Molecular Breeding for Improved Berry Quality in Ribes ...archive.northsearegion.eu/files/repository/20140106094846_UK-Encl… · Raspberry Volatiles Analysis Season Field 2006 Field

Molecular Breeding for Improved Berry Quality in Ribes, Rubus and Vaccinium

Rex Brennan

Fruit Breeding Group

Vaccinium & Other Superfruits Symposium Maastricht, June 2012

Fruit Breeding at JHI

Commercially-funded breeding

programmes

Raspberry, blackberry

Blackcurrant

New techniques for selecting the

plants we need

Marker–assisted breeding strategies

Faster and more specific cultivar development

Breeding techniques Expensive to run breeding programmes:

Lengthy timescales

Some traits take a long time to screen for, others are impossible to screen on a high-throughput basis

Field/glasshouse costs

Timescales need to be reduced and efficiency needs to be increased

Time to cv. currently 12-15 years

Extensive phenotyping in field, glasshouse and CE rooms

Establish link between genotype and phenotype

Molecular Breeding Faster identification of genetically superior individuals in breeding

populations

Can be utilised in situations where:

Assessment in field takes a long time

Pest resistance (some)

Assessment can only be done on mature plants over time

Fruit quality

Basic research development costs relatively high, deployment costs

low

No environmental effects

Must be associated with detailed phenotyping

Existing markers in Rubus and Ribes Markers linked to pest/disease resistances

Phytophthora root rot (raspberry)

Cecidophyopsis gall mite (blackcurrant)

SSR marker (root rot)

PCR-based marker (gall mite)

Time saved compared to field infestation plots (ca. 4 years) for screening of new lines from breeding programme

Markers now routinely deployed in JHI breeding programmes as a selection tool

Brennan et al., 2009. TAG 118: 205-212Graham et al., 2011. TAG 123: 585-601

Blackcurrant Breeding Objectives

Fruit quality

High Brix/acid ratio

Low total acidity

Anthocyanins

Delphinidins preferentially selected

Vitamin C (AsA)

> 140 mg/100 ml

Sensory traits

Berry size

1 g minimum

Fresh market – 2 g minimum

Agronomic

Environmental resilience

Winter chill levels

< 2000 h/7.2oC

Pest resistance for low-input

growing

Acceptable crop yield

> 6 t/ha

Juice yield also quantified

Trait associations – Ribes fruit quality I

Measurements across reference 9328 mapping

population (ca. 300 plants) for 4 years at JHI

SNP-based linkage map developed using

transcriptome-based 2GS 454 sequencing

Individual traits placed on genetic linkage map

Fruit size

Anthocyanins

Associated molecular markers identified for

validation in other germplasm

CL1028Contig1_5220.0

CL609Contig2_26587.3CL2096Contig1_42914.3CL1694Contig2_35316.2CL1830Contig1_45621.4CL1323Contig1_64926.7CL222Contig2_43229.9g1_K0438.7CL79Contig5_33739.4g2_P03a40.4g1_O1741.6CL181Contig3_11642.2CL1Contig17_183444.0g2_P03b44.7CL105Contig1_120245.5e1_O2047.2gr2_J05_18348.0g2_P1748.1CL124Contig2_89849.4CL1199Contig1_69951.0CL1Contig181_88051.5CL1463Contig2_25651.9g2_D0552.5CL2643Contig1_46854.2g1_P0554.9CL1484Contig1_38255.5g1_M0757.7CL1092Contig1_97176.0CL177Contig2_44576.8CL1060Contig1_48881.3CL139Contig3_84693.3

