QTL Analysis for Morphological Traits and Capsaicinoids Contents in Capsicum annuum

저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.

A THESIS FOR THE DEGREE OF MASTER OF SCIENCE

QTL Analysis for Morphological Traits and

Capsaicinoids Contents in Capsicum annuum

고추의

형태학적 특성 및 캡사이시노이드 함량을

조절하는

양적 형질 유전자좌 분석

FEBRUARY, 2014

KOEUN HAN

MAJOR IN HORTICULTURAL SCIENCE

DEPARTMENT OF PLANT SCIENCE

QTL Analysis for Morphological Traits

and Capsaicinoids Contents in

Capsicum annuum

KOEUN HAN

DEPARTMENT OF PLANT SCIENCE

THE GRADUATE SCHOOL OF SEOUL NATIONAL

UNIVERSITY

ABSTRACTS

Agriculturally important traits for breeding such as growth habit, yield, and pungency level are controlled by quantitative trait loci (QTLs) in pepper (Capsicum spp.). In this study QTLs were detected which are responsible for morphological traits and capsaicinoids contents. F6-F9 recombinant inbred lines

(RILs) derived from a cross between pungent Perennial (C. annuum) and non-pungent Dempsey (C. annuum) were constructed. A total 28 morphological traits and capsaicinoids contents were evaluated for parents and RILs grown in three

different environments. An ultra-high density linkage map containing 3,300 single nucleotide polymorphism (SNP) markers defined from whole genome sequencing of two parents and 120 RILs was used for QTL analyses. Using evaluated data and the linkage map, QTL analyses were performed for 27 morphological traits and capsaicinoids contents. Detected QTLs were consolidated to find the major QTLs controlling traits in multiple environments. 402 QTLs were detected for plant architecture, leaf, flower, fruit, and seed related traits. Consolidated 21 QTLs were mapped in chromosome 1, 2, 3, 4, 6, 7, 9, 10, and 11 which were found commonly from two or three environments. Among them, 4 QTL regions were associated with multiple traits. Major QTLs explained 50.9 and 61.4% of phenotypic variation for leaf length and fruit weight respectively. QTL analyses for capsaicin, dihydrocapsaicin and total capsaicinoids were performed and 72 QTLs were detected. Seven consolidated QTLs were distributed to chromosomes 1, 6, 9, and 11. QTLs in chromosome 6 controlling capsaicin and total capsaicinoids and in chromosome 11 controlling dihydrocapsaicin and total capsaicinoids were located in the same region. For major QTLs and the QTLs controlling more than two traits, SNP markers were located less than 1 cM. Therefore genes located in these QTL regions could be identified by SNP markers. Furthermore, SNP markers near the QTLs can be used to predict the values of quantitative trait after validation of the markers.

(RIL), single nucleotide polymorphism (SNP), quantitative trait locus (QTL)

CONTENTS

ABSTRACT ... i

CONTENTS ... iv

LIST OF TABLES ... vi

LIST OF FIGURES ... vii

LIST OF ABBREVIATIONS ... viii

INTRODUCTION ... 1

LITERATURE REVIEW QTL analysis of morphological traits in Capsicum ... 5

Capsaicinoids in pepper... 7

Measurement of capsaicinoids ... 9

Biosynthesis of capsaicinoids ... 9

QTL analysis of capsaicinoids ... 10

MATERIALS AND METHODS Plant materials ... 13

Phenotype evaluation ... 13

Measurement of capsaicinoids contents by HPLC analysis ... 15

Genetic mapping and QTL analysis ... 16

RESULTS Evaluation of morphological traits ... 18

Construction of linkage map ... 23

QTL analysis for morphological traits ... 23

Plant architecture ... 25

Leaf length and width ... 25

Flower traits ... 28

Fruit traits ... 28

Seed traits ... 29

Measurement of capsaicinoids contents ... 29

QTL analysis for capsaicinoids contents ... 32

DISCUSSION ... 35

REFERENCES ... 40

ABTRACT IN KOREAN ... 49

LIST OF TABLES

Table 1. Morphological traits evaluated for RILs ... 14

Table 2. Average values of quantitative traits evaluated for QTL analysis ... 19

Table 3.Pearson correlation between morphological traits of RILs ... 22

Table 4. Distribution of RILs with different grades in qualitative traits ... 24

Table 5. QTLs controlling morphological traits detected from more than two environments ... 27

Table 6. Capsaicinoids contents of Perennial, Dempsey, and RILs ... 30

Table 7. Consolidated QTL controlling capsaicinoids contents detected from more than two environments ... 33

LIST OF FIGURES

Fig.1. Fruit shape of parental lines and RILs ... 21

Fig.2. Variations of quantitative traits in RILs ... 22

Fig.3. Consolidated QTLs controlling morphological traits ... 27

Fig.4. Pun1genotypes of RILs for identifying pungency ... 35

LIST OF ABBREVIATIONS

AFLP Amplified fragment length polymorphism

BC Backcross

Bcat Branched-chain amino acid transferase Comt Caffeic acid O-methyltransferase

DH Double haploid

Kas -ketoacyl ACP synthase

NIL Near isogenic line

pAMT Putative aminotransferase

QTL Quantitative trait locus

RAPD Random amplified polymorphic DNA RFLP Restriction fragment length polymorphism

RIL Recombinant inbred line

SNP Single nucleotide polymorphism

INTRODUCTION

Most of yield and fruit quality related traits are regulated by multiple

genes that are prone to be influenced by environmental conditions. Peppers

(Capsicum spp.), one of the first domesticated vegetables (Paran and van der

Knaap 2007), show enormous variations in yield and quality of fruits.

Morphological trait includes traits such as plant architecture, leaf size, fruit

size. Variation of plant height, leaf size, fruit shape, and other morphological

traits for yield-related traits were observed in diverse pepper accessions

(Eshbaugh 1980; Sudré et al. 2010; Thul et al. 2009).

Representative example for quantitative trait is contents of

capsaicinoids. Capsaicinoid is a unique compound produced only by pepper.

Due to the pungency and pharmacological effects of capsaicinoids, pepper is

consumed worldwide as a fresh fruit, sauce, and pharmaceuticals

(Aza-Gonzalez et al. 2011). The presence/absence of pungency is thought to be

controlled by the Pun1 gene (Stellari et al. 2010; Stewart et al. 2005).

However, capsaicinoids contents have been demonstrated to be a

quantitative trait because of pungency levels among pungent peppers can

controlled by multiple loci and environmental conditions (Collins et al. 1995;

Sanatombi and Sharma 2008).

Genetic regions control quantitative traits were found by QTL. QTL

analysis uses genetic map and phenotyping data of quantitative traits. For

QTL study, suitable populations should be constructed from parents with

contrast phenotypes for the target trait.

