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A THESIS FOR THE DEGREE OF MASTER OF SCIENCE

Molecular Mapping of ChiVMV (Chili veinal mottle virus) Resistance

Genes in Hot Pepper

AUGUST, 2015

JOUNG-HO LEE

MAJOR IN HORTICULTURAL SCIENCE

DEPARTMENT OF PLANT SCIENCE

THE GRADUATE SCHOOL OF SEOUL NATIONAL UNIVERSITY

Molecular Mapping of ChiVMV

(Chili veinal mottle virus) Resistance

Genes in Hot Pepper

JOUNG-HO LEE

DEPARTMENT OF PLANT SCIENCE

THE GRADUATE SCHOOL OF SEOUL NATIONAL UNIVERSITY

ABSTRACT

Chili veinal mottle virus (ChiVMV) induces severe loss to pepper production in

Asia and Africa. In this study, four different pepper accessions (CV3, CV4, CV8

and CV9) known to have ChiVMV resistance genes, were used to explore

inheritance modes and their chromosomal positions. To confirm the allelic

relationship among these genes, molecular marker mapping approach was used and

then each gene was named. Among the accessions, resistance of CV3 and CV8

followed the single dominant inheritance pattern. Molecular marker study revealed

that the resistance in CV3 and CV8 may be originated from the same gene and

named as Cvr1. Resistance of CV4 appeared to be controlled by two independent

complementary genes. These two genes were named as Cvr2-1 and Cvr2-2.

Resistance of CV9 was turned out to be controlled by a single recessive gene which

was nominated as cvr4. To map Cvr2-1 and Cvr2-2 genes, single nucleotide

polymorphic marker (SNP) based linkage map was constructed using by

genotyping-by-sequencing (GBS) and an F2 population derived from a cross ‘CV4

x Jeju’. Genetic mapping showed one of the two genes is located on pepper

chromosome 6. The results of this study will accelerate the ChiVMV resistance

research.

.

Keywords: Chile veinal mottle virus (ChiVMV), ChiVMV resistance genes,

Genotyping by sequencing (GBS), Single nucleotide polymorphism (SNP)

Student number: 2013-23231

CONTENTS

ABSTRACT ..........................................................................................................

CONTENTS ..........................................................................................................

LIST OF TABLES .................................................................................................

LIST OF FIGURES ...............................................................................................

LIST OF ABBREVIATIONS ................................................................................

INTRODUCTION ................................................................................. 1

LITERATURE REVIEW

Plant immunity ................................................................................ 4

Plant immunity against non-viral pathogen ................................ 5

Plant immunity against virus ....................................................... 6

Plant virus resistance ...................................................................... 7

The recessive virus resistance ..................................................... 7

The dominant virus resistance ..................................................... 8

Potyvirus resistance genes in pepper .............................................. 9

MATERIALS AND METHODS

Plant materials .................................................................................. 11

Plant virus inoculation and Virus detection assay ........................... 11

Genomic DNA extraction and genotyping-by-sequencing (GBS)

library preparation............................................................................ 12

Sequence data analysis and SNP identification ............................... 13

Construction of the linkage map and genetic mapping of ChiVMV

resistance in 'CV4'............................................................................ 14

RESULTS

ChiVMV resistance in ‘CV3’, ‘CV4’, ‘CV8’ and ‘CV9’Inheritance

study of ChiVMV resistance sources ............................................... 15

Segregation analysis in the mapping population of ‘CV3’ and

‘CV8’

.................................................................................................. 17

Segregation analysis in the mapping population of ‘CV9’ ......... 18

Segregation analysis in the mapping population of ‘CV4’ ......... 18

Identification of ChiVMV resistance source in ‘CV3’, ‘CV4’, ‘CV8’

and ‘CV9’ ........................................................................................ 21

Allelism test for confirming dominant ChiVMV resistance locus

.................................................................................................. 21

Identification of dominant resistance source with genotyping . 23

Genetic mapping of the ChiVMV resistance loci in 'CV4' .............. 27

Genotyping by sequencing of F2 plants .................................... 27

Linkage map construction and comparison of physical map of

pepper ....................................................................................... 29

Mapping the ChiVMV resistance loci ...................................... 34

DISCUSSION ............................................................................................ 37

REFERENCES ........................................................................................ 40

ABSTRACT IN KOREAN ............................................................. 48

LIST OF TABLES

Table 1 Segregation analysis of resistant sources against ChiVMV

resistance ..................................................................................................... 20

Table 2 Genotyping summary and information of CVMV2 and CVMV3

marker analysis in F2 populations of ‘CV3’, ‘CV4’, and ‘CV8’ ................ 26

Table 3 Number of SNPs from GBS data per chromosome ....................... 28

Table 4 Distribution of SNP markers on the linkage groups....................... 30

Table 5 Information of candidate ChiVMV resistance region .................... 36

LIST OF FIGURES

Figure 1 Resistant responses to ChiVMV in parental lines, ‘CV3’, ‘CV4’,

‘CV8’, ‘CV9’ and ‘Jeju’ .............................................................................. 16

Figure 2 Detection of ChiVMV accumulation in ‘CV3 x CV4’, ‘CV3 x

CV8’ and ‘CV4 x CV8’ by DAS-ELISA ..................................................... 22

Figure 3 Cleaved amplified polymorphism sequence (CAPS) analysis of

ChiVMV dominant resistance locus-linked markers on pepper chromosome

6. ................................................................................................................ 25

Figure 4 SNPs from GBS distribution on 12 pepper chromosomes to link

between genetic and physical map .............................................................. 33

Figure 5 Genetic mapping result for ChiVMV resistance in “CV4 x Jeju” F2

population .................................................................................................... 35

LIST OF ABBREVIATIONS

ChiVMV Chili veinal mottle virus

Pvr Potyvirus resistance gene

Cvr ChiVMV resistance locus

GBS Genotyping by sequencing

SNP Single nucleotide polymorphism

NB-LRR Nucleotid binding-leucine rich repeat

CP Viral coat protein

eIF4E Eukaryotic inititation factor 4E

eIF(iso)4E Eukaryotic initation factor(iso)4E

NGS Next generation sequencing

GBS Genotyping by sequencing

cM Centi Morgan (the unit of genetic distance)

LOD Logarithm of the odds

INTRODUCTION

ChiVMV is included in the Potyvirus genus belonged to the Potyviridae

family, which is one of the most important limiting factors for the production of

Solanaceous plants worldwide. ChiVMV is transmitted by several aphid species or

mechanical inoculation (Green and Kim 1994). ChiVMV was first reported in

Malaysia and, now days, it has become a major problem for pepper reduction in

Asia and Africa (Green et al. 1999; Lee et al. 2013; Ong et al. 1979). According to

the Asian vegetable research and development center (AVRDC), a total of 30%

pepper production was reduced because of ChiVMV infection in 16 Asian

countries (www.avrdc.org). Although most of the Solanaceae species such as,

Capsicum annuum, Solanum melongena and Nicotiana spp., are included in the

host range of ChiVMV, ChiVMV most severely affects pepper growth inducing

symptoms of leaf mottling, vein-clearing and chlorosis of leaves (Tsai et al. 2008).