CL175C

ontig

2_839,

83%

CL2516C

ontig

1_469,

98%

CL495C

ontig

3_954,

89%

CL1913C

ontig

1_419,

57%

CL1636C

ontig

1_1112,

66%

CL115C

ontig

4_555,

78%

CL186C

ontig

2_1502,

84%

CL1247C

ontig

1_287,

73%

SCRI9328 LG1

CL609Contig2_2658 0.0CL2096Contig1_429 2.4CL1694Contig2_353 5.1

CL1830Contig1_456 16.6

CL1323Contig1_649 22.3

CL222Contig2_432 28.1

CL1Contig17_1834 38.4CL105Contig1_1202 39.1



CL1247Contig1_287 60.0

CL1060Contig1_488 65.9

CL139Contig3_846 79.2

CL2516C

ontig

1_469, 9

9%

CL186C

ontig

2_1502, 7

0%

CL124C

ontig

2_898, 9

8%

MP7 LG1

Russell et al., BMC Plant Biology 2011, 11:147

Blackcurrant berry weight



0

5

10

15

SCRI9328 LG1

HBW_08

HBW_09

HBW_10

Smaller effects on LG4 and LG5, but less consistent

Blackcurrant – Ascorbic acid



0

2

4

6

8

10

SCRI9328 LG1

e3_B020.0CL2142Contig1_4259.2CL917Contig1_21320.5CL1Contig926_233 CL1Contig385_91425.4CL1Contig323_123 CL121Contig2_310g2_N20

26.3

CL152Contig3_156526.4CL1Contig968_64 CL1Contig525_204CL125Contig2_1119

26.5

CL1Contig279_332 CL1243Contig1_47626.9CL1Contig16_44227.3CL158Contig3_103427.4CL1121contig1ssr CL1Contig872_24328.6CL351Contig1_63330.0g1_H0930.6g1_L1232.1g1_A0132.5CL662Contig1_69139.1CL168Contig1_153943.2CL199Contig1_79645.2CL1Contig727_45845.4g1_O0246.8CL17Contig1_54547.3CL1464Contig1_81749.9CL4457contig1ssr52.0CL10Contig3_792 CL754Contig1_75858.3CL103Contig5_49158.7

CL2036Contig1_67385.4

0

2

4

6

8

10

SCRI9328 LG5

Ascorbate_08

Ascorbate_09

Ascorbate_08

Ascorbate_09

Blackcurrant total anthocyanins

e4_D030.0CL1397Contig1_47510.8CL2859Contig1_446 CL1Contig889_53423.4CL1259Contig1_11726.8CL1097Contig1_79127.7CL234Contig1_60834.5CL176Contig1_23039.5CL951Contig1_19044.1CL61Contig1_237246.6CL2001Contig1_30448.1CL192Contig3_48048.2CL1343Contig1_57448.9CL1167Contig2_549 CL135Contig1_992CL193Contig1_501 CL1212Contig1_1333CL1033Contig2_690 CL196Contig1_344CL657Contig2_887 CL1061Contig1_121CL1590Contig1_819 CL1Contig109_936CL126Contig2_235 CL1057Contig1_870CL6584contig1ssr

49.3

CL1278Contig2_825 CL1653Contig1_40249.7CL836Contig1_101750.0g2_B2050.3g2_M19_30350.7CL227Contig2_1171 CL1Contig931_1929CL1Contig285_845

50.9

CL1529Contig1_61551.5g2_M19_29351.8e3_M04a54.3CL138Contig1_37156.2CL830Contig1_10060.4CL1488Contig1_19662.3CL1Contig714_20196.0CL1974Contig1_21197.0CL1Contig973_658102.7CL1Contig291_268104.9

0

2

4

6

SCRI9328 LG3

Anthocyanin_08

Anthocyanin_09

Also major QTLs for citrate in this area. Also anthocyanin (pomace) in both years, and Brix