F2, recombinant inbred lines (RILs),

near isogenic lines (NILs), backcross populations (BCs), and double haploid lines (DHs) are generally used for QTL analysis (Kearsey 1998)

. For construction of a

genetic map,

restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) markers, and microsatellite (SSR) markers have been widely used (Barchi et al. 2007; Ben-Chaim et al. 2001; Kang et al. 2001; Rao et al. 2003). However, for accurate detection and characterization of QTLs, high-density genetic map is required. Pepper GeneChip with 30,815 unigenes was used for genotyping a RIL population (Hill et al. 2013).

QTL analysis of morphological traits in pepper was performed using intraspecific and interspecific population (Ben-Chaim et al. 2001; Chaim et al. 2003; Rao et al. 2003; Zygier et al. 2005). Most of the QTL analyses for morphological traits were concentrated on fruit associated traits. fs3.1 and fs10.1, were detected for fruit shape (ration of fruit length to fruit width) (Ben-Chaim et al. 2003; Ben-Chaim et al. 2001). Conservation of the QTL region between pepper and tomato were found and two QTLs controlled cell elongation (Ben-Chaim et al.

2003; Chaim et al. 2003). Also major QTLs controlling fruit weight were detected in chromosome 2 and 4, and by comparison with tomato genetic map, some of the QTLs were found to be conserved between two crops (Rao et al. 2003; Zygier et al. 2005). QTL analyses for plant height, leaf area, stem length, and other traits were also performed (Ben-Chaim et al. 2001; Rao et al. 2003; Yarnes et al. 2013), but no major QTLs were detected and the QTLs were not characterized.

Compare to morphological traits of pepper, limited number of QTLs controlling capsaicinoids contents were detected. QTL analyses for capsaicinoids contents were performed using interspecific populations (Ben-Chaim et al. 2006; Blum et al. 2003; Yarnes et al. 2013). cap explaining 34-38% of phenotypic variation were detected (Blum et al. 2003) from the population derived from a cross between non-pungent Maor (C. annuum) and pungent BG 2816 (C.

frutescens). This QTL was also detected in another population derived from two

pungent parents (Ben-Chaim et al. 2006). In this population, cap7.2 (same region with cap) and other five QTLs were also detected in chromosome 3, 4, and 7. Twelve QTLs were found in recent study (Yarnes et al. 2013). Using high-density map intervals of each QTL (0.6-14.1) became shorter than previous researches. From these three studies, some QTLs were co-localized with the putative genes related to capsaicinoid biosynthesis pathway (Ben-Chaim et al. 2006). However for characterization of QTLs, fine mapping should be performed.

In this study, QTLs controlling morphological traits and capsaicinoids contents were identified using RILs derived from a cross between Perennial (C.

annuum) and Dempsey (C. annuum). Furthermore, SNP markers linked to the

LITERATURE REVIEW

1. QTL analysis of morphological traits in Capsicum

Pepper (Capsicum spp.) is one of the earliest domesticated vegetables (Paran and van der Knaap 2007) and consists of more than 20 different species (Walsh and Hoot 2001). Various morphological traits including fruit size and color, flower color, stem pubescence and seed color were observed among species. Morphological traits often show broad range of distribution and affected by environments. Most of agronomically important morphological traits of pepper are known as quantitative traits. Quantitative trait locus (QTL) controls quantitative traits like fruit yield, plant height, fruit shape, taste, and disease resistance.

QTL analysis was performed to find the QTL controlling morphological traits ultimately to assist the pepper breeding. For QTL analysis, segregated population like F2, recombinant inbred lines (RILs), near isogenic lines (NILs), backcross

populations (BCs), and double haploid lines (DHs) are constructed (Kearsey 1998). Also genetic map and phenotyping data are required. QTL analysis of morphological traits in pepper was performed using intraspecific population crossed between C. annuum and C. annuum and interspecific population crossed between C. annuum and C. chinense, C. annuum and C. frutescens, and C. chinense and C. frutescens (Ben-Chaim and Paran 2000; Chaim et al. 2003; Rao et al. 2003; Zygier et al. 2005).

Most of QTL analysis of morphological traits were concentrated on fruit associated traits. F3 population derived from a cross between Maor (C. annuum)

and Perennial (C. annuum) was grown and evaluated of 14 traits including fruit diameter, length, weight, and shape (Ben-Chaim et al. 2001). A total 177 restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) markers were used to construct genetic map. By QTL analysis, 55 QTLs were identified and a major QTL fs3.1 for fruit shape (ratio of fruit length to fruit diameter) was detected. Conservation of fs3.1 was elucidated in other two interspecific populations and tomato introgression lines (Ben-Chaim et al. 2003). By comparison of backcross inbred lines (BILs), fruit elongation function of fs3.1 was demonstrated. Another major QTL for fruit shape was fs10.1 and it was shown that this QTL is highly linked to A locus which controls anthocyanin accumulation of pepper (Chaim et al. 2003). Like fs3.1, fs10.1 also controls fruit elongation and the function was characterized in an interspecific population and a mutant population of Maor (C.

annuum) (Borovsky and Paran 2011). Major QTLs controlling fruit weight were

detected in chromosome 2 and 4, and by comparison with tomato genetic map, some of the QTLs were found to be conserved between two crops (Rao et al. 2003; Zygier et al. 2005). Genetic maps used in previous researches had relatively low density and average distance between markers often exceeded 10 cM. However high-density map was constructed using 489 markers (Barchi et al. 2007), and this map was used for QTL analysis of morphological traits (Alimi et al. 2012; Barchi

et al. 2009). And another high-density map was used for QTL analysis by Yarnes et al. (Yarnes et al. 2013). In this research, QTLs for plant, leaf, flower and fruit related traits and capsaicinoids contents were detected.

Importance of genotyping for QTL analysis is well known (Kearsey 1998; Kearsey and Farquhar 1998). Also many researches evaluated traits more than two locations or consecutive years to understand environmental effects. For precise analysis of QTL, accurate measurement of quantitative traits is also needed. To reduce the error of evaluation by man, and improve the efficiency of managing data, software tools have been developed (Fiorani and Schurr 2013). Tomato Analyzer was first developed for tomato to measure fruit size, size, and color (Brewer et al. 2006; Darrigues et al. 2008), and it was also used for pepper fruit related traits (Yarnes et al. 2013). Another system was developed to measure plant and leaf related traits in pepper (Van der Heijden et al. 2012). This system records plant figure using multiple cameras follow the row of plants. By reconstruction the features in 3D, traits were measured automatically. However this system could find less number of QTLs compare to manual measurement.

2. Capsaicinoids in pepper

Capsaicinoid is a unique compound of hot peppers and it gives burning sensation called pungency. Aromatic moieties and acyl groups determine the diverse structure of capsaicinoids (Mazourek et al. 2009). Two predominant

capsaicinoids are capsaicin and dihydrocapsaicin which have one different bond in acyl moiety (Kozukue et al. 2005). However not all peppers contain capsaicinoids (Stellari et al. 2010; Stewart et al. 2005; Stewart et al. 2007), and capsaicinoids contents vary among different accessions (Collins et al. 1995; Sanatombi and Sharma 2008).

