocalised in high-copy number on either side of thecentromeres of all eight Brassica B-genome chromosomesand appears with low frequency or is absent in theinterstitial and telomeric regions. This is the first reportconfirming a B-genome-specific marker that is distributedacross all B-genome chromosomes.

The PCR sub-fragment from pBNBH35 resolved as aunique single band on agarose gel at the expected size of329 bp, and two independently cloned products from thisband produced identical sequences with the predicted sizeof 329 bp. The pBNBH35 sub-fragment has the propertiesof a highly conserved dispersed tandem repeat sequence,and there was no evidence for sequence homology withany known retro-element. Strong signals surrounding thecentromeres of the chromosomes indicate high-copynumbers in pericentromeric heterochromatin, howeverthere are virtually no signals towards the telomericregions. Such a distribution, frequently observed ingenomic in situ hybridisation (GISH) with Brassica

Fig. 3 Fluorescence in situ hybridisation to B. juncea mitoticmetaphase chromosomes with Cy3-labelled B-genome-specificsequence pBNBH35 (red). Green signals show a FITC-labelled A-genome-specific repeat sequence (Snowdon, unpublished results)that hybridises to four A-genome chromosomes in B. juncea.Chromosomes are counterstained with the blue fluorescent dyeDAPI

Fig. 4 B. napus probed with Cy3-labelled, B-genome-specificsequence. pBNBH35: no red labels

920

hybrids (Fahleson et al. 1997; Skarzhinskaya et al. 1998;Snowdon et al. 2000), reflects the generally low number ofdispersed repeat sequences in the interstitial and telomericchromosome regions of Brassica and related genera(Heslop-Harrison and Schwarzacher 1996).

A variety of applications can be envisaged for this PCR-based, B-genome-specific marker. The marker could beused for detecting the B-genome in the progeny of wideinterspecific and intergeneric crosses where this has notbeen possible for studies in the past (Roy 1984; Prakashand Chopra 1988; Chèvre et al. 1991; Rao et al. 1993)where it may be present in whole chromosomes (additionlines) or introgressions. This marker would complementexisting randomly amplified polymorphic DNA markerswhere linkage to B-genome alleles have been useful inaiding the selection of beneficial traits in Brassica widecrosses (Chèvre et al. 1997). Previous work has demon-strated the utility of GISH to distinguish Brassica B-genome chromosomes from those of the A- and C-genomes (Snowdon et al. 1997). However GISH givesonly cytogenetic information on anonymous genome-specific repeat sequences. In contrast, the availability of aPCR-based tandemly repeated marker enables a combina-tion of exact cytological characterisation by FISH withmolecular genetic analyses. The latter can give consider-ably more insight into the presence or absence of a givensequence or its near homologues and enables a much moreaccurate estimation of repeat copy numbers. Furthermore,tandem repeats tend to remain phylogenetically wellconserved after they are amplified to high-copy numbersin genomes. Hence this B-genome-specific marker willalso be useful in comparative mapping and phylogeneticstudies among the Brassicaceae. For example, there hasbeen speculation from cytological studies that B. nigramight be more closely related to Sinapis species than it isto the Brassica A- and C-genome diploids. The avail-ability of PCR-based repeat sequence markers will allowdetailed investigation of the sequences and distributions ofgenome-specific tandem repeats throughout differentcrucifer genera and give new molecular phylogeneticinformation on Brassica genome evolution.

Acknowledgements This research was funded by an AustralianResearch Council—Strategic Partnership Industry Research Train-ing grant with co-funding provided by the Export Grains Centre Ltdand the Council of Grain Grower Organisations Ltd. We thankMichael Francki and Matthew Nelson for support and advice invarious stages of this research.

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34

Chapter 4 – Published paper

Schelfhout CJ, Snowdon R, Cowling WA, Wroth JM (2006) Tracing B-genome

chromatin in Brassica napus x B. juncea interspecific progeny. Genome 49,

1490-1497.

Tracing B-genome chromatin in Brassica napus �B. juncea interspecific progeny

C.J. Schelfhout, R. Snowdon, W.A. Cowling, and J.M. Wroth

Abstract: We used polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques to demon-strate the presence of Brassica B-genome chromosomes and putative B-genome introgressions in B. napus � B. juncea in-terspecific progeny. The B-genome - specific repeat sequence pBNBH35 was used to generate PCR products and FISHprobes. The highest frequencies of viable progeny were obtained when B. napus was the maternal parent of the interspe-cific hybrid and the first backcross. B-genome - positive PCR assays were found in 34/51 fertile F2 progeny (67%), whichwas more than double the proportion found in fertile BC1 progeny. Four B-genome - positive F2-derived families and 1BC1-derived family were fixed or segregating for B. juncea morphology in the F4 and BC1S2, respectively, but in only 2of these families did B. juncea-type plants exhibit B. juncea chromosome count (2n = 36) and typical B-genome FISHsignals on 16 chromosomes. The remaining B. juncea-type plants had B. napus chromosome count (2n = 38) and no B-genome FISH signals, except for 1 exceptional F4-derived line that exhibited isolated and weak B-genome FISH signalson 11 chromosomes and typical A-genome FISH signals. B. juncea morphology was associated with B-genome - positivePCR signals but not necessarily with 16 intact B-genome chromosomes as detected by FISH. B-genome chromosomestend to be eliminated during selfing or backcrossing after crossing B. juncea with B. napus, and selection of lines contain-ing B-genome chromatin during early generations would be promoted by use of this B-genome repetitive marker.

Key words: B genome, introgression, FISH, PCR, Brassica napus, Brassica juncea, canola, oilseed rape.

Resume : Les auteurs ont employe des techniques PCR et de l’hybridation in situ en fluorescence (FISH) pour demontrerla presence de chromosomes du genome B de Brassica et d’introgressions putatives de tels chromosomes dans des proge-nitures interspecifiques B. napus x B. juncea. La sequence repetitive pBNBH35, specifique du genome B, a ete employeepour generer des produits PCR et des sondes FISH. Les plus grandes frequences de progenitures viables ont ete obtenueslorsque le B. napus etait employe comme parent maternel de l’hybride interspecifique et du premier retrocroisement. Desresultats positifs en PCR ont ete obtenus pour 34/51 (67 %) de la progeniture F2 fertile, ce qui est plus de deux fois la pro-portion trouvee au sein de la progeniture BC1 fertile. Quatre familles derivees en F2 et une derivee en BC1, toutes positivespour le genome B, etaient fixees ou en segregation pour la morphologie de type B. juncea au sein de la F4 et de la BC1S2

respectivement. Dans seulement deux de ces familles, les plantes de type B. juncea affichaient le nombre chromosomiqueattendu (2n = 36) et des signaux FISH typiques du genome B sur 16 chromosomes. Les autres plantes de type B. junceapresentaient un nombre chromosomique typique du B. napus (2n = 38) et aucun signal FISH du genome B, a l’exceptiond’une plante derivee en F4 qui montrait des signaux FISH faibles et isoles sur 11 chromosomes et des signaux FISH typi-ques du genome A. La morphologie B. juncea etait associee a des produits PCR specifiques du genome B, mais pas neces-sairement avec 16 chromosomes intacts du genome B tels que detectes par FISH. Les chromosomes du genome B tendenta subir une elimination suite a l’autofecondation ou au retrocroisement dans les progenitures issues de croisements entre leB. juncea et le B. napus. Ce marqueur de l’ADN repete du genome B pourrait faciliter la selection de lignees contenant dela chromatine du genome B au cours des premieres generations.

Mots cles : genome B, introgression, FISH, PCR, Brassica napus, Brassica juncea, colza, colza oleagineux.

[Traduit par la Redaction]

Received 13 March 2006. Accepted 9 August 2006. Published on the NRC Research Press Web site at http://genome.nrc.ca on30 January 2007.

C.J. Schelfhout. School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 StirlingHwy, Crawley WA, 6009, Australia.R. Snowdon. Institut fur Pflanzenbau und Pflanzenzuchtung I, Justus Liebig Universitat, Heinrich-Buff-Ring 26-32, D-35392 Giessen,Germany.W.A. Cowling1 and J.M. Wroth. School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of WesternAustralia, 35 Stirling Hwy, Crawley WA, 6009, Australia; Canola Breeders Western Australia Pty Ltd, Locked Bag 888, Como WA6952, Australia.

1Corresponding author (e-mail: wcowling@cyllene.uwa.edu.au).

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IntroductionStable introgression of Brassica B genome into rapeseed

(Brassica napus L., also known as canola or oilseed rape)through wide crossing has been an objective for many rape-seed breeders as a means to improve the core germplasm.The Brassica B genome is associated with many valuabletraits, such as nonshattering siliqua or disease resistance,which are not found in the A or C genomes of B. napus.Brassica juncea (L.) Czern. et Coss, an amphidiploid withA and B genomes, is often used as the source of B genomein interspecific crossing with B. napus.

There have been several attempts to introgress resistanceto blackleg disease (caused by the fungus Leptosphaeriamaculans [Desm] Ces. & De Not.) from the B genome ofB. juncea into B. napus (Roy 1984; Gerdemann-Knorck etal. 1995; Chevre et al. 1997; Dixelius and Wahlberg 1999).Siliqua shatter resistance has been transferred from B. jun-cea into B. napus (Prakash and Chopra 1988).

Problems with cross incompatibility and hybrid sterilityhave limited the success of Brassica interspecific hybridiza-tion, and successful crossing outcomes vary, depending onthe species, subspecies, or varieties used (Roy 1980a).Strong reciprocal effects were observed in Brassica interspe-cific crosses (Roy 1980a) and seed set was higher whenB. juncea was used as the maternal parent in crosses withB. napus. While B. juncea was the more successful maternalparent, Roy (1980b) reported a very low frequency of prog-eny with B. juncea morphology from this cross.

Cytogenetic and molecular studies of the Brassica ge-nomes have supported the view that the 3 diploid species(B. rapa L., B. nigra [L.] Koch, and B. oleracea L.) evolvedfrom a common progenitor species (Warwick and Black1991; Prakash and Chopra 1993). However, contrasting mo-lecular studies (Song et al. 1988; Yanagino et al. 1987;Truco et al. 1996) suggest a biphyletic origin of the diploidBrassicas, in which B. rapa (A genome) and B. oleracea (Cgenome) evolved from 1 lineage, and B. nigra (B genome)evolved separately along another lineage. The A and C chro-mosomes of B. napus pair readily with their respective Aand C homologues in B. oleracea and B. rapa (Parkin andLydiate 1997). Support for the biphyletic origin was pro-vided by Axelsson et al. (2000) based on the behaviour ofthe A and B genomes in B. juncea interspecific crosses.Nevertheless, the 3 diploid Brassica spp. are united in line-age back to an Arabidopsis progenitor 14–24 million yearsago, and up to 90% of the B. napus genome can be tracedto 21 conserved blocks from the Arabidopsis genome(Parkin et al. 2005).

