Construction of a Genetic Map and QTL Mapping for Turnip Tuber Traits Using an Recombinant Inbred Line Population from a Cross

between Turnip and Rapid Cycling in Brassica rapa

Minor Thesis Report (PBR-80424)

Temesgen Menamo (Reg. No. 860717-557-050)

Supervisors: Dr. Ningwen Zhang

Dr. Guusje Bonnema

Laboratory of Plant Breeding, Wageningen University

The Netherlands, Wageningen

January, 2013

Construction of a Genetic Map and QTL Mapping for Turnip Tuber Traits Using an Recombinant Inbred Line Population from a Cross

between Turnip and Rapid Cycling in Brassica rapa

Minor Thesis Report (PBR-80424)

Temesgen Menamo (Reg. No. 860717-557-050)

Supervisors: Dr. Ningwen Zhang

Dr. Guusje Bonnema

Examiners: Dr. Guusje Bonnema

Dr. Ningwen Zhang

Laboratory of Plant Breeding, Wageningen University

The Netherlands, Wageningen

October-January, 2013

i

Acknowledgement

I would like to thank the Brassica group leader Guusje Bonnema for giving me this opportunity to join

in this group. I would like to thank Ningwen for support, guidance and encouragement in doing each

part.

I thank to Johan Bucher for his support in lab work.

I would like to acknowledge NFP for financially supporting until finishing of my study.

Last but not least for all Brassica group members for support by ideas.

Abstract This study described on construction of a genetic linkage map based on 110 SNPs and 14 InDels

markers on Brassica rapa. It also described turnip tuber related traits QTLs for the Brassica rapa. An

recombinant inbreed line population consists of 106 F6/7 individual lines derived from a cross

between VT-115 and RC-144 were used for the present study. The Kosambi mapping function was

used for linkage map to convert recombination frequencies into map distances (cM). A total of 61

markers were mapped over 13 newly constructed linkage groups. Linkage group 01, 02 and 09 had

two linkage groups. The alignment and orientation of the newly constructed linkage groups were

agreed with existing B. rapa linkage maps from chiifu genome. The map covered a total of 639.5 cM

of the genome with an average linkage length (coverage) of 49.2 cM and an interval distance of 10.5

cM. The length of linkage groups ranged from 1.0 cM to 183.8 cM for L01_1 and L09_1 respectively.

The turnip tuber related QTLs was made by Kruskal- wallis Mapping. A total of 94 turnip related

QTLs were detected at significant level of ≤0.1 in all newly constructed linkage groups except L01_1

and L04. Out of the total 18 for Brix (total Soluble Sugar), 29 for turnip tuber fresh weight, 26 for

turnip tuber dry weight and 21 for turnip tuber dry mass. Dry weight per turnip tuber, fresh weight

turnip tuber and weight per turnip co-located together on linkage group A02 at flowering QTL loci.

this paper would like to recommend making linkage and QTLs mapping with more number of

individuals and markers. It gives accurate and precise linkage groups and QTLs maps.

Keywords: Brassica rapa, SNPs, InDels, linkage map, QTL

Table of Contents

Acknowledgement .................................................................................................................................... i

Abstract ....................................................................................................................................................ii

1. Introduction ..................................................................................................................................... 1

1.1. Background on Brassica ........................................................................................................... 1

1.2. Economic importance .............................................................................................................. 2

1.3. Morphological description ...................................................................................................... 3

1.4. Brassica rapa breeding Objectives .......................................................................................... 3

1.5. Molecular Markers and Development of Genetic Linkage Maps ............................................ 3

1.6. Scope of the thesis .................................................................................................................. 5

2. Method and Material ...................................................................................................................... 6

2.1. Parental material ..................................................................................................................... 6

2.2. DNA extraction ........................................................................................................................ 6

2.3. LightScanner ............................................................................................................................ 7

2.4. Pre-Screening markers with polymorphism ............................................................................ 8

2.5. Markers and Genotyping ......................................................................................................... 8

2.6. Linkage map construction ....................................................................................................... 9

2.7. Phenotype and QTLs mapping ................................................................................................. 9

3. Results and discussion ................................................................................................................... 10

3.1. Pre-Screening of marker with polymorphism and Genotyping ............................................ 10

3.2. Linkage map construction ..................................................................................................... 12

3.3. Frequency distribution of turnip tuber traits ....................................................................... 17

3.4. QTL analysis ........................................................................................................................... 18

4. Conclusion and recommendation ................................................................................................. 23

References ............................................................................................................................................. 24

Appendixes ............................................................................................................................................ 27

1

1. Introduction

1.1. Background on Brassica

Brassica has a large plant family with 338 genera and 3,700 species, including a wide range of

horticulture crops, which have great economic value and grown as vegetables, fodders, sources of

oils and condiment (Al-Shehbaz et al., 2006; Campo, 1999; Zhao et al., 2010). The model plant

Arabidopsis is also closely related to the genus Brassica (Ostergaard and King, 2008) which can be

exploited to find orthologous genes from Arabidopsis with known function. This is more

advantageous for genetic study for Brassica genus. This Brassica genus has six species including: B.

rapa, B. nigra, B. oleracea, B. juncea, B. napus and B. carinata. The three diploid Brassica (B. rapa, B.

nigra and B. oleracea) species forming the Triangle of U (figure 1), have hybridized in all possible

combinations to produce the three allotetraploid species; B. juncea, B. napus and B. carinata

(Nagaharu, 1935; Ostergaard and King, 2008). Brassica Rapa species has a diploid (2n=20) genotype

and is a morphological diverse including vegetable and oilseed crops ( turnip types, leafy types and

oil types) such as: Chinese cabbage (Brassica Rapa subsp. pekinensis), non-heading pak choi (Brassica

rapa subsp. Chinensis), turnip (Brassica rapa subsp. rapa L.), turnip rape ( Brassica rapa), and Sarson

(Brassica rapa L. var. trilocularis) (Song et al., 1995). Turnip (Brassica rapa subsp. rapa L.) is

domesticated in very ancient time and was used for human consumption and animal feed (Vogl-

Lukasser et al., 2008). It believes that the turnip was the first domesticated Brassica species and

naturally grown from the Western Mediterranean region to central Asia (Campo, 1999). Europe and

Eastern Asia are one of the center of diversity for B. rapa subsp. rapa. It is grown popular in Europe

starting from 2500-2000 BC, the Middle East is starting 1000 BC and Asia starts around 1800 B.C

(Vogl-Lukasser et al., 2008).

2

Figure 1. The "Triangle of U" diagram, showing the genetic relationships of six species in the genus Brassica (Nagaharu, 1935).

1.2. Economic importance

Brassica rapa is one of important crop among the six economically cultivated Brassica species of U’s

triangle. It is cultivated all over the world as a vegetable for food and fodder for animals. Brassica

rapa var. Chinese cabbage and var. chinensis (leaf cabbage) are extensively grown as important

market garden vegetables in China. The main economic important part of the turnip is hypocotyl with

tap root but the leaves are also used for fodder as well as for food. The turnip greens (tops) are

excellent chopped in salads or cooked briefly as a side dish. Turnip also used for flavor in ingredient

soups and a winter salad. It also used in different dishes in some country like India and Pakistan. The

nutrient composition of turnip (USDA, 2011), it has more calcium and sodium mineral, particularly

rich in vitamin C, very low fat and proteins (USDA, 2011). Brassica vegetables are rich in

glucosinolates, a chemical which can block the growth of human cancer cells (He et al., 2012; Kushad

et al., 1999; Rosa et al., 1997).

3

1.3. Morphological description

Flower agronomic traits are important for vegetative as well as for turnip which related to the

quantity and quality of turnip tuber formation. The flowers are small, yellowish color and clustered

at the top of the raceme. It produces pea-like pods which contain seeds. Turnip is a biennial crop but

most commonly grown as an annual. The first year the plants grow to be used for human

consumption in their hypocotyl with tap root and the second year produce flowers and set seed.

Traditionally, turnip is called root crop but actually it is not a root at all. It consists mainly of the

hypocotyl as well as main root. Hypocotyl is plant part that lies between the main root and the first

seedling leaves (cotyledons) which grows below or above the surface of the soil. It also has little or

no neck and a distinct tap root( Brassica group, Wageningen University).

1.4. Brassica rapa breeding objectives

Brassica rapa has a wide genetic variability, it helps to important traits and improvement in this

crop. Recent advanced genetic marker techniques can supplement for improving of the agronomical

important traits of this crop since conventional genetic analyses and breeding also could success in

the improvement of many traits. Genetic analysis and identification of chromosomal regions which

containing agronomic important morphological traits would be helpful in breeding for desired traits.

Many root crops or root like crops can undergo considerable root radial growths which would be

suitable for food. Turnip is one of fleshy and edible hypocotyl crop. So, it is important to improve the

quality and quantity of the turnip yields.

1.5. Molecular Markers and Development of Genetic Linkage Maps

Most of agriculturally important traits (yield, flowering time and some of disease resistance) are

controlled by many genes, called quantitative traits (polygenic) which contain genes associated with

a particular quantitative trait are known as quantitative trait loci (QTLs) (Collard et al., 2005). Brassica

Rapa has a large number of morphological differences including flowering time and turnip tuber

formation. Vegetative turnips have a very high morphological diverse among turnip genotypes

(Yildirim et al., 2010) including; flowering, turnip onset, turnip shape, size and so on.