Several potyvirus resistance genes are known in Capsicum species. These

potyvirus resistance genes are abbreviated as pvr (potyvirus resistance) (Kyle and

Palloix 1997). Most of pvrs (pvr1, pvr3, pvr5, pvr6 and pvr8) show recessive

inheritance patterns because resistance is resulted from mutations of host factors

required for virus infection (Kang et al. 2005a; Yeam et al. 2007). The pvr1 gene is

located on pepper chromosome 3 and confers resistance to several Tobacco etch

virus (TEV) strains and Potato virus Y (PVY) (Kang et al. 2005a). The pvr2 locus

was confirmed to be identical with pvr1, so the allele names were given as pvr11

and pvr22 (Yeam et al. 2005). Furthermore, pvr5 was also turned out to be another

allele of pvr1 (Yeam et al. 2005). The pvr3 gene renders resistance to Pepper

mottle virus (PepMoV) by preventing long-distance movement (Parrella et al.

2002). However, the position of pvr3 has not known yet. The pvr6 gene located on

chromosome 9 confers resistance to Pepper veinal mottle virus (PVMV) together

with pvr11 or pvr12 (Hwang et al. 2009). While most of potyvirus resistance genes

show recessive inheritance pattern, Pvr4 shows dominant inheritance. Pvr7 is

located on chromosome 10 very closely linked to Pvr4 and exhibits resistant to

PepMoV (Grube et al. 2000).

Several genetic studies on ChiVMV resistance have been reported previously.

First ChiVMV inheritance study using doubled haploid (DH) lines derived from a

cross between C. annuum ‘Perennial’ and C. annuum ‘Yolo Wonder’ revealed the

requirement of two independent dominant genes for resistance (Caranta and Palloix

1996). In addition, the study demonstrated clear dominant effect of resistance in F1

plants. Moreover, the pvr loci also confer ChiVMV resistance in pepper.

Simultaneous mutations in pvr12 and pvr6 show recessive resistance to ChiVMV

(Hwang et al. 2009). The interaction between ChiVMV VPg (Viral protein

genome-linked) and eukaryotic initiation factor 4E (eIF4E) encoded by pvr12 or

eIF(iso)4E encoded by pvr6 was demonstrated in this study (Hwang et al. 2009).

Independent mutation in pvr12 or pvr6 does not confer ChiVMV resistance. More

recently, a single dominant ChiVMV resistance gene was reported (Lee et al. 2013).

This dominant resistance gene is located on the short arm of pepper chromosome 6

(peppergenome.snu.ac.kr). Molecular markers linked to this gene were developed

but positions of markers are rather far from ChiVMV resistance gene (3 cM, 4 cM

and 7 cM) (Lee et al. 2013).

With the advent of next-generation sequencing (NGS) technologies, the

paradigm of marker technology have been changed to analyze thousands of

markers in a single experiment (Davey et al. 2011). Among NGS sequencing-based

marker discovery methods, reduced-representation sequencing and restriction site-

associated sequencing (RAD-seq and GBS) depend on restriction enzyme for

reducing representation and complexity of a genome (Davey et al. 2011; Elshire et

al. 2011). These methods enable to reduce sequencing cost (Poland and Rife 2012).

To reduce genome complexity further, two restriction enzymes or selective

nucleotides in PCR amplification step were used (Poland et al. 2012 and Truong et

al. 2012).

In this study, inheritance patterns of several ChiVMV resistance genes were

analyzed and molecular markers were developed linked to ChiVMV resistance

genes. To achieve this goal, four ChiVMV resistant lines were evaluated by

inoculation of a ChiVMV strain. In order to identify the resistance loci, genome-

wide single nucleotide polymorphism (SNP) markers were developed by GBS

technology and a genetic map was constructed.

LITERATURAL REVIEW

1. Plant immunity

The innate immunity system of plants is more complex than that of animals for

compensating their lack of a somatic adaptive immune system and mobile defender

cells (Dodds and Rathjen 2010; Jones and Dangl 2006). The name of immunity is

derived from similar protein motifs of nucleotide binding (NB) and leucine rich

repeat (LRR) domain and from analogous response of these NB-LRR protein from

pathogen-derived protein activation between plants and animal kingdoms (Lelpe et

al. 2004; Ting and Davis 2005). For example, plants rely on the autonomous

immune events of each cell or systemic signal from infection site (Jones and Dangl

2006). The representative innate immune systems of plants are disease resistance

(R) protein involved system and RNA silencing based system (Jones and Dangl

2006; Voinnet 2001). Most fungal and bacterial pathogen resistance have been

researched from a view of R protein based immunity, but most viral pathogen

resistance have been studied on RNA silencing based immunity (Pumplin and

Voinnet 2013). However, recent studies revealed these two immune systems could

be considered as small RNAs for important regulators of R gene expression

(Shivaprasad et al. 2012; Zhai et al. 2011) or integrated view for plant immunity

(Staiger et al. 2013).

1.1. Plant immunity against non-viral pathogen

To protect from microbial pathogen attack, plant have two different types of

innate immune systems (Jones and Dangl 2006). One immune system is induced by

pathogen-associated molecular patterns (PAMPs) and use receptor protein called

pattern recognition receptors (PRRs) for recognition of PAMPs (Chisholm et al.