The Brassica B genome appears to be excluded in favourof homologous and homoeologous pairing of A and C ge-nomes in interspecific crosses among Brassica species(Meng et al. 1998). The B genome may be closer to the Cgenome than the A genome, as the substitution of B-genomechromosomes by C-genome chromosomes in interspecificprogeny has been observed (Banga 1988). Attia andRobbelen (1986) observed similar trends in chromosomalbehaviour among the 3 genomes but also noted that the per-ceived distance of the B genome may be a product of geniccontrol.

Detection of alien DNA in wide crosses has been

achieved by quantification of chromosome content by flowcytometry (Sabharwal and Dolezel 1993) and tracing ofchromosome and DNA transfer with molecular markers(Quiros et al. 1991; Chevre et al. 1991). Visualization ofalien chromatin in interspecific hybrids using in situ hybrid-ization techniques (Fahleson et al. 1997; Skarzhinskaya etal. 1998; Snowdon et al. 1997, 2000) enables pinpointing ofintrogressions to specific chromosomes. A B-genome-specific repeat sequence, pBNBH35 (Gupta et al. 1992),was developed to trace and identify B genome using poly-merase chain reaction (PCR) and fluorescence in situ hy-bridization (FISH) (Schelfhout et al. 2004).

The aim of this study was to demonstrate the potential ofpBNBH35 (Schelfhout et al. 2004) to act as a marker for B-genome chromosomes and chromosomal introgressions inthe self and backcross progeny of an interspecific hybridpopulation between B. napus and B. juncea. The markerwould help to enrich the population for B genome and coun-ter the tendency for B chromosomes to be eliminated inearly generations (Meng et al. 1998). The potential for intro-gression of B-genome chromatin into the A and C genomesof B. napus would be improved by selecting early-generation progeny with B-genome chromosomes. We usedPCR amplification and FISH of pBNBH35 to detect B-genome chromatin in F2 and BC1 progeny and in subsequentgenerations. We discuss the stability and fate of B-genomechromosomes and putative introgressions in these interspe-cific hybrids.

Materials and methods

Production of interspecific hybridsReciprocal crosses 99X022 and 99X055 were made be-

tween B. napus canola ‘Mystic’ and B. juncea near-canolaquality line ‘JN29’ in 1999 (Fig. 1). Twelve viable F1 inter-specific hybrid seeds from each cross were sown underglasshouse conditions in January 2000. The F1 lines wereself-pollinated by hand to generate F2 seed or backcrossedto both parents by hand-pollination in reciprocal combina-tions (Fig. 1). More than 1000 F2 and BC1 seeds were sownin seedling trays under glasshouse conditions in July 2000,of which 725 germinated (Table 1). Leaf tissue was takenfrom each seedling after 3 weeks and immediately stored at–80 8C for later use of DNA in PCR amplification studies.After 4 weeks, seedlings were transplanted directly into soilin an insect-proof screenhouse. Plants were maintained withslow release fertilizer and controlled irrigation. Before flow-ering the primary inflorescence of every plant was coveredwith a pollination bag, and bags were regularly shaken topromote self-pollination.

Fertile F2 and BC1 plants were harvested at the end of2000 (total 358, Table 1), and those that had sufficient seedwere sown in rows (20 seeds per row, with 2 replicates) inthe field under normal agronomic management for canola atShenton Park and Merredin, Western Australia, in June2001. There were 59 F2-derived F3 and 229 BC1S1 lines inthe field in 2001. Agronomically desirable plants werebagged at flowering to prevent crosspollination. The pres-ence of B. napus morphology and B. juncea morphologywas noted within rows. Lines were assessed for agronomictraits, blackleg disease resistance (measured as percentage

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plant survival under field disease conditions), seed oil con-tent, protein, and total glucosinolate content. Single plantsand row-bulks with near-canola quality were advanced tothe F4 and BC1S2 generation for further field testing atShenton Park in 2002.

Total genomic DNA extraction and quantificationA rapid DNA extraction protocol was used to isolate total

genomic DNA from leaf material of interspecific progenyfor the PCR assays (Schelfhout et al. 2004).

PCR assayFrozen leaf material from fertile F2 and BC1 interspecific

progeny, grown in the field in 2001 as F3 and BC1S1 rows,was screened using a Brassica B-genome - specific markerfollowing the protocol described in Schelfhout et al. (2004).DNA was isolated from 248 of the 288 fertile progeny thatshowed reasonable agronomic attributes in 2001. PCR prod-ucts were screened on 1.5% agarose gels to test for the pres-ence of a single band at 329 bp that was confirmed to be theB-genome - specific marker pBNBH35 (Schelfhout et al.

Fig. 1. Breeding strategy with all possible reciprocal combinations in the cross B. napus ‘Mystic’ � B. juncea ‘JN29’, including BC1

families and subsequent self-fertilized generations. In the first cross, the maternal parent is listed first. In the backcrosses, (,) denotes thematernal parent and (<) denotes the paternal parent.

Table 1. F2 and BC1 progeny from the interspecific cross Brassica napus ‘Mystic’ � B. juncea ‘JN29’: number of viableseeds and fertile plants generated from the 2 reciprocal crosses and 8 backcross families.

F2 or BC1

Cross pedigreeCrossnumber

No. of F1

seedsharvestedJan. 2000

No. ofseedsharvestedJune 2000

No. ofviable seedsgerminatedJuly 2000

No. of fertileplants withmature seedDec. 2000

Mean massof harvestedseed (g/plant)

ParentsB. napus ‘Mystic’ (Mys) — — — 40 40 4.8B. juncea ‘JN29’ — — — 46 46 5.2

B. napus , primary hybridSelf (Mys/JN29) 99X022 >50 250 148 58 3.9BC1 (Mys//Mys/JN29) 00X011 — 440 299 161 4.5BC1 (Mys/JN29//Mys) 00X012 — 160 157 131 4.6BC1 (Mys/JN29//JN29) 00X013 — 68 67 2 <0.1BC1 (JN29//Mys/JN29) 00X014 — 3 0 0 0Total progeny B. napus , 921 671 352

B. juncea , primary hybridSelf (JN29/Mys) 99X055 >50 50 22 1 0BC1 (JN29/Mys//JN29) 00X015 — 26 4 5 4.5BC1 (JN29//JN29/Mys) 00X016 — 47 9 0 1.1BC1 (JN29/Mys//Mys) 00X017 — 26 15 0 0BC1 (Mys//JN29/Mys) 00X018 — 9 4 0 0.6Total progeny B. juncea , 158 54 6

Note: Refer to Fig. 1 for an explanation of cross numbers.

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2004). Positive (B. juncea ‘JN29’) and negative (B. napus‘Mystic’) control reactions along with a size marker ladderwere included in all electrophoresis gel assays to confirmthat the PCR fragment was the B-genome - specificpBNBH35 subfragment.

FISH assayTen PCR-positive lines were selected from F2-derived

families and 18 from BC1-derived families for FISH assay.The F2-derived families were tested as F2-derived F4 bulksor F4-derived F5 single plant-selections. The BC1-derivedfamilies were tested as BC1S2 bulks or BC1S2-derivedBC1S3 single-plant selections. The families selected forFISH assay included those segregating for B. napus andB. juncea morphology, or those with outstanding or unusualagronomic or seed quality attributes. The B. napus andB. juncea parents were included as controls.

FISH analysis followed the protocol described inSchelfhout et al. (2004). The pBNBH35 subsequence probewas directly labelled with the fluorochrome Cy3–11-dUTP(Amersham Biosciences, Piscataway, N.J.) by PCR. An A-genome - specific repeat sequence that hybridizes to be-tween 4 and 6 A-genome chromosomes (R. Snowdon, un-published results) was directly labelled with FITC-11-dUPT(Amersham Life Science) and included in the hybridizationmix as a control FISH reaction and to confirm the presenceof A-genome chromosomes. No cell debris or cytoplasmwas present in the preparations, and no unlabelled blockingDNA was required. Chromosomes were counterstained with4’,6-diamidino-2-phenylindole, and fluorescence was visual-ized using a Leica DM-R microscope (Leica Microsystems,Wetzlar, Germany). At least 5 well-hybridized metaphases,free from nonspecific background hybridization signals,were examined for each plant investigated. Digital imageswere recorded using a Cohu 4912 uncooled CCD camera(Cohu Inc., San Diego, Calif.) and Leica QFISH software.

Results

Production of interspecific progenyThe majority of F1 seeds from the reciprocal crosses be-

tween B. napus � B. juncea germinated precociously in thepods before seed maturity, but several viable F1 seeds wereharvested from each cross (Table 1). The F1 hybrid plantswere clearly distinguished from parent plants by the pres-ence of galls at the root–shoot junction and other morpho-logical differences, including poor seed set and infertility.B. napus ‘Mystic’ was the more successful maternal parent(cross 99X022) with more viable F1 seeds and more fertileF2 plants than B. juncea ‘JN29’ as the maternal parent (cross99X055) (Table 1). There were more failed self-pollinationsand siliqua with aborted seeds in cross 99X055. Only 1 F3line from 99X055 survived to the F4 generation for furthertesting (99X055-004S).

BC1 families gave similar results: B. napus ‘Mystic’ and99X022-F1 were more successful maternal parents thanB. juncea ‘JN29’ and 99X055-F1 (Table 1). BC1 familieswith lower fertility also produced seed with lower viability.Only 4/8 backcrosses produced viable BC1S1 seed for fieldtrials in 2001, and 3 of these involved B. napus or 99X022-F1 as the maternal parent to produce the F1 or BC1 (Table 1).

Only 3 families expressed greater than 33% fertility(00X011-BC1, 00X012-BC1, and 99X022-F2).

For those families that survived to BC1S1 and F3, the fer-tility rate was high from that generation onwards. Five ofthe 51 F2:3 progeny of 99X022 and 7 of the 194 BC1S1 prog-eny of 00X011 and 00X012 in the field in 2001 containedplants with B. juncea morphology. The remaining familieshad stable B. napus morphology. The sole surviving F2-derived progeny of 99X055 (B. juncea as maternal parent)contained plants with B. juncea and B. napus morphology.Four of the selected F4 families and 1 of the selected BC1S2families segregated for B. juncea morphology in the field in2002 (Table 2).