There are several molecular linkage maps in B. rapa have been developed using RFLP, RAPD,

AFLP, and SSRs molecular markers and mapping populations such as F2, DH, RIL. In Arabidopsis (El-

Assal et al., 2001) and some of Brassica rapa (Lou et al., 2007) studied at the co-location of QTL for

4

floral and other growth-related traits. Several studies are observed in QTL flowering time and turnip

tuber formation in different populations in the Brassica rapa at Wageningen University by Brassica

group members. The genetic of turnip tuber formation studied by SSR and InDel molecular markers

to profile an F2 population derived from a cross between a Vegetable Turnip (VT-115) and a Wutacai

(PC-105) (Wang, 2011). He also studied morphological traits of the corresponding F3 population in

the field and the reliability of InDel markers and LightScanner. He observed phenotypic variation in F3

population even unexpected variation also. For instance, red or purple color in leaves and/or turnips,

since both of the parents (VT-115 and PC-105) didn’t have this type of trait. He found ten markers to

be associated with leaf traits eight of which are associated with leaf number, spreading over linkage

group A01, A02, A03, A05, A09, and A10.

Vernalization caused delays of turnip onsets which unvernalized turnips are caused by the

earlier turnip swelling onset (Visser, 2010). This study also showed that a strong correlation between

turnip traits (diameter and width) and the swelling onset as significantly delayed by vernalization. In

addition to vernalization, the study also had shown the turnip swelling onset different between

genotypes. The study used three turnip growth candidate genes but only one marker (nw_60) was

associated with turnip traits (diameter and width).

A total of 27 QTL detected which were affecting 20 morphological traits from population F2/3

crossing Rapid cycling vs. Chinese cabbage; double haploid from Yellow sarson, Pak choi, and a

Japanese vegetable turnip variety. From those of it, eight linkage groups are detected for flowering

time on R01, R02, R03, R06, R07, R08, and R10 in all populations ( F2/3, DH-38, DH-30, and BC1) in

different growing seasons (Lou et al., 2007). It shows the major flowering time QTL (FLQTL-2)

detected in all populations on linkage group R02 co-localized with BrFLC2. This study also detected a

single QTL for the turnip traits at the top of R02 which co-localizes with the major flowering time QTL

(FLQTL-2).

The major flowering time QTL detected in different Brassica rapa populations on linkage

group R02 co-localized with BrFLC2. FLOWERING LOCUS T (FT) also identified in Arabidopsis as a

main component of the florigen or mobile flowering promoting signal produced in the leaves

(Abelenda et al., 2011). This locus has a homologous gene in Brassica rapa. For instance, BrFLC5 gene

homologous to FLC regions to Arabidopsis (Schranz et al., 2002).

5

1.6. Scope of the thesis

The two parental lines crosses VT-115 x RC-144 originated from Japan (VT-115) and USA (RC-144).

Between these two parental lines there are different flowering time which RC-144 is an early

flowering type than VT-115 (discussion with Ningwen) and they are different morphotype (VT-115

vegetative turnip; RC-144 oil type). The frequency distribution of this population had shown similar

pattern in F2 and F3 progenies (Rémi, 2010). Phenotyping in F2 population could not repeated and

the genotype comes from the combination of two parents while RILs are homozygous lines each

harbours different combination of two parental genome fragments. QTLs mapping using of

dominant F2 population is less accurate when compared to RILs (Ferreira et al., 2006).

The scope of this thesis was:

to construct a genetic map of 106 lines of an RIL population (F6/7)from a cross between VT-

115 and RC-144

to detect turnip tuber related QTLs to the constructed linkage groups

6

2. Method and Material

2.1. Parental material

The mapping population have been consisted of 106 RILs F6/7 populations developed by single-

seed descent from the cross Brassica rapa accessions; Vegetable turnip VT-115 as female and Rapid

cycling line RC-144 as male from a laboratory of Plant breeding, Brassica group, Wageningen

University. VT-115 is vegetable turnip which originated from Japan and RC-144 is rapid cycling which

originated from the USA. Between these accessions, the flowering time is different (discussion with

Ningwen) which RC-144 line has earlier flowering time than VT-115 (Zhao et al., 2005).

This population (36RIL) is flowering time between 30 and 78 days after sowing which it gives

very early turnips formation. During F2 QTLs detection, it showed that all markers the average turnip

size of plants with the “a” genotype (VT-115) was always bigger than the group with the “h”

genotype, “h” heterozygous progenies have been always bigger than the “b” genotype (PC-144) (Vos,

2009). So, this indicates the co-dominance of turnip tuber related genes.

2.2. DNA extraction

The samples of 150 populations RIL36 F6/7 were collected from greenhouse grown condition. The

protocol for DNA isolation (Gert van Arkel & Maarten Nijenhuis) was adapted from laboratory of

plant breeding, Wageningen University. The leaf samples already were collected and put in the 96

micronic tubes with 2 steel balls in a blue holder in liquid nitrogen. Two hundred ml of isolation

buffer was prepared with 4mg/ml of RNase. The isolation buffer was put in water bath with 65OC

until used. The samples were fixed in the RETCH machine with the blue holder without the

transparent lid into a black and white adapter. The samples Milled for 30 seconds at 30 Hz for three

times with the changing of the direction of the holders. After 1 ½ minute the entire blue holder with

the tubes was back into liquid nitrogen. The boxes were Centrifuge briefly in order to get the powder

in the bottom of the tube. The isolation buffer (400 µl) was added to each the tube. For safety

purpose each chemical added in the fumehood. The blue holder with samples and without the

transparent lid placed in the specially designed clamp and tight the 4 nuts (This will prevent that the

lids not pop off after incubation at 65°C) for incubate at 65°C in a water bath for 60 minutes. The

clamp containing the tubes cooled down for 15 minutes on ice-water. Four hundred µl of chloroform:

isoamyalcohol (24: 1) was added to the tubes and mixed by inversion for 5 minutes by machine. The

phases separated by centrifuging at 4600 rpm for 40 minutes. The water phases were Pipettes

7

2X175µl in to the new tubes and 280 µl isopropanol was added. It had mixed carefully by the

inversion blue holder with tubes. The DNA pelleted by centrifuging for 25 minutes at 4600 rpm. The

supernatant was discarded and 300µl of 70% ethanol was added. The samples again centrifuge for 20

minutes at 4600rpm for pellet the DNA. The supernatant was discarded and the pellet dried

overnight in the fumehood. The DNA concentration was checked by NANOdrop. The quality of DNA

was also checked on a 1% agarose gel by putting of the 2µl DNA sample, 8µl MilliQ and 2µl of loading

buffer; total 12µl.

2.3. LightScanner

The LightScanner machine is high-resolution DNA melting curve (HRM) which provides post-PCR

detection of mutations SNPs in genomic DNA. It detects high-density information of the fluorescence

as a function of temperature which contain double-strand DNA binding dye LCGreen Plus. LCGreen

has the ability to detect the presence of heteroduplexes formed during PCR if the sample is

heterozygous for a particular mutation when compared to other DNA dye ( e.g. SYBR Green). The

main advantage is faster (1.5 hour for PCR and half an hour for measuring and analysis) when

compare to Li-Cor procedure (3 hours for PCR and 2 hours Li-Cor) which eliminating the need for

DGGE gels. SNP genotype scoring by high-resolution melting analysis is required only PCR, a DNA dye

(LCGreen), and melting instrumentation which completed within 5 minutes after completion of PCR.

It is simple, rapid, inexpensive and no-need of post PCR reagents. It also detects the SNPs (sequence

variations) without the need for dye-labelled probes.

The Lightscanner has specific master mix and PCR program. The master mix was prepared:

MillQ water 4.5 µl, reaction buffer for phire 2 µl, dNTP 0.4µl, primer (diluted) forward and reverse 0.5

µl, LC-green 1µl, and Phire enzyme 0.1µl per master mix. Finally, 9 µl of master mix, 1 µl DNA and 20

µl mineral oil were added into PCR plate in black-white with 96-wells for amplification of the DNA.

The mineral oil helps to avoid evaporation of the samples during Hi-Res Melting.

Hi-Res Melting is shown a melting curve of the DNA which changes of the shape of the curve

within one marker, it is indicating of the presence of sequence variances in the PCR product. The first

step in the analysis defined subsets which define by DNA markers. Negative filter also was done to

identify any samples that were included on the plate but failed PCR reactions. It helps to avoid the

possibility of generating false positive data during the next step, normalization. Normalization is

important before doing of analysis of the data. The two regions were selected one before and one

after the major transition. The region shouldn’t show any melting curves difference after the selected

region. It should be shown a straight line out of selected regions. The final step is grouping of the

DNA templates according to melting curves. The grouping of genotyping was made to automate

8

clustering when different curve shapes can be clearly distinguished. However, if confusing curve

shapes happen, the automated clustering changed to manually select samples that share similar

curve shapes based on difference temperature of melting peaks .

2.4. Pre-Screening markers with polymorphism

Pre-screening of the markers was made by LightScanner; testing the polymorphism of the markers.

Before starting genotype scoring of the whole RIL36 F6/7 population, pre-screening of the

polymorphic marker test was made on 6 progenies with parents (VT-115 X PC-144). A total of 137

markers were pre-screened. Those markers were developed Dong Xiao to detect flower related QTL

on DH-68 population of Brassica rapa subspp rapa . Simultaneously, genotype scoring of those of 6

progenies was done when they were shown a polymorphic for specific markers.