2006; Dodds and Rathjen 2010; Jones and Dangl 2006). Usually, PAMPs are

essential and conserved molecules of whole class of pathogens, such as bacterial

Ef-Tu, bacterial flagellin or fungal chitin (Chisholm et al. 2006; Dodds and Rathjen

2010; Zipfel and Felix 2005). After perception of PAMPs in PRRs, it leads towards

pathogen-triggered immunity (PTI) for basal defense. This PTI sometimes is

accompanied with MAP kinase signaling, transcriptional induction and production

of reactive oxygen species to prevent microbial growth (Chisholm et al. 2006). To

overcome plant PTI response, pathogens secret effectors proteins which are

pathogen virulence molecules for successful infection and this phenomenon is

called to effector triggered susceptibility (ETS) (Jones and Dangl 2006). To

overcome this susceptibility, plants use R protein for specifically recognizing a

given effector. In this step, most R proteins contain NB-LRR motif for interacting

with effectors. This immunity response using R protein called effector-triggered

immunity (ETI). Usually, ETI response is more severe than PTI, so it induces local

cell death called hypersensitive response (HR) at the infection site (Jones and

Dangl 2006). PTI and ETI response of the plants explain not only pathogen host

interaction clearly but it also gives details about co-evolution relationship of plant-

microbe interactions. (Jones and Dangl 2006).

1.2. Plant immunity against virus

Plants use mostly NB-LRR protein-based immunity against viruses, but RNA-

silencing mediated antiviral mechanism is more popular immunity mechanism in

plants. (Voinnet 2001). When virus entered into host cell, viral double stranded

RNA is processed by dicer-like protein (DCL) family or cleaved by ARGONAUTE

protein (AGO) family (Pumplin and Voinnet 2013). After processing by DCL

family, virus induced small RNA could translocate to other cell or replicates into

more longer dsRNA with RNA-dependent RNA polymerase for delivering signal to

nucleus or inducing antiviral RNA silencing mechanism to other plant cells

(Nakahara and Masuta 2014; Pumplin and Voinnet 2013). To overcome antiviral

RNA silencing, viral RNA obtain viral suppressors of RNA silencing (VSRs)

evolutionarily (Voinnet et al. 1999). For example, Turnip crinkle virus protein P38

binds and inhibits AGO protein function or tombusviral protein P19 sequesters 21-

nt size of small RNAs to prevent RNA silencing disturbing interaction between

small RNAs and AGO proteins (Mallory and Vaucheret 2009; Pumplin and Voinnet

2013). In recent researches, miRNAs revealed a switch to control the NB-LRR

motif genes based resistance mechanism, so it could explain how hosts and plant-

microbes processed co-evolution successively (Li et al. 2012).

2. Plant virus resistance

Numerous approaches are used to differentiate the resistance mechanism to the

plant viruses; such as non-host or host resistance, analyzing function of virus

resistance gene and exploring the inheritance mode of virus resistance gene (Kang

et al. 2005b). According to categorization of inheritance mode, virus resistance can

be divided into two types, dominant and recessive resistance (Kang et al. 2005b).

More than 80% of plant viral resistance loci are inherited monogenically and, most

of them have recessive locus to virus resistance (Truniger and Aranda 2009).

2.1. The recessive virus resistance

Since plant virus genome contains a small number of genes, virus uses host

proteins, called host factor, for completion of their life cycles (Nagy and Pogany

2012). These host factors are required for viral replication, cell-to-cell movement

and systemic development (Kang et al. 2005b). Most of the recessive virus

resistance concept are originated from host factors for viral infection (Truniger and

Aranda 2009). If mutations or deletions are occurred in host factors, it can confer

durable virus resistance, called recessive resistance (Truniger and Aranda 2009). In

nature, mainly virus resistance prevailed as a single recessive resistance gene

mechanism (Kang et al. 2005b). For example, pepper eIF4E gene is required for

several potyvirus infection because of interaction between potyviral genome linked

protein (VPg) and eIF4E protein product (Robaglia and Caranta 2006). Therefore,

several mutations in eIF4E region can induce potyvirus resistance in pepper (Kang

et al. 2005a).

2.2. The dominant virus resistance

Most plant R genes were isolated based on gene-for-gene interactions

accompanied with HR in usual or extreme resistance (ER) responses in rare

occasions (de Ronde et al. 2014) . Dominant R genes can be grouped into two

classes, those encoding NB-LRRs and non-NB-LRRs (Gururani et al. 2012). The

major class of R genes encodes NB-LRR motif with three domains, the Nucleotide

Binding (NB) site, a Leucine Rich Repeat (LRR) at the C-terminus and a Coiled-

coil (CC) or Toll Interleukin-1 Receptor (TIR) domain at the N-terminus of

resistance protein (Collier and Moffett 2009). NB domain is consisted of nucleotide

binding site and ARC-domain for binding and hydrolyzing ATP (van der Biezen

and Jones 1998). LRR domains are the most variable domains in R gene motifs

determining host specificity by recognizing target proteins (de Ronde et al. 2014).

Finally, TIR domain, one of the C-terminus domains, shares homology with PRRs

in animal innate immune systems. However, CC domain has no common structure.

These three domains are responsible for interaction with other R proteins and

induction of resistance responses (Lukasik and Takken 2009; Slootweg et al. 2010).

Except R gene containing NB-LRR motif, only a few researchs have been done

such as Ty-1 resistance gene (Verlaan et al. 2013). Majority of non NB-LRR type R

genes recognize pathogen indirectly (Collier and Moffett 2009).

3. Potyvirus resistance genes in pepper

The nomenclature of potyvirus resistance genes were revised for discriminating

clearly the genetic and biological relationships among potyvirus resistance genes in

pepper (Kyle and Palloix 1997). Since then, several potyvirus resistance genes

were grouped to the name of pvr (potyvirus resistances) by their resistance

spectrum and sources (Kang et al. 2005b). To date, five potyvirus resistance genes

were studied in detail. Since, potyviral VPg require to interact with host factors for

their complete infection in host, mutations or deletions of essential host factor can

confer resistance to several species of potyvirus (Kang et al. 2005a). Thus, most

pvr genes are corresponded to recessive resistance (Kang et al. 2005b). The gene

pvr1 is located on pepper chromosome 3 and known as coding region of eIF4E

(Yeam et al. 2005). The pvr1 confers resistance to TEV strains, PepMoV and PVY

(Kang et al. 2005a). In addition, pvr1 is also known as the identical locus of pvr2

but several mutations in eIF4E coding region confer somewhat different resistance

spectrum of viral strain (Kang et al. 2005a; Ruffel et al. 2002). Therefore, the name

of pvr2 was changed to pvr11 and pvr12 for allelic relationship with pvr1 (Kang et

al. 2005a). The pvr6 is another recessive resistance gene for potyvirus (Ruffel et al.