Capsaicinoids are presented to be synthesized to protect fruits from disease like Fusarium (Tewksbury et al. 2008) and to disperse the seeds by the birds which cannot feel the pungency and do not harm the seeds like other mammals (Tewksbury and Nabhan 2001). But humans use pungent pepper as a vegetable, sauces, and other foods (Aza-Gonzalez et al. 2011). Also capsaicinoids are used for pharmaceuticals, medicines, and cosmetics due to their pharmacological effects (Aza-Gonzalez et al. 2011; Luo et al. 2011). Transient receptor potential vanilloid subfamily member 1 (TRPV1) is known as a receptor of capsaicinoids (Caterina et al. 1997). This receptor is a non-selective cation channel and associated with other pains (Caterina et al. 1999). Relationship between sensation of pungency and other pains is thought to reduce the inflammatory heat and pain from rheumatoid arthritis (Fraenkel et al. 2004). Anticancer activities for prostate cancer, breast cancer and detoxification of carcinogens were also reported (Mori et al. 2006; Singh et al. 2001; Thoennissen et al. 2010). Anticancer effects are closely related to the prevention of cell proliferation and migration (Luo et al. 2011). Thermogenesis of capsaicinoids thought to be related anti-obesity effects (Joo et al. 2010; Luo et al. 2011). Capsaicinoids also affect cardiovascular function (Peng and Li 2010). Even

though many researches showed the benefits of capsaicinoids, there were also lots of studies about side effects of long-term capsaicinoid consumption (Bode and Dong 2011).

2.1 Measurement of capsaicinoids

As pungency is one of the most important traits in pepper breeding, measurement of capsaicinoids contents was studied for a long time. Scoville unit is widely used for rough measurement, and for accurate measruement thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometer (GC-MS) methods is being used (Collins et al. 1995; Iwai et al. 1979; Spanyar and Blazovich 1969). However the methods using chromatography usually take long time for capsaicinoid extraction from the fruit samples and detection in the machine. Therefore a simple capsaicinoid measurement method was developed based on Gibb’s reagent (Jeong et al. 2012).

2.2 Biosynthesis of capsaicinoids

Capsaicinoids are synthesized in the placenta of pepper and accumulate in the blisters located on the placenta (Stewart et al. 2007). Accumulation of capsaicinoids increases until 20-30 days post anthesis (dpa) and starts to decrease or stay still after fruits mature (Stewart et al. 2005; Sukrasno and Yeoman 1993). Two pathways are involved in capsaicinoid synthesis. One is phenylpropanoid

pathway starting from phenylalanine and resulting in vanillylamine at last (Curry et al. 1999). The other pathway synthesizes fatty acid from leucine or valine (Curry et al. 1999). Capsaicin is synthesized when vanillylamine and 8-methyl-6-nonenoyl-CoA are condensed by capsaicin synthase (CS). And the Pun1 gene is thought to encode the CS (Stewart et al. 2005). As the Pun1 gene is associated with the last step of capsaicinoid synthesis, mutation in Pun1 causes non-pungency of pepper (Stellari et al. 2010; Stewart et al. 2005; Stewart et al. 2007). Using the normal

Pun1 and mutated pun1 sequence, molecular markers to identify pungent and

non-pungent pepper were developed (Garces-Claver et al. 2007; Lang et al. 2006; Lee et al. 2005). Other candidate genes associated with capsaicinoid synthesis pathway including -ketoacyl ACP synthase (Kas), caffeic acid O-methyltransferase (Comt), and aminotransferase (pAmt) were cloned and mapped to pepper genetic map (Curry et al. 1999; Mazourek et al. 2009; Stewart et al. 2005).

2.3 QTL analysis of capsaicinoids

Capsaicinoids contents are controlled by qualitative and quantitative genes. The Pun1 gene is responsible for presence/absence of pungency in pepper (Stewart et al. 2005). Capsaicinoids contents in pepper vary due to multiple genetic factors and environmental conditions. QTL analyses of capsaicinoids contents were performed in several interspecific populations (Ben-Chaim et al. 2006; Blum et al. 2003; Yarnes et al. 2013). In 2003, bulked segregant analysis (BSA) was used for

QTL analysis to identify the QTLs (Blum et al. 2003). Low-pungent and high-pungent plants were selected from the F2 population derived from a cross between

non-pungent Maor (C. annuum) and pungent BG 2816 (C. frutescens) and screened with 400 RFLP markers. Capsaicin and dihydrocapsaicin were extracted from dried fruits and measured their contents in two seasons. Three markers linked to genes controlling capsaicinoids contents, and all three markers were located in chromosome 7. This QTL named cap explained 34-38% of phenotypic variation.

Another interspecific population was analyzed. Mildly pungent NuMex RNaky (C. annuum) and highly pungent BG2814-6 (C. frutescens) were crossed and F3 populations were grown in three different environments (Ben-Chaim et al.

2006). A total 728 SSR, AFLP and RFLP markers were used to construct a genetic map. Contents of capsaicin, dihydrocapsaicin and nordihydrocapsaicin were measured from whole fruits and fruit weights were also measured together in each environment. Fruit weight was negatively correlated to capsaicinoids contents. QTL analyses were performed using each trait (capsaicin, dihydrocapsaicin, nordihydrocapsaicin, and total capsaicinoids), and five, four, one, and five QTLs were detected respectively. Those QTLs were distributed on chromosome 3, 4, and 7. cap7.2 had the highest effect on capsaicin content, and it was orthologous to cap that was detected in the previous research (Blum et al. 2003).

The most recent study on capsaicinoid controlling QTL was also performed using an interspecific population. RILs derived from a cross between BG2814-6 (C.

conditions (Yarnes et al. 2013). Of 30,815 unigenes in Pepper GeneChip, 16,188 unigenes were mapped and used to construct genetic map. Capsaicin, dihydrocapsaicin and nordihydrocapsaicin contents were measured. QTLs were detected in chromosome 3, 4, 5, 6, 7, 10, and 11. QTL on chromosome 4 was also found in F2 population derived from the same parents (Ben-Chaim et al. 2006).

Several QTLs were detected in three different researches. However to use the QTL region for pepper breeding, finding interaction between QTLs, characterization of QTLs, and development of marker highly linked to the QTL still need to be performed.

MATERIALS AND METHODS

Plant materials

F6 – F10 Recombinant inbred lines (RILs) crossed between pungent ‘Perennial’

(Capsicum annuum) and non-pungent ‘Dempsey’ (C. annuum) were constructed. RILs were grown for two consecutive years (2011 and 2012) in order to reduce the environmental effects on detecting quantitative trait locus (QTL). Plants were grown under greenhouse condition in two places (location 2011 and 2012a; Anseong, Korea and location 2012b; Suwon, Korea). In 2011 and 2012a, plants were grown in the ground while plants were planted to the pot in 2012b. Seeds were disinfected using 2% sodium chlorate and 10% trisodium phosphate. Five plants per line were planted and fruits were harvested separately.