Trait associations – Ribes fruit quality II

Use of gene expression data from ripening

fruit linked to metabolomic analyses

Fruit quality analysed at various stages

Gene expression monitored across stages

using Agilent microarrays

Key genes mapped, markers identified for the

various quality and nutritional traits

Environmental influences on gene expression

http://upload.wikimedia.org/wikipedia/commons/e/e7/L-Ascorbic_acid.s

http://upload.wikimedia.org/wikipedia/commons/0/06/Glucose_chain_structure.svg

Reduction of seedling numbers using marker-assisted breeding - Ribes

Marker for gall mite resistance2012

Marker for berry size Est. 2013

Markers for anthocyanins, sugars, vitamin CEst. 2015

Reduced seedling numbers –but increased relevance to industry needs

Faster field selections and cv. releases

Raspberry quality traits

Commercial traits assessed across seasons and environments:

Ripening

Cropping season

Colour

pH

Anthocyanins

Berry size

Sensory traits

Brix

Volatiles

Raspberry ketone

Experimental outline

Glen Moy x Latham reference mapping population

3 seasons and 3 environments

Assessments of trait of interest

QTL mapping

Candidate gene analysis

Microarray to examine changes in gene expression during

fruit development

Ripening QTLs for the ripening stages were identified across four chromosomes

2, 3, 5 and 6.

Each of the groups had markers that had a significant effect at various ripening stages.

The work identified genetic markers associated with early or late bud break and short or long ripening periods.

ERubLR_SQ06_E010RiMADS_015P14M60-1187Bac25D1026215P13M55-28018P13M40-11421E40M43-15722P12M31-10325P12M61-13726P14M39-23827P13M95-191R29P14M60-11031E40M50-25135ERubLR_SQ01_M2037P13M95-19539P13M40-30940P14M61-22341E40M58-24944P13M40-11546P13M58-24250P13M95-193R53P13M95-287R58P13M95-294R59E40M43-11261ERubLR_SQ19_A0563ERubLR_SQ05_E0266P13M39-147R70P13M40-24971ERubLR_SQ01_I20P12M121-169

73

Rubpara_SQ005_K2377P13M60-321R80P13M60-199R84

F_

PC

O1

(LO

D 4

.7)

F_

Op

en

(LO

D 7

.5)

F_

Se

t (LO

D 6

.4)

F_

PC

O4

(LO

D 3

.8)

F_

Gre

en

(LO

D 4

.2)

He

ight (L

OD

4.1

)

Chromosome 5

E40M50-1670P14M39-2326E40M61-2778P13M95-117R11P14M61-217P13M40-187

14

Rub43a17Rub1b19Rub123a24Rubnebp41929

E41M39-15551P13M40-12952P13M58-26557P13M40-8567P14M39-38368P12M31-14570P13M60-158R72P13M39-109RP14M61-164

74

P14M61-15676E40M43-205E41M31-156

77

Rub118b78E40M43-8279E41M40-31580LEAF10282LEAF9783P13M58-23084P13M55-31590

F_

PC

O2

(LO

D 7

.9)

F_

PC

O4

(LO

D 3

.1)

F_

Gre

en

/Red

(LO

D 6

.0)

Chromosome 6

Anthocyanins in Raspberry Anthocyanins in mapping progeny and parents:

cyanidin-3-sophoroside (C3S)

cyanidin-3-glucoside (C3G)

cyanidin-3-glucosylrutinoside (C3GR)

cyanidin-3-rutinoside (C3R)

pelargonidin-3-sophoroside (P3S)

pelargonidin-3-glucoside (P3G)

pelargonidin-3-glucosylrutinoside (P3GR)

pelargonidin-3-rutinoside (P3R)

Major QTL identified for all anthocyanins across seasons and sites

Several transcription factors underlying QTL:

bHLH, FruitE4 encoding a basic leucine zipper (bZIP) transcription factor

Rub119 encoding a NAM (no apical meristem)-like transcription factor

Markers linked to QTL under validation

QTL for anthocyanin production

P14M60-830.025D10SSR044.625D10_end6.7RUB210a11.6RUB257a13.9RUB137a21.3RUB124a27.9Rubpara_SQ008_D0431.5E40M43-9342.2E40M60-10647.9E40M60-12558.8P13M58-8663.4P13M60-11766.0Rubnebp4c8b67.4E40M43-11668.4P13M40-13170.4ERubLR_SQ07_3_C0775.2E40M43-268 E41M42-14879.6RUB47a88.2P14M39-40290.4P12M95-9592.9P12M31-17196.6E41M60-31597.5P14M39-196100.8E41M39-125101.0RUB262b103.2RUB256e107.2bHLH109.3RUB243a RUB119a111.4Rub232b116.0RUB166b119.2Rub244a125.3