2006). The research involved in pvr6 was not performed by single gene effect but

as polygenic effect with pvr1 or pvr12. The pvr12 with pvr6 is known to be

effective in resistance to Pepper veinal mottle virus (PVMV) and ChiVMV

(Hwang et al. 2009). The pvr6 is located on pepper chromosome 9 (Hwang et al.

2009). On the other side, the Pvr4 is dominant potyvirus resistance gene and

located on pepper chromosome 10 (Grube et al. 2000). It has been reported that

Pvr4 is tightly linked to Tsw gene, which is designated as Tomato spotted wilt virus

(TSWV) resistance gene (Grube et al. 2000). Furthermore, Pvr4 is responsible for

PepMoV resistance in pepper (Grube et al. 2000). In potyvirus resistance gene

study, several reported genes are overlapped with previously reported genes

because of under taking different resistant sources in each study. Recently, with the

advent of genomics approaches, several potyvirus resistance genes have been

revealed in detail, so the nomenclature of potyvirus resistance genes is now

organized thoroughly by more researches.

MATERIALS AND METHODS

Plant materials

Four pepper lines, C. annuum ‘CV3’, ‘CV4’, ‘CV8’ and ‘CV9’, were provided

by Clover Seed Ltd. (Hong Kong) and used as ChiVMV resistance sources. A local

landrace of Korea C. annuum ‘Jeju’ was used as a susceptible parent for ChiVMV.

To perform genetic analysis of the ChiVMV resistances, F2 populations were

constructed by self-pollination of F1 plants derived from crosses between each

ChiVMV resistant line (‘CV3’, ‘CV4’, ‘CV8’, ‘CV9’) with ‘Jeju’. For resistance

gene mapping, over three hundreds F2 seeds were obtained from each population

and used.

To reveal allelic relationship of ChiVMV resistance genes, diallele crosses were

made between parents containing a dominant resistance gene: 'CV3 x CV4’, ‘CV3

x CV8’, ‘CV4 x CV8' (Hoang et al. 2013). Fifteen F1 individuals from each cross

were screened by ChiVMV for the allelism test.

Plant virus inoculation and virus detection assay

For virus inoculum preparation, ChiVMV frozen stocks were used which was

stored at -80 . Ten days prior to disease screening, frozen ChiVMV inocula were

inoculated to Nicotiana benthamiana. Fronzen inocula were ground in 0.1M

potassium phosphate buffer, pH 7.0, mixed with 400-grit carborundum, and rubbed

on the 3rd leaves of N. benthamiana. After 10-20 minutes of inoculation, leaves

were washed with distilled water (Hull 2009).

To inoculate the pepper plants, two pairs of cotyledons leaves were inoculated.

Infected pepper plants were grown in the walk-in-chamber (16 hours light, 23

night and day under fluorescent light), and ChiVMV symptoms were observed by

naked eye on non-inoculated upper leaves at 21 – 30 days of post-inoculation (dpi).

At 21-30 dpi, double antibody sandwich enzyme-linked immunosorbent assay

(DAS-ELISA) was performed for detecting coat protein (CP) of ChiVMV Three

replicates of non-inoculated and infected leaves of parental lines were selected for

the use of positive and negative controls in ELISA test.

According to manufacturer’s protocol (Agdia, Elkhart, IN, USA), absorbance

value of 405 nm was measured in microplate reader (Anthon zenith 340 micro

plate reader, UK).

Genomic DNA extraction and genotyping by sequencing

(GBS) library preparation

Genomic DNA was sampled from young true leaf tissues of each plant and

isolated by modified cetytrimethylammonium bromide (CTAB) method in a 1.5ml

micro tube as described by (Prince et al., 1997). After DNA isolation, DNA

concentration and quality was measure by NanoDrop (NanoDrop Technologies,

Inc., Wilmington, DE, USA) and gel electrophoresis. DNA was eluted to adjust

final concentration at 80ng/ l by 0.1 M TE (Tris-EDTA) buffer (pH 7.0) for genetic

marker analysis and genotyping-by-sequencing (GBS) library preparation.

To construct the compact representation libraries for Illumina sequencing, a

total 400 ng of genomic DNA was used. For multiplex sequencing, PstI primer was

constructed with PstI adapter and six nucleotides of individual samples ID tags

while MseI primer was prepared with MseI primer and two selective nucleotides,

guanidine and thymidine (Truong et al. 2012). Genomic DNA digestion and the

adapter ligation was performed according to SBG 100-kit version 2.0.2 manual

(Keygene, Wageningen, The Netherlands). The size check of PCR-amplified

library fragments was performed by DNA 1000 chip of the BioAnalyzer 2100

(Agilent Technologies, Santa Clara, CA, USA). In addition, library quantity was

checked by qPCR according to Illumina qPCR quantification guide book and

calculated by Roche’s Rapid library standard Quantification solution and calculator.

Sequence data analysis and SNP identification

Sequencing was performed by Illumina HiSeq 2000. Raw reads were de-

multiplexed in accordance with individual barcodes and, the adapter and barcode

sequences were removed using commercially available CLC genomics workbench

software (version 6.5). Trimmed reads were mapped to pepper chromosome

version 1.55 (http://peppergenome.snu.ac.kr/) by Burrows-Wheeler Aligner (BWA).

For grouping and sorting these reads by chromosomal order, Picards and SAMtools

program were used. To call SNPs in whole chromosome, The Genome Analysis

Toolkit (GATK) program was used for SNP calling. All markers data was arranged

using Perl program before running each step and finding polymorphic SNP markers.

Construction of the linkage map and genetic mapping of

ChiVMV resistance in 'CV4'

Identified and arranged polymorphic SNP markers were grouped to linked

markers by LOD (Logarithm of the odds) threshold 3.0 and distance threshold 40

cM using CarthaGene software (version 1.2). The Kosambi mapping function was

used for converting genetic distances between markers. After assigning linkage

groups, MapChart (version 2.2) was executed for constructing genetic map of

SNPs from GBS data. For the comparison of genetic map made by SNPs data and

physical map of pepper (peppergenome.snu.ac.kr), GBS browser (147.46.217.95)

developed in Seoul National University was used. WinQTL carthographer (version

2.5) was used to detect ChiVMV resistance locus and composite interval mapping

(CIM) function was practiced for fine mapping.