Phenotype evaluation

A total of 28 horticultural traits were evaluated for Perennial, Dempsey and RILs based on the descriptions of RDA-genebank, Korea. Two to three plants per line were evaluated for each trait. Qualitative traits were described by grade according to the descriptions (Table 1). Quantitative traits like length and weight were measured in cm and g respectively and average values were calculated. Traits related to plant architecture, leaf and flower were evaluated 9 weeks after t r a n s p l a n t i n 2 0 11 , a n d 1 0 w e e k s a f t e r i n 2 0 1 2 a a n d 2 0 1 2 b .

Table 1. Morphological traits evaluated for RILs

Phenotype Description

Plant Plant habit 1: Dwarf, 2: Intermediate, 3: Erect

Plant height (cm) From soil to head of the plant

Plant width (cm) Wide part of the plant

Main stem length (cm) From soil to the first branch

Stem thickness (cm) Thickness of basal stem

Stem color 1: Green, 2: Green with purple, 3: Purple

Stem pubescence 1: Absent, 2: Moderate, 3: Heavy

Lateral branch number Basal lateral-branch number before the first branch Internode length (cm) Length of internode between the 3rdand 4th node

Leaf Leaf length (cm) Length of completely grown leaf

Leaf width (cm) Width of completely grown leaf

Flower Stamen number 1: Four, 2: Five, 3: Six

Flower size 1: Small, 2: Intermediate, 3: Big

Flowering time Flowering time (days after transplant)

Fruit Fruit position 1: Erect, 2:Pendant, 3: Intermediate

Fruit length (cm) Average length of fruit

Fruit diameter (cm) Average width of fruit

Fruit shape Ratio between fruit length and fruit diameter

Calyx shape 1: Cup-shaped, 2: Intermediate, 3: Saucer-shaped

Immature fruit color 1: Light-green, 2: Green, 3: Dark-green, 4: Yellow Mature fruit color 1: Orange, 2: Red, 3: Dark-red, 4: Yellow

Fruit weight (g) Average weight of fresh fruit

Fruit surface smoothness 1: Very smooth, 2: Smooth, 3: Medium, 4: Rough, 5: Very rough

Locule number (mode) Most frequent number of locule Stalk length (cm) Stalk length from fruit calyx to node

Stalk width (cm) Stalk width above the calyx

Seed Seed number Average number of seeds per fruit

Fruit related traits were evaluated after harvest. For precise measurement of the fruit length and width, the image of hemisected fruits was scanned (Epson V30) and analyzed Tomato Analyzer 3.0 (Brewer et al. 2006). Phenotyping data for 27 traits were used for QTL analysis. Mature fruit color was all red, so was not used for QTL analysis. Pearson correlation coefficients were used to analyze the relation between traits evaluated in 2012a.

Genomic DNA extraction and the Pun1 genotype analysis

Fresh and young leaves were sampled and DNA was extracted with CTAB method (Doyle and Doyle 1987). Concentration of the DNA was measured with NanoDrop machine (NanoDrop Technologies, Inc., Wilmington, DE, USA) and diluted to 20 ng/µL by adding 0.1 M TE buffer (pH 7.0).

For genotyping of the Pun1 gene, PCR was performed with two pairs of primers (Han et al. 2012). PCR products were electrophoresed and genotype was determined by the pattern of the bands. Sample with two bands (459 bp and 348 bp) was determined as dominant homozygous Pun1/Pun1. And one band (348 bp) was genotyped as recessive homozygous pun1/pun1.

Measurement of capsaicinoids contents by HPLC analysis

For HPLC analysis, three to five pepper fruits from individual plants of the RILs were harvested when the fruits were fully matured. The placenta tissue of

each fruit was separated from the fruits and freeze dried. Freeze-dried powder was extracted with 7.5 ml of ethyl acetate and acetone mixture (6:4) by shaking at 37 oC for 24 h, and 3 ml of supernatant was evaporated in an Automatic Environmental SpeedVac System AES1010 (Operon, Gimpo, Korea). The pellet was dissolved in 1 ml 99.9% methanol (Honeywell Burdick & Jackson, Muskegon, MI, USA), and a 10 µl aliquot was injected for HPLC analysis. HPLC was performed on a SP LC (Shiseido, Tokyo, Japan) with two connected CAPCELL PAK C18 UG120 S-5 columns (240 mm × 4.6 mm, 5 µm) (Shiseido, Tokyo, Japan). Both capsaicinoid and capsinoid were monitored for by a photodiode array detector operating at 280 nm. Capsaicin and dihydrocapsaicin were purchased from Sigma-Aldrich (M2028 and M1022, respectively). The composition of mobile phase was 50:50 (0.1% formic acid in water : 70% methanol in water) for the first 1 min, gradually changed to 10:90 until 6 min, remained for the next 5 min, and gradually changed to 50:50 for the last 10 min. The flow rate was 1 ml/min. Average content of capsaicin and dihydrocapsaicin was used for QTL analysis.

Genetic mapping and QTL analysis

Perennial, Dempsey and 120 RILs were sequenced by high-throughput sequencing method. Parents were sequenced to 6-fold coverage and RILs were sequenced to 1-fold coverage. SNP calling and genotyping was performed by Prof. Choi (Horticultural Crop Genomics Lab., Seoul National University). A total of 3,300 polymorphic contigs were used to construct high-density genetic map by

Carthagene program (De Givry et al. 2005). Kosambi mapping function was used to calculate the distance (cM) of each contigs. Twelve linkage groups were drawn with MapChart 2.2 program (Voorrips 2002).

The ultra-high density genetic map was used for QTL analysis. Composite interval mapping was performed by Windows QTL Cartographer 2.5 (Wang et al. 2012). Mean value of each traits in three locations were analyzed separately to detect QTLs. Pungent 56 lines with Pun1/Pun1 genotype were used for QTL analysis to detect QTLs controlling capsaicinoids contents. LOD threshold was determined by applying 1,000 permutation tests with 5% probability for each chromosome and each trait. The largest LOD among 12 chromosomes was selected for threshold LOD in each trait. The proportion of phenotypic variation explained by each QTL was estimated using R2 (%) value. The closest contig to the QTL with the highest LOD was selected to represent each QTL. QTLs with overlapped 99% confidence interval were consolidated to one QTL. Consolidation was performed for each morphological traits, capsaicin, dihydrocapsaicin and total capsaicinoids contents, respectively.

RESULTS

Evaluation of morphological traits

Mean values of quantitative traits were calculated for each growing environment (Table 2). Plant was higher and narrower, and leaf was smaller in Perennial compared to Dempsey. Perennial had small, thin, and pungent fruits whereas Dempsey had bell-type, and non-pungent fruits (Fig.1). Quantitative traits contrasting between parents showed transgressive segregation in RILs (Fig.2). ANOVA results estimated that the mean value of each traits were different in each locations (data not shown). Comparison of 2011 and 2012a demonstrated that plants grown in the same place in different year showed different phenotypic value. Although plant height and width were measured in the same place and at the same growth stage, both values were much higher in 2012a than those in 2011. Also most of the traits values were larger in 2012a than those in 2012b maybe due to different growing conditions.