LG 1

07 P

T0

7 O

F0

6 O

F

C3S**

07 P

T0

7 O

F0

6 O

F

C3GR**

07 P

T0

7 O

F0

6 O

F

C3G**

07 P

T0

7 O

F0

6 O

F

C3R**

QTL Peak

5 > LOD > 20

P3S* P3G* P3R*

06 O

F

07 P

T0

7 O

F

07 P

T

06 O

F0

7 O

F

P3GR*

06 O

F

07 P

T0

7 O

F

07 P

T

06 O

F0

7 O

F



LG 1

07 P

T0

7 O

F0

6 O

F

C3S**

07 P

T0

7 O

F0

6 O

F

C3GR**

07 P

T0

7 O

F0

6 O

F

C3GR**

07 P

T0

7 O

F0

6 O

F

C3G**

07 P

T0

7 O

F0

6 O

F

C3G**

07 P

T0

7 O

F0

6 O

F

C3R**

07 P

T0

7 O

F0

6 O

F

C3R**

QTL Peak

5 > LOD > 20

P3S* P3G* P3R*

06 O

F

07 P

T0

7 O

F0

6 O

F

07 P

T0

7 O

F0

6 O

F

07 P

T0

7 O

F

07 P

T

06 O

F0

7 O

F0

7 P

T

06 O

F0

7 O

F0

6 O

F0

7 O

F

P3GR*

06 O

F

07 P

T0

7 O

F0

6 O

F

07 P

T0

7 O

F

07 P

T

06 O

F0

7 O

F

P13M55-1250.0P13M39-450R9.4P14M61-9724.8GalSNP30.6RUB126b33.0P14M39-9834.1Rub278b39.8Rub236b44.3P13M40-23946.0P12M31-20046.9P13M55-25948.9RUB57a51.7RUB153a52.2P13M60-172R54.2P12M61-16554.5E40M43-30055.7FRUITE458.0P13M39-303R60.7P13M58-28873.2Rub212a78.1E41M40-20581.9Rub264b298.9ERubLR_SQ19_3_G09100.4ERubLR_SQ01_B06101.3RubEndoSQ004_N23102.6ERubLRSQ07_2_H02103.3RUB280a103.8RUB167aR110.5RUB260a113.7E41M41-185118.2

LG 4

QTL Peak

3 > LOD > 7.3

07 P

T0

7 O

F0

6 O

F

C3R**

07 P

T0

6 O

F

C3G**

07 P

T0

6 O

F

C3S** P3S*

06 O

F

P3R*

06 O

F

P3GR*

07 P

T0

7 O

F0

6 O

F

P3G*

06 O

F



LG 4

QTL Peak

3 > LOD > 7.3

07 P

T0

7 O

F0

6 O

F

C3R**

07 P

T0

6 O

F

C3G**

07 P

T0

6 O

F

C3S** P3S*

06 O

F

P3R*

06 O

F

P3GR*

07 P

T0

7 O

F0

6 O

F

P3G*

06 O

F

Kassim et al., 2009

Raspberry Volatiles Analysis

Season Field 2006 Field 2007 Polytunnel 2007

Sample Progeny Progeny Progeny

Volatile Mean± SEM Min-Max Mean ± SEM Min-Max Mean ± SEM Min-Max

b-damascenone 72.29 ± 4.02 0 – 463.8 87.10 ± 6.82 0 – 872.10 64.88 ± 4.71 3.17 - 368.8