RESULTS

Confirmation of ChiVMV resistance in resistance sources

To confirm resistance of four parental lines, ‘CV3’, ‘CV4’, ‘CV8’ and ‘CV9’,

ten plants of each parental line were inoculated with a ChiVMV strain. All four

parental lines, ‘CV3’, ‘CV4’, ‘CV8’ and ‘CV9’, showed no ChiVMV symptoms

after 14 dpi of ChiVMV inoculation. By contrast, vein clearing and curling leaf

symptoms were established on the uninoculated upper leaves at 10 dpi in a

susceptible control, ‘Jeju’. After 21 dpi, ChiVMV symptoms became more severe

with chlorosis and veinal mottling in ‘Jeju’ (Figure 1A). To firmly confirm the

visual evaluations, DAS-ELISA was performed at 26 dpi to detect the ChiVMV

coat protein (CP) accumulation in uninoculated upper leaves of parental lines

(Figure 1B). In quantitative ELISA test, a minimal ChiVMV CP accumulation was

detected in ‘CV3’, ‘CV4’, ‘CV8’ and ‘CV9’, whereas high levels of ChiVMV CP

accumulation were observed at 405 nm absorbance in ‘Jeju’. These results

confirmed that ‘CV3’, ‘CV4’, ‘CV8’ and ‘CV9’ contain resistance genes to

ChiVMV.

A

B

Figure 1. Resistant responses of parental lines, ‘CV3’, ‘CV4’, ‘CV8’, ‘CV9’ and‘Jeju’ to ChiVMV strain. (A) Symptoms of ChiVMV in C. annuum ‘CV3’, ‘CV4’,‘CV8’, ‘CV9’ and ‘Jeju’ at 14 dpi. Resistant peppers ‘CV3’, ‘CV4’, ‘CV8’, ‘CV9’showed no disease symptoms, but susceptible line ‘Jeju’ showed typical symptoms ofChiVMV (leaf mottle and dark green vein-banding pattern) (B) Accumulation ofChiVMV coat protein in non-inoculated upper leaves of parental lines using ELISA testsat 26 dpi. Error bars indicate standard deviation of the mean absorbance value in 5replicates and asterisks mean significant difference between resistant and susceptible

lines [Analysis of variance (ANOVA) P 0.05].

Inheritance study of ChiVMV resistance in different sources

Genetic analysis of resistance in ‘CV3’ and ‘CV8’: Eight F1 plants derived

from ‘CV3 x Jeju’ showed complete resistance to ChiVMV in uninoculated upper

leaves at 27 dpi (Table 1). To confirm ChiVMV resistance quantitatively, ELISA

tests were performed at 27 dpi. According to results, a minimal quantity of CP

accumulation was detected in F1 plants. For further investigation of inheritance

pattern, a total of 146 ‘CV3 x Jeju’ F2 plants were screened by ChiVMV

inoculation. Segregation ratio of resistance and susceptibility in the F2 population

fitted to an expected ratio of 3:1 (susceptible vs. resistant plant number and

probability). These results demonstrate that the ChiVMV resistance in ‘CV3’ is

controlled by a monogenic dominant resistance locus. We named Cvr1 to the

ChiVMV resistance gene in ‘CV3’.

The resistance responses of F1 plants of the ‘CV8 x Jeju’ cross were similar to

those of F1 plants derived from ‘CV3 x Jeju’. These results confirmed, ‘CV8’ also

contains dominantly inherited resistance. Again, a total of 140 F2 plants were

screened to reveal the inheritance pattern of ChiVMV resistance in ‘CV8’. The

segregation ratio of ‘CV8 x Jeju’ F2 population also fitted to 3 : 1 ratio (susceptible

vs. resistant plant number and probability). The inheritance of resistance gene in

‘CV8’ is also controlled by a single dominant gene (Table 1). Consequently, this

ChiVMV resistance gene in ‘CV8’ was designated as Cvr3.

Genetic analysis of resistance in ‘CV9’: To determine the inheritance pattern

of ChiVMV resistance in ‘CV9’, resistance responses were observed in eight ‘CV9

x Jeju’ F1 plants. It was difficult to determine ChiVMV symptoms by naked eyes,

but ChiVMV CP accumulation in all eight F1 was three times higher than those of

the negative control and ‘CV9’ in ELISA at 27 dpi (Table 1). To reveal the

inheritance mode of ChiVMV resistance in ‘CV9’, a total of 85 ‘CV9 x Jeju’ F2

plants were screened with ChiVMV. As a result, the segregation ratio of resistance

and susceptibility in the F2 population was 23 resistance : 62 susceptibility which

corresponds to an 1:3 ratio (Table 1). These segregation data indicated that

ChiVMV resistance in ‘CV9’ is controlled by a monogenic recessive inheritance.

We named the recessive resistance gene in ‘CV9’ cvr4.

Genetic analysis of resistance in ‘CV4’: When eight plants were tested for

segregation analysis, all F1 plants exhibited complete resistance to ChiVMV. In

ELISA analysis, a minimal ChiVMV CP accumulation equivalent to those of the

negative control was detected in all F1 plants (Table 1) and ‘CV4’. ChiVMV

resistance in ‘CV4’ was also inherited by a dominant manner. For detailed genetic

analysis, ChiVMV was challenged to ‘CV4 x Jeju’ F2 population comprising of 450

individuals. Phenotype evaluation showed that 260 plants were resistant whereas

190 were susceptible (Table 2). This segregation ratio is in agreement with a 9:7

ratio of resistance and susceptibility, which indicates that two independently

inherited dominant loci are responsible for ChiVMV resistance in ‘CV9’. These

two independent dominant resistance genes were named Cvr2-1 and Cvr2-2. In the

previous study, two unlinked loci affecting ChiVMV resistance were reported in

the doubled haploid (DH) population obtained by crossing C. annuum ‘Perennial’

and C. annuum ‘Yolo Wonder’ (Caranta and Palloix 1996). However, the exact

position of ChiVMV resistance genes has not been reported yet.

In the summary, the resistances in ‘CV3’ and ‘CV8’ are determined by a

monogenic dominant resistance gene, whereas the resistance in ‘CV9’ is controlled

by a monogenic recessive resistance gene and the resistance ‘CV4’ is controlled by

two dominant resistance genes. These ChiVMV resistance genes were designated

as Cvr1 in ‘CV3’, Cvr2-1 and Cvr2-2 in ‘CV4’, Cvr3 in ‘CV8’, and cvr4 in ‘CV9’.

Tabl

e 1.