Correlation coefficients between quantitative traits are listed in Table 3. Positive and negative values mean positive and negative correlation, respectively. High correlations between different environments were observed. Main stem length and lateral branch number, plant height and internode length, leaf length and width, and stalk width and fruit weight had positive correlations. Fruit weight and diameter evaluated in 2011 showed the highest correlation with r = 0.84. As fruit

Table 2. Average values of quantitative traits evaluated for QTL analysis

Phenotype Perennial Dempsey RIL (F5 - F10)

2011 2012a 2012b 2011 2012a 2012b 2011 2012a 2012b

Plant Plant height (cm) 132.0 207.5 157.0 53.5 128.0 88.7 139.0 ± 2.12* 163.7 ± 2.08 127.8 ± 3.13

Plant width (cm) 46.0 64.0 68.3 30.0 85.5 63.3 82.5 ± 1.07 88.8 ± 1.45 70.8 ± 0.77

Main stem length (cm) -** 40.5 33.3 - 20.0 20.7 - 24.6 ± 0.57 23.9 ± 0.55

Stem thickness (cm) - 2.2 1.4 - 2.6 1.5 - 1.9 ± 0.03 1.5 ± 0.01

Lateral branch number - 21.5 20.0 - 12.0 6.7 - 12.8 ± 0.2 13.0 ± 0.24

Internode length (cm) - 9.3 16.3 - 9.8 3.8 - 9.5 ± 0.19 6.4 ± 0.19

Leaf Leaf length (cm) 12.3 11.5 7.6 12.8 18.0 13.6 8.0 ± 0.16 12.0 ± 0.16 11.6 ± 0.33

Leaf width (cm) 4.7 6.8 4.9 8.4 9.5 7.9 4.4 ± 0.12 7.0 ± 0.1 6.4 ± 0.08

Flower Flowering time - - 33.0 - - 39.0 - - 39.1 ± 0.34

Fruit Fruit length (cm) 1.8 - 3.1 5.2 - 7.9 6.7 ± 0.17 6.4 ± 0.17 5.7 ± 0.15

Fruit diameter (cm) 1.8 - 0.7 5.3 - 8.0 2.4 ± 0.06 2.2 ± 0.05 1.9 ± 0.06

Fruit shape 2.3 - 4.2 0.7 - 1.0 3.1 ± 0.11 3.2 ± 0.13 3.4 ± 0.14

Fruit fresh weight (g) 1.4 0.7 0.9 - 90.0 115.5 10.9 ± 0.62 11.9 ± 1.05 7.5 ± 0.4

Locule number (mode) - 2.0 - - 3.0 - - 2.9 ± 0.04 -

Stalk length (cm) - 3.2 - - 4.5 - - 4.0 ± 0.06 -

Stalk width (cm) - 0.3 - - 0.6 - - 0.4 ± 0.01 -

Seed Seed number - - 137.0 - - 105.0 - - 55.5 ± 1.81

weight of 1,000 Seeds - - 3.3 - - 7.2 - - 5.0 ± 0.05

* Standard error

Fig.1. Fruit shape of parental lines and RILs. Perennial and Dempsey are

conical and paprika-type fruit shape, respectively. RILs have various fruit shapes from small and round to long and thin.

Fig.2. Variations of quantitative traits in RILs. Quantitative morphological traits

showed broad distribution. Mean and variation was diverse among the different locations due to environmental effects.

Table 3.Pearson correlation between morphological traits of RILs

shape is the ration between fruit length and diameter, it correlated positively with fruit length and negatively with diameter.

Ten traits were evaluated as grade (Table 4). Most of the traits showed the same grade in parents in each environment. All traits except for fruit color were evaluated in RILs when parents showed different phenotypes of that trait. Fruit color did not show difference between parents, however, RILs grown in 2012b showed segregation of immature fruit color.

Construction of linkage map

An ultra-high density map consist of 3,300 SNP markers was constructed. 66-440 markers were distributed to twelve linkage groups. Chromosome 7 and chromosome 4 had the smallest and largest number of markers, respectively. Total genetic distance was 2,423.1 cM and average interval of markers was 0.74 cM.

QTL analysis for morphological traits

Seven quantitative traits including plant height and width, leaf length and width, fruit length and diameter, and fruit weight, and 4 qualitative traits including plant habit, calyx shape, and immature and mature fruit color were measured in three different environments. Another 11 quantitative traits were evaluated in one or two environments. Twenty seven traits were analyzed to detect QTLs (Table 1). A total 402 QTLs were detected (Appendix 1) and some QTLs for different traits

Table 4. Distribution of RILs with different grades in qualitative traits

Trait Year Parameter* Number of RILs

Perennial Dempsey 1 2 3 4 5 Plant habit 2011 3 2 2 3 135 0 0 2012a 3 2 0 1 163 0 0 2012b 3 2 3 70 87 - - Stem color 2011 2 1 108 32 0 - - 2012b 2 1 87 35 38 - - Stem pubescence 2012b 2 1 121 39 0 - - Stamen number 2011 2 3 0 94 46 - - 2012b 2 3 0 106 54 - - Flower size 2011 2 3 2 124 14 - - 2012b 1 3 40 120 0 - - Fruit position 2011 1 2 51 81 8 - - 2012a 1 2 63 77 20 - - Calyx shape 2011 1 3 11 74 56 - - 2012a 1 3 14 80 60 - - 2012b 1 3 35 68 57 - -

Immature fruit color 2011 2 2 0 140 0 0 -

2012a 2 2 0 156 0 0 -

2012b 2 2 87 70 3 0 -

Mature fruit color 2011 2 2 0 140 0 0 -

2012a 2 2 0 156 0 0 -

2012b 2 2 0 160 0 0 -

Fruit surface

smoothness 2011 3 2 0 127 0 15 0

2012a 4 2 40 88 27 4 0

were detected in the same chromosome region. Those QTLs were consolidated when 99% confidence interval overlapped for each trait. Consolidated 21 QTLs were named by the abbreviation of trait name and chromosome number (Fig.3; Table 5). Those consolidated QTLs were detected more than two environments. FW-1 and FD-1, FW-4 and FD-4,

CS-4 and FS-CS-4, and FW-6 and FD-6 were located in same region (Fig.3).

Plant architecture

Plant architecture related traits including plant height and width, main stem length, stem thickness, lateral branch number, and internode length were evaluated. Through QTL analysis 151 QTLs for plant architecture related traits were detected (Appendix 1). Three QTLs associated with plant height were found in common from two environments.

PH-7 and PH-11 were detected in 2011 and 2012a and PH-9 was detected in 2011 and

2012b. Especially PH-9 could explain 12.1% to 32.7% of phenotypic variation. MSL-2 and MSL-10 controlling main stem length were observed in 2012a and 2012b.