2.5. Markers and Genotyping

Flowering traits

A total of 137 markers were selected by Xiao Dong for his study on flowering traits of F1 double

haplod from crossing yellow Sarson (YS-143) and Pak Choi (PC-175) population(Xiao et al.,

Unpublished). He develop the markers based on arabidopsis genes which are involved in flowering

time control. He also BLAST the Arabidopsis those genes sequences in Chinese cabbage Chiifu

genome sequence; Brassica genome database; to get paralogues in Brassica rapa. I screened those

137 markers on 144 RILs C36 populations.

InDels and SNPs

Genome-wide Brassica rapa short insertion/deletion length polymorphisms (InDels) (<5 bp) were

detected by comparing whole genome re-sequencing data from two B. rapa accessions, L144 and

Z16, to the reference genome sequence of Chiifu-401-42. Previously tested 14 InDel polymorphisms

(4~5 bp in length) between L144 and Z16 also showed polymorphisms between VT-115 and RC-144,

and therefore included in the map construction of this study. SNPs markers were generated from re-

sequencing data which compared five re-sequenced genomes (Chiifu, DH-VT-117, RC144, DH-YS143

and a Wutaicai DH line) and detected SNPs between each of the two genomes. The genotyping using

these SNP markers was done through KARSPar SNP genotyping system from Kbioscience

(Hoddesdon, Hertfordshire, UK). Genotyping data of the same RIL population with 51 SNPs that

randomly distributed in B. rapa genome was included in the map construction in this study as well.

9

2.6. Linkage map construction

For construction of a genetic linkage map a total of 124 markers (SNPs & InDels) in 106 F6/7

population were used. Before linkage analysis, chi-square tests (X2) were performed on both all

markers for goodness of fit to the expected Mendelian 1:1 segregation ratio of each locus. Linkage

analysis was performed with the Joinmap 4.1 and the Kosambi mapping function (Manly et al., 2001)

was used to convert recombination frequencies into map distances (cM). Initially, the maximum of

0.20 recombination frequency was used to identify linkage groups. Linkage maps were drawn using

MapChart software (Voorrips, 2002). A framework physical map of FT markers was previously

constructed by Xiao dong, which corresponds to the linkage groups of Brassica genome. The 51 SNPs

and 14 InDels markers also incorporated into the frame based on their physical position in the

reference genome of Chiifu-402 retrieved from BRAD database.

2.7. Phenotype and QTLs mapping

Seeds were sown on 30 July 2012 in greenhouse and plants were transplanted into field on 21 August

2012. For each line, three blocks with five plants in each block according to a complete randomized

block design. Harvesting took place in three patches according to their flowering time. Because

turnip growth turns slower after flowering and it might not be fair to harvest plants all in once. So

harvest made in three patches to allow different lines reaching the maximal size while not losing

much of fresh weight and not differ too much for other metabolic changes. First harvest was on 1-oct

2012, second time was on 12-oct and the last harvest was on 31-oct in the same year. The flowering

turnip only harvested the first two harvests. The lastly, all leftover plants harvested, including the

ones that were not flowering yet. QTL map was made, nonparametric mapping method, it uses the

rank sum test of Kruskal- wallis MapQTL6 mapping statically software (Ooien et al., 1996) with 0.1 a

significant level of ≤0.1. Four turnip tuber traits were used for QTLs.

10

3. Results and discussion

3.1. Pre-Screening of marker with polymorphism and Genotyping

Construction of a genetic linkage map using a bi-parental segregating population needs to develop

many molecular markers that show polymorphism between the parents as well as in the progeny. In

laboratory of the plant breeding WUR, Dong Xiao has developed and mapped 137 flowering related

markers using a doubled haploid population DH68 which was derived from a cross between YS-143

(oil type) and PC-175 (leafy type). Those 137 markers were developed from B. rapa sequences with

high homolog (>75% nucleotide sequence identity) to flowering time related genes in Arabidopsis

thaliana (Xiao et al., Unpublished). Polymorphism test was made on those 137 flowering markers on

6lines from F6/7 RIL36 population. The DNA of the real parents was missing. The DNA of a plant from

another accession of the parents was used as references which the parents consider as genetic

similarity. These parents are representative but not really identical with the real parent genotypes.

Therefore, selection of markers was made based on screening of two parents representatives and six

RIL progenies. As a result, 59 markers out of 137 showed polymorphism in the RIL population of a

cross between VT-115 and RC-144 (Appendix 1). The polymorphism was identified when two groups

were formed on those 6 progenies and parents. For instance, BrSPA1P2a was one of marker showed

polymorphic between F6/7 progenies (Figure 2).

Figure 2. pre-screening of BrSPA1P2a marker which shows a polymorphic within 6 progenies and

parents( brown from VT-115 parent and red from RC-144parent ).

11

Genotyping of the polymorphic markers during pre-screen as well as on all RIL population was

made. However, the genotyping was divided into three parts: easy, medium and too difficult to

genotype scoring (Figure 3). The easy and too difficult one was rear but most of the markers were

medium; there were manual scoring of the genotype. The lightScaner give the scoring directly for

the easiest markers. The lightScaner sometimes gives different colour but they look like belong to

one group ( Figure 3B). Figure 3B shows that BrPHYAP1g markers had 3 colours. The green colour was

belong to red colour. This type of marker/s made some correction manually such as making green

colour belong to red colour. Some markers also showed polymorphic but not easy to score e.g.

BrRGAP2c (F9gure 3C).

Figure 3. genotyping style A) easy, B) medium and C) difficult to make genotype scoring.

12

3.2. Linkage map construction

A genetic linkage map was constructed based on 124 markers in 36RILs populations of 106 F6/7

progeny lines. In total, 79 markers (60% of total markers) were scored as 1:1 ratio (5 to 2%

heterozygous loci) with equal contribution from both parents. A total of 11 InDels and 34 SNPs

showed significant distortion from the expected Mendelian gene frequencies. The chi-square tests

confirmed that on those 11 InDels and 34 SNPs markers the frequencies of individual parental alleles

in the F6/7 population indicated a significant deviation (P≤0.05) from the expected 1:1 ratio.

Theoretically, F6 population is considered a RIL and presents an exception of 1:1 ratio for allele

segregation among individuals with 3% of individuals should show as heterozygous. As a result, 9

InDels and 17 SNPs markers were distributed to the skewed segregation with highly significance at P

value 0.0001. In most of Brassica rapa linkage maps skewed segregation of markers is a common

feature (Choi et al., 2007; Xinhua, 2011; Kapoor et al., 2009). Some researchers were reported that

might be due to loss of chromosomes, the presence gene conversion event and homologous

recombination.

The markers which showed distorted segregation were distributed randomly along all the

linkage groups. In total 61 markers were grouped into 13 linkage groups, with the number of markers

per linkage group ranged from 2 (linkage 1,2 and 10) to 12 (linkage 9), and covered a total of 639.5

cM of the genome (Figure 3). This might be due to insufficient linkages and lack of polymorphism

spanning those regions. Lower number of individuals showed higher stress values and higher

distance variances as well as wide deviation from estimates of the genome size(Ferreira et al.,

2006).The thirteen linkage groups had the average interval distance of 10.5 cM. The number of

markers which used for mapping per linkage ranged from 6 (linkage 4) to 24 (linkage 9), with an

average of 11 markers per linkage group. On linkage L02_1, L02_2 and 10 have been shown only 2

markers were found per linkage group. The length of linkage groups ranged from 1.0 cM for group

L01_1 to 184 cM for group L09_1. Markers BrPHYAP1g, BrVRN1P1b and BrRGAP2c showed

polymorphism in the population, but were excluded during mapping analysis as the scoring of light

scanner output was too difficult and not reliable.

All linkage groups contained region which the adjacent marker larger than 15 cM except in

linkage groups L01_1, L06, L09_2and L10 (Table 1). The linkage groups placement and order for 124

of the SNPs and InDels markers were in good agreement with Brassica rapa flowering markers map

(Xiao dong, DH68). However, markers PV_Br_SNP_0125 were placed on linkage group L07, while it

was predicted to locate on A02 in the reference Chiifu genome. It might be many reasons, such as

13

small population size, lack of recombination between the limited number of markers, true inversion

or translocation of between genotypes.

Two linkage group was observed in linkage 1 and 9. However, the first group was only

contained two markers in linkage group L01. On linkage group 2 also showed two linkage groups with

only two markers per linkage group. On linkage groups 3, 4, 6, 8 and 10 were from only SNPs marker.

The two linkage groups on L09 the placement and order were agreed with the physical map of

Brassica rapa physical map which developed from chiifu genome except loci BrSVPP1c and

PV_Br_SNP_0796 ( Figure 3).

Table 1. Summary of a genetic linkage map construction on Brassica rapa using SNPs and InDels

markers.

Linkage group Number of markers assigned

Number of intervala

Number of gapsb

Length of linkage groups(cM)

L01_1 2 0 0 1.0 L01_2 6 6 2 65.3 L02_1 2 2 2 33.5 L02_2 2 2 2 24.8 L03 3 3 3 50.3 L04 3 3 3 63.8 L05 6 6 2 43.4 L06 4 4 0 23.0 L07 6 6 2 56.0 L08 7 7 1 41.8 L09_1 12 12 7 183.8 L09_2 6 6 0 39.2 L10 2 2 0 12.4 aAdjacent markers > 1cM

bDistance between adjacent markers 15cM (Choi et al., 2007; Kapoor et al., 2009b;

Wang et al., 2011)

14

Figure 3. Comparing of newly constructed linkage groups (right side(Lx)) to Brassica rapa physical map(left side(Ax)); The length of each genetic linkage

groups are indicated in centimorgans (cM) and the length for the physical map is in million base pairs (Mbp, A01-A10) .The cM of LX linkage groups was

0.1cM instead of 1CM to make it easy; because the physical map was constructed based on Mbp with maximum 50. The genetic map was greater than

50cM. The colour markers indicate the newly SNPs(rose) and InDels (green) markers.

