genome-assisted marker development for disease resistance ...€¦ · genome-assisted marker...

Horticultural Crop Breeding and Genetics Lab

Genome-assisted Marker Development for Disease Resistance in Pepper

2017. 2. 24

Seoul National University

Byoung-Cheorl Kang

Seoul National University_ Lab. of Horticultural Crop Breeding and

Genetics

Overview of this talk

Marker development & map-based cloning

- High-density map based on reference genome

- Map-based cloning of disease resistance genes (Tsw)

QTL mapping & GWAS

- Development of a core collection

- Genome-wide association studies

Fruit traits and Phytophthora blight resistance


Genetics

The most important vegetable crop in Korea (The 3rd most important vegetable crop in the world)

Consists of approximately 22 wild species and five domesticated species (C. annuum, C. chinense, C. frutescens, C. pubescens, C. baccatum)

Use: spices, vegetables, ornamental, pharmaceutical and industrial applications

A member of the family Solanaceae that includes tomato, potato, eggplant, tobacco, and petunia

Genome size: about 3.5 Gb (x=12)

Molecular markers are available for major disease R genes

Genome sequence and high density maps are available

Capsicum (hot and sweet peppers)


Genetics

Molecular markers developed in my lab

Ultimate goal: molecular cloning of resistance genes (functional MARKER)

Trait Gene Marker type Reference

Cucumber mosaic virusCmr1

cmr2Linked marker (about 2 cM)

TAG 120:1587

On going

Pepper mild mottle virus L1 , L2, L3, L4Gene-based marker

Mol Breeding 24:433

Mol Breeding 30:819

Chile veinal mottle virusPvr1, pvr6

Cvr1

Gene-based marker

Linked marker

Mol Cells 27:329

Mol Breeding (in press)

Tomato spotted wilt virus

Pepper mild mottle virus

Tsw

Pvr4Gene-based marker New Phytol 213:886

Phytophthora capsici Phyto-QTL5 QTL-linked marker TAG 127:2503

Nematode Me7 Linked marker On-going (fine mapping)

Leveillula taurica PMR1 Linked marker On-going (fine mapping)

Restorer of fertility Rf Linked marker TAG 129:2003

Pungency Pun1 Gene-based marker Mol Breeding 30-889

Capsiate pAMT Gene-based marker Mol Breeding 35:226


Genetics5

Next Generation Sequencing

Sequencing capacity


Genetics

Evolution of molecular markers


Genetics

Genotype-by-sequencing(Reduced representation sequencing)

(Myles, Trends in Genetics, 2013)

Digestion with

restriction enzymes

Adapter ligation –

barcode / common

Pooling libraries and

sequencing

Data analysis


Genetics

Advantages of GBS

Reduced complexity of analysis

Enough number of high-quality SNPs

Resequencing GBS

LD blockLD block*

Large number of SNPs

Low read depth/SNP

2-4 SNPs per LD block

Smaller number of SNPs

High read depth/SNP

1 SNPs per LD block

*LD block: linkage disequilibrium block


Genetics

Genotype-by-sequencing

9

Sequencing• Whole-genome skim sequencing

Sequencing 18× for parents and 1× for RILs

• Genotyping-by-sequencing

Restriction enzyme digestion (PstI/MseI & EcoRI/MseI)

Genotyping• SNP calling

C. annuum ‘CM334’ reference genome

BWA-mem, SAMtools, Picard, and GATK UnifiedGenotyper

SNPs with minimum QUAL 30 and depth 3

Bin map• Sliding window approach

Imputation of missing data

Window length: physical length (PD RIL) or SNP number (TH RIL)

Association

analysis

• Identification of loci controlling traits

• Composite interval mapping

WinQTL Cartographer


Genetics

Imputation by sliding window approach

10

Missing dataSequencing / genotyping error

(Huang et al, 2009)

Bin map

Increased accuracy

Useful for QTL analysis

physical length (PD RIL) or SNP number (TH RIL)


Genetics

Whole-genome skim sequencing

Chr. Number of SNPs Number of binsPhysical length of bin (Mb) Genetic distance of bin (cM)

Mean Total Mean Total

1 82,966 390 0.7 272.6 0.5 210.9

2 80,141 207 0.8 171.1 0.5 108

3 87,793 279 0.9 257.9 0.4 118

4 54,657 224 1.0 222.5 0.5 117.2

5 82,413 201 1.2 233.4 0.5 100.2

6 107,015 234 1.0 236.9 0.4 102.2

7 84,339 180 1.3 231.9 0.5 86.3

8 24,383 224 0.6 144.8 0.7 145.6

9 275,842 179 1.4 252.7 0.5 89.7

10 230,360 160 1.5 233.6 0.6 102.1

11 252,765 202 1.3 259.7 0.4 83.8

12 68,540 233 1.0 235.7 0.4 94.9

Total 1,431,214 2,713 1.0 2,752.8 0.5 1,358.9

(DNA Resear Han et al., 2016)

