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Correcting population stratification in pepper core collection for genome-wide association studies (GWAS)

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2016 한국육종학회-차세대BG21사업단-GSP사업단 공동심포지엄 Gene, Genome & New Technology for Plant Breeding 2016년 6월 29일(수) ~ 7월 1일(금), 라마다플라자 청주호텔 1일째 [2016. 6. 29. 수] 19:00~ 이사회의 및 조직위원회의 2일째 [2016. 6. 30. 목] 09:00~09:50 공동심포지엄 학술발표회 등록 및 포스터 부착 09:50~10:00 개회식 개회사 - 정영수 교수 (조직위원장, 동아대학교) 환영사 - 조용구 교수 (회장, 충북대학교) << 1부 Plenary Session >> - 좌장 : 고희종 교수 (서울대학교) 10:00~10:40

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2일째 [2016. 6. 30. 목] << 2부 한국육종학회 분과발표 & 포스터 발표 >> 15:40~17:40 ▸분과발표 OA 수량 및 저항성육종 - 좌장 : 김용호 교수 (순천향대학교), 조영찬 박사 (국립식량과학원) ▸분과발표 OB 품질육종 및 유전변이 - 좌장 : 김보경 과장 (국립식량과학원) 강성택 교수 (단국대학교) ▸분과발표 OC 분자육종 및 유전공학 - 좌장 : 강권규 교수 (한경대학교), 박용진 교수 (공주대학교) 17:40~18:00 한국육종학회 정기총회 18:00~18:20 포스터 발표 18:20~ 특별공연 및 간친회 3일째 [2016. 7. 1. 금] << 3부 Concurrent Session >> 주요 작물에서 유전체 정보활용 육종가 친화형 인터페이스 연구 소개 (농생물게놈활용연구사업단) - 좌장 : 유의수 박사 ((주)파이젠) 09:00~09:25 ▸가지과 유전체 활용을 위한 생물정보분석 파이프라인 및 데이터베이스 TGsol 현황 - 조성환 박사 ((주)씨더스) 09:25~09:50 ▸두과작물 유전체정보 기반 분자육종 활용을 위한 플랫폼 개발 - 최홍규 교수 (동아대학교)

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▸Versatile application of CRSPR/Cas9 system in plant research - 배상수 교수 (한양대학교)

▸Transgenic plants producing green-vaccine for CSFV(classical swine fever virus) lead on plant biotechnology-based product on market

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iii 3일째 [2016. 7. 1. 금] << 3부 Concurrent Session >> - GSP 채소종자사업단 & GSP 원예종자사업단 ▸좌장 : 임용표 단장 (채소종자사업단) 09:00~10:40 ▸육성가 권리보호와 종자산업의 발달 - 이승인 박사 (국립종자원) ▸종자검정서비스 확대를 위한 국립종자원의 전략 - 소은희 박사 (국립종자원) ▸좌장 : 노일섭 단장 (원예종자사업단)

▸Breeding for Pyramiding Target-genes and Selection of F1 Hybrids by Marker Assisted Selection in Tomato

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▸Gene Identification, Expression Analysis and Breeding for Enhanced Glucosinolate Biosynthesis in Brassica

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Tolerance in the Philippines - Dr. Norvie Manigbas (PhilRice)

▸Rice production and the change of major diseases during the period of climate change in Vietnam

- Dr. Dung Laitien (Plant Protection Research Institute, Vietnam) 10:40~11:00 휴식

<< 4부 Plenary Session >>

▸좌장 : 서용원 교수 (고려대학교)

11:00~11:40 ▸CRISPR RNA-guided Genome Editing in Human Stem Cells, Animals, and Plants - 김진수 교수 (서울대학교)

11:40~12:20 ▸CRISPR Genome Editing in Outcrossing Woody Perennials - Dr. CJ Tsai, University of Georgia, USA