PCR-based assayA single band at 329 bp was confirmed to be the B-

genome - specific PCR fragment pBNBH35 by sequenceanalysis in previous work (Schelfhout et al. 2004) and byits presence in the positive control (B. juncea ‘JN29’) andabsence in the negative (B. napus ‘Mystic’) control reac-tions. The fragment was also present in other B-genome -containing Brassica species (as shown in fig. 2 ofSchelfhout et al. 2004). The proportion of fertile F2 or BC1progeny with B genome detected by the PCR-based assaywas lower in progeny of backcrosses 00X011 (23 of 80lines, or 29%) and 00X012 (29 of 114 lines, or 25%) thanin the selfed progeny of line 99X022 (34 of 51 lines, or67%). In all these crosses, B. napus was the maternal parentin the interspecific hybrid (Table 1). B-genome reactionswere detected in each of the progeny when B. juncea wasthe maternal parent, in backcross 00X013 (2 lines) and F2progeny of 99X055 (1 line) (Table 1).

FISH assayThe B. juncea and B. napus parents (‘JN29’ and ‘Mystic’,

respectively) showed FISH signals typical of the species.‘JN29’ was characterized by 16 chromosomes with B-genome FISH signals that were strong around the centro-mere but weak in the interstitial regions. ‘Mystic’showedthe A-genome - specific repeat sequence in 4–6 chromo-somes. B-genome FISH signals were detected in plantsfrom 2 of the 10 selected F2-derived F4 bulks (99X022–044and 99X022–058) but in none of the 18 selected BC1-derived families.

All plants from the F2-derived F4 bulk and F4-derived F5lines from family 99X022-058 were typical B. juncea mor-photypes with B. juncea chromosome count (2n = 36), posi-tive PCR signals, and strong B-genome FISH signals on 16chromosomes (for example, line 99X022-058M2-SP02 inFig. 2a). Plants in F2-derived F4 progeny of 99X022-044segregated for B. napus and B. juncea morphology and var-ied in FISH signals (Table 2): one plant (99X022-044-13035) showed strong B-genome signals on 16 chromo-somes and had typical B. juncea morphology and chromo-some number (not pictured), whereas another (99X022-044-14011) showed no B-genome signals, B. napus chromosomecount, typical A-genome FISH signals, and B. napus mor-phology (Fig. 2b).

One exceptional line, F4-derived F5 line 99X022-044M2-SP02, displayed B. juncea phenotypic traits, including leafmorphology, shatter-resistant siliqua, yellow seed coats, and

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moderate blackleg resistance, but a diploid chromosomecount of 38. B-genome FISH signals were observed on 11chromosomes with the B-genome - specific pBNBH35 probe(Fig. 2c). Six chromosomes exhibited FISH signals with theA-genome - specific repeat sequence. This pattern of FISHsignals was confirmed in several cells in the preparation.The line was not as resistant to blackleg as the B. juncea pa-rent ‘JN29’. Another F2-derived F4 family with B. junceamorphology (2n = 38), 99X022-093, showed positive B-genome PCR but was negative for B-genome FISH signalsand was not resistant to blackleg (Table 2).

None of the PCR-positive BC1 interspecific progenyshowed visible B-genome introgressions with FISH,although some segregated for B. juncea morphological traits,for example, family 00X011-076 (Table 2).

DiscussionSubstantial F1 sterility was observed in the cross

B. napus � B. juncea in both cross directions, but in ourcase B. napus was the more successful maternal parent.This indicates unbalanced chromosome behaviour amongthe Brassica A, B, and C genomes especially when B. jun-cea was used as the maternal parent (Fig. 1, Table 2). Incontrast to our observation, Roy (1980a) reported thatB. juncea was more successful as the maternal parent thanB. napus. However, like Roy (1980b), we observed a lowfrequency of interspecific progeny with B. juncea morphol-ogy. The favoured direction of the initial hybridization ap-pears to be genotype specific, but from that point on thefavoured chromosome complement is B. napus, and B-ge-nome chromosomes tend to be discarded (Meng et al. 1998).

Genotype-specific infertility in the interspecific hybridplant may be caused by pre- or post-zygotic barriers at thestigma, style, or ovary. Luu et al. (1998) suggest that mostinterspecific pre-zygotic barriers in Brassicaceae occurwithin the style after the pollen grains have germinated.There may be further genetic factors that contribute to hy-brid infertility, including interactions with cytoplasmic ge-netic factors of the maternal parent, and these interactionsmay be genotype dependent.

B-genome chromatin was detected by PCR at twice thefrequency in selfed progeny (67%) as in progeny back-crossed to B. napus (25%–29%). More opportunities for ho-

moeologous pairing of the B genome with A or C genomeoccur with selfing. We conclude that selfing of early-generation progeny that react positively in the B-genomePCR assay, followed by selection of progeny that retainPCR-positive signals, is a promising way to increase the fre-quency of B-genome introgression into B. napus. Intercross-ing PCR-positive sibling lines may be even more successfulthan selfing to achieve this goal.

The strong homology between amphidiploid Brassica ge-nomes and their diploid progenitors indicates that very littlechange has occurred since the formation of the amphidi-ploids in agricultural times (Parkin and Lydiate 1997;Axelsson et al. 2000). In the cross B. napus � B. juncea, A-genome chromosomes are expected to pair normally atmeiosis and are unlikely to pair with B- or C-genome chro-mosomes. Many of the unmatched B- and C-genome chro-mosomes (especially B chromosomes) will be lost duringmeiosis, as observed by Meng et al. (1998). Some nonpair-ing chromosomes will become incorporated into the gametesand be carried on to the following generation. C-genomechromosomes appear to be retained preferentially over B-genome chromosomes in this process.

The frequency of introgression of B genome into the A orC genome may be increased if the selfed population is en-riched for B genome through use of the B-genome - specificmarker. Whole chromosome substitutions may occur, as hasbeen observed previously in Brassica interspecific crosses(Banga 1988).

It was expected that backcrossing to B. juncea would al-low homologous pairing of the B-genome chromosomes inthe F1, thus favouring the retention of the B genome. As ob-served in this study, B genome was present in all 3 surviv-ing backcrosses to B. juncea (00X015), but unfortunatelyfertility was low, and few of these lines survived past theBC1 generation. It is not clear why viable seeds are rarelyproduced from backcrossing to B. juncea or why the pre-dominant progeny have B. napus morphology and chromo-some constitution, but similar observations have been madein the past (Roy 1980b).

FISH was effective at identifying B. juncea-type progenywith a normal complement of B-genome chromosomes(Fig. 2a), but no B-genome FISH reactions were observedfor the majority of B. napus-type progeny with positive B-

Table 2. FISH assay and chromosome count in parents, F2-derived, and BC1-derived families from the cross B. napus � B. napus that werefixed or segregating for B. juncea morphology and were positive for B-genome - specific markers by PCR assay.

GenerationFamily no. orcultivar Morphotype

Blackleg re-sistance*

B-genomePCR assay

B-genomeFISH assay

Chromosome no.(2n)

B. napus parent ’Mystic’ B. napus – – – 38B. juncea parent ‘JN29’ B. juncea + + + (16) 36BC1-derived 00X011-076 Segregating B. napus and

B. juncea– + – Segregating 38 and

36F2-derived 99X022-044 Segregating B. napus and

B. juncea+ + +/– Segregating 38 and

36F2-derived 99X022-058 B. juncea + + + (16) 36F2-derived 99X022-093 B. juncea – + – 38F2-derived 99X055-004 Segregating B. napus and

B. juncea– + – Segregating 38 and

36

*Assessment of blackleg resistance occurred in the field at Shenton Park in 2001 and 2002, and is summarized by ‘‘+’’ for moderate to high resistance and‘‘–’’ for susceptible.

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genome PCR signals. One F4-derived F5 progeny withB. juncea morphology (99X022-044M2-SP02) showed posi-tive B-genome FISH signals and putative B-genome intro-gressions on 11 chromosomes, as well as A-genome FISHsignals and a chromosome complement of 2n = 38(Fig. 2c). None of the chromosomes carried both A-genome -

specific and B-genome - specific FISH signals, which indi-cates that the introgression of B genome did not occur inthese particular A chromosomes.

The majority of B-genome signals in this genotype wereless intense, narrower, and not concentrated around the cen-tromeres as in B. juncea control lines or other B. junceamorphotypes. Some of the stronger B-genome signals in99X022-044M2-SP02 may represent whole B chromosomesor A/C introgression into B chromosomes (Fig. 2c). How-ever the small size and low intensity of the majority of sig-nals favours the conclusion that they result from B-genomeintrogression into B. napus A- or C-genome chromosomes.

The small size of Brassica chromosomes and the confine-ment of heterochromatic FISH and genomic in-situ hybrid-ization signals to centromeric regions (Harrison and Heslop-Harrison 1995; Snowdon et al. 1997) prevent exact localiza-tion and cytological characterization of the introgressionswithout the use of additional molecular techniques. In line99X022-044M2-SP02, there was an absence of B-genomesignals on the 6 A-genome chromosomes identified by anA-genome specific sequence (Fig. 2c). Nevertheless, the in-trogressions may involve the remaining A-genome or C-genome chromosomes. Line 99X022-044M2-SP02 providesphysical evidence that B. juncea morphology can occur ininterspecific progeny without all of the B genome presentand with a chromosome complement similar to that ofB. napus (2n = 38). Support for this contention is found infamily 99X022-093, which has B. juncea morphology andB. napus chromosome complement (2n = 38), is positive forB-genome PCR, and negative for B-genome FISH signals(Table 2).

The pBNBH35 repeat sequence was detected exclusivelyin pericentromeric heterochromatin flanking the centromeresof B-genome chromosomes (Schelfhout et al. 2004), whereit most likely occurs in high copy numbers. Most of thelines that were positive for B genome by PCR assay werenegative for B genome by FISH assay. These lines may con-

Fig. 2. (a) FISH patterns on prometaphase chromosomes of99X022-58M2-SP02, an F4-derived F5 line of B. napus ‘Mystic’ �B. juncea ‘JN29’ with 16 B-genome chromosomes (labelled redwith a probe derived from the B-genome - specific repeat sequencepBNBH35) and a total chromosome count of 2n = 36. The greenprobe hybridized specifically to 4–6 A-genome chromosomes.Plants showing patterns of this type were morphologically B. jun-cea type and tested positive for B-genome chromatin by the PCRassay. (b) FISH patterns on prometaphase chromosomes of99X022-044-14011, a plant from an F2-derived F4 bulk of B. napus‘Mystic’ � B. juncea ‘JN29’ with no B-genome chromosomes ap-parent by FISH assay but positive signals for pBNBH35 by PCRassay and a total chromosome count of 2n = 38. Four chromosomesare labelled with the green A-genome - specific probe (circled).Plants showing patterns of this type were morphologically B. napustype. (c) FISH patterns on prometaphase chromosomes of 99X022-044M2-SP02, an F4-derived F5 plant from B. napus ‘Mystic’ �B. juncea ‘JN29’. B-genome - specific signals from the pBNBH35subfragment (red) are visible on 11 chromosomes (circled). Sixchromosomes carry A-genome - specific signals (green). The totalchromosome count is 2n = 38, indicating a B. napus type with B-genome introgressions. However, the morphology of the plant re-sembled B. juncea. The bar in each photomicrograph represents10 mm.