Leaf length and width

Leaf length and width were measured in three environments. Correlation coefficients between two traits were 0.67, 0.74 and 0.33 in 2011, 2012a and 2012b indicating significant positive correlation. There were 27 and 25 QTLs for leaf length and leaf width, respectively (Appendix 1). LW-6 and LW-9 were commonly detected for leaf

Fig.3. Consolidated QTLs controlling morphological traits. QTLs controlling same

morphological traits with overlapped 99% confidence interval (CI) were consolidated to one region. Consolidated QTLs detected from more than two environments are shown in the right side of each chromosome. Abbreviation of each QTL is on Table 5.

Table 5. QTLs controlling morphological traits detected from more than two environments

Trait Year QTL* Chr. Location (cM) LOD R2 (%)

Plant height 2011, 2012a PH-7 7 56.6-64.0 2.6-5.2 8.9-21.3

2011, 2012b PH-9 9 94.4-97.2 3.4-8.1 12.1-32.7

2011, 2012a PH-11 11 111.5-120.8 2.6-3.3 9.0-11.4

Main stem length 2012a, 2012b MSL-2 2 21.6-34.0 2.8-3.1 10.5-10.6

2012a, 2012b MSL-10 10 5.4-13.0 3.5-5.7 12.8-16.5

Leaf width 2011, 2012b LW-6 6 105.7-108.9 2.8-2.9 8.7-23.1

2012a, 2012b LW-9 9 150.7-168.5 2.5-2.8 9.8-10.4

Stamen number 2011, 2012b STN-4 4 114.3-121.7 2.6-4.7 9.6-15.2

Fruit length 2011, 2012b FL-9 9 125.5-134.9 3.0-3.9 10.6-13.8

Fruit diameter 2012a, 2012b FD-1 1 80.6-83.3 3.9-4.3 14.1-15.5

2012a, 2012b FD-4 4 203.1-208.7 3.2-3.8 12.5-13.0

2011, 2012a, 2012b FD-6 6 161.5-179.1 3.3-4.0 11.0-13.9

Fruit shape 2011, 2012b FS-3 3 104.4-108.5 4.8-7.3 15.5-22.4

2011, 2012b FS-9 9 111.5-116.6 3.5-5.0 11.8-15.9

2011, 2012b FS-4 4 83.7-91.6 3.4-4.7 12.0-15.8

Calyx shape 2012a, 2012b CS-2 2 50.2-62.1 2.8-4.8 11.1-17.3

2011, 2012b CS-4 4 83.7-87.4 2.6-4.1 8.6-13.7

2011, 2012b CS-10 10 74.3-77.7 2.9-3.2 10.0-10.9

Fruit weight 2012a, 2012b FW-1 1 79.0-83.3 2.8-3.2 10.3-24.9

2011, 2012b FW-4 4 196.0-204.3 2.6-2.7 8.4-10.3

2011, 2012b FW-6 6 167.0-178.4 5.0-6.3 16.2-20.3

* QTLs were consolidated when 99% confidence intervals were overlapped in each trait respectively. QTL name represent abbreviation of trait and the chromosome.

width (Table 5) in 2011 and 2012b, and 2012a and 2012b. However majority of QTLs were not detected at the same region in different environments. This result demonstrates that other QTLs are highly influenced by environmental conditions.

Flower traits

Stamen number, flower size and flowering time were evaluated for QTL analysis. Eight, seven, and three QTLs were detected for each trait (Appendix 1). Only one QTL controlling stamen number, STN-4, was detected in 2011 and 2012b (Table 5). 9.6% to 15.2% of phenotypic variation was explained by STN-4. As flowering time was analyzed only one environment (2012b), evaluation of flowering time and QTL analysis should be performed again to validate the effect of these QTLs.

Fruit traits

A total of 169 QTLs associated with fruit were detected (Appendix 1). There were 13 QTLs for fruit related traits detected at the same region in different environments (Table 5). Fruit diameter and fruit weight had common QTL region in chromosome 1, 4, and 6. Detection of same QTL in different environments ensures the effect of QTL and indicates the relation between traits. Fruit diameter and fruit weight had positive correlation (correlation coefficient 0.84, 0.53, and

0.65 in 2011, 2012a, and 2012b; Table 3). Fruit shape and calyx shape had common QTL region on chromosome 4. Those QTLs were detected in 2011 and 2012b, which had negative correlation coefficient -0.59 and -0.57 (Table 3). Limited numbers of QTLs were detected for locule number, stalk length and stalk width measured in one environment (2012a).

Seed traits

Twelve QTLs were detected for seed number and seed weight (Appendix 1). QTLs were located in five chromosomes associated with seed number, and two chromosomes associated with seed weight.

Measurement of capsaicinoids contents

Capsaicin and dihydrocapsaicin content were measured in Perennial, Dempsey and RILs by HPLC analysis. Capsaicinoids contents in Perennial was 38.0 mg/gDW (mg per g dry weight of placenta) in 2011, 31.5 mg/gDW in 2012a, and 81.3 mg/gDW in 2012b (Table 6). Dempsey did not contain capsaicinoid. To identify the pungency of RILs, Pun1 genotyping was performed (Fig.4) using dominant markers. Perennial and pungent lines had 348 bp and 459 bp bands while Dempsey and non-pungent lines had 348 bp. Out of 170, 86 lines were pungent in 2011 and of 162, 83 lines were pungent in 2012a and 2012b. Average capsaicinoids contents of pungent lines and standard error were calculated (Table 6).

Table 6. Capsaicinoids contents of Perennial, Dempsey, and RILs

Trait Perennial Dempsey RIL (F5 - F10)*

2011 2012a 2012b 2011 2012a 2012b 2011 2012a 2012b

Capsaicin 25.0 ± 0** 17.0 ± 1.37 42.8 ± 8.73 ND ND ND 10.5 ± 1.21 7.8 ± 1.14 14.8 ± 1.75

Dihydrocapsaicin 13.0 ± 0 14.5 ± 1.44 38.5 ± 5.46 ND ND ND 7.6 ± 0.95 6.5 ± 0.88 12.4 ± 1.39

Total 38.0 ± 0 31.5 ± 2.81 81.3 ± 14.19 ND ND ND 18.1 ± 2.12 14.2 ± 1.99 27.0 ± 3.07

All content was measure as mg/gDW (mg of compound per g dry weight of placenta) * Average content of pungent lines

** Standard error. Zero standard error value means only one sample was evaluated

Fig.4. Pun1genotypes of RILs for identifying pungency. Two sets of markers were used for Pun1 genotyping. Pungent RIL

lines including perennial (Pun1/Pun1) showed two bands with a 459 bp and a 348 bp while non-pungent lines with Dempsey (pun1/pun1) showed only a 348 bp band. Per Perennial, Dem Dempsey

Capsaicinoids contents of Perennial and RILs were higher in 2012b than other two environments. Transgressive segregation was shown in RILs for capsaicinoids contents, but the content was skewed to low content (data not shown).