Bin map of ‘120 PD RILs’


Genetics

High density linkage maps in Capsicum

Population SequencingNumber of

SNP

Genetic

map size

(cM)

Average

distance of

markers (cM)

C. annuum ‘Perennial’

x C. annuum ‘Dempsey’ RIL

Whole genome

resequencing1,431,214 1,358 0.5

C. annuum ‘MicroPep’

x C. annuum ‘Jeju’ F2

GBS (PstI/MseI) 2,612 1,731 1.8

C. annuum ‘TF68’

x C. chinense ‘Habanero’ RILGBS (PstI/MseI) 8,587 1,127 1.0

C. annuum ‘YCM334’

x C. annuum ‘Tean’ RIL

GBS (PstI/MseI,

EcoRI/MseI)2,335 1,063 1.2

C. annuum ‘ECW30R’

x C. annuum ‘CM334’ F5

GBS (PstI/MseI) 4,621 1,537 2.7


Genetics

Pipeline of Map-based Cloning of R genes

Fine mapping using WG database

Chromosome walking using BAC library

Identification of candidate genes

Validation of candidate genes

BAC library 2D pool

Gap closing the target region

Whole Genome Sequence

Inheritance analysis & Linkage mappingHigh density SNP LG map

Bulked segregant analysis (RNA seq)

Expression analysis of candidate genes

VIGS to test candidate gene function


Genetics

Map-based cloning of Tsw

Tomato spotted wilt virus

Member of genus Tospovirus

Genome: tripartite RNA genomes

Broadest host range: about 900 plant species

Transmitted by thrips

Causing yield and quality losses in sweet pepper

The Tsw gene

Source: C. chinense PI15225, PI159236

Resistance resposne: HR

Tightly linked to potyvirus resistance gene Pvr4

resistant susceptible

(New Phytol Kim et al., 2017)


Genetics

Map-based cloning of Pvr4

(New Phytol Kim et al., 2017)


Genetics

Overview of this talk

Marker development & map-based cloning

- High-density map based on reference genome

- Map-based cloning of disease resistance genes (Tsw)

QTL mapping & GWAS

- Development of a core collection

- Genome-wide association studies

Fruit traits and Phytophthora resistance


Genetics

Importance of

quantitative

traits

Molecular breeding has been focused on monogenic traits

Agriculturally important traits including yield, contents of

secondary metabolites and disease resistance are under the

control of quantitative genes

Studies on quantitative traits are very limited

Opportunities

&

challenges

Diverse molecular markers have been developed

Whole-genome sequencing was reported

Difficulties in improving quantitative traits

QTL mapping in peppers

17


Genetics

Cross combination Generation # of lines Trait

C. annuum ‘Long sweet’ x C. annuum ‘AC2212’

F6-7 207Vit C and EFruit color

C. annuum ‘35001’ x C. annuum ‘35009’

F7-9 176 Carotenoid content

C. annuum ‘Perennial’x C. annuum ‘Dempsey’

F6-11 166Pungency

Morphological traits

C. annuum ‘TF68’x C. chinense ‘Habanero’

F10-11 92Pungency

Yield

C. annuum ‘YCM334’x C. annuum ‘Tean’

F9-11 176Pungency

Phytophthora

F6-7 250 Disease resistanceC. annuum ‘CM334’x C. annuum ‘ECW123’

QTL mapping and RIL populations


Genetics

QTL for capsaicinoids content High-density genetic map by re-sequencing and GBS

Major QTLs detected from both RILs

19


Genetics

20

Genome-wide association study

Biparental

population

(F2, RIL)

Natural

population

(+ NAM, MAGIC)

Long haul to build a population (F2, RIL)

Low resolution (>5cM) low practical use of markers

Detecting only two allelic difference in locus

Impossible to use diverse natural variation

Utilization of natural individual variation

High resolution makes practical use of markers to

discriminate differences at molecular level

Detecting more than two allelic differences at locus

Enormous number of markers are necessary depending on LD

Higher potential false positive markers

Approaches Strength and weakness

(Zhu et al, 2008)


Genetics

Scheme for GWAS in Capsicum

21

Experimental

population

Genotype

Phenotype

Association

mapping

Validation

Natural population

Proper sequencing

method for pepper

Interesting traits useful

for breeding

Diverse model approach

Using bi-parental

population and previous

researches

General Linear Model (GLM)

Mixed Linear Model (MLM)Core collection

GBS

Plant architecture

Pungency

Fruit color

Plant disease


Genetics

Capsicum germplasm collections

22

South America

128

Central America

56

North Ameraica

197 Europe

730Asia

1,814

Africa

37

Oceania

11

Unknown origin

1,679

C.annuum 97%

C.annuum 93%

93%

C.annuum

89%

57%

47%25%

25%

C.annuum 80%

(4613)