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tain small chromosomal inserts from interstitial or telomericregions of B-genome chromosomes. Alternatively, therewere simply insufficient copy numbers of introgressedpBNBH35 to generate a visible FISH signal. While it wasnot an aim of this research to determine the frequency of B-genome introgression, this is clearly a rare event, and selec-tion based on the FISH assay alone will not be sufficient toenhance the frequency of progeny with B-genome introgres-sions.

Theoretically, a single copy of pBNBH35 is all that is re-quired for the PCR assay to generate a positive result.pBNBH35 could be used to develop marker-assisted selec-tion of relevant traits, such as blackleg resistance or non-shattering siliqua, in introgressions derived from the B-genome in advanced breeding lines of B. napus. A combina-tion of a forward primer in the pBNBH35 sequence with re-verse amplified fragment length polymorphism primers, forexample, should generate sufficient polymorphisms to mapthe introgressions in segregating populations and identifylinkage to relevant traits.

Genome-specific and standard molecular markers mayhelp to identify chromosomal sites where A or C genomehas been substituted by B genome. The latter approach wasused by Howell et al. (1996) to identify intervarietal substi-tution lines and could easily be adapted to interspecies or in-tergeneric crosses.

Some progeny with B. napus or B. juncea chromosomecount and morphology had improved agronomic traits, suchas pod shatter resistance, blackleg resistance, and yellowseed colour. In B. napus-type progeny, B-genome retentionis not the only explanation for these changes, which mayarise also from exotic A-genome alleles derived from B. jun-cea. The extent of A-genome exchange will be relativelysimple to estimate using molecular markers. Such progenyhave the potential, both in B. juncea and in B. napus, to in-crease the allelic diversity for a range of important traits.New A- and B-genome alleles may also be of interest forbreeding heterotic pools in the respective crops.

AcknowledgementsThis research was funded by an Australian Research

Council – Strategic Partnership Industry Research Traininggrant, with co-funding provided by the Council of GrainGrower Organisations Ltd and the Export Grains CentreLtd. Near-canola quality B. juncea ‘JN29’ was kindly pro-vided for our research by Wayne Burton, Victorian Depart-ment of Primary Industries, Horsham, Victoria, Australia.We thank Drs. Guijun Yan, Michael Francki, and MatthewNelson for support and advice in various stages of this re-search.

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Chevre, A.M., Barret, P., Eber, F., Dupuy, P., Brun, H., Tanguy, X.,and Renard, M. 1997. Selection of stable Brassica napus-B. juncearecombinant lines resistant to blackleg (Leptosphaeria maculans).1. Identification of molecular markers, chromosomal and genomicorigin of the introgression. Theor. Appl. Genet. 95: 1104–1111.

Dixelius, C., and Wahlberg, S. 1999. Resistance to Leptosphaeriamaculans is conserved in a specific region of the Brassica Bgenome. Theor. Appl. Genet. 99: 368–372. doi:10.1007/s001220051246.

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Gupta, V., Lakshmisita, G., Shaila, M.S., Jagannathan, V., andLakshmikumaran, M.S. 1992. Characterization of species-specific repeated DNA sequences from B. nigra. Theor. Appl.Genet. 84: 397–402.

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Howell, P.M., Marshall, D.F., and Lydiate, D.J. 1996. Towards de-veloping intervarietal substitution lines in Brassica napus usingmarker assisted selection. Genome, 39: 348–358.

Luu, D.T., Passeleque, E., Dumas, C., and Heizmann, P. 1998.Pollen-stigma capture is not species discriminant within theBrassicaceae family. C. R. Acad. Sci. Ser. III, 321: 747–755.

Meng, J., Shi, S., Gan L., Li, Z., and Qu, X. 1998. The productionof yellow-seeded Brassica napus (AACC) through crossing in-terspecific hybrids of B. campestris (AA) and B. carinata(BBCC) with B. napus. Euphytica, 103: 329–333. doi:10.1023/A:1018646223643.

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43

Chapter 5 – In press paper. As submitted for publication in Australian Journal

of Agricultural Research (in press, 2008)

Enhancement of genetic diversity in canola-quality Brassica napus and B.

juncea by interspecific hybridisation

C. J. SchelfhoutA,C, J. M. WrothA,B, G. YanA and W. A. CowlingA,B,D

ASchool of Plant Biology, Faculty of Natural and Agricultural Sciences, The

University of Western Australia, 35 Stirling Highway, Crawley, WA 6009,

Australia BCanola Breeders Western Australia Pty Ltd, Locked Bag 888, Como, WA

6952, Australia CCurrent address: Department of Agriculture and Food Western Australia, PO

Box 432, Merredin WA 6415, Australia DCorresponding author. Email: wcowling@cyllene.uwa.edu.au

Abstract

Reciprocal crosses were made between Brassica napus cv. Mystic (canola) and

B. juncea JN29 (near canola quality). The F1 hybrids were selfed and

backcrossed in all possible combinations to parent plants. The greatest number

of selfed fertile progeny were obtained when Mystic was the maternal parent,

and its F1 was most successful in backcrosses to Mystic or JN29 as maternal or

paternal parent. The predominant morphological type of fertile progeny was B.

napus, but several B. juncea morphological types occurred in F2 and BC1

derived lines. F2:3 and BC1S0:1 progeny showed transgressive segregation for

agronomic and seed quality traits in two contrasting field environments. Several

of the B. juncea-type progeny had improved seed quality (lower total seed

glucosinolates and higher percent oleic acid) than the B. juncea parent. Selfing

of interspecific hybrids between canola quality B. napus and B. juncea has the

potential to greatly enhance genetic diversity in canola quality progeny of both

species, without the loss of donor alleles that normally occurs with repeated

backcrossing.

44

Additional keywords:

Oilseed rape, Indian mustard, interspecific crossing

Introduction

Oilseed rape or canola (Brassica napus L.) is grown in southern cropping

regions of Australia with winter dominant rainfall. Traditionally, canola was

grown in the higher rainfall cropping areas with up to 700 mm annual average

rainfall, but shorter season varieties are now available for regions with 325 mm

rainfall (Potter et al. 1999). Canola is a term introduced by the Canola Council

of Canada to describe oilseed rape with <2% erucic acid in the seed oil and less

than 30 µmoles/g glucosinolates in the meal (Anon. 2007). Canola seed has

approximately 60% oleic acid in the oil.

The genetic diversity of Australian breeding populations of B. napus has

narrowed in the past 30 years during a period of rapid genetic improvement in

agronomic traits and conversion to canola quality (Cowling 2007). Several

canola varieties released in the late 1990s had a coefficient of ancestry greater

than full-sib mating in animals, and the population coefficient of inbreeding after

30 years of closed recurrent selection was 0.21 (Cowling 2007). While this is

not a problem with regard to inbreeding defects or lethality, the loss of genetic

diversity may limit future gains in adaptation, yield and disease resistance. A

similar diminution of genetic diversity existed in Canadian B. napus after

decades of seed quality improvement (Downey and Rimmer 1993, Juska et al.

1997). Likewise, three international canola-quality B. juncea breeding programs

have strong genetic similarities within and between breeding programs, and

canola quality B. juncea is relatively distant from traditional Indian mustards

(Burton et al. 2004).

The Brassica secondary gene pool has been a valuable source of genes

for improvement of B. napus. Traits introgressed into B. napus from closely

related species include shatter resistance (Prakash and Chopra 1988),

earliness (Rao et al. 1993a, 1993b) and blackleg resistance (Chèvre et al. 1997,

Roy 1978, 1984). The Brassica B-genome has been a useful source of alleles

for a number of disease traits in B. napus (Plieske et al. 1998). The benefits

have flowed in both directions, and recombination with the A genome of B.

napus may have been responsible for the very low glucosinolates in one B.

juncea-type progeny of an interspecific cross between a low glucosinolate B.

45

napus and a mustard variety of B. juncea (Love et al. 1990). However, in most

cases, benefits have been derived from introgression of major genes from one

species to the other by repeated backcrossing.

Kirk and Oram (1981) identified low erucic acid B. juncea lines and

combined them with the low glucosinolate lines from Love et al. (1990) to

develop low erucic, low glucosinolate B. juncea in Australia (Oram et al. 1999).

Breeding of canola-quality B. juncea continues in Australia with increases in

oleic acid to canola standards through crossing with Canadian lines (Burton et

al. 2004).

B. juncea would be a valuable source of new genetic diversity in B.

napus, and vice versa, if extensive backcrossing was not necessary to restore

seed quality and agronomic traits. Selfing of interspecific hybrids should

promote recombination among the shared A genomes of the two species, and

enhance genetic diversity for complex traits, even if the selfed progeny quickly

reverted back to one or the other parental genomes. In a previous study, it was

shown that selfed progeny of interspecific hybrids between B. napus × B.

juncea were predominantly B. napus genotypes, some with visible

introgressions of B genome, and a minority had a full set of B chromosomes

and B. juncea chromosome number (Schelfhout et al., 2006).

The aim of this work was to demonstrate that genetic diversity for

morphological and agronomic traits increased in the selfed and first backcross

progeny of B. napus × B. juncea, while maintaining or improving canola quality.

Interspecific progeny were tested as F2-derived and BC1S1-derived progeny in

low and high rainfall environments, where transgressive segregants were

selected with B. napus and B. juncea morphology, and tested for seed quality.

Materials and Methods

Crossing regime

F1 plants from reciprocal crosses between B. napus cv. Mystic and B. juncea

JN29 were selfed and backcrossed to both parents in all possible combinations,

with crossing codes and outcomes as described in Schelfhout et al. (2006).

Field trials

F2 and BC1 progeny were grown in an insect-proof screenhouse at Shenton

Park field station, The University of Western Australia, in 2000 (site code

46

SP2000), and inflorescences were covered with pollination bags to prevent

cross-pollination, as described in Schelfhout et al. (2006). Measurements of

agronomic traits on F2 and BC1 individuals at SP2000 included plant biomass

(air dry weight of above ground dry matter) at harvest, seed yield per plant,

number of primary branches, days to anthesis (first open flower), height at

harvest and harvest index (seed yield/biomass x 100).