Bra0110240.0VEL10.9FLP11.4KNAT51.6PV_Br_SNP_00021.9ATH12.1AGL162.5CIB12.6FDP FD3.1PV_Br_SNP_10483.3AP23.6COP33.8T28I19.1603.9CKB2 CKB14.5PHYE4.8JMJ145.8PDF26.3EBS6.5AGL14 AGL196.9AT4G233407.1SLY17.7AGL24 PGI7.9GA20OX1 GA20OX28.3FWA8.4CIP79.6DFL19.7TOR19.8NS110.5HAT410.6FIP2 FCA10.7CKL1311.2FAR112.0ELIP212.2LUX/PCL113.5ARP413.8PV_Br_SNP_005514.0VIP516.6GA20OX4 YAP16916.7PV_Br_SNP_008017.0GA20OX1 GA20OX2PGM

17.4

CKL1318.7TIC19.5AT3G2132020.0FIS320.4ATFYPP320.8HAF2 RTV1VRN1

21.0

PV_Br_SNP_009821.2SPL521.6SPA4 SPA321.7NFYB3/HAP3C21.9TCTP22.2

A01

PV_Br_SNP_0002 0.0PV_Br_SNP_1048 0.1

L01_1

BrPHYEP2c 0.0

BrEBSP1b 1.3

BrNS1P2c 4.0

PV_Br_SNP_0055 5.8PV_Br_SNP_0080 6.0PV_Br_SNP_0098 6.5

L01_2 Bra0283860.0CDF20.3BNQ10.4FLC FLM/MAF1MAF2

1.2

TCP111.5GA20OX4 YAP1691.7CPD1.9HY52.8EMF12.9NFYA1/HAP2A3.1COL13.9TFL24.3NPH45.1CAM15.3MIR172(EAT) TOE25.9AT1G10588 AT5G59845ZAT12

6.0

VIP2 FDPSRR1

6.1

COL56.7VIN36.8FT TSF7.7RGL17.8EMF28.1GA20OX1 GA20OX28.3CAL AP19.0TEM29.2DFL19.5XAL110.1CSTF6410.2TFIIIA10.4HAP2C10.8GASA611.5GA2OX113.0PV_Br_SNP_017213.5PRE13.6GA3ox2 GA3OX413.8CUL416.5NFYB2 RAV117.6HSP88.117.8SEPALLATA418.0ICU218.1FUS319.3PV_Br_SNP_014320.1TEL120.6BR6OX222.8K19B1.17 CDF123.4LFY23.7FLM/MAF1 MAF4MAF5 FLM/MAF1

25.0

RGL225.5ATVGT125.6Bra03202825.7

A02

BrFTP1c 0.0

PV_Br_SNP_0172 3.4

L02_1

BrTSFP2c 0.0

PV_Br_SNP_0143 2.5

L02_2

Bra0056570.0LAC80.2CPD1.0GA20OX4 YAP1691.3CHE1.5FLC1.8EMF12.0NFYA1/HAP2A2.3FY2.5AGL152.6AT5G149202.8BNQ2 GASA42.9SEPALLATA23.0VIP24.7SEF5.0VIN35.2DFL15.9FRI6.8CBF2 FLC7.1SPL38.1NFYB19.4ELF49.8CAM110.1CKB310.9SOC111.0protein_coding11.3ELF4-L111.4TOE111.5ATC11.6pEARLI13.1PDF213.4CLPS3 WNK115.0HAP2B15.2COL915.7FLD16.1SPY16.4PV_Br_SNP_027417.3FADB NFYB3/HAP3C17.5TCTP17.8RTV1 VRN118.2ATFYPP318.5DDL18.6AT3G2132018.8TIC19.2CKB2 CKB120.7TOC122.3PV_Br_SNP_029422.4DFL223.1PDF224.1CBF2 CBF3GA20OX2

25.2

PGI AGL2425.5AT4G2334026.1PV_Br_SNP_030926.2AGL14 AGL1926.4EBS26.7KNAT527.0FLP1 ELF927.3SKB127.4VEL127.8AT4G33280 AGL1629.0ATH129.2FDP FD29.7SNZ AP230.2COP330.4Bra01790531.2

A03

PV_Br_SNP_0274 0.0

PV_Br_SNP_0294 2.7

PV_Br_SNP_0309 5.1

L03

15

Continuo Figure 3. Comparing newly constructed linkage groups (right side(Lx)) to Brassica rapa physical map(left side(Ax)); The length of each genetic

linkage groups are indicated in centimorgans (cM) and the length for the physical map is in million base pairs (Mbp, A01-A10) .The cM of LX linkage groups

was 0.1cM instead of 1CM to make it easy; because the physical map was constructed based on Mbp with maximum 50. The genetic map was greater than

50cM. The colour markers indicate the newly SNPs(rose) and InDels(green) markers.

Bra040431 0.0CAM1 CML24

SPL9 GAr3AT5G28450

0.1

CIP1 CKB1 0.2SOC1 0.8

GA2OX3 0.9CDF2 PIF4 1.0

SHP1 1.4SPL15 1.7

AGL18 INO80 1.9AMP1 3.2

AP3 3.4COP9 5.5

BR6OX2 7.1CKB2 7.3CKB2 7.7

AGL21 9.1PV_Br_SNP_0338 9.5

CBFs 9.6

COP3 11.3

FRI 12.8

PV_Br_SNP_0353 16.2

PV_Br_SNP_0372 18.7Bra040486 18.8

A04

PV_Br_SNP_0338 0.0

PV_Br_SNP_0353 3.2

PV_Br_SNP_0372 6.4

L04

Bra0044090.0RFI20.1CCA1 PRR90.5SPA10.7SPL30.9SPL91.3SHP2 SHP11.5FPA1.7CKB4 CKB32.2HUB12.3SAP18 AGL6SOC1

2.5

ELF42.9NFYB13.3FHY13.8NFYA45.2GA2OX35.4FES1 SPL35.7COP16.1ARR38.2LD9.1PHYC9.3CIR19.6SPA4 SPA3SPL4 PV_Br_SNP_0413

10.2

GA2ox7 PV_Br_SNP_042411.5VIP111.9GA2OX412.4RTV1 VRN113.0TIC15.2PV_Br_SNP_046415.5AT3G2132015.8FIS3 FIS316.8ATFYPP317.3BrTCTPP2b17.5PV_Br_SNP_1051 PHYBPHYD

17.6

PV_Br_SNP_049218.3RTV1 VRN118.7NFYB3/HAP3C FADB19.5SPA4 SPA319.8SPL519.9PIE121.0SPY MYB65MYB33

21.5

PRMT4B PRMT4a21.9COL922.1LCL122.6GAr223.3MBD923.5SEPALLATA223.7Bra03918323.8

A05

PV_Br_SNP_0464 0.0

PV_Br_SNP_0413 1.8PV_Br_SNP_0424 2.1PV_Br_SNP_1051 2.7PV_Br_SNP_0492 2.8

BrTCTPP2b 4.3

L05Bra0379540.0SPL40.5HAP5C NFYC9(NF-YC3)0.8CBFs1.2F14G24.73.1DDF1 DDF24.2ICE14.3RAV14.5AT1G105884.9OTS2 OTS1ARR3

5.0

DRB15.3PHYA PIF35.4PV_Br_SNP_10406.3GA3OX16.6UBC17.1NPH47.4PV_Br_SNP_05458.2UBP269.2NFYC1/HAP5A9.7REF69.9BNQ310.3CDF310.5LUX/PCL1 BOA11.0CAM112.2UBP2613.9LFY14.0NFYC1/HAP5A14.3ALX814.5PV_Br_SNP_056514.9LIP115.0FLM/MAF1 MAF2MAF3 MAF5MAF4

15.1

AT5G6554015.4APRR517.1FKF1 FPF117.3TNY GA317.7GAr2 GAr1AT5G27230

18.3

hst WNK118.4LSH2 LSH118.5BFT18.8YAP16919.0CDF119.2ELF519.4PV_Br_SNP_057819.7PRMT4B PRMT4a21.1ADG121.4HSP88.121.6RAV121.8TEL122.0PKS123.4UBC123.5GAI RGA23.9RPI224.1DOG124.8BIN125.4Bra03375926.2

A06

PV_Br_SNP_1040 0.0

PV_Br_SNP_0545 0.7

PV_Br_SNP_0578 1.8PV_Br_SNP_0565 2.3

L06

Bra036411 0.0

HXK1 0.9LKP2 FKF1 1.1

F17F8.15 2.7VIN3-L 4.1ADG1 5.0

FIP1 5.6SEP3 5.7

GA2OX2 7.0TOE1 7.3

ATC 7.8CAM1 8.0

CYP72B1 8.1ICU2 8.6

PV_Br_SNP_0637 11.6CAM1 12.1

AGL18 12.2SPL15 12.4

NFYC2/HAP5B 12.6SHP2 SHP1 12.7CKB4 CKB3 13.0AT5G28450 13.2

PIL2 13.3GAr3 13.5PRE 13.8NUA 14.0MIF1 15.0

HAP2C 15.4BrID101075 TFIIIA 15.5

CSTF64 15.6XAL1 15.7MMP 15.9CDF5 16.1VIM3 16.6

FT TSF 16.8CML24 CAM1 17.1

PV_Br_SNP_0663 17.8CAL AP1

BrAP3P1b18.1

EFS 18.7BrFTP2b 18.9

HAP2C 20.4XAL1 20.8UBC1 21.6

GA2OX1 21.8NUA 22.1

GA2/KS 22.2Bra035206 22.5

A07

PV_Br_SNP_0125 0.0

PV_Br_SNP_0637 0.7BrAP3P1b 1.1

BrID101075 2.8

BrFTP2b 4.2

PV_Br_SNP_0663 5.6

L07

16

Continue Figure 3...