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PC-53 Characterization of Cytochrome P450 (CYP) Genes Related to Saponin Biosynthesis

in

Platycodon grandiflorum

214

Sohyeon Park, Jemin Yoo, Yurry Um, Ok Tae Kim, Chang Pyo Hong, Seong-Cheol Kim, Yi Lee

PC-54 Analysis of Genetic Diversity in 5

Codonopsis

Species Based on SSR Markers 215

Sohyeon Park, Serim Kim, Jinsu Gil, Yurry Um, Hee Chung, Ok Tae Kim, Ho Bang Kim, Seong-Cheol Kim, Yi Lee

PC-55 The CRISPR/Cas9-mediated Genome Editing in Banana Cells 216

Youjin Shin, Jin-Soo Kim, Jae-Young Yun

PC-56 Development of Chloroplast InDel Markers to Distinguish

Angelica

Species 217

Sohyeon Park, Sangik Park, Jinsu Gil, Yurry Um, Hee Chung, Seong-Cheol Kim, Yi Lee

PC-57 High-throughput transcriptome analysis for identifying genes determining flowering time 217

Cheol-Won Lee, Yong Weon Seo

PC-58 Candidate Gene Analysis for the Genes Controlling the Yellow Color in

Capsicum annuum

Cultivar Micropep 218

Ayoung Jung, Juhun Lee, Jin-Kyung Kwon, Suna Kim, Byoung-Cheorl Kang

PC-59 Identification of New Resistance Sources for

Cucumber mosaic

virus

New Isolate-P1 (CMV-P1)

in the Germplasm Collection of Capsicum spp. 219

Seula Choi, Myeong-Sook Han, Jin-Kwan Jo, Joung-Ho Lee, Eun-Ho Son, Byoung-Cheorl Kang

PC-60 Genetic Mapping of Resistance Sources Against ChiVMV (

Chili veinal mottle virus

) in Hot Pepper 220

Joung-Ho Lee, Jeong-Tak An, Koeun Han, Seula Choi, Muhammad Irfan Siddique, Byoung-Cheorl Kang

PC-61 Correcting Population Stratification in Pepper Core Collection for Genome-wide Association

Studies (GWAS) 221

Hea-Young Lee, Koeun Han, Jin-Kyung Kwon, Byoung-Cheorl Kang

PC-62 Hormone Related Transcriptome Analysis of Wheat During Pre-harvest Sprouting

and Exogenous ABA Treatment 222

Yong Jin Lee, Jae Yoon Kim, Yong Weon Seo

PC-63 Probing High-yield Traits of Soybean by Transforming Senescence-delay Genes 223

Hyun Suk Cho, Jin Ho Yang, Jin Sol Park, Hye Jeong Kim, Yoon Jeong Lee, Jae Seong Kim, Hyun Hee Im, Ki Jung Lee, Dong Hee Lee, Young Soo Chung

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221

PC-61 ◆1

Correcting Population Stratification in Pepper Core Collection for Genome-wide Association

Studies (GWAS)

Hea-Young Lee*, Koeun Han, Jin-Kyung Kwon, Byoung-Cheorl Kang*

Department of Plant Science and Vegetable Breeding Research Center CALS, Seoul National University, Seoul 151-921, Korea

Genome-wide association study (GWAS) is an effective approach for identifying genetic variants associated to useful agronomic traits. GWAS has emerged as a powerful approach for identifying genes underlying complex diseases or morphological traits at an unprecedented rate. In such studies, it is very important to correct for population stratification, which refers to allele frequency differences between cases and controls due to systematic ancestry differences. Population stratification can cause false positive findings if not adjusted properly. As we are planning to perform GWAS for various agronomic traits in pepper, a genotyping-by-sequencing (GBS) approach was used to provide dense genome-wide marker coverage (>19,000 SNPs) for a 250 pepper core collection. Using GBS platform, high density haplotype map was constructed and various stratification methods, including distance based phylogenetic methods, principal component analysis (PCA), and bayesian phylogenetic methods (STRUCTURE) were performed to show the genetic diversity and population stratification. These results will not only find genetic variants among pepper accessions but also provide powerful evidence for reducing first positive error to perform GWAS in large scale studies.