Selfed seed (from inside pollination bags) from fertile F2 and BC1 families

were advanced in 2001 as F2:3 and BC1S0:1 plots in field trials with 2 replicates

at 2 sites as described in Schelfhout et al. (2006), with 20 seeds per replicate at

Merredin, Western Australia (site code MD2001) and more than 20 seeds at

Shenton Park (site code SP2001) where sufficient seed was available. The

MD2001 site was located at the Department of Agriculture and Food Western

Australia Research Station at Merredin, 250 km east of Perth (31.50 oS, 118.22 oE). This location was a low rainfall cropping site with annual average rainfall

313 mm with 75 per cent falling from April to October (95-year average data

from Bureau of Meteorology site number 010093, available on-line at

http://www.bom.gov.au/climate/averages/tables/cw_010093.shtml). The

SP2001 site was located at The University of Western Australia Field Station, 6

km west of Perth (31.96 oS, 115.79 oE). This site has an annual average rainfall

of 705 mm with 90% falling from April to October (39-year average data from

Bureau of Meteorology site number 009151, available on-line at

http://www.bom.gov.au/climate/averages/tables/cw_009151.shtml). High

blackleg disease (causal agent Leptosphaeria maculans) pressure was

promoted by distributing previous-years infected canola stubble (from B. napus

cv. Pinnacle) evenly around the entire trial area approximately 4 weeks after

seeding. Included in each 2001 trial were 18 selfed individuals from B. juncea

JN29 and 19 selfed individuals from B. napus Mystic from SP2000.

At SP2001 and MD2001, the number of leaves per plant was counted at 4

weeks from sowing, and the average recorded for each plot. At SP2001,

blackleg disease survival was calculated by subtracting the number of plants

per plot at harvest from the number of plants emerged at 4 weeks, and

calculating the percentage survival. At maturity, 6 open-pollinated plants were

bulk-harvested from within each plot at each site to calculate the average grain

yield and biomass (air dry weight of above ground plant matter) per plant.

47

Seed quality was analysed on lines with superior agronomic traits and

blackleg disease resistance at SP2001 and MD2001. Seed from the bulk

harvest of selected plots, or from up to 4 selfed plants per plot, was analysed for

oil, protein, glucosinolate and oleic acid content by near infrared radiation (NIR)

assay (Foss NIRS 5000) using standard samples for calibration.

Lines were selected at the end of 2001 on the basis of yield, blackleg

resistance, seed quality and superior agronomic traits, and promoted to the next

generation at Shenton Park in 2002 (SP2002) as F2:4 open-pollinated line bulks,

F3:4 selfed F3-derived lines, BC1S2 open-pollinated line bulks, or BC1S1:2 selfed

BC1S1-derived lines. The SP2002 trial followed the design in SP2001 and

included two replicates, with 100 seeds per replicate. Segregation for B. juncea

and B. napus morphology was recorded in the SP2002 field trial. The main

purpose of the SP2002 trial was to harvest superior agronomic lines and

analyse harvested seed for oil, protein, and glucosinolates by NIR and fatty acid

profile by gas chromatography (GC) on a GC-17A V3 (Shimadzu) equipped with

flame-ionization detector and flow splitter. Results for fatty acid content from

GC were integrated by GC solution software (Shimadzu).

Analysis of field trial data

Column scatter plots were used to illustrate the mean and range of variation in

the parent populations and F2 and BC1 families in SP2000. Fisher’s protected

least significant difference test was used to demonstrate similarities or

differences between family means for each agronomic trait.

Principal components analysis (PCA) was used to summarize

associations among progeny and parents based on the mean value across

replicates of agronomic traits measured on plots at SP2001 and MD2001. Data

were standardized for mean and variance before analysis by Genstat software

(VSN International, UK).

Results

Morphological attributes of parental lines

B. napus cv. Mystic was characterised by medium plant stature, dark green

ovoid shape leaves with smooth serrations, and long non-constricted siliqua

with black seeds. B. juncea JN29 plants were 26% taller on average than

Mystic (Fig. 1), with light green elongated leaves and pronounced serrations.

48

Siliqua of JN29 were more constricted around the seed than Mystic, with

mustard yellow seeds, and were resistant to shattering at maturity. The

flowering time of JN29 was similar to Mystic however the harvest index was

considerably lower (Fig. 1).

Agronomic traits of F2 and BC1 progeny

F1 plants of cross 99X022 (Mystic/JN29) were more fertile on selfing and

backcrossing than F1 plants of the reciprocal cross 99X055 (JN29/Mystic)

(Schelfhout et al. 2006). One backcross family (00X014) failed to germinate in

July 2000 (Schelfhout et al. 2006), and did not contribute further to results.

The dominant morphological type of the F2 and BC1 progeny in SP2000

was that of the B. napus parent Mystic. However, in later generations, some B.

juncea morphological types were observed in a minority of selfed (99X022 and

99X055) and backcross progeny. Backcross family 00X013

(Mystic/JN29//JN29) showed only B. juncea-type morphology. Lines from

00X013 had high scores for height and low scores for harvest index, similar to

the B. juncea parent JN29. However, these lines were low in fertility and did not

survive to future generations (Schelfhout et al. 2006). Lines segregating for B.

juncea morphology at SP2002 were:

00X011-022, -076, -127, -140 (BC1-derived ex Mystic/JN29//Mystic)

00X012-046, -141, -152 (BC1-derived ex Mystic//Mystic/JN29)

99X022-10, -44, -53, -58, -93 (F2-derived ex Mystic/Jn29)

99X055-04 (F2-derived ex Jn29/Mystic)

49

Figure 1. (caption next page)

0

100

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mas

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dbcd bcd cd

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11 12 13 15 16 17 18 22 55 JN29 Mys

50

Figure 1. Column scatter plots of agronomic measurements on F2 or BC1

plants from the interspecific hybridisation of B. napus cv. Mystic × B. juncea

JN29 at SP2000. Agronomic traits included number of days to anthesis, mature

plant biomass (g/plant), harvest index (%), and height at maturity (cm). The

mean of each family is indicated by the horizontal bar in the vertical column of

data points from each family, and letters adjacent to the mean indicate

differences among the means according to Fisher’s protected least significant

difference test (P = 0.05). Codes for cross families are abbreviated from

Schelfhout et al. (2006), where “11” = 00X011, “12” = 00X012, “13” = 00X013,

“15” = 00X015, “16” = 00X016, “17” = 00X017, “18” = 00X018, “22” = 99X022,

“55” = 99X055, and “Mys” = Mystic.

F2 and BC1 families at SP2000 varied significantly in dry biomass at maturity,

harvest index, height at maturity, and days to flowering (Fig. 1). Plant seed

yield differences were also observed among families (Schelfhout et al. 2006).

The selfed family with the highest mean biomass was 99X022 (Mystic/JN29)

and its backcross progeny, especially 00X012 (Mystic/JN29//Mystic), which had

the highest mean biomass (greater than the parents and self or backcross

families) and showed transgressive segregation above the parents (Fig. 1). F2

family 99X055 (JN29/Mystic) and its backcrosses tended to be low yielding or to

produce unviable seed (00X017), except for 00X015 (JN29/Mystic//JN29) which

had similar mean yield and biomass per plant to the parents, but only had 6

surviving plants (Fig. 1).

B. napus cv. Mystic plants had greater harvest index on average (14.6%)

than B. juncea JN29 (11.6%). Mean harvest index was between 10 and 15

percent for most F2 and BC1 families except those with poor fertility.

B. juncea JN29 plants were 40 cm taller on average than B. napus cv.

Mystic plants in SP2000, with large variation in height in both parental lines (Fig.

1). Most F2 and BC1 families had a mean height between the two parents,

except for 00X013 (Mystic/JN29//JN29) which was 40 cm taller on average than

the B. juncea JN29 and 80 cm taller on average than B. napus cv. Mystic.

Flowering date was significantly later in all F2 and BC1 families than in the

parents, and the range of flowering dates among B. juncea JN29 plants (67 –

51

101 days) was much wider than among B. napus cv. Mystic plants (69 – 86

days) (Fig. 1).

Agronomic traits in F2:3 and BC1S0:1 field trials

Strong positive correlations between yield per plant and biomass per plant in

SP2001 and MD2001 are reflected by the close association of vectors for these

traits in PCA plots for SP2001 (Fig. 2) and MD2001 (Fig. 3). The vectors for

yield and harvest index at both sites were less strongly associated, and there

was a weak negative association between yield and flowering date at SP2001.

Yield was independent of height at both sites, and independent of blackleg

disease resistance at SP2001 (as indicated by perpendicular vectors).

The ellipses on the PCA graphs in Fig. 2 (SP2001) and Fig. 3 (MD2001)

indicate the range of positions for most of the selfed plants of B. juncea JN29

and B. napus cv. Mystic, and there was no overlap between the two parents.

Two control varieties of B. napus, cv. Surpass 400 and cv. Monty, fell within the

range of values recorded for B. napus cv. Mystic (Figs. 2 and 3). JN29 plants

were associated with the height vector (taller than Mystic plants), had more

leaves per plant at 4 weeks, and tended to have more biomass than Mystic

plants, but both species had a similar range in yield at both locations. B. juncea

JN29 plants had lower harvest index than B. napus cv. Mystic plants, were later

flowering, and had stronger blackleg resistance at SP2001 (Fig. 2).

F2:3 and BC1S0:1 progeny tended to be distributed between the two parent

ranges, with some inside each range, at SP2001 (Fig. 2) but most tended to

cluster within the B. napus parent range at MD2001 (Fig. 3) where blackleg

resistance and flowering date were not measured (very little blackleg was

observed at MD2001). F2:3 progeny from cross 99X022 at SP2001 were

associated with the vectors for longer days to flowering and lower yield (Fig. 2).

Backcross progeny appeared to be more strongly associated with yield and

harvest index (towards the B. napus range) in both SP2001 and MD2001 trials.

The best yielding lines in 2001 were predominantly BC1S0:1 progeny with

few F2:3 progeny in these regions of the PCA graphs. Some BC1S0:1 and F2:3

progeny were higher yielding than the parental lines, especially in the drought-

stressed environment of MD2001.