(163)

(1)

(28)

(122)

(4)

(152)

(2)

(5)

(11)

(1)

4,650 accessions (11 Capsicum species)


Genetics

Analysis of population structureNeighbor-joining tree based on distance based clustering method

from STRUCTURE

Most C. baccatum, and C. frutescens accessions

C. baccatum, C. frutescens, C. pubescens,

C. cardenasii, C. chacoense, C. eximium,

C. praetermissum, C. tovarii accessions mostly

from South America and Europe countries

Most C. chinense accessions

C. annuum accessions with one

C. galapagoense mostly from

Europe countries

C. annuum accessions mostly from Asia countries

A

B

E

D

C

C. galapagoense

23

(BMC Gentics, Lee et al, 2016)


Genetics

Base core collection

Addition of more other

Capsicum species

Addition of accessions with

useful traits

Capsicum germplasms

used for GWAS

ChiVMV

CMV

PepMoV

TMV

Anthracnose

Powdery Mildew

C. annuum 218

C. baccatum 47

C. chinense 46

C. frutescens 25

others 5

Total 351

240

60

51

GWAS population in Capsicum

24


Genetics

Scheme for GWAS in Capsicum

25

Experimental

population

Genotype

Phenotype

Association

mapping

Validation

Natural population

Proper sequencing

method for pepper

Interesting traits useful

for breeding


Using bi-parental

population and previous

researches


Mixed Linear Model (MLM)Core collection

GBS

Plant architecture

Pungency

Fruit color

Plant disease


Genetics

Summary of GBS

26

Raw SNPs(9,074,059)

Pre-filtered raw SNPs(2,114,946)

Imputation SNPs(2,114,946)

Filtered SNPs(451,148)

Pre-imputation filtering (Criteria: SNP coverage >0.1, tri-allelic SNP removed)

Imputation (Criteria: hapSize= 1000, minSites= 25, minPres= 250)

Filtering (Criteria: MAF >0.05, SNP coverage >0.6, IF >0.8)

PstI-MseI(4,352,133)

EcoRI-MseI(4,977,434)


Genetics

27

LD= 26.8 kb

LD decay= 84kb (slow)

At least 112,275 markers are needed

Calculation of LD in Capsicum

LD was calculated using SNPs obtained from GBS of the PstI-MseI library

Average LD block size was 26.8 kb

For GWAS, it is estimated that at least 112,275 markers are required.

(Lee et al., Unpulished)


Genetics

Overview of GWAS in Capsicum

28

Genotype

Phenotype

Association

mapping

Validation

Natural population

Fast genotyping method

Various morpjological


Using bi-parental

populations and previous

studues

350 Core set

GBS

Fruit traits

Disease resistance

4,600 accessions


Genetics

29

Distribution of fruit traits for GWAS

0

50

100

150

200

250

300

0

30

60

90

120

150

0

50

100

150

200

250

0

2

4

6

8

10

Fruit length (mm) Fruit width (mm)

Fruit weight (g) Pericarp thickness (mm)

Accessions Accessions

Accessions Accessions

Mean values of 2 years with 3 replications in each year (mean ± SE. P < 0.05)

7.8~262.3 mm

0.2~167.6 g0.04~7.8 mm

5~108.4 mm



Genetics

Phytophthora blight (phenotyping)

1 week 2 weeks 3 weeks 4 weeks

Highly resistant ( 0~1 ) 44 22 19 15

Resistant ( 1~2 ) 32 17 13 9

Moderate resistant ( 2~3 ) 20 19 14 12

Susceptible ( 3~4 ) 41 22 17 14

Highly susceptible ( 4~5 ) 201 258 275 288

350 CC population + Phytophthora capsici (KPC7)

Inoculation of 10 seedling per accession

Disease scale: 0 to 5

Evaluation resistance of CC to Phytophthora


Genetics

Overview of GWAS in Capsicum

31

Genotype

Phenotype

Association

mapping

Validation

Natural population

Fast genotyping method

Various morpjological


Using bi-parental

populations and previous

studues


Mixed Linear Model (MLM)350 Core set

GBS

Fruit traits

Phytophthora

4,600 accessions


Genetics

Bayesian model based clustering methods (STRUCTURE)

K=2

GWAS

population

Other species C. annuum

C. baccatum

C. chinense

C. frutescensHot pepper type Bell pepper typeSub populations

K=2 K=2

0

200

400

600

800

1000

1 2 3 4 5 6 7 8 9 10 11 12

Sub population 2

K

0

200

400

600

800

1000

1200

1 2 3 4 5 6 7 8 9 101112131415

Sub population 1

K

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6 7 8 9 101112131415

GWAS population

K

ΔK

Genomic structure of the population

32

Principal component analysis (PCA)