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Hea-Young Lee

1

, Koeun Han

1

, Jin-Kyung Kwon

1

, and Byoung-Cheorl Kang

1*

1

Department of Plant Science and Vegetable Breeding Research Center CALS, Seoul National University, Seoul 151-921, Korea,

*Corresponding author Byoung-Cheorl Kang

bk54@snu.ac.kr

+82-2-880-4563

Correcting population stratification in pepper core collection for

genome-wide association studies (GWAS)

Genome-wide association study (GWAS) is an effective approach for identifying genetic variants associated to useful agronomic traits. GWAS has emerged as a powerful approach for

identifying genes underlying complex diseases or morphological traits at an unprecedented rate. In such studies, it is very important to correct for population stratification, which

refers to allele frequency differences between cases and controls due to systematic ancestry differences. Population stratification can cause false positive findings if not adjusted

properly. As we are planning to perform GWAS for various agronomic traits in pepper, a genotyping-by-sequencing (GBS) approach was used to provide dense genome-wide marker

coverage (>33,000 SNPs) for a 250 pepper core collection. Using GBS platform, high density haplotype map was constructed and various stratification methods, including distance

based phylogenetic methods, principal component analysis (PCA), and bayesian phylogenetic methods (STRUCTURE) were performed to show the genetic diversity and population

stratification. These results will not only find genetic variants among pepper accessions but also provide powerful evidence for reducing first positive error to perform GWAS in large

scale studies.

ABSTRACT

OBJECTIVES

MATERIALS & METHODS

Detection of genome-wide SNPs among pepper GWAS population using

genotyping-by-sequencing (GBS) approach

Construction of high density haplotype map

Population structure analysis using various stratification methods

A pepper GWAS population including 9 species, consisting of 351 accessions

was constructed by combining three different collections.

Capsicum

species

included in this population are shown in figure 1.

RESULTS

This work was carried out with the support of "Cooperative Research Program

for Agriculture Science & Technology Development (Project No.

PJ011204012016)" Rural Development Administration, Republic of Korea

ACKNOWLEDGEMENT

Genomic structure of pepper GWAS population

Figure 3. Population structure of the

Capsicum

core collection (CC250) using GBS data. ΔK

reached its maximum value when K=2 following the

ed-hoc

method. Subpopulations were

grouping by Q. Each subpopulation was separated in to two subgroups.

REFERENCE

SNP observation in high density haplotype map

Based on the Bayesian phylogenetic methods, whole population showed two subpopulations

as

C. annuum

and the other species. The first subpopulation which contains the other species

was also divided in two subgroups as

C. baccatum

and the other species. The second

subpopulation which contains all the

C. annuum

was tend to separate by fruit shape as hot

pepper type and bell pepper type (Figure 3).

Distribution of pepper GWAS population

Plant material

Genotyping-by-sequencing (GBS)

To better understand the genetic diversity of germplasm, phylogenetic analysis

and PCA were performed by DARwin 6.0.9 (Perrier and Jacquemoud-Collet, 2006).

Population structure was identified using STRUCUTRE 2.3.4 software.

Overall 3,000,000 SNPs were detected among pepper GWAS population. SNPs with > 50%

missing data and monomorphic SNPs were dropped from the data set. After strong SNP

filtering, 33,843 SNPs were remained with call rates > 0.5.

SNP observation and haplotype map construction

Population structure and genetic diversity analysis

Pepper GWAS population

Pepper core collection (250)

Accessions with additional

useful traits (51)

ChiVMV CMV PepMoV TMV Anthracnose Powdery Mildew

Core collection in other

Capsicum species (50)

C. annuum

226

C. baccatum

47

C. chacoense

2

C. chinense

46

C. frutescens

25

C. eximium

2

C. galapagoense

1

C. praetermissum

1

C. pubescens

1

Total

351

Figure 1. Pepper GWAS population using in this study. A total of 351 accessions were

placed in this population constructed by combining three different pepper

collections.