52

-6

-3

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-6 -3 0 3 6-1

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-1 -0.5 0 0.5 1

00X011

00X012

99X022

B. napusMystic

B. junceaJN29

S400

Monty

HIYld

BM

Lvs

HtBL

DtF

Figure 2. Principal components analysis biplot of line means for agronomic and

disease traits measured on interspecific hybrid progeny at SP2001. Progeny

data points are plotted on the primary x-y axes and trait vectors are plotted on

the secondary x-y axes. Abbreviations: “Yld” = yield (g) per plant, “HI” =

harvest index, “DtF” = days to first flowering, “BL” = blackleg resistance rating,

“Ht” = height at maturity, “Lvs” = leaf number 4 weeks after sowing, “BM” =

whole plant biomass at harvest (g), “S400” = B. napus cv. Surpass 400.

53

-6

-3

0

3

6

-6 -3 0 3 6

-1

-0.5

0

0.5

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-1 -0.5 0 0.5 1

00X011

00X012

99X022

B. junceaJN29

B. napusMystic

S400

HI

Yld

BMHt

Lvs

Figure 3. Principal components analysis biplot of line means for agronomic and

disease traits measured on interspecific hybrid progeny at MD2001. Progeny

data points are plotted on the primary x-y axes and trait vectors are plotted on

the secondary x-y axes. Abbreviations: “Yld” = yield (g) per plant, “HI” =

harvest index, “Ht” = height at maturity, “Lvs” = leaf number 4 weeks after

sowing, “BM” = whole plant biomass at harvest (g), “S400” = B. napus cv.

Surpass 400.

Seed quality analysis

Seed harvested from parent selections, F2:3 and BC1S0:1 lines at SP2001

consistently produced higher seed oil, higher oleic acid, lower seed protein and

lower glucosinolates in seed than those in the MD2001 trial (Table 1). There

was a strong inverse relationship between protein and oil content at the two

sites, but the combined seed {oil + protein} was consistent between sites, with

B. napus cv. Mystic selections achieving the same value of 70.5% {oil + protein}

at both sites (Table 1).

B. napus cv. Mystic lines were 3-4% higher in seed oil, 2-3% lower in

seed protein, 1% higher in seed {oil + protein}, 12% higher in oleic acid, and

54

significantly lower in glucosinolate content than B. juncea JN29 at the two trial

locations (Table 1). At MD2001, some individuals in families 00X011 and

00X012 had higher oil, higher protein and higher oleic acid contents than

Mystic, with glucosinolate content equal to or lower than Mystic, and at both

MD2001 and SP2001 some individuals exceeded the seed {oil + protein}

percentage of Mystic. Lines harvested from the SP2001 trial did not show the

same extremes in quality values as those harvested from MD2001.

55

Table 1. Mean and range of seed composition (NIR assay) of families from the

interspecific cross B. napus cv. Mystic × B. juncea JN29 at SP2001 and

MD2001. Seed composition was assessed on harvested samples from the

most promising progeny at each site, based on agronomic observations before

harvest.

Site MD2001 Site SP2001 FamilyA No. lines Mean Range No. lines Mean Range

Seed oil (% dry weight)

Mystic 4 45.1 43.6 – 45.8 4 53.9 52.3 – 55.2 JN29 4 40.4 37.5 – 43.3 4 50.3 48.7 – 50.9 99X022 11 41.7 38.1 – 45.0 10 51.6 46.9 – 54.7 99X055 2 38.8 36.5 – 41.1 1 51.5 00X015 3 43.5 42.4 – 45.1 3 53.2 49.9 – 55.3 00X013 1 45.5 1 53.0 00X012 44 42.9 38.8 – 47.5 46 50.9 42.8 – 55.4 00X011 28 43.9 41.3 – 47.1 29 52.0 46.9 – 55.9

Seed protein (% dry weight)

Mystic 4 25.4 24.8 – 26.5 4 16.6 16.0 – 17.8 JN29 4 29.3 26.7 – 31.6 4 18.3 17.6 – 19.3 99X022 11 27.8 25.3 – 28.9 10 18.1 15.7 – 20.1 99X055 2 29.5 28.6 – 30.4 1 18.2 00X015 3 27.1 26.2 – 28.1 3 17.2 15.8 – 19.2 00X013 1 25.6 1 17.3 00X012 44 27.2 24.7 – 29.2 46 18.7 15.2 – 24.6 00X011 28 26.7 24.5 – 29.4 29 17.9 15.2 – 21.8

Seed oil + protein (% dry weight)

Mystic 4 70.5 70.1 – 70.8 4 70.5 69.9 – 71.2 JN29 4 69.7 69.1 – 70.0 4 68.6 68.0 – 69.6 99X022 11 69.5 66.4 – 72.9 10 69.7 67.0 – 71.2 99X055 2 68.3 66.9 – 69.8 1 69.7 00X015 3 70.6 70.3 – 70.5 3 70.4 69.0 – 71.1 00X013 1 71.1 1 70.3 00X012 44 70.1 66.2 – 73.8 46 69.6 66.3 – 73.0 00X011 28 70.6 68.1 – 72.6 29 69.9 68.5 – 71.4

Seed glucosinolates (µmol/g)

Mystic 4 8.1 3.2 – 11.3 4 2.5 0.7 – 3.4 JN29 4 44.5 34.3 – 57.6 4 43.8 37.8 – 48.8 99X022 11 11.7 4.7 – 37.6 10 3.9 1.8 – 7.2 99X055 2 8.2 6.6 – 9.7 1 4.2 00X015 3 6.1 4.5 – 7.0 3 2.3 0.6 – 3.5 00X013 1 2.3 1 2.2 00X012 44 7.8 1.1 – 13.9 46 4.0 1.4 – 7.0 00X011 28 7.4 2.3 – 12.1 29 5.1 1.3 – 8.8

Oleic acid (%fatty acid composition)

Mystic 4 59.2 56.7 – 61.4 4 61.1 60.1 – 61.8 JN29 4 46.3 42.2 – 49.1 4 49.4 48.7 – 50.9 99X022 11 53.8 47.7 – 59.6 10 58.7 51.0 – 62.5 99X055 2 50.9 46.0 – 55.7 1 60.6 00X015 3 57.6 54.8 – 62.4 3 59.7 52.2 – 63.6 00X013 1 57.6 1 65.1 00X012 44 57.8 50.8 – 65.8 46 59.0 48.7 – 65.7 00X011 28 61.1 54.4 – 66.7 29 62.6 56.2 – 66.8

AThe full cross pedigrees for families are provided in Schelfhout et al. (2006)

56

Of the 6 highest yielding interspecific progeny in MD2001, all were B. napus

morphotypes, 5 were BC1-derived and 1 was F2-derived (Table 2). These lines

out-yielded the parent lines, and had equal or higher oil, oleic acid and protein

contents than Mystic and lower glucosinolate content than JN29 (Table 2).

Two lines harvested from the BC1S2 and F4 generation at SP2002

(99X022-093-3 and 99X055-004-5) were high-yielding B. napus morphotypes

that had high glucosinolates similar to the B. juncea parent (Table 2). Some B.

juncea morphotypes in SP2002 (00X011-022-4, 00X012-141-3, and 99X022-

058M2-3) had canola quality seed composition with low glucosinolate levels

(Table 2). All lines in Table 2 had zero detectable erucic acid content.

Genetic segregation for seed quality traits within families was also

evident. F2:3 line 99X022-044 (B. juncea morphotype) had low oil, high protein

and high glucosinolates in seed harvested from MD2001, whereas selfed single

plant selections from MD2001 (99X022-044-1 and 99X022-044-3) were

segregating for oil, protein and glucosinolates in seed harvested from SP2002,

but retained their B. juncea morphotype (Table 2).

57

Table 2. Seed composition (NIR assay) of notable individual interspecific

progeny of B. napus cv. Mystic × B. juncea JN29 from SP2001, MD2001 and

SP2002. Values for parent lines are as listed in Table 1.

AThe full cross pedigrees for families are provided in Schelfhout et al. (2006) BMorphology relates to the parent-type. For B. juncea, this includes B. juncea parental leaf shape and colour, plant morphology, and seed colour (yellow). For B. napus, the parental leaf shape and plant morphology were accompanied by B. napus seed colour (black or dark brown).

Discussion

Enhanced genetic variation for key agronomic and seed quality traits was

present in selfed progeny of interspecific hybrids between B. napus (canola) ×

B. juncea (near-canola quality). B. napus was the predominant morphotype in

selfed and backcrossed progeny, and most of these lines retained their canola

Selections within

familiesA

Generatio

n

Site and

year Oil (%)

Protei

n (%)

Oil +

protein

(%)

Oleic

acid

(%)

Glucosinolates

(µmol/g)

MorphologyB

High yielding accessions at MD2001:

00X011-127 BC1S1 MD2001 42.8 29.4 72.2 61.7 6.6 B. napus

00X011-084 BC1S1 MD2001 47.1 24.8 71.9 66.7 1.7 B. napus

00X011-116 BC1S1 MD2001 46.5 25.4 71.9 62.7 2.3 B. napus

00X012-037 BC1S1 MD2001 46.8 25.4 72.2 63.5 6.3 B. napus

00X012-105 BC1S1 MD2001 45.5 25.5 71.0 60.4 7.0 B. napus

99X022-147 F3 MD2001 44.6 28.3 72.9 59.6 4.9 B. napus

Accessions with unusual traits at SP2001, SP2002 and MD2001:

99X022-093-3 F4 SP2002 40.7 25.9 66.6 58.1 47.0 B. napus

99X055-004-5 F4 SP2002 40.4 25.4 65.8 50.8 42.0 B. napus

00X011-022-4 BC1S2 SP2002 48.6 19.0 67.6 46.4 2.0 B. juncea

00X012-141-3 BC1S2 SP2002 48.8 18.9 67.7 63.0 2.0 B. juncea

99X022-058M2-

3

F4 SP2002 49.8 18.6 68.4 46.1 8.0 B. juncea

99X022-044 F3 MD2001 43.5 27.7 71.2 53.2 37.6 B. juncea

99X022-044-1 F4 SP2002 46.0 22.2 68.2 43.7 40.0 B. juncea

99X022-044-3 F4 SP2002 49.2 18.5 67.7 47.0 18.0 B. juncea

58

quality. Genetic variation was present also among rare B. juncea morphotypes

among the selfed interspecific progeny, and the seed quality of some B. juncea-

type progeny was improved beyond the B. juncea parent with lower

glucosinolates and higher oleic acid. All progeny had zero detectable erucic

acid. Transgressive segregation for agronomic and quality traits was evident

among the selfed and backcross interspecific progeny.

The most successful primary cross direction was with B. napus as the

maternal parent, and the most successful backcross parent was B. napus. This

contrasts with results of the similar interspecific crosses by Roy (1980), and

agrees with the conclusion of Patil et al. (2003), that the most successful

primary cross direction is genotype dependent.