QTL analysis for capsaicinoids contents

Capsaicinoids contents were measured in RILs grown in three environments. QTL analysis was performed for each environment and 22, 28, and 22 QTLs controlling capsaicin, dihydrocapsaicin and total capsaicinoid were detected (Appendix 1). QTLs for capsaicinoids content were consolidated and a total of 7 QTLs were mapped (Table 7; Fig.5). For capsaicin, CAP-1, CAP-9, and CAP-11 were detected and CAP-1 had slightly higher R2 value than other QTLs. DICAP-1 and DICAP-6 were detected for dihydrocapsaicin and TOTAL-6 and TOTAL-11 for total capsaicinoid. As total capsaicinoid is the sum of capsaicin and dihydrocapsaicin content, QTLs controlling total capsaicinoid were co-located with the QTLs controlling capsaicin and dihydrocapsaicin. Although there was no QTL controlling capsaicin, dihydrocapsaicin and total capsaicinoid together, maximum 22.2 and 20.7 phenotypic variation could be explained by QTLs located in chromosome 6 and 11. Therefore CAP-11 and DICAP-6 were thought to be major QTLs.

Table 7. Consolidated QTL controlling capsaicinoids contents detected from more than two environments

Trait Year QTL* Chr. Location (cM) LOD R2 (%)

Capsaicin 2011, 2012a CAP-1 1 184.1-191.4 3.1-3.3 16.9-23.4

2011, 2012a CAP-9 9 96.4-115.1 2.5-3.1 15.6-21.7

2011, 2012b CAP-11 11 27.6-35.5 3.3-3.4 20.3-20.7

Dihydrocapsaicin 2012a, 2012b DICAP-1 1 136.9-148.6 2.7-3.1 15.7-16.8

2011, 2012a, 2012b DICAP-6 6 145.2-160.7 2.9-4.8 14.8-22.1

Total capsaicinoid 2011, 2012a TOTAL-6 6 145.6-160.9 2.6-5 13.4-22.2

2011, 2012b TOTAL-11 11 26.1-35.6 2.6-3.1 16.5-19.2

* QTLs were consolidated when 99% confidence intervals were overlapped in each trait respectively. QTL name represent abbreviation of trait and the chromosome.

Fig.5. Location of QTL controlling capsacinoids contents in genetic map. QTL

locations for capsaicinoids are shown in twelve chromosomes. Horizontal lines in chromosome refer SNP marker used for construction of genetic map and QTL analysis. Vertical lines indicate 99% confidence interval of each QTL. QTLs were named using the abbreviation of capsaicin, dihydrocapsaicin, and total capsaicinoid (CAP, DICAP and TOTAL), and chromosome number.

DISCUSSION

This study is the first in QTL analysis using ultra-high density genetic map in pepper (Capsicum spp.). Morphological traits related to plant, leaf, and fruit was evaluated in three environments. Also pungency was evaluated by measuring capsaicinoids contents of RILs grown in three different environments. Using high resolution genetic map and phenotype data evaluated from two consecutive years, large number of QTLs were identified.

Two Capsicum annuum accessions, Perennial and Dempsey, were selected to construct the RIL population due to their contrasting morphological traits (Table 2; Table 4; Fig.1). Perennial has small, thin, and pungent chili pepper whereas Dempsey bears bell-type sweet pepper. Perennial and Dempsey have different plant type, flower size, anthocyanin accumulation, fruit type, and other morphological traits. Those contrasting traits of Perennial and Dempsey are known to be related to fruit yield and quality and important for pepper breeding (Ben-Chaim and Paran 2000; Yarnes et al. 2013).

Morphological traits were evaluated in three different environments. Even though some traits were evaluated only one environment, most of the traits were evaluated at least two environments. Due to growing conditions, quantitative traits showed large variations between environments. Comparing 2012a and 2012b, most of the phenotypic values of morphological traits were larger in 2012a. Plants were

grown on the pot in 2012b. Therefore temperature and moisture of the root and whole plant could fluctuate wildly. Also the greenhouse of 2012a had shade clothes which may reduce light intensity during the daytime. Low light caused less photosynthesis, and plants could not grow as big as plants in 2012b. Low light intensity may also affect immature fruit color. Even though parents showed green immature fruits, RILs grown in 2012b had light-green immature fruits. Effects of light quantity on chlorophyll contents were well known in plants (Portepr 1937). Also there was a research about the QTLs controlling chlorophyll content of pepper fruit using the population crossed between dark-green C. annumm line and light-green C. chinense line (Brand et al. 2012). The QTL is pc8.1 (Brand et al. 2012) which are located on different chromosome fro the QTL detected in this study. Repetitive evaluation in low light condition will confirm the effect of newly detected QTLs controlling immature fruit color. Capsaicinoids contents also measured in three different environments. Average capsaicinoids contents of Perennial and pungent RILs grown in 2012b were the highest. As all the environmental conditions including temperature, light, nutrient or other environmental factors gave stress to pepper plant, the level of capsaicinoids synthesis and accumulation were varied.

Most of QTLs associated with morphological traits were detected in each environment separately. As traits used for QTL analysis are quantitative traits and affected by environments excessively, different QTLs are detected in diverse environments. In this study QTLs explaining high percentage of fruit phenotype

variation were detected in 84.4-101.5 cM on chromosome 11 and 85.5-86.04 cM on chromosome 12. But those QTLs were detected only one environment. These QTLs could be minor QTLs with amplified effects due to environment. Therefore consecutive growth and evaluation of traits are essential to detect minor QTLs and to reduce errors from environmental effect.

QTLs detected at the same region in different environments were located on chromosome 1, 4, and 6. QTLs controlling fruit diameter and fruit weight were co-located. Through correlation test, high correlation coefficient between two traits was observed. Maybe the same genes controlling cell elongation or expansion located at QTL regions. In the previous research, fs10.1 controlling fruit shape of pepper was characterized (Borovsky and Paran 2011). fs10.1 affects cell shape in the first two weeks after anthesis, and at last influences fruit shape. Until now the genes located on the QTLs controlling fruit diameter and weight were not identified. Cell elongation may be one possible function of the QTLs as the fruit diameter, shape and weight are highly correlated between each other.

In tomato (Solanum lycopersicum), a major QTL fw3.2 controlling fruit weight was characterized and candidate gene was cloned (Chakrabarti et al. 2013). A cytochrome P450 increases cell layers of the fruit, and controls fruit mass. This QTL region was overlapped with pepper fw3.2 which controls fruit weight of pepper fruit. Although there was no major QTL on chromosome 3 detected in RILs derived from a cross between Perennial and Dempsey, using orthologous gene informations, and comparative mapping between pepper and tomato, candidate

genes could be identified.

The other co-location of QTLs was detected on chromosome 4. CS-4 for calyx shape and FS-4 for fruit shape were located in the same region. Calyx shape of the fruit is affected by the fruit diameter. When the fruit becomes wider, calyx shape seems to be like more saucer-shaped. This makes negative correlation between calyx shape and fruit shape (Table 3). CS-4 is the first QTL controlling calyx shape of pepper. There was no genetic analysis for calyx shape. But it is one trait for description of new cultivars and could affect the quality of pepper fruit.