DNA of germplasm was extracted by CTAB method. Two restriction enzymes

(

PstI-MseI

), and a compatible set of 96 barcode were used to prepare the GBS

library. Single end sequencing was performed on four lanes of an Illumina HiSeq

2000 at the Macrogen Inc (Seoul, Korea).

The CLC Genomics Workbench was used to check sequencing quality (QC) and

trim the sequence reads. Two software tools, BWA and GATK were used for the

processing of Illumina sequence read trimmed data. Haplotype map was

constructed using FILLIN in TASSEL 5 (Figure 2).

SNP calling

CLC Genomics Workbench

•Quality trimming and demultiplexing using barcode

BWA

•BWA-MEM (0.7.12)

GATK

•GATK Unified Genotyper

•Filtering SNPs with QUAL >= 30, and minimum depth 3

Library construction &

Sequencing

GBS library

Pst

I and

Mse

I double digestion

HiSeq 2000

•Run mode: 101 single end

Imputation

TASSEL FILLIN

•Construction of haplotype map

•Imputation of missing SNPs by haplotype map

High-quality SNPs

Figure 2. Workflow of SNP calling and haplotype map construction.

K=2

Other species

C. annuum

C. baccatum

C. chinense

C. frutescens

Hot pepper type

Bell pepper type

K=2

K=2

0 2000 4000 6000 8000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 500 1000 1 2 3 4 5 6 7 8 9 10 11 12 0 500 1000 1500 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

K

ΔK

Whole population

Sub population 1

Sub population 2

C. frutescens

+

1

C. eximium

(Group 2)

C. chinense

+

1

C. praetermissum

(Group 3)

C. annuum

(Group 4)

C. annuum

(Group 5)

C. annuum

(Group 6)

C. annuum

(Group 7)

C. annuum

(Group 8)

Figure 4. Genetic relatedness among the core collection assessed by PCA (A) and neighboring

joining method (B) after haplotype imputation. Eight groups (Group 1-8) were distinguished

by distance between branches; group 1 included

C. baccatum

;

group 2 included

C. frutescens

with 1

C. eximium

;

group 3 included

C. chinense

with 1

C. praetermissum

;

most of

C. annuum

species spread among group 4 to 8; group 5 and 6 consist of hot pepper type; group 7 and 8

consist of bell pepper type.

Axis 1: 47.26%

A

xi

s

2:

27

%

C. baccatum

C. chinense

C. frutescens

C. annuum

To understand the population stratification in pepper GWAS population, whole accessions

were plotted in distance metrics. The first and second axes explained 47.26% and 27% of the

genotypic variance, respectively, that clearly separated

C. annuum

from

C. baccatum

and the

other species. In right below of matric,

C. frutescens

and

C. chinense

showed admixture slightly.

Overall, PCA analysis showed that four distinct sub population was existent in GWAS

population (Figure 4A). For an unrooted phylogenetic tree, eight groups were figure out

among the whole accessions. And the distribution of clusters showed similar patterns of PCA

results that groups were divided in species level (Figure 4B).

(A)

(B)

1. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, et al. A

robust, simple genotyping-by-sequencing (GBS) approach for high diversity

species. PLoS One. 2011;6(5):1–10.

2. Liu L, Zhang D, Liu H, Arendt C. Robust methods for population stratification

in genome wide association studies. BMC Bioinformatics. 2013;14(1):132.

3. Perrier X, Jacquemoud-Collet, JP. 2006. DARwin software

4. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using

multilocus genotype data. Genetics. 2000;155(2):945–59.

GWAS of various fruit traits

Figure 5. Manhattan plots of association p-values over the 12 pepper chromosome. MLM (K+Q)

model was used to screen for association between genotype and (A) Fruit length, (B) Fruit

width, (C) Fruit weight, and (D) Pericarp thickness.

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