Interspecific hybridisation has been used to introduce new genes for

shatter resistance, earliness and blackleg disease resistance into B. napus from

B. juncea or B. carinata and other Brassica species (Prakash and Chopra 1988,

Rao et al. 1993a, 1993b, Chèvre et al. 1997, Roy 1978, 1984). Resynthesis of

the amphidiploid B. napus from the two diploid progenitor species (B. rapa, B.

oleracea) also introduced new genetic variation into B. napus (Chen and

Heenan 1989). The usefulness of interspecific crossing in Brassica is often

limited by low fertility in progeny, and embryo rescue or other interventions are

often required (Kumar et al. 1988). However, the major problem for canola

breeders is the lack of canola quality progeny in such wide crosses. Fertile,

canola-quality and genetically stable B. napus-type progeny are rare, and

breeders generally undertake extensive backcrossing to the B. napus parent in

order to restore canola quality.

In these experiments, genetic variation was introduced into selfed B.

napus-type canola quality progeny following wide crossing with B. juncea –

when the B. juncea parent was near canola-quality. This new genetic diversity

from B. juncea was immediately available for exploitation in B. napus breeding.

This increases the potential to retain potentially useful alleles from the donor

parent, compared with continued backcrossing to the B. napus parent. The

majority of selfed interspecific progeny were B. napus morphology, fertile with

high seed yield, biomass and harvest index (Fig. 1). A minority of F2-derived or

BC1-derived progeny had B. juncea morphology, and some of these had better

seed quality (higher seed oil, higher oleic acid and lower glucosinolates) than

59

the B. juncea parent. Benefits from interspecific crossing with canola quality

parents of B. juncea and B. napus will be possible in both species.

Some selfed progeny had B. juncea morphology (leaf shape and colour,

seed colour, plant morphology), a chromosome count of 2n = 38 and evidence

for B-genome introgression, but lacked complete B-genome chromosomes

(Schelfhout et al. 2006). These results indicate that B. juncea morphology does

not depend on the presence of a complete set of B genome chromosomes.

Our data on seed quality in selections from interspecific crossing were

obtained from two locations in 2001, and further years and locations would be

necessary to obtain definitive information on seed quality of particular

selections. However, there were many consistencies in the ranking of lines for

quality across the two sites, and some general conclusions can be drawn. The

seed quality of B. juncea parent JN29 was close to canola (Anon. 2007) but with

elevated glucosinolates and lower oleic acid% in oil (Tables 1 and 2). The level

of glucosinolates in JN29 is above the recommended level for use of the term

“canola” (Anon. 2007). Interspecific crossing with canola (B. napus) cv. Mystic

resulted in B. juncea-type progeny with lower levels of seed glucosinolates and

higher levels of oleic acid than JN29 (Table 2). B. napus was a useful source of

seed quality alleles for B. juncea in this interspecific crossing program.

There was evidence for transgressive segregation for important

agronomic and seed quality traits in both species following this cross. Evidence

was presented for a B-genome repeat sequence (Schelfhout et al. 2004) or

partial introgressions in B. napus-type selfed progeny (Schelfhout et al. 2006).

Another source of variation is recombination between the A-genome

chromosomes of B. juncea and B. napus during meiosis in the F1 interspecific

hybrid. F3-derived progeny 99X055-004-5, which was derived from B. napus

cv. Mystic as pollen parent and B. juncea JN29 as maternal parent, had B.

napus morphology and B. juncea seed composition (Table 2).

Breeders should avoid the temptation to eliminate lines following

interspecific crossing based on visual observations or seed quality assessment

in early generations. Potentially valuable lines may have their potential masked

by epistatic or dominance effects, and continued selfing and selection is

necessary to reveal the full potential of these crosses. For example, progeny

99X022-044 in the F3 generation at MD2001 had elevated glucosinolates and

60

low seed oil, but segregation occurred between two F3:4 progeny at SP2002 for

glucosinolates and both had higher seed oil than the parent line (Table 2).

Principal component analysis was a useful tool for displaying trends in

agronomic traits in interspecific progeny versus parents, and for the selection of

elite lines in target environments. It also showed the range of attributes among

selfed lines from B. juncea JN29 and B. napus cv. Mystic. B. juncea is

considered a potentially useful species for low rainfall and higher temperature

environments (Woods et al. 1991), and therefore work is underway to improve

this species for Australian cropping systems (Burton et al. 1999, 2004).

Traditionally B. juncea is grown in India under dry and heat stress conditions,

and we anticipated that B. juncea would be a source of useful alleles for low

rainfall environments in Australia, which suffer from moisture and heat stress.

This was shown to be true in B. napus-type interspecific progeny at MD2001,

where some progeny showed greater tolerance of drought stress than the B.

napus parent Mystic.

These results demonstrate that it is possible to make agronomic gains

from interspecific crossing without compromising the quality of oilseed Brassica.

The yellow seeded B. juncea used in this study is likely to be derived from a

similar lineage to the ZEM 1 and ZEM 2 lines reported by Kirk and Oram (1981).

The tall stature and yellow seeds of JN29 are characteristic of the China /

Eastern Europe group identified by Vaughan (1963). B. juncea JN29 is

inefficient at converting biomass to yield. This is reflected in the low harvest

index values, and tall growth. Future genetic selections of canola quality B.

juncea with shorter stature and drought and heat tolerance may provide more

useful alleles for B. napus canola breeding than described in this paper.

B. juncea accessions from the India / Pakistan group (Vaughan, 1963)

are better adapted to shorter seasons and higher temperatures than their more

northern relatives, but are not canola quality. Access to these alleles in B.

napus may be possible via interspecific crossing with canola quality B. juncea,

but this would require more diverse germplasm in canola quality B. juncea than

currently exists. This flow of alleles across species boundaries will be

accelerated by selfing of interspecific hybrids and reselection of canola quality

progeny.

61

Acknowledgements

This research was funded by an Australian Research Council – Strategic

Partnership Industry Research Training grant with co-funding provided by the

Export Grains Centre Ltd and the Council of Grain Grower Organisations Ltd.

Near-canola quality Brassica juncea line JN29 was kindly provided for our

research by Wayne Burton, Victorian Department of Primary Industries,

Horsham, Victoria, Australia. Milton Sanders, Kylie Edwards and Michael Blair

at The University of Western Australia, Graham Walton and staff at the

Department of Agriculture and Food Western Australia, and staff at the

Merredin Research Station provided assistance with the management of field

trials. Seed quality analysis of 2001 trial seed by NIR was generously provided

by Norddeutsche Pflanzenzucht Hans-Georg Lembke KG, Germany, and

analysis of 2002 trial seed by NIR and GC was conducted by the Chemistry

Centre of WA.

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of Australian canola quality Indian mustard breeding lines. In ‘Proceedings of

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September 1999 (Eds N Wratten and PA Salisbury). (Groupe Consultatif

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chromosomal and genomic origin of the introgression. Theoretical & Applied

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glucosinolate mustard. Canadian Journal of Plant Science 70, 419-424.

Oram R, Salisbury P, Kirk J, Burton W (1999) Brassica juncea breeding. In

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McDonald AG Green) pp 37-40. (Organising Committee of the 10th

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Plieske J, Struss D, Röbbelen G (1998) Inheritance of resistance derived from

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Potter T, Marcroft S, Walton G, Parker P (1999) Climate and soils. In ‘Canola in

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Congress: Canberra)

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65

Chapter 6 - General Discussion

The aim of this study was to introduce new genetic variation for important

agronomic and quality traits into the B. napus gene pool from B. juncea, without

losing canola quality and basic agronomic adaptation of B. napus under

Australian conditions. Simultaneous transfer of interesting traits in the reverse

direction, from B. napus to B. juncea, was also tested. Reciprocal interspecific

crosses were made between canola quality B. napus and near-canola quality B.

juncea. Selfed progeny of such crosses were shown to retain their canola

quality traits in the seed, as well as to display transgressive segregation for

agronomic and seed quality traits (Chapter 5). Some progeny showed

improved adaptation to low rainfall conditions. Most of the selfed progeny were

B. napus in morphology, but some displayed B. juncea morphology. To monitor

germplasm improvement, interspecific progeny were subject to a series of

agronomic assessments. Molecular and cytological marker tests were

developed to identify and visualise B-genome transfer from B. juncea to B.

napus (Schelfhout et al. 2004, 2006; Chapters 3 and 4).

This research has demonstrated that new oligo- and polygenetic

variation for economically important agronomic and seed quality traits can be

transferred between B. napus and B. juncea by interspecific crossing, without

sacrificing canola quality. Carver and Taliaferro (1992) commented there is a

preference for the backcross method for the introgression of complex

characters from wild or distant relatives of self-pollinated crops. Recurrent

backcrossing to B. napus allows transfer of readily detected Mendelian genes

from the donor species, such as major genes for disease resistance, but also

reduces potentially valuable polygenic variation for agronomic and quality traits

from the donor species. It would be preferable not to backcross, but rather to

self from the interspecific F1 and recover fertile B. napus or B. juncea selfed

progeny with useful characters as well as canola quality in the seed oil. The

chances of obtaining useful genetic variation in selfed interspecific progeny will

be increased if the donor species is also canola quality. Research presented in

this thesis supports the use of canola quality parents in interspecific transfer of

useful genetic variation from B. juncea to B. napus, and vice versa.

66

Genetic restrictions in the Australian canola crop

Traditionally, rapeseed and canola (B. napus) were grown and bred in higher

rainfall regions of southern Australia. Early Australian canola cultivars were

developed in the 1970’s from Canadian and European ‘single-low’ cultivars

(Salisbury and Wratten 1999), with major contributions from Asian B. napus and

B. juncea types (rapeseed quality) introduced in 1970 (Cowling 2007). Blackleg

resistance was a main goal of genetic improvement in the Australian canola

breeding programs 1970 - 2000, and only a mild vernalisation requirement

could be tolerated. After 30 years of closed recurrent selection, the Australian

gene pool had become moderately inbred, with an effective population size of

approximately 11 and a population coefficient of inbreeding of 0.21 (Cowling

2007).

Rapeseed (B. napus) itself was subject to severe genetic bottlenecks as

a result of its origins as an agricultural species, following rare natural

interspecific crosses between agricultural varieties of B. rapa and B. oleracea,

and separation into winter and spring gene pools each with relatively narrow

genetic diversity (Becker et al. 1995). In order to meet market demands and

quality standards, the gene pool of B. napus was further restricted by the

conversion from rapeseed quality to canola quality in Canada in the 1970s

(Downey and Rimmer 1993, Juska et al. 1997). This is the background to the

narrow gene pool of spring canola introduced to Australia in 1970, following

which the crop was bred in relative isolation from the rest of the world’s canola

gene pool.