Axis 1: 47.26%

Ax

is 2

: 2

7%

C. baccatum

C. chinense

C. frutescens

C. annuum

The GWAS population was divided in 4 subgroups both in STRUUCTURE and PCA(Lee et al., Unpulished)


Genetics

33

QTLs associated with fruit traitsGLM (PCA)

FL-3.2 (Han)

FD-1 (Han)

FD-3.2 (Han)

FD-3.1 (Han)

FW-3 (Han)

38 candidate QTLs were detected among 4 fruit related traits

(10 QTLs) (12 QTLs)

(7 QTLs) (9 QTLs)



Genetics

34

Validation of QTLs from GWAS

0

1

2

3

4

5

6

7

8

1.2

5

2.7

5

5.5

5

7.1

5

7.9

5

13.0

5

13.7

5

14.8

5

18.6

5

20.7

5

21.8

5

23.8

5

25.7

5

27.8

5

27.9

5

29.4

5

33.4

5

34.4

5

45.6

5

75.3

5

156.7

5

107.2

5

179.0

5

199.7

5

202.5

5

207.0

5

211.2

5

214.9

5

216.4

5

217.3

5

219.1

5

221.5

5

222.6

5

223.3

5

227.3

5

230.8

5

230.9

5

232.7

5

237.1

5

237.8

5

238.9

5

239.6

5

242.3

5

244.3

5

246.5

5

247.2

5

250.1

5

252.9

5

254.2

5

254.3

5

255.2

5

256.9

5

LOD

Chr. 3

0

1

2

3

4

5

6

7

8

1.3

5

3.2

5

5.5

5

9.0

5

10.9

5

14.7

5

16.6

5

21.3

5

29.9

5

34.1

5

39.3

5

41.0

5

43.4

5

60.6

5

127.5

5

151.9

5

157.8

5

161.1

5

165.9

5

170.6

5

175.6

5

181.5

5

186.0

5

187.2

5

202.1

5

207.2

5

219.6

5

223.4

5

225.8

5

230.4

5

235.0

5

240.3

5

242.2

5

243.0

5

244.5

5

247.8

5

247.9

5

248.5

5

251.3

5

265.6

5

265.5

5

268.6

5

271.5

5

LOD

Chr. 1

C. annuum cv. Perennial

x C. annuum cv. Dempsey

120 RILs (Han, 2016)

FD-1 (Han)

FD-3.2 (Han)

FD-3.1 (Han)

Chromosome 1 Chromosome 3

2015 20152015

All

All

FD-1 (Han)

All

2015

2015

All

All

FD-3.1 (Han)

FD-3.2 (Han)

Manhattan plot

QTL map


Genetics

Phytophthora blight (GWAS)

Total 51 SNPs located in

chromosome 1,2,5,6,10,11, and

12 were highly correlated with

Phytophthora blight resistance

CHR 5 CHR 11 CHR 12

28Mb 77Mb

109Mb

85Mb 38Mb 211Mb

QQ plot

Major QTL 5-2(27~ 33Mb)



Genetics

Validation of QTLs from GWAS

1 2 3 4 5 6 7 8 9 10 11 12

Origin of QTLs:

Populations

JC CC YC

PP HV PYResistance for

Stem rot

Root rot

Foliar blight

Phyto-5

Phyto-6

Phyto-9Phyto-11


Genetics

To expedite the breeding of peppers, our group has developed molecular

markers linked to major genes including disease resistance using various

genomics tools. Some of the genes have cloned (are being) by the

genome-assisted method.

For QTL mapping of horticultural traits and disease resistance, RILs and

CC were developed and genotyped by the genotype-by-sequencing

methods.

GWAS using CC revealed candidate QTLs for four fruit traits and

Phytophthora blight resistance, and these QTLs were corresponded to

previously detected QTLs.

Summary

37


Genetics

38

Acknowledgements

SNUDr. Jin-Kyung Kwon

Dr. Won-Hee Kang

Hea-Young Lee

Koeun Han

Irfan Siddique

Jong-Ho Lee

Jin-Kwan Jo

Si-Young Jang

Seula ChoiIrfan SiddiqueAmornrat Chingkwian

Funded byMinistry of Agriculture & Fishery and Rural Development Administration, Republic ofKorea

RDADr. Jung-sook Sung

On-Sook Hur

Ho-Cheol Go

Horticultural Crop Breeding and Genetics Lab

Thank you for your attention

Construct a Core Collection in Plant germplasm _ Lab. of Horticultural Crop Breeding

and Genetics

genome-assisted marker development for disease resistance ...€¦ · genome-assisted marker...

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