Bra0362260.0GA10.1APRR51.5FKF1 FPF11.6TNY1.7AT5G272302.1WNK12.2PV_Br_SNP_10832.9K19B1.173.0TOC13.2VIP43.3LIP13.6BrID1010213.7PV_Br_SNP-10714.5BrFDPP2a4.6PV_Br_SNP_07954.9BrSVPP1c ORTH2VIM3

5.0

AAT25.4HXK15.8FVE5.9DDF26.0GA20OX4 YAP1698.5PV_Br_SNP_08268.7OTS18.9ARR39.0ATFYPP39.9ATXR710.3PV_Br_SNP_084813.5HSP88.114.5SEPALLATA414.7PKS1 UBC115.0GAI RGA15.3NFYB215.6AT5G4691015.9CUL416.5DOG116.7AGL14 AGL1916.9pEARLI17.2AGL1517.5PCFS4 BrPDF2a17.7F17F8.15 SUF420.0BrFRL2P1b FRL120.5

GA2OX222.0T22J18.14 BrGIP1dBrGIP2b

22.8

CUL323.8EDF1 TEM2BrPFT1P2c BrPFT1P4f

24.2

AP327.0AMP127.1SNZ SMZ27.3CAM128.1INO80 AGL1828.4SHP2 SHP128.9PIL629.0FPA29.1CKB4 CKB329.4BrID10108329.9PIL230.1BrGAr3P1a30.4ELF330.6ORTH2 BrVIM3P3b30.8AGL17 AGL2131.5PV_Br_SNP_090232.1BrFVEP3a32.3RAV134.1LWD1/ATAN1134.3CBFs34.8HAP5C NFYC9(NF-YC3)PIF3

35.1

STO35.9CKL1336.2PSAT136.3Bra03267036.9

A09

BrID101083 0.0

BrGAr3P1a 0.8

BrVIM3P3b 1.7

BrSVPP1c 2.6

PV_Br_SNP_0902 3.3

BrFVEP3a 3.9

L09_2

PV_Br_SNP_0795 0.0

PV_Br_SNP_1083 2.5

BrID101021 5.3

PV_Br_SNP_1071 7.1

BrFDPP2a 8.7

PV_Br_SNP_0826 11.7

PV_Br_SNP_0848 12.4

BrFRL2P1b 14.1

BrGIP1d 15.5BrGIP2b 15.7

BrPFT1P2c 17.1

BrPFT1P4f 18.4

L09_1

Bra0379250.0PSAT11.5CRY2 CKL131.7UBC12.1STO2.6CO T1N6_23.9LHY SIN14.2GA2OX64.4CCR25.1MYB65 MYB335.7AAT27.0PI7.2NPH47.4CAM17.7MIR172(EAT) TOE2PRR3 AT5G59845

8.6

AT1G10588 VIP28.7SRR18.8SEF9.3COL59.6FRL2 FRL1ELF9

13.1

COL1 COSEPALLATA2

13.3

BNQ213.5AGL1513.9NFYA1/HAP2A14.2HY514.7AT5G1062514.8FLC15.0CPD15.3PV_Br_SNP_098515.7GA20OX4 YAP16915.9HUA2 CHE16.2ELF6 PV_Br_SNP_099616.8TFL116.9LMI117.0LCL1 PRR717.2ELF717.3FHL PNY17.4Bra00964617.6

A10

PV_Br_SNP_0985 0.0

PV_Br_SNP_0996 1.2

L10

Bra030807 0.0ARP6 0.1

PV_Br_SNP_0696 HUB2 0.4MED9 0.6TFL2 1.3RGL2 1.4

GA20OX5 1.8PV_Br_SNP_0709 3.1

RTV1 VRN1 3.6ATFYPP3 4.1

RPI2 4.7PV_Br_SNP_1066 5.1

VIP1 5.6ATR4 5.8

UFO SUF4 6.2PV_Br_SNP_0721 6.3

ESD4 6.7HAF2 7.8

COP9 PV_Br_SNP_0743 9.0SPA2 9.5

AGL14 AGL19 10.0EBS 10.2

PDF2 10.4GA2OX8 10.5GA3OX3 10.6

PV_Br_SNP_0754 11.2FIP2 11.8

VRN2 11.9FLP1 13.2SKB1 13.3VEL1 13.6VIP3 13.7

CBF2 CBF1 14.5FDP FD 14.7

COP3 15.3T28I19.160 AGL17

AGL2115.5

BIN1 15.8PV_Br_SNP_0776 15.9

CKL13 16.1GA2OX2 16.4

SEP3 17.1PFT1 EDF1 17.4

CAL AP1 17.5CUL3 17.6NPH4 18.7

MFT 18.9ATCSTF77 19.0

ICE1 19.6DDF2 19.7CRY2 20.4

HAP5C NFYC9(NF-YC3) 21.2DRB1 Bra030806 21.4

A08

PV_Br_SNP_0696 0.0PV_Br_SNP_0709 0.8PV_Br_SNP_1066 1.0PV_Br_SNP_0743 1.4PV_Br_SNP_0721 1.6

PV_Br_SNP_0754 2.8

PV_Br_SNP_0776 4.2

L08

17

3.3. Frequency distribution of turnip tuber traits

Average turnip tuber fresh weight, dry weight, dry mass and brix ( total soluble sugar) were showed

no normal distribution, the skewedness was shifted to positive ( Figure 4). A distribution has values

of skew which the results were above 0 . This indicates a deviation from normal distributions. The

mean result of turnip tuber fresh weight, dry weight and dry mass were indicated most of progenies

(population F6/7) shifted to VT-115 parent (Appendix 4 ).

Figure 4. Frequency distribution of turnip tuber morphological traits in 36RIL F6/7 population.

18

3.4. QTL analysis

Four turnip tube related traits were evaluated; average turnip tuber fresh weight, dry weight, dry

mass and brix (Total soluble Sugar). Non linkage group also included in QTLs detection. Some markers

were unlinked but they have contribution for QTLs (Figure 5). A total of 94 turnip tuber related traits

QTLs were detected at significant level ≤0.1 on 13 newly constructed brassica rapa linkage groups

except linkage group L01_1 and L04 (Figure 5). As a result, 18 Brix (total soluble sugar) related QTL

was detected on all newly constructed linkage groups except L02_1, L02_2 and L10. Average turnip

tuber fresh weight related QTLs(29) were also detected on all linkage group except L03. Twenty six

turnip tuber dry weight and 21 turnip tuber dry mass percentage QTLs were also detected on all

newly constructed linkage groups except L08. The interesting thing was in linkage group 2, turnip

tuber dry weight and fresh weight co-located together which all were affected by BrFT loci. Turnip

related traits QTLs were detected on the top of A02 in DH-30 and the BC1 population which were co-

located with flowering QTL ((Lou et al., 2007)). In F2 segregation population study, it showed turnip

width and flowering markers correlated each other on linkage groups of A02 and A10 (Vos, 2009).

Average turnip tuber fresh weight, dry weight and dry mass were co-located on linkage group L02,

L05, L06, L07, L09 and L10. F2 population study on linkage group L02 didn’t show any turnip related

traits QTLS but in this study some QTLs were detected. Only linkage groups A01, A05 A06 A08 and

A10 were detected turnip related QTLs F2/3 of this population (RIL 36C) (Rémi, 2010). The explained

variation per QTL was less than 16.5%.

I was working major thesis on turnip tuber traits genes expression in vitro condition. I have

found some genes which differentially expressed related to turnip tuber formation. For instance,

DDF1(delay and dwarf flowering 1), VTuni_02 and VTuni_14 (unique for DHVT-117 turnip) genes

were differentially expressed in turnip tuber induced medium. The physical position of DDF1 and

VTuni_02 is A09 at 34.51Mbp and 36.99Mbp, respectively on Chiifu genome. The newly constructed

L09_2 linkage map had 14 turnip tuber related QTLs. The mega base pair size was not far from DDF1

and VTuni_02 genes. If those genes included in the linkage map, they might be contribute in turnip

tuber QTLs.

19

Figure 5. Turnip tuber related QTLs on Brassica rapa linkage groups on flower time related markers of physical map including SNPs and InDels markers . The

coloured loci shows significantly(<0.001) for correspondence QTL; red colour markers for brix QTLs, brown colour for fresh weight QTLS, blue for dry weight

and green for dry mass percentage; stars with markers indicates the significant different(*=0.1, **=0.05, ***=0.01, ****=0.005, *****=0.001 and ******=

0.0005, *******=0.0001) from hypothesis( no QTLs for linkage group); colour and underline indicates the markers contribute for more than one QTLs; black

colour and underline indicated the markers which didn’t assigned any linkage group but it contribute for correspondence QTL.