b-ionone 13.65 ± 0.53 0.46 – 47.88 9.83 ± 0.44 0.61 – 44.33 6.43 ± 0.36 0.25 - 33.02

a-ionone 7.31 ± 0.19 1.65 – 17.16 3.86 ± 0.14 0.79 – 13.82 5.16 ± 0.24 0.22 - 21.61

a-ionol 2.24 ± 0.09 0.16 – 7.28 1.83 ± 0.09 0.24 – 11.68 5.96 ± 0.30 0.16 - 29.97

Linalool 4.72 ± 0.32 0.67 – 22.26 2.90 ± 0.17 0.33 – 14.52 4.39 ± 0.20 0.90 - 29.28

Geraniol 2.64 ± 0.08 0.68 – 8.87 1.82 ± 0.07 0.44 – 10.74 3.68 ± 0.14 0.11 - 15.46

(Z)-3-hexenol 22.35 ± 0.34 0.71 – 28.15 9.06 ± 0.29 0.63 – 28.18 16.70 ± 0.67 0.07 - 54.03

Acetic acid 1.39 ± 0.06 0.06 – 8.26 0.64 ± 0.05 0.01 – 4.05 0.72 ± 0.03 0.01 - 3.54

Hexanoic acid 6.54 ± 0.35 0.89 – 41.68 7.97 ± 0.30 1.71 – 22.88 7.04 ± 0.34 0.28 - 30.84

Acetoin 1.02 ± 0.05 0.09 – 4.74 1.03 ± 0.04 0.10 – 4.79 0.80 ± 0.05 0.02 – 4.95

Benzyl alcohol 0.59 ± 0.03 0.15 – 2.18 1.07 ± 0.04 0.09 – 3.16 2.67 ± 0.11 0 - 9.94

E41M60-1840.0FRUITE8OMT9.3ERUB271PR11.8ERubLR_SQ01_P18unk14.4ERubLR_SQ01_FG23Pgl15.2Ri26Sprot16.5RiSnf117.3ERubLR_SQ07_1_E10TF18.5RUB279a20.0ERubLR_SQ13.2C12IPPI20.8RubnebH1522.3ERubLR_SQ12.4_A04DMQ22.4ERubLR_SQ4.2_A08LTP23.5P13M58-11231.1P12M121-12733.7P13M55-11235.4RUB20a37.1P13M55-9838.7P12M121-18647.2ERubLR_SQ13.2_E09Exp53.2454C6570_ISPH57.8P12M31-15564.0Rub242a68.2E41M31-15370.0E41M31-14771.7P13M55-25173.2RiM01573.3RiCTR1 454C2985_PhysynRub177a1

73.7

E40M55-17574.5ERubLR_SQ07_4_D05unk77.0RUB19a77.9Rub2a179.6RUB2a280.5E41M31-16084.3RUB233a85.2ERubLRcont74PME-I87.1E41M31-16788.3P13M95-214R88.4P14M61-12489.6RißGal191.7Ri9022_RZnf93.5Ri9022_BRX93.9RiMYB95.7Ri9022SSR0196.4E40M43-9597.7Ri9022_GTPbindp98.7Ri9022_AP2_prot100.3454C3991_PME102.3P13M60-223R102.9E41M40-136104.4P14M39-145108.2P14M60-131117.1ERubLath2_cont24119.1ERubLath2_cont21119.6ERubLR_SQ12.2_C05Acon126.2P14M60-129130.7ERubLR_SQ10.2_E02SAM135.7ERubLR_SQ05.3_D11AOC136.4E41M39-138140.8P13M40-203147.6Rub120a150.6RUB223a153.0P14M39-92157.5Rub238h161.2Rub259b168.3RUB259f180.7