Seg

rega

tion

anal

ysis

of C

hiV

MV

resi

stan

ce u

sing

diff

eren

t pop

ulat

ions

Popu

latio

n To

tal

Res

ista

nt (R

) Su

scep

tible

(S)

Exp

ecte

d

ratio

(R

:S)

2 P-

valu

e

N

o.

EL

ISA

N

o.

EL

ISA

‘Jej

u’

10

0 -

10

3.17

781

0 : 1

-

- ‘C

V3’

8

8 0.

0962

1 0

- 1

: 0

- -

‘CV

3 x

Jeju

’ F1

10

10

0.12

7751

0 -

1 : 0

-

- ‘C

V3

x Je

ju’ F

2 1

46

102

0.13

941

44

3.16

41 3

: 1

1.79

0 0.

1809

‘C

V4’

8

8 0.

1571

0 -

1 : 0

-

- ‘C

V4

x Je

ju’ F

1 10

10

0.

1014

1 0

- 1

: 0

- -

‘CV

4 x

Jeju

’ F2

450

260

0.13

821

190

3.77

881

9 : 7

0.

367

0.54

47

‘CV

8’

8 8

0.11

51 0

- 1

: 0

- -

‘CV

8 x

Jeju

’ F1

10

10

0.15

3671

0 -

1 : 0

-

- ‘C

V8

x Je

ju’ F

2 14

0 10

9 0.

1294

1 30

3.

5922

1 3

: 1

0.68

8 0.

4068

‘C

V9’

10

10

0.

1288

1 0

- 1

: 0

- -

‘CV

9 x

Jeju

’ F1

8 0

- 8

3.72

4251

0 : 1

-

- ‘C

V9

x Je

ju’ F

2 85

23

0.

1686

1 62

3.

6831

1 : 3

0.

098

0.75

42

1 Ave

rage

val

ue o

f ELI

SA w

ith re

pres

enta

tive

five

repl

icat

es in

non

-inoc

ulat

ed u

pper

leav

es

Investigation of relationships of ChiVMV resistance genes

Allelism test by diallele analysis: Inheritance studies showed that ChiVMV

resistances in ‘CV3’ and ‘CV8’ are controlled by single dominant resistance genes

whereas the resistance in ‘CV4’ is governed by dominant resistance loci (Table 1).

To reveal allelic relationships of these dominant genes, three F1 populations

obtained from crosses ‘CV3 x CV4’, ‘CV3 x CV8’, and ‘CV4 x CV8’ were

constructed. As expected, all F1 plants showed no noticeable ChiVMV symptoms

in the both inoculated and non-inoculated leaves. Therefore, it could be inferred

that ChiVMV resistance in ‘CV3’ and ‘CV8’ may be controlled by the same or

tightly linked locus. However, further study using segregating populations

developed by self-pollination of F1 plants of ‘CV3 x CV4’ and ‘CV4 x CV8’ are

required to investigate genetic relationships of resistance genes in 'CV4' and those

of others.

Figure 2. Detection of ChiVMV coat protein accumulation in inoculated and non-inoculated upper leaves of ‘CV3 x CV4’, ‘CV3 x CV8’ and ‘CV4 x CV8’ using ELISA tests at 30 dpi. Error bars indicate for standard deviation of the mean absorbance value in five replicates and asterisks mean significant difference between resistant and susceptible lines [analysis of variance (ANOVA) P 0.05].

Allelism test using molecular markers: A single dominant ChiVMV

resistance gene located on pepper chromosome 6 was reported in the previous

study (Lee et al. 2013). To reveal allelic relationships of the dominant ChiVMV

genes, previously developed cleaved amplified polymorphic sequence (CAPS)

markers (CVMV2 and CVMV3) linked to a ChiVMV resistance gene was tested

(Lee et al. 2013). First, these SNP markers were tested to survey polymorphisms

among the resistance lines. The genotyping of CVMV2 showed that all resistant

lines (CV3, CV4, and CV8) had the same genotype that was distinct from that of

the susceptible line 'Jeju' (Figure 3). By contrast, the CVMV3 genotypes of ‘CV3’

and ‘CV8’ were distinct from that of 'Jeju' whereas 'CV4' had the same genotype as

'Jeju' (Figure 3).

In order to test if the markers are linked to ChiMV resistance, two molecular

markers were mapped in F2 populations segregating ChiVMV resistance (Table 2).

The molecular CVMV2 and CVMV3 were reported to be located at 3 cM and 4 cM

away from ChiVMV resistance, respectively (Lee et al. 2013). In this study, we

observed more recombinant events in ‘CV3 x Jeju’ F2 population (Table 2). These

results demonstrated that the Cvr1 gene is the same gene as the previously reported

ChiMV resistance gene or linked to the gene and located on pepper chromosome 6.

In ‘CV8 and Jeju' F2 population, CVMV2 and CVMV3 markers showed similar

genetic distances to the resistance gene as previously reported. Therefore, it could

be assumed that Cvr3 in 'CV8' may be the same resistance gene as that in ‘NW3’

(Lee et al. 2013) and may be the same gene as Cvr1 in ‘CV3’. When we mapped

CVMV2 in ‘CV4 x Jeju’ F2 population more distant genetic distance were observed

than previous study (Table 2). It indicated that other gene may be also involved in

ChiVMV resistance although the gene on chromosome 6 plays a major role (Table

2).

Figure 3. Cleaved amplified polymorphic sequence (CAPS) analysis of ChiVMV dominant resistance locus-linked markers on pepper chromosome 6. ‘CV3’, ‘CV4’ and ‘CV8’ are resistant parental lines and ‘Jeju’ is the susceptible line. In CVMV2, all resistant line showed about 900 and 700 bp of restricted fragments clearly discriminated from susceptible line ‘Jeju’. All ChiVMV resistant lines showed polymorphism in CVMV2. However, CVMV3 showed about 300 and 400 bp of restricted fragments in ‘CV3’ and ‘CV8’. ‘CV4’ and ‘Jeju’ have no polymorphism in CVMV3.

Tabl

e 2.