Bra0110240.0

PV_Br_SNP_00021.9

PV_Br_SNP_10483.3

Bra04052027.7

L01_1

Bra0110240.0

PV_Br_SNP_10483.3

PHYE**/**/**4.8

EBS**/***6.5

BrID101119**/**/**9.9

PV_Br_SNP_0055*/*14.0

PV_Br_SNP_008017.0

VRN1****21.0PV_Br_SNP_0098*/**21.2

Bra04052027.7

BrixQ

TL1

BrixQ

TL2

FWQTL

FWQTL

FWQTL

FWQTL

FWQTL

DWQTL

DWQTL

DWQTL

DWQTL

DMQTL

DMQTL

L01_2

Bra028386 0.0

FT*****/*****/*** TSF****/***/** 7.7

PV_Br_SNP_0172 13.5

Bra032028 25.7

FWQTL

DWQTL

DMQTL

L02_1

Bra0283860.0

FT*****/*****/*** TSF****/***/**7.7

PV_Br_SNP_017213.5

PV_Br_SNP_014320.1

Bra03202825.7

FWQTL

DWQTL

DMQTL

L02_2

20

Continue Figure 5. ...

Bra005657 0.0

FY* 2.5

PV_Br_SNP_0274 17.3

PV_Br_SNP_0294 22.4

PV_Br_SNP_0309* 26.2

Bra017905 31.2

BrixQ

TLDMQTL

L03

Bra004409 0.0

BrID10131* 2.1BrID10133*/**/** 2.5

FES1****/* 5.7

PV_Br_SNP_0413**** 10.2

PV_Br_SNP_0424*****/* 11.5

PV_Br_SNP_0464**** 15.5

BrTCTPP2b**/* 17.5PV_Br_SNP_1051******/* 17.6

PV_Br_SNP_0492****/* 18.3

Bra039183 23.8

BrixQ

TL

BrixQ

TLBrixQ

TL

BrixQ

TLBrixQ

TL

BrixQ

TL

BrixQ

TLBrixQ

TL

FWQTL

FWQTL

DWQTL

DWQTL

DMQTL

DMQTL

DMQTL

DMQTL

A05

Bra037954 0.0PV_Br_SNP_0517* 0.3

PV_Br_SNP_1040 6.3

PV_Br_SNP_0545* 8.2

PV_Br_SNP_0565***/**/** 14.9

BrID101035 19.0PV_Br_SNP_0578 19.7

RGA 23.9

PV_Br_SNP_1069*** 25.2

Bra033759 26.2

BrixQ

TL

FWQTL

DWQTL

DMQTL

DMQTL

DMQTL

DMQTL

DMQTL

A06

21

Continue Figure 5...

Bra036411 0.0PV_Br_SNP_0125 0.5

FKF1* 1.1

BrID10103** 7.8

PV_Br_SNP_0637****/****/*** 11.6

BrID101075**/** 15.5

FT** 16.8PV_Br_SNP_0663** 17.8

AP3*****/****/**** 18.1

PV_Br_SNP_0691*/* 22.4Bra035206 22.5

BrixQ

TLBrixQ

TL

BrixQ

TL

FWQTL

FWQTL

FWQTL

FWQTL

FWQTL

DWQTL

DWQTL

DWQTL

DWQTL

DMQTL

DMQTL

A07

Bra0308070.0PV_Br_SNP_0696*0.4

PV_Br_SNP_0709*3.1

PV_Br_SNP_10665.1

PV_Br_SNP_07216.3

PV_Br_SNP_07439.0

PV_Br_SNP_075411.2

PV_Br_SNP_077615.9

PV-Br_SNP_1037*20.7Bra03080621.4

BrixQ

TLBrixQ

TL

DWQTL

A08

Bra036226 0.0

PV_Br_SNP_1083* 2.9BrID101021**/**/* 3.7

PV_Br_SNP-1071**/** 4.5FDP**/** 4.6

PV_Br_SNP_0795**/* 4.9BrSVPP1c**/** 5.0

FVE* 5.9

PV_Br_SNP_0826 8.7

PV_Br_SNP_0848 13.5

BrFRL2P1b 20.5

BrGIP1d BrGIP2b 22.8

BrPFT1P2c* BrPFT1P4f 24.2

Bra032670 36.9

BrixQ

TL

FWQTL

FWQTL

FWQTL

FWQTL

FWQTL

FWQTL

DWQTL

DWQTL

DWQTL

DWQTL

DWQTL

DWQTL

DMQTL

DMQTL

L09_1

22

Continue Figure 5...

Bra0362260.0

BrSVPP1c**/**5.0

BrID101083*/*29.9BrGAr3P1a30.4BrVIM3P3b**30.8PV_Br_SNP_0902*/**32.1BrFVEP3a*/*32.3GA3OX133.4

Bra03267036.9

FWQTL

FWQTL

FWQTL

FWQTLFW

QTL

DWQTL

DWQTL

DWQTL

DWQTLDWQTL

L09_2

Bra037925 0.0

CRY2** 1.7

FRL1****/****/*** 13.1

PV_Br_SNP_0985**/** 15.7

PV_Br_SNP_0996 16.8Bra009646 17.6

FWQTL

FWQTL

DWQTL

DWQTL

DMQTL

DMQTL

DMQTL

A10

23

4. Conclusion and recommendation

A genetic linkage map provides the basic and essential information to study the B. rapa genome and

to conduct marker-assisted selection in genetics and breeding. Genotype scoring was not easy for

some of the markers which excluded during mapping. I was able to locate more than ten linkage

groups to ten corresponding Brassica rapa linkage groups. The size of population were reduced from

144 to 106 to make correlation with different markers. Size and number of marker can affect linkage

group and QTLs analysis. If there are more markers included, those loci might be linked. After

running the JoinMap program for each group, a linkage map on each linkage group was determined

based on recombination frequency.

A total 61 markers were grouped into 13 newly constructed linkage groups. The orders and

orientations of the markers on each 13 newly constructed linkage groups were the same as physical

the map map of Brassica rapa from chiifu genome except BrTSFp2c (A02), Pv_Br_SNP_0464 (A05),

PV_Br_0578 (A06), BrID101075 (A07), PV_Br_SNP_0663 (A07), PV_Br_SNP_0743 (A08),

PV_Br_SNP_0795(A09) and BrSVPP1c (A09). Of the 124 markers assigned to the linkage groups, 49

showed a deviation from the expected segregation ratios 1:1. This might be due to common feature

markers skewed segregation Brassica linkage maps. Generally, 90% of loci had homozygous

genotypes, 4% heterozygous and 6% missing values during scoring or unamplified genotypes. I also

observed the differential genes expressed in tuber formation and physical position of those gene to

related to the newly constructed QTLs. It showed some of genes (e.g. VTuni_02) were not far from in

L09 linkage groups. This supported turnip tuber traits are polygenic. A total of 94 turnip related QTLs

were detected in all linkage groups; 18 for brix(Total soluble Sugar), 29 for turnip tuber fresh weight,

26 for turnip tuber dry weight and 21 for tuber dry mass percentage. Dry weight and fresh weight co-

located together on linkage group A02 at flowering QTL loci. The linkage map reported here is

therefore help as a resource in undertaking future linkage mapping on C36 RIL population. Finally, I

would recommend to make linkage and QTLs mapping with higher number of individuals and

markers. It gives accurate and precise linkage groups and QTLs maps.

24

References

Abelenda, J. A., C. Navarro and S. Prat (2011). "From the model to the crop: genes controlling tuber

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27

Appendixes

Appendix 1. SNPs FT markers;

Abbreviation

Markers

Polymorphism in RIL36 (VT-115 X PC-144)

estimated position

DH-68 mapped position

C36 mapped position*

Annotation

AGL15 BrAGL15P2a No unassigned A02 unassigned AGL15 (AGAMOUS-Like 15) is a member of the MADS domain family of regulatory factors.

AGL24 BrAGL24P1b No A03 A03 unassigned MADS-box protein

AGL24 BrAGL24P2c No unassigned A03 unassigned MADS-box protein

AP2 BrAP2P1d No A01 A01 unassigned Encodes a floral homeotic gene

AP2 BrAP2P2a No unassigned A03 unassigned Encodes a floral homeotic gene

AP3 BrAP3P1b Yes A07 A07 A07 Floral homeotic gene encoding a MADS domain protein

APRR5 BrAPRR5P1a No A09 A09 unassigned Encodes a pseudo-response regulator whose mutation affects various circadian-associated biological events

APRR5 BrAPRR5P1b No A09 unassigned Encodes a pseudo-response regulator whose mutation affects various circadian-associated biological events

AS1 BrAS1P1c Yes A05 A03 ungrouped Encodes a MYB-domain protein involved in specification of the leaf proximodistal axis

AS1 BrAS1P2a Yes unassigned A03 ungrouped Encodes a MYB-domain protein involved in specification of the leaf proximodistal axis

AT1G18450 Br450P1a No A06 A06 unassigned Encodes a gene similar to actin-related proteins in other organisms

AtBRM BrAtBRMP1d No A03 A03 unassigned Encodes a SWI/SNF chromatin remodeling ATPase

ATVGT1 BrATVGT1P1a No A03 A01 unassigned Encodes a vacuolar membrane-localized glucose transporter

BFT BrBFTP1a No unassigned A02 unassigned brother of FT and TFL1 protein (BFT

BIN1 BrBIN1P2c Yes A08 A08 ungrouped Encodes a plasma membrane localized leucine-rich repeat

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receptor kinase

CAL BrCALP1g No A07 A02 unassigned encoding a MADS domain protein homologous to AP1

CAL BrCALP2a Yes unassigned A02 ungrouped encoding a MADS domain protein homologous to AP1

CBFs BrCBFsP2b No unassigned A06 unassigned transcription factor activity

CCA1 BrCCA1P1c Yes A03 A09 ungrouped Encodes a transcriptional repressor

CCA1 BrCCA1P3b Yes unassigned A05 ungrouped Encodes a transcriptional repressor

CDF1 BrCDF1P2e Yes unassigned A06 ungrouped Dof-type zinc finger

CO BrCOP1f No A10 A02 unassigned Similar to zinc finger

COL1 BrCOL1P1a No unassigned unassigned Homologous to the flowering-time gene CONSTANS.