a-io

nol_

07 9

0%

a-io

nol_

06 9

0%

a-io

none_06 9

5%

a-io

none_07 P

T 9

5%

b-io

none_07 9

0%

b-io

none_07P

T 9

0%

dam

ascenone_06 9

5%

dam

ascenone_07P

T 9

0%

b-a

lcohol_

06 9

5%

b-a

lcohol _

07P

T 9

0%

hexanoic

acid

07P

T 9

0%

hexenol_

06 L

OD

3.0

hexenol_

07P

T L

OD

3.0

rasp k

eto

ne_07P

T 9

5%

3

Volatiles QTL and some of the underlying genes:Phytoene synthasePMRISPHCTR1 IPPI

Vaccinium

Mapping of key traits

Mapping population developed (Draper

x Jewel), segregating for:

Fruit size

Firmness

Flavour

Sugar/acid ratios

Tetraploid mapping developed at JHI for

potato used to map traits

Linkage Analysis and QTL Mapping Separate markers into linkage groups, based on independent

segregation

Order markers to obtain linkage maps

Relate marker data to phenotypic traits to identify regions

containing QTLs

• Putative quality-related QTL mapped using TetraploidMapsoftware

• Identification of linked markers in progress

• Berry Weight• Total Anthocyanins (Cyanidin-3-glucoside equivalents mg/L)• Sugar:Acid ratio

Sc6Ms4f07c0.0ScCo2784.0Jo36828a18.5Jo50936b29.7Jo116433.7ScCA344b34.2Jo43043.2JoCon78c60.7Pb-CA85564.3NbVac28857366.7ScContig600b69.1Pb-CA34469.7Jo1107a86.9Jo50936a91.8Jo53769b92.6Jo38168c100.0ScCA344a115.4NbVc110398a148.7NbVc123749151.1Sc5ms-2C09a157.7Jo53769a174.6Jo50936c181.2Jo94595b181.9JoCon78a183.3ScContig600a188.0Jo1030b195.7Sc5ms-2C09b202.0Jo628a210.9ScCA325a220.0ScCo646221.9NbVc124930223.3ScCa106223.4PbCA94b224.5ScCA325b230.0Jo1030a231.5Sc2Ms2a02a234.6Jo628b247.2Jo94595a281.1Jo536b307.3NbVc110398b313.8Nb6ms2c10315.2Jo991a331.7ScCo505338.5Jo799b355.5Sc6Ms4f07a359.6Pb-NA172365.2Pb-CA61385.9ScVc.287779c418.4Jo73891c431.1NbVac123022434.3JoSL282b442.9Jo1107b459.7JoNA619b471.4Pb-NA1778482.3Jo36828b521.5Pb-NA800582.7

Wdth

(P<

0.0

01)

Wdth

(P<

0.0

1)

Wgt(P

<0.0

1) Anth

o(P

<0.0

1)

Anth

o (P

<0.0

1)

Sug:A

cd

(P<

0.0

1)

Scar (P

<0.0

1)

12

Vaccinium Fruit Quality QTL

Summary Marker-assisted breeding in berry fruit

offers potential for faster cultivar development and reduction in both time and costs of breeding programmes

Exploitation of genomics resources now possible even for minor crops

Focus must be on quality traits important to industry and end-users

Combining markers for both quality and agronomic traits is the long-term aim

Improved phenotyping methods are essential

Acknowledgements

James Hutton InstituteChristine Hackett Rob HancockDerek StewartPete Hedley

RubusJulie GrahamKay SmithMary WoodheadSandie WilliamsonAlastair Patterson (University of Strathclyde)

RibesSandra Gordon Dorota JarretJoanne RussellLinzi Jorgensen

VacciniumSusan McCallumJulie GrahamNahla Bassil (USDA, OR)Jim Hancock (Michigan State U)

Documents

Molecular Breeding for Improved Berry Quality in Ribes ...archive.northsearegion.eu/files/repository/20140106094846_UK-Encl… · Raspberry Volatiles Analysis Season Field 2006 Field