Gen

otyp

ing

sum

mar

y an

d m

arke

r ana

lysi

s inf

orm

atio

n of

CV

MV

2 an

d C

VM

V3

in F

2 pop

ulat

ions

of ‘

CV

3’, ‘

CV

4’,

and

‘CV

8’

Popu

latio

n C

VM

V2

Tota

l

Rec

ombi

nant

s R

R

Rr

Rr

'CV

3 x

Jeju

' F2

22

46

18

86

10

'CV

4 x

Jeju

' F2

69

117

43

229

36

'CV

8 x

Jeju

' F2

18

56

11

85

3

Popu

latio

n C

VM

V3

Tota

l

Rec

ombi

nant

s R

R

Rr

Rr

'CV

3 x

Jeju

' F2

24

48

16

88

7

'CV

4 x

Jeju

' F2

- -

- -

-

'CV

8 x

Jeju

' F2

16

57

12

85

4

Genetic mapping of the ChiVMV resistance loci in 'CV4'

Genotyping by sequencing of F2 plants: To develop molecular markers and

find ChiVMV resistance loci more precisely at the entire chromosomal level,

genotyping by sequencing (GBS) approach was performed using ‘CV4 x Jeju’ F2

plants consisting of 80 individuals. In GBS analysis, we captured SNPs distributing

in whole genome. Initially, we obtained 64,515 SNPs polymorphic to the reference

genome sequence (Table 3). After trimming missing data, a total of 7,126 high

quality SNPs were obtained (Table 3). A total of 581 SNPs which are polymorphic

between ‘CV4’ and ‘Jeju’ were obtained (Table 3). The SNP data subsequently

filtered to select markers with the expected Mendelian inheritance at Chi-square

significance threshold at P < 0.05. Finally, a total of 525 SNPs were proved as

useful SNP markers for constructing an SNP linkage map (Table 3). After selecting

SNPs in GBS data, non-polymorphic SNPs between parental lines were accounted

almost 90% of original SNP candidates (from 7,126 SNPs to 581 SNPs). These

results indicated that the genomic sequences of ‘CV4’ and ‘Jeju’ are very similar

each other.

Tabl

e 3.

Num

ber o

f SN

Ps id

entif

ied

from

GB

S da

ta p

er c

hrom

osom

e

Chr

omos

ome

Num

ber o

f

Pre-

proc

esse

d SN

Ps

SNP

trim

min

g st

ep 1

1 SN

P tri

mm

ing

step

22

Fina

l

Proc

esse

d SN

Ps3

Num

ber o

f SN

Ps

in li

nkag

e gr

oup4

1 6,

016

804

638

45

27

2 4,

651

568

519

56

54

3 5,

508

860

683

84

83

4 3,

968

474

395

32

32

5 5,

266

618

507

61

56

6 5,

910

711

563

56

56

7 5,

160

643

528

61

60

8 2,

888

408

330

23

14

9 6,

696

803

649

28

23

10

5,53

8 1,

103

961

19

10

11

7,83

1 93

2 79

9 32

26

12

5,08

3 70

9 55

4 84

84

Tota

l 64

,515

8,

634

7,12

6 58

1 52

5

1 Trim

min

g st

ep 1

rem

oved

if S

NPs

wer

e no

n-ca

lled

in p

aren

tal l

ine,

‘CV

4’ o

r ‘Je

ju’.

2 Trim

min

g st

ep 2

rem

oved

if S

NPs

wer

e ca

lled

hete

rozy

gous

in p

aren

tal l

ine,

‘CV

4’ o

r ‘Je

ju’.

3 Trim

min

g st

ep 3

rem

oved

if sa

me

SNPs

wer

e ob

serv

ed b

etw

een

‘CV

4’ a

nd ‘J

eju’

(non

-pol

ymor

phic

SN

Ps).

4 Link

age

SNPs

wer

e gr

oupe

d fo

llow

ing

thre

shol

d, L

OD

scor

e 3

and

map

dis

tanc

e 50

cM

Linkage map construction and comparison with physical map of pepper:

Using identified 525 SNPs, an SNP linkage map of the ‘CV4 x Jeju’ F2 population

was constructed. The map is consisted of 24 linkage groups with the average

distances between SNPs of 8.125 cM. The cumulative Kosambi mapping distance

of the map was 3929.4 cM with diverse linkage group size ranging from 32.6 cM

(LG 20) to 339.2 cM (LG 5) (Table 4). The number of SNPs in each linkage group

was varied from 6 (LG 20and LG 21) to 50 (LG 5). As the expected pepper

genome size is about 3.5 Gb and the average recombination rate in ‘CV4 x Jeju’ F2

population was calculated to be 1.122 cM/Mb. The mapping distances among SNP

markers in this study is much higher than that of other linkage map of pepper based

on EST markers (Park et al., 2014). It might be from SNP gaps in some

chromosomal regions.

The SNP information was aligned to the pepper genome using a GBS browser

to compare the genetic and physical maps, and to observe SNP distribution patterns

in the linkage groups (Figure 4). SNPs in each linkage group were aligned to 12

pepper chromosomes. Most of the linkage groups were aligned well with all

chromosomes except for chromosome 1, 8, 9 and 10. Highly repetitive sequences

located in chromosome 1 and 8 might affect calling only few SNPs in these regions

(Park et al. 2014). In chromosome 9 and 10, the total number of SNP markers were

much smaller as compared to other chromosomes (Table 3). Therefore, it indicated

that that genomic sequences of chromosome 9 and 10 might be very similar

between 'CV4, and 'Jeju'.

Table 4. Distribution of SNP markers among the linkage groups

Linkage group Chromosome

Number of SNP

markers

Length (cM)

Average distance between markers

(cM) LG 1 1 8 88.7 11.1 LG 2 1 17 77.8 4.6 LG 3 2 27 224 8.3 LG 4 2 24 181.1 7.5 LG 5 3 50 339.2 6.8 LG 6 3 36 232.8 6.5 LG 7 4 18 171.8 9.5 LG 8 4 14 163.7 11.7 LG 9 5 19 129.2 6.8 LG 10 5 7 48.1 6.9 LG 11 5 29 189.1 6.5 LG 12 6 23 226.3 9.8 LG 13 6 34 199.8 5.9 LG 14 7 26 249.1 9.6 LG 15 7 35 259.9 7.4 LG 16 8 14 101.7 7.3 LG 17 9 10 114.9 11.5 LG 18 9 13 102.3 7.9 LG 19 10 9 131.4 14.6 LG 20 11 6 32.6 5.4 LG 21 11 6 43.4 7.2 LG 22 11 15 175.5 11.7 LG 23 12 42 216.1 5.1 LG 24 12 43 230.9 5.4

31

Figure 4. Alignment of SNP linkage map with reference genome sequence of using a GBS browser. Left bars is SNP genetic map and blue right bars mean physical map of pepper. Black lines mean aligning between genetic map and physical map of pepper.