One consequence of this restricted gene pool in Australia is the lack of

genetic variation to continue improving canola cultivars for adaptation to low

rainfall Mediterranean-type environments in Australia. Genetic variation for

early vigour, pod (siliqua) shatter resistance, drought tolerance, early flowering,

and blackleg resistance may no longer be available in the Australian canola

gene pool, which poses a dilemma for future genetic progress in adaptation to

low rainfall environments.

67

Choice of germplasm donor

B. juncea was chosen as an interspecific germplasm donor because it carries a

number of agronomically beneficial traits such as shorter duration to anthesis,

non-shattering siliqua (Prakash and Chopra 1988), better tolerance to abiotic

stress (Lewis and Thurling 1994), and other traits that would be of benefit to B.

napus canola grown in low rainfall Mediterranean environments. Amongst

Brassica species, B. juncea appears to be more resistant to blackleg disease

than B. rapa, B. carinata and B. napus (Roy 1978, Helms and Cruikshank 1979,

Saharan 1992).

Field trials in Victoria and Western Australia showed the B. juncea line

used in this study (JN29) had tall plant stature, yellow seed coats, non-

shattering pods, and high levels of resistance to blackleg (W. Burton and W A

Cowling, personal communications). This line was sourced from the Agriculture

Victoria breeding program in Australia (Burton et al. 2004) and bears similarities

to the Chinese – Eastern European group of B. juncea accessions described by

Vaughan (1963). JN29 (as described in Chapter 5) produced lower oil and

higher protein than the B. napus parent (Mystic) when grown in the same trials.

The oil profile of JN29 exhibited zero erucic acid but the oil-free meal had

elevated glucosinolate content of approximately 45 µmol/g, which exceeds the

value of 30 µmol/g for canola quality (Anon. 2007). This “near-canola quality” of

JN29 was critical to the value of this interspecific crossing program – canola

quality progeny were obtained by selfing the primary interspecific cross

(Chapter 5) and this circumvented the need for extensive backcrossing to

eliminate deleterious quality alleles. JN29 was the source of increased genetic

variation in B. napus progeny for various agronomic and seed quality traits,

including adaptation to a low rainfall environment (Chapter 5). However, JN29

possessed some shortcomings when grown in the target low rainfall

environment. At Merredin in 2001, JN29 consistently grew above 1.5 m tall,

almost twice the height and with half the yield of some B. napus interspecific

progeny. Its height made it more susceptible to lodging caused by wind and

less suitable for direct harvesting. As discussed in Chapter 5, the harvest index

of JN29 was also low as a result of low seed yield and high biomass. However,

JN29 had excellent early vigour and this trait was improved in some B. napus

interspecific progeny derived from crosses with JN29 (Chapter 5).

68

Further improvement of B. napus in crosses with B. juncea may be

possible by using the India - Pakistan group of B. juncea (Vaughan 1963),

which may be better adapted to the low rainfall Mediterranean climates of

southern Australia.

The India - Pakistan group have brown seed coats rather than yellow.

Canola quality has been reported in B. juncea of the India – Pakistan type

(Kaushik and Agnihotri 1996) but it appears as though this particular material

may not have been used for improvement of Australian germplasm. Burton et

al. (2004) report that Indian types of B. juncea have been used in Australian

breeding programs to reduce height and duration to anthesis. It would be

interesting to breed canola quality B. juncea from JN29 (used in this study) with

the short stature and heat/drought tolerant India - Pakistan B. juncea. Based on

the approach outlined in this thesis, new canola quality B. juncea with promising

traits from the India – Pakistan group could be used to introduce desirable

alleles for improved agronomic, quality and disease traits into B. napus¸ and

canola quality would be maintained in selfed progeny of such a cross.

Fecundity of cross direction

In Chapter 4 (Schelfhout et al. 2006) and Chapter 5, it was shown that the

direction of the B. napus – B. juncea interspecific cross greatly affected the

fecundity of the cross. In this study, B. napus was the most successful maternal

parent. In contrast, Roy (1980a) found seed set was markedly higher when B.

juncea was used as the female parent. Roy (1980a) conducted interspecific

crosses between several Brassica oilseed species. He found that B. juncea as

the female parent improved the success of the cross with B. juncea × B. napus

and B. juncea × B. campestris (syn. rapa) crosses. In a closely related project,

J Wroth (personal communication) made similar observations to Roy (1980a)

with higher F1 seed set from crosses where B. juncea was the maternal parent.

However subsequent F2 seed set was found to be higher in crosses which

involved B. napus as the maternal parent (J Wroth, personal communication). It

is clear that the genotype of parents chosen for interspecific hybridisation, and

the direction of crossing, can greatly influence the fecundity of the hybridisation

event.

69

Characteristics of interspecific hybrid progeny

In this study, the morphology of interspecific progeny tended to reflect

morphology of the maternal parent. Consequently, there was a greater number

of progeny generated with B. napus morphology than B. juncea morphology,

since B. juncea JN29 was not a successful maternal parent. Roy (1980a,

1980b) found that progeny of crosses where B. juncea was the maternal parent

(and B. napus and B. rapa the paternal parents) produced progeny with

predominantly B. juncea morphology. It may be concluded that B. napus

maternal parents give rise to B. napus-type progeny predominantly, and B.

juncea maternal parents give rise to B. juncea-type progeny predominantly.

This may be the result of maternal control of chromosome arrangements during

meiosis in the F1. The fertility of such progeny is determined independently of

the most fecund cross direction in the parents.

Roy (1980b) stated, with respect to his interspecific crosses, that the

chance of B. juncea acquiring genes for low erucic acid or low glucosinolate

from B. napus (or B. campestris - syn B. rapa) appeared to be remote. In this

study, seed quality improvements were apparent in both B. juncea and B. napus

interspecific progeny derived from the interspecific cross. Some B. juncea

interspecific progeny had lower glucosinolates than the B. juncea parent JN29

(Chapter 5), and some B. napus interspecific progeny had higher oil in seed that

the B. napus parent (Chapter 5). In effect, genetic improvement and genetic

diversity was apparent in both morphological types of offspring - B. napus and

B. juncea.

Interspecific crossing and selfing of the F1 appears to be a valuable

method for introducing genetic diversity into B. napus from B. juncea, and into

B. juncea from B. napus, when both parents are canola quality. Further studies

will identify the specific genetic control of the new allelic diversity in B. napus

found in this study – whether the donor alleles from B. juncea were derived from

the A or B genomes. The opportunity exists now to fix the new alleles by selfing

and assessing their origin and function.

70

B-genome specific markers

The B-genome specific marker, developed in this study as a PCR marker

(Schelfhout et al. 2004, Chapter 3), facilitated the selection of interspecific

progeny carrying putative B-genome introgressions (Schelfhout et al. 2006,

Chapter 4). The B-genome marker was used in a PCR-based assay and as a

hybridisation probe in both Southern and in situ hybridisations as demonstrated

in Schelfhout et al. (2004) (Chapter 3). The repetitive B-genome marker

pBNBH35 was present on all B-genome chromosomes (Schelfhout et al. 2004,

Chapter 3). Large chromosomal fragments or chromosomal substitutions,

additions and introgressions of whole chromosome arms were observed in

fluorescence in-situ hybridisation (FISH) studies (Schelfhout et al. 2006,

Chapter 4). pBNBH35 was dispersed primarily about the centromeres of the

eight Brassica B-genome chromosomes with very little signal in the distal

regions of the chromosomes (Schelfhout et al. 2006, Chapter 4). Low copy

numbers on distal regions may limit FISH hybridisation studies, however the

PCR-based assay was efficient in detecting low copies, although it did not

provide a visual image of the site of introgression. PCR and in situ studies are

best used in combination (Schelfhout et al. 2006, Chapter 4). The initial screen

for the presence of B-genome in early generation progeny may be performed

using the PCR based assay. Once accessions are identified as B-genome

positive, they can be selected – thus “filtering” progeny for presence of B-

genome and avoiding unintentional losses. Later generation progeny may be

tested by the FISH assay and, if introgressions are large enough, introgressions

may be located within the genome using additional molecular and cytogenetic

techniques based on the pBNBH35 probe.

In this study, interspecific progeny with positive FISH signals for B-

genome were of B. juncea morphology, whereas progeny that did not have a

positive FISH B-genome signal were of B. napus morphology (Schelfhout et al.

2006, Chapter 4). On one occasion a hybrid was found with the full

complement of B. napus chromosomes but expressing a B. juncea phenotype

(Schelfhout et al. 2006, Chapter 4). FISH images showed this line to have B-

genome signals embedded in a full B. napus genome complement (Schelfhout

et al. 2006, Chapter 4). It appears that these putative introgressions are

carrying alleles controlling B. juncea morphology. The majority of interspecific

progeny tested positive to B-genome in the PCR assay but did not generate

71

visible signals in the FISH assay. These results indicate minor introgressions

may have occurred without massive structural changes to chromosomes. The

B-genome specific marker, after conversion to a PCR-based marker, provided a

relatively simple assay to detect putative B-genome introgressions, and

potentially promising lines were readily identified in early generations.

This marker may be useful in selection of B. napus lines carrying

Brassica B-genome traits such as blackleg resistance, shatter resistance and

numerous other beneficial traits. It may be used to investigate phylogenetic

relationships within Brassicaceae.

Summary

Significant levels of new genetic variation were added to the gene pool of

canola quality B. napus from B. juncea, and vice versa, through the use of

reciprocal interspecific crossing. Despite some limitations in fertility of progeny

and cross direction, this breeding approach permits successful expansion of

genetic diversity for the benefit of both species. The key to this expansion is the

use of canola quality parents, so that repeated backcrossing to restore canola

quality is not necessary.

In the low rainfall environment of Merredin, Western Australia,

interspecific progeny were identified that outperformed both parental lines in

growth, yield, seed quality and harvest index. B. juncea parent JN29 failed to

yield well due to its physiological and morphological limitations. Transgressive

segregation in interspecific progeny resulted in higher yield and improved seed

quality of B. napus-type progeny under the water-limiting environment of

Merredin. The results suggest that there has been an introduction of minor

alleles from B. juncea that contribute to a transgressive improvement in yield

and quality when B. napus cv. Mystic is the maternal parent.

This study has combined molecular, cytogenetic and agronomic studies

to demonstrate the benefits of interspecific crossing with near-canola quality B.

juncea to improve B. napus canola. This process has further potential to

improve canola quality B. juncea or to expand the gene pool of either of these

species to accommodate desirable agronomic, disease resistance and quality

traits.

72

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