COL1 BrCOL1P1c No unassigned A10 unassigned Homologous to the flowering-time gene CONSTANS.

COL2 BrCOL2P3aF Yes unassigned A01 ungrouped homologous to the flowering-time gene CONSTANS (CO) encoding zinc-finger proteins

CPD BrCPDP1a No A10 A10 unassigned Encodes a member of the CP90A family,

CRY1 BrCRY1P2d No unassigned A09 unassigned Blue-light photoreceptor

CRY1 BrCRY1P1c No unassigned A09 unassigned Blue-light photoreceptor

CRY2 BrCRY2P1a Yes A10 A10 ungrouped Blue-light photoreceptor

CRY2 BrCRY2P4b No unassigned A04 unassigned Blue-light photoreceptor

CRY2 BrCRY2P2c Yes unassigned A08 ungrouped Blue-light photoreceptor

CRY2 BrCRY2P3a Yes unassigned A08 ungrouped Blue-light photoreceptor

CUL3 BrCUL3P1a No A08 A08 unassigned Cullin, putative, similar to Cullin homolog 3

DDF1 BrDDF1P3a No unassigned A06 unassigned Encodes a member of the DREB subfamily A-1 of ERF/AP2 transcription factor family (DDF1).

DDF1 BrDDF1P2b No unassigned A09 unassigned Encodes a member of the DREB subfamily A-1 of ERF/AP2 transcription factor family (DDF1).

DRB1 BrDRB1P1d Yes unassigned A06 ungrouped Encodes a nuclear dsRNA binding protein delayed flowering

DRL1 BrDRLP3b Yes unassigned A08 ungrouped Encodes a homolog of the yeast TOT4/KTI12 protein. Yeast TOT4/KTI12

DRL1 BrDRLP1c No A09 A09 unassigned Encodes a homolog of the yeast TOT4/KTI12 protein. Yeast TOT4/KTI12

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DRL1 BrDRLP2b No A08 A08 unassigned Encodes a homolog of the yeast TOT4/KTI12 protein. Yeast TOT4/KTI12

EBS BrEBSP2d Yes A08 A02 ungrouped Putative plant chromatin remodeling factor

EBS BrEBSP1b Yes A01 A01 A01 Putative plant chromatin remodeling factor

EDF1 BrEDF1P2c No unassigned A08 unassigned Encodes a member of the RAV transcription factor family that contains AP2 and B3 binding domains.

ELF3 BrELF3P1c No A09 A09 unassigned Encodes a novel nuclear protein

ELF5 BrELF5P1a No unassigned A06 unassigned Nuclear targeted protein

ELF8 BrELF8P1b No A03 A03 unassigned Encodes a yeast CTR9 homolog

ESD4 BrESD4P1a Yes A03 A06 ungrouped encodes a SUMO protease

ESD4 BrESD4P2b Yes unassigned A08 ungrouped encodes a SUMO protease

FD BrFDP1g No A01 A01 unassigned bZIP transcription factor

FDP BrFDPP2a Yes unassigned A09 ungrouped bZIP transcription factor

FDP BrFDPP1a No unassigned A07 unassigned bZIP transcription factor

FES1 BrFES1P2c Yes unassigned A05 ungrouped Zinc finger

FES1 BrFES1P3b No unassigned A05 unassigned Zinc finger

FKF1 BrFKF1P1c No unassigned A07 unassigned F-box protein

FKF1 BrFKF1P2d Yes unassigned A07 ungrouped F-box protein

FKF1 BrFKF1P3c Yes unassigned A07 ungrouped F-box protein

FLD BrFLDP1c No A10 A03 unassigned Encodes a plant homolog of a SWIRM domain containing protein

FLK BrFLKP2b No unassigned A03 unassigned Nucleic acid binding

FLM/MAF1 BrFLMP1b Yes A02 A02 ungrouped MADS domain protein

FLM/MAF1 BrFLMP2d No unassigned A02 unassigned MADS domain protein

FPA BrFPAP1c No A09 A09 unassigned regulates flowering time in Arabidopsis via a pathway that is independent of daylength

FPF1 BrFPF1P1d No A02 A06 unassigned Small,12.6kDa protein

FPF1 BrFPF1P2a No unassigned A06 unassigned Small,12.6kDa protein

FRI BrFRIP2c No unassigned A10 unassigned Vernalization response factor

FRL1 BrFRL1P2a Yes unassigned A10 ungrouped FRI-related gene

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FRL2 BrFRL2P1b Yes unassigned A03 ungrouped FRI-related gene

FRY1 BrFRY1P1b Yes unassigned A09 ungrouped Encodes a bifunctional protein that has 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase

FT FT-P2a No A07 A02 unassigned TFL1 homolog;antagonist of TFL1

FT BrFTP1c Yes A02 A02 A02 TFL1 homolog;antagonist of TFL1

FT BrFTP2b Yes A07 A07 A07 TFL1 homolog;antagonist of TFL1

FUL BrFULP1b Yes A09 A02 ungrouped MADS box gene negatively regulated by APETALA1

FVE BrPVEP2a No unassigned A07 unassigned Controls flowering

FVE BrPVEP3a Yes unassigned A09 A09 Controls flowering

FY BrFYP1a Yes A03 A02 ungrouped Encodes a protein with similarity to yeast Pfs2p

GA1 BrGA1P2c Yes A03 A03 ungrouped Gibberellin biosynthesis

GAr1 BrGAr1P1d No unassigned A06 unassigned GA receptor homolog

GAr3 BrGAr3P1a Yes A03 A09 A09 GA receptor homolog

GAr3 BrGAr3P2b No unassigned A09 unassigned GA receptor homolog

GI BrGIP2b Yes unassigned A09 ungrouped Circadian clock gene

GI BrGIP1d Yes A09 A09 ungrouped Circadian clock gene

GI BrGIP3b No unassigned A09 unassigned Circadian clock gene

HOS1 BrHOS1P2a No unassigned A04 unassigned RING finger E3 ligase

HUA2 BrHUA2P1b Yes A02 A06 ungrouped Transcription factor

LKP2 BrLKP2P1a No unassigned A07 unassigned encodes a member of F-box proteins that includes two other proteins in Arabidopsis (ZTL and FKF1).

MAF2 BrMAF2P1b No A10 A02 unassigned Originally published as Agamous like MADS-box protein AGL31

MAF4 BrMAF4P1d No A02 A02 unassigned Encodes MADS-box containing FLC paralog

MAF4 BrMAF4P2a No A02 A02 unassigned Encodes MADS-box containing FLC paralog

MAF5 BrMAF5P1a Yes unassigned A06 ungrouped Is upregulated during vernalization and regulates flowering time.

MFT BrMFTP1b No A08 A08 unassigned Nuclease

MFT BrMFTP2b No unassigned A08 unassigned Nuclease

NS1 BrNS1P2c Yes A01 A03 A01 Asparaginyl-tRNA synthetase protein

NS1 BrNS1P1d No A08 A08 unassigned Asparaginyl-tRNA synthetase protein

31

PFT1 BrPFT1P2c Yes unassigned A09 ungrouped Transcription coactivator

PFT1 BrPFT1P4f Yes unassigned A09 ungrouped Transcription coactivator

PFT1 BrPFT1P3b Yes unassigned A08 ungrouped Transcription coactivator

PHYA BrPHYAP1g Yes A08 A09 ungrouped G-protein coupled red/far red photoreceptor

PHYA BrPHYAP3b No unassigned A04 unassigned G-protein coupled red/far red photoreceptor

PHYA BrPHYAP3c No unassigned A04 unassigned G-protein coupled red/far red photoreceptor

PHYE BrPHYEP1b Yes A01 A06 ungrouped G-protein coupled photoreceptor

PHYE BrPHYEP1a No A01 A10 unassigned G-protein coupled photoreceptor

PHYE BrPHYEP2c Yes unassigned A01 A01 G-protein coupled photoreceptor

PI BrPIP2a No unassigned A02 unassigned Floral homeotic gene encoding a MADS domain transcription factor

PI BrPIP1b Yes A10 A02 ungrouped Floral homeotic gene encoding a MADS domain transcription factor