Mapping the ChiVMV resistance loci: Using the SNP linkage map and

ChiVMV phenotypes of F2 plants, the positions of ChiVMV resistance genes were

mapped using WinQTL (version 2.5). Two major candidate loci associated with

ChiVMV resistance were identified in the mapping analysis (Figure 5; Table 5).

Resistance loci were mapped to pepper chromosome 6 with LOD peak of over 2.5.

One candidate locus, Cvr2-1, was located between v.1.55.6-199095961 and

v.1.55.6-190901930. The LOD score of this locus was 3.224616 and R2 value was

12.3098. The other locus, Cvr2-2, was positioned between v.1.55.6-184956691 and

v.1.55.6-188128083), with 2.547121 LOD score and 22.4793 R2 value (Table 5).

The expected position of Cvr2-1 and Cvr2-2 were far from ChiVMV resistance

genetic markers (CVMV2 and CVMV3) which is located on short arm of pepper

chromosome 6 (Figure 5). This results conflicted with genetic study (Table 1)

which revealed two independent resistance genes in ‘CV4’.

In the segregation analysis of ChiVMV resistance in ‘CV4’ (Table 1), it was

hypothesized that ‘CV4’ carries two independent dominant resistance loci.

However, only two closely linked loci were detected on chromosome 6 in genetic

mapping study. As indicated above, one of the two resistance loci of could be Cvr1

(or Cvr3) on chromosome 6 (Figure 5).

Figure 5. Genetic mapping ChiVMV resistance in ‘CV4 x Jeju’ F2 population. Comparing with pepper physical map, expected region of ChiVMV resistance is closely linked to each other at the long arm of pepper chromosome 6. The black box means expected region of Cvr2-1 and Cvr2-2.

Table 5. Target candidate resistance regions Information of ChiVMV

1CVMV_R means the trait of ChiVMV resistance.

Trait Linkage group Chromosome Name of closest SNP LOD R2

CVMV_R1 12 6 v.1.55.6-199095961 v.1.55.6-190901930 3.224616 12.3098

CVMV_R1 13 6 v.1.55.6-188128083 v.1.55.6-184956691 2.547121 22.4793

DISCUSSION

To date, only two types of ChiVMV resistance have been reported in pepper.

One type of resistance is mediated by two complementary recessive resistance

genes, pvr12 and pvr6. In this case, plants show resistance when two homozyous

recessive resistance are present (Hwang et al. 2009). The other is a monogenic

dominant resistance located on chromosome 6 for which several closely linked

markers were developed (Lee et al. 2013). Genetic distances of these three markers

to ChiVMV resistance gene were ranged 3 to 7cM. In this study, four resistance

locus were analyzed and named as Cvr1, Cvr2-1, Cvr2-2, Cvr3, and cvr4.

Noticeably, cvr4 is first reported as a single recessive resistance locus in current

findings although its position required be identified.

Single dominant genes Cvr1 and Cvr3 were identified in C. annuum ‘CV3’ and

‘CV8’. To determine the chromosomal positions of the genetic locus of Cvr1 and

Cvr3, and to reveal allelic relationship between the previously identified ChiVMV

gene, F2 populations of CV3 and CV8 were tested with previously reported genetic

markers (CVMV2 and CVMV3). Genetic marker screening results showed that

Cvr1 and Cvr3 are closely linked or same locus in pepper chromosome 6.

Moreover, Cvr2-1 in ‘CV4’ appears to be located at the same position on pepper

chromosome 6. It is possible that one of the dominant resistance loci to ChiVMV

originated from the same ancestor gene on pepper chromosome 6. To test whether

Cvr1 and Cvr3 are same gene or not, construction of F2 population crossed between

‘CV3 and CV8’ is required for allelism test (Hoang et al. 2013). However, this

allelism test would not be enough to explain relationship between Cvr1 and Cvr3.

To reveal the relationship between genes, screening of a large number of F2

individuals is required. In tomato, explaining the same issue of allelic relationship

between Ty-1 and Ty-3 was resolved by screening large F2 population (Verlaan et al.

2013; Verlaan et al. 2011).

To locate Cvr2-1 and Cvr2-2 position in ‘CV4’, GBS technology was utilized in

the F2 population of ‘CV4 x Jeju’. GBS is an efficient tool for high throughput

marker development with thousands of markers using NGS technologies(Elshire et

al. 2011). However, in the present study, only 512 SNP markers were obtained in

‘CV4 x Jeju’ F2 population. The reason of obtaining low numbers of SNPs was that

thousands of SNPs were removed after SNPs trimming step 3 (Removing SNPs

that are identical between ‘CV4’ and ‘Jeju’). The genomic sequence similarities

between ‘CV4’ and ‘Jeju’ might be the reason of obtaining only 512 informative

SNP markers in CV4 x Jeju’ F2 population. Typically, inter-specific cross is used

for the construction of high resolution genetic linkage map for gene interaction

study with thousands of SNPs (Kang et al. 2001). However, this research revealed

the genetic locus harboring ChiVMV resistance was located at the long arm of

pepper chromosome 6. Although GBS results revealed two closely linked genes,

Cvr2-1 and Cvr2-2 in ‘CV4’ (Figure 5), genetic study showed two independent

genes affect the ChiVMV resistance by complementary relationship. It indicated an

error in virus screening experiment or GBS results found only one gene in

chromosome 6 between two complementary genes. To find complementary

relationship between Cvr2-1 and Cvr2-2, further research is required.

For finding cvr4 gene, genetic mapping analysis was needed. However single

recessive ChiVMV resistance gene has not been reported yet, so it makes more

difficult to find cvr4 gene. Because pvr1 is known as a broad spectrum resistance

gene against diverse potyvirus, molecular markers linked to pvr1could applied to

be sure whether cvr4 is one of the allele of pvr1 or an independent locus (Yeam et

al. 2005).

In this study, we performed an inheritance analysis and hypothesized that

ChiVMV resistance sources followed the Mendelian genetic inheritance pattern.

Identification of ChiVMV resistant sources was carried out by exploiting the

traditional and molecular breeding strategies. To detect the genetic markers linked

to ChiVMV resistance, the revolutionary GBS technology was used to find the

SNP markers. The current finding discovered various ChiVMV resistance sources

and it opens new avenues for the crop improvement against Chili veinal mottle

virus.

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: (ChiVMV), ChiVMV ,

(GBS), (SNP)

: 2013-23231