RGA BrRGAP2c Yes A06 A09 ungrouped VHIID/DELLA transcription factor

RGA BrRGAP1a Yes A06 A06 ungrouped VHIID/DELLA transcription factor

RGA BrRGAP1c Yes A06 A06 ungrouped VHIID/DELLA transcription factor

SEP3 BrSEP3P2a Yes unassigned A07 ungrouped Member of the MADs box transcription factor family

SEP3 BrSEP3P1a No A09 A07 unassigned Member of the MADs box transcription factor family

SLY1 BrSLY1-P1b No A01 A01 unassigned F-box protein involved in GA signaling

SNZ BrSNZP2b No unassigned A09 unassigned Encodes a AP2 domain transcription

SOC1 BrSOC1-P1a No A01 A01 unassigned Transcription factor

SPA1 BrSPA1P1a Yes A03 A05 unassigned Encodes a member of the SPA (suppressor of phyA-105) protein family (SPA1-SPA4

SPA1 BrSPA1P2a Yes unassigned A05 ungrouped Encodes a member of the SPA (suppressor of phyA-105) protein family (SPA1-SPA4

SPL5 BrSPL5P1a No A03 A01 unassigned Encodes a member of the SPL

SPL5 BrSPL5P2bF No unassigned A05 unassigned Encodes a member of the SPL

SVP BrSVPP1c Yes A09 A09 A09 Transcription factor

SVP BrSVPP2d Yes unassigned A04 ungrouped Transcription factor

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T1N6_2 T1N6_2P1c No A09 A09 unassigned Putative role in flower development

T1N6_2 T1N6_2P2a Yes A04 ungrouped Putative role in flower development

TCTP BrTCTPP1b No A03 A03 unassigned Encodes a protein homologous to translationally controlled tumor protein (

TCTP BrTCTPP2b Yes unassigned A05 A05 Encodes a protein homologous to translationally controlled tumor protein (

TFL1 BrTFL1P2c No A02 A02 unassigned Phosphatidylethanolamine binding

TFL2 BrTFL2P4b No unassigned A10 unassigned Chromatin maintenance protein

TFL2 BrTFL2P1c No A02 A10 unassigned Chromatin maintenance protein

TFL2 BrTFL2P2a No unassigned A10 unassigned Chromatin maintenance protein

TOC1 BrTOC1P2b No mapped A09

A09 unassigned Pseudo response regulator involved in the generation of circadian rhythms.

TOC1 BrTOC1P1b No mapped A03

A03 unassigned Pseudo response regulator involved in the generation of circadian rhythms.

TSF BrTSFP2c Yes unassigned A02 A02 FT homolog

VEL1 BrVEL1P1e Yes A01 A08 Ungrouped Encodes a protein with similarity to VRN5 and VIN3.

VIM3 BrVIM3P3b Yes A09 A09 A09 Encodes the VIM3/ORTH1 protein that is similar to VIM1. This protein has an N-terminal PHD domain and two RING

VIN3 BrVIN3P1a A10 A10 unassigned Homeodomain protein

VIN3 BrVIN3P2b No unassigned A02 unassigned Homeodomain protein

VIN3 BrVIN3P3a No unassigned A03 unassigned Homeodomain protein

VIP4 BrVIP4P1b Yes A09 A09 ungrouped Encodes highly hydrophilic protein

VIP4 BrVIP4P2b Yes unassigned A09 ungrouped Encodes highly hydrophilic protein

VRN1 BrVRN1P1b Yes A01 A05 ungrouped Required for vernalization.Essential for the complete vernalized plants.

VRN1 BrVRN1P3b Yes unassigned A03 ungrouped Required for vernalization.Essential for the complete vernalized plants.

VRN2 BrVRN2P4b Yes A08 A08 ungrouped encodes a nuclear-localized zinc finger protein with similarity to Polycomb group (PcG) proteins

*unassigned =the markers didn’t show polymorphism; ungrouped =markers showed polymorphism but didn’t have linkage groups

33

Appendix 2. SNPs markers

SNP_ID Estimated linkage group

VT-115X RC-144

Turnip vs. Flanking-200bp SNPs

PV_Br_SNP_0002 A01 A01 L144,L143,WTC 0,0,0

PV_Br_SNP_0055 A01 A01 WTC,L143 0,0,0

PV_Br_SNP_0080 A01 A01 L144,L143 0,0,0

PV_Br_SNP_0098 A01 A01 L144,WTC 0,0,0

PV_Br_SNP_0125 A02 A07 L144,WTC 0,0,0

PV_Br_SNP_0143 A02 A02 L144,L143 0,0,0

PV_Br_SNP_0172 A02 A02 WTC,L143 0,0,0

PV_Br_SNP_0242 A03 Ungrouped L144,L143 0,0,0

PV_Br_SNP_0274 A03 A03 L144,L143 0,0,0

PV_Br_SNP_0294 A03 A03 L144,L143 0,0,0

PV_Br_SNP_0309 A03 A03 L144,L143 0,0,0

PV_Br_SNP_0319 A04 Ungrouped WTC,L143 0,0,0

PV_Br_SNP_0338 A04 A04 L144,L143 0,0,0

PV_Br_SNP_0353 A04 A04 L144,WTC 0,0,0

PV_Br_SNP_0372 A04 A04 L144,L143 0,0,0

PV_Br_SNP_0413 A05 A05 L144,L143 0,0,0

PV_Br_SNP_0424 A05 A05 L144,L143 0,0,0

PV_Br_SNP_0464 A05 A05 L144,L143,WTC 0,0,0

PV_Br_SNP_0492 A05 A05 L144,L143 0,0,0

PV_Br_SNP_0517 A06 Ungrouped L144,L143 0,0,0

PV_Br_SNP_0545 A06 A06 L144,L143 0,0,0

PV_Br_SNP_0565 A06 A06 WTC,L143 0,0,0

PV_Br_SNP_0578 A06 A06 L144,L143 0,0,0

PV_Br_SNP_0637 A07 A07 L144,L143 0,0,0

PV_Br_SNP_0663 A07 A07 L144,WTC 0,0,0

PV_Br_SNP_0691 A07 Ungrouped L144,L143 0,0,0

PV_Br_SNP_0696 A08 A08 L144,L143 0,0,0

PV_Br_SNP_0709 A08 A08 L144,L143,WTC 0,0,0

PV_Br_SNP_0721 A08 A08 L144,L143 0,0,0

PV_Br_SNP_0743 A08 A08 L144,L143 0,0,0

PV_Br_SNP_0754 A08 A08 L144,L143 0,0,0

PV_Br_SNP_0776 A08 A08 L144,L143 0,0,0

PV_Br_SNP_0790 A08 Ungrouped L144,WTC 0,0,0

PV_Br_SNP_0795 A09 A09 L144,L143 0,0,0

PV_Br_SNP_0826 A09 A09 L144,L143 0,0,0

PV_Br_SNP_0848 A09 A09 L144,L143 0,0,0

PV_Br_SNP_0902 A09 A09 L144,WTC 0,0,0

PV_Br_SNP_0918 A09 Ungrouped L144,L143 0,0,0

PV_Br_SNP_0921 A10 Ungrouped L144,L143 0,0,0

PV_Br_SNP_0985 A10 A10 L144,L143 0,0,0

PV_Br_SNP_0996 A10 A10 L144,L143 0,0,0

34

PV_Br_SNP_1037 A08 L143 0,0,0

PV_Br_SNP_1040 A06 A06 L144 0,0,0

PV_Br_SNP_1041 A08 Ungrouped L144 0,0,0

PV_Br_SNP_1048 A01 A01 L143 0,0,0

PV_Br_SNP_1051 A05 A05 L144 0,0,0

PV_Br_SNP_1066 A08 A08 L144,L143 0,0,0

PV_Br_SNP_1067 A10 Ungrouped L144 0,0,0

PV_Br_SNP_1069 A06 Ungrouped L143 0,0,0

PV_Br_SNP_1071 A09 A09 L144 0,0,0

PV_Br_SNP_1082 A03 Ungrouped L144,WTC 0,0,0

PV_Br_SNP_1083 A09 A09 L143,WTC 0,0,0

Appendix 3. InDels markers

InDel_ID Estimated linkage group

VT-115 X RC-144

Start Stop

BrID10075 A10 Ungrouped 10184370 10184527

BrID10081 A10 Ungrouped 8588747 8588898

BrID10091 A10 Ungrouped 5042697 5042828

BrID101021 A09 Ungrouped 3696434 3696561

BrID101025 A05 Ungrouped 9323883 9324018

BrID10103 A07 Ungrouped 13590527 13590696

BrID101035 A06 Ungrouped 18959379 18959513

BrID101075 A07 A07 15492626 15492755

BrID101083 A09 A09 29890041 29890177

BrID101119 A01 Ungrouped 9895949 9896088

BrID10113 A07 Ungrouped 16124793 16124947

BrID10131 A05 Ungrouped 2101631 2101797

BrID10133 A05 Ungrouped 2506072 2506218

BrID10253 A06 Ungrouped 17658487 17658580

Appendix 4. Average mean of turnip tuber related traits in RIL F6/7 population

brix FW DW DM

No. of lines 94 94 94 94

Mean 4.49 9.36 1.00 0.127

Maximum 6.67 130.97 11.94 0.22

Minimum 2.33 0.24 0.06 0.06

VT-115 4.5 0.26 0.06 0.15

RC-144 5.33 130.97 11.94 0.09

Skewness 0.22 5.73 5.49 0.74

Std. Error of Skewness 0.25 0.25 0.25 0.25