QTL analysis for capsaicinoids contents was performed using three populations (Ben-Chaim et al. 2006; Blum et al. 2003; Yarnes et al. 2013). They found QTLs in chromosome 7 in common and 3, 4, 5, 6, 10, and 11 additionally. And it was demonstrated that there is no major QTL for capsaicinoids contents. In this study major QTLs were identified in chromosomes 1, 6, 9, and 11. In the previous studies, capsaicinoids contents were measured using the extracts of whole fruit. Capsaicinoids are synthesized and accumulated in placenta (Stewart et al. 2007). If capsaicinoids extracted from whole fruit, content can be affected by the fruit related traits like fruit size and pericarp thickness. Therefore in this study capsaicinoids were extracted from placenta to measure capsaicinoids contents precisely. Using an ultra-high density genetic map and precisely measured capsaicinoids contents data, specific region of QTLs were identified. Genes related to capsaicinoids biosynthesis pathway, Pun1 and ketoacyl-ACP synthase (Kas), and putative aminotransferase (pAMT), and phenylalanine ammonia-lyase (PAL) were

known to locate in chromosome 2, 3, and 9 respectively (Mazourek et al. 2009). Until now, it was not confirmed that genes locate on the same region with detected QTLs. While in 2006, branched-chain amino acid transferase (Bcat) was co-localized with cap4.1 (Ben-Chaim et al. 2006). Fine mapping is required to find the genes.

SNP marker located in the QTL region could be used to expect the capsaicinoids contents without the knowledge of which gene the QTL is. By Pun1 genotyping, pungent and non-pungent pepper can be easily distinguished. But to evaluate pungency levels, plant should be grown until the fruit matures. So if the molecular markers for capsaicinoids contents developed, it will reduce the time and effort to grow the pepper plants. Multiple interval mapping (MIM) will be performed to measure the epistasis between QTLs and heritability of each QTL (Kao et al. 1999). Because QTLs were detected from RILs composed of half of non-pungent lines, there will be other QTLs that could not be detected in this study. Therefore another RIL population derived from pungent parents was developed (data not shown). SNP markers highly linked to QTLs will be validated in this population and other cultivars to find out that the expected capsaicinoids value well fitted to real capsaicinoids contents. As capsaicinoids contents are affected by environment a lot, many markers will be needed for accurate expectation.

REFERENCES

Alimi NA, Bink MCAM, Dieleman JA, Nicolaï M, Wubs M, Heuvelink E, Magan J, Voorrips RE, Jansen J, Rodrigues PC, Heijden GWAM, Vercauteren A, Vuylsteke M, Song Y, Glasbey C, Barocsi A, Lefebvre V, Palloix A, Eeuwijk FA (2012) Genetic and QTL analyses of yield and a set of physiological traits in pepper. Euphytica 190:181-201

Aza-Gonzalez C, Nunez-Palenius HG, Ochoa-Alejo N (2011) Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.). Plant Cell Reports 30:695-706

Barchi L, Bonnet J, Boudet C, Signoret P, Nagy I, Lanteri S, Palloix A, Lefebvre V (2007) A high-resolution, intraspecific linkage map of pepper (Capsicum

annuum L.) and selection of reduced recombinant inbred line subsets for fast

mapping. Genome 50:51-60

Barchi L, Lefebvre V, Sage-Palloix AM, Lanteri S, Palloix A (2009) QTL analysis of plant development and fruit traits in pepper and performance of selective phenotyping. Theoretical and Applied Genetics 118:1157-1171

Ben-Chaim A, Borovsky Y, Falise M, Mazourek M, Kang BC, Paran I, Jahn M (2006) QTL analysis for capsaicinoid content in Capsicum. Theoretical and Applied Genetics 113:1481-1490

shape QTL conserved in Capsicum. Genome 46:1-9

Ben-Chaim A, Paran I (2000) Genetic analysis of quantitative traits in pepper (Capsicum annuum). Journal of the American Society for Horticultural Science 125:66-70

Ben-Chaim A, Paran I, Grube RC, Jahn M, Wijk Rv, Peleman J (2001) QTL mapping of fruit-related traits in pepper (Capsicum annuum). Theoretical and Applied Genetics 102:1016-1028

Blum E, Mazourek M, O'Connell M, Curry J, Thorup T, Liu K, Jahn M, Paran I (2003) Molecular mapping of capsaicinoid biosynthesis genes and quantitative trait loci analysis for capsaicinoid content in Capsicum. Theoretical and Applied Genetics 108:79-86

Bode AM, Dong Z (2011) The two faces of capsaicin. Cancer Research 71:2809-2814

Borovsky Y, Paran I (2011) Characterization of fs10.1, a major QTL controlling fruit elongation in Capsicum. Theoretical and Applied Genetics 123:657-665 Brand A, Borovsky Y, Meir S, Rogachev I, Aharoni A, Paran I (2012) pc8.1, a

major QTL for pigment content in pepper fruit, is associated with variation in plastid compartment size. Planta 235:579-588

Brewer MT, Lang L, Fujimura K, Dujmovic N, Gray S, van der Knaap E (2006) Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species. Plant Physiology 141:15-25

Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D (1999) A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398:436-441

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816-824

Chaim AB, Borovsky Y, Jong WD, Paran I (2003) Linkage of the A locus for the presence of anthocyanin and fs10.1, a major fruit-shape QTL in pepper. Theoretical and Applied Genetics 106:889-894

Chakrabarti M, Zhang N, Sauvage C, Munos S, Blanca J, Canizares J, Diez MJ, Schneider R, Mazourek M, McClead J, Causse M, van der Knaap E (2013) A cytochrome P450 regulates a domestication trait in cultivated tomato. Proceedings of the National Academy of Sciences 110:17125-17130

Collins MD, Wasmund LM, Bosland PW (1995) Improved method for quantifying capsaicinoids in Capsicum using highperformance liquid chromatography. Hortscience 30:137-139

Curry J, Aluru M, Mendoza M, Nevarez J, Melendrez M, O’Connell MA (1999) Transcripts for possible capsaicinoid biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum spp. Plant Science 148:47-57

Darrigues A, Hall J, Knaap Evd, Francis DM (2008) Tomato Analyzer-color test: a new tool for efficient digital phenotyping. Journal of the American Society for

Horticultural Science 133:579-586

De Givry S, Bouchez M, Chabrier P, Milan D, Schiex T (2005) CARHTA GENE: multipopulation integrated genetic and radiation hybrid mapping. Bioinformatics 21:1703-1704

Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19:11-15

Eshbaugh WH (1980) The taxonomy of the genus Capsicum (Solanaceae). Phytologia 47:153-166

Fiorani F, Schurr U (2013) Future scenarios for plant phenotyping. Annual Review of Plant Biology 64:267-291

Fraenkel L, Bogardus ST, Concato J, Wittink DR (2004) Treatment options in knee osteoarthritis: the patient's perspective. Archives of Internal Medicine 164:1299-1304

Garces-Claver A, Fellman SM, Gil-Ortega R, Jahn M, Arnedo-Andres MS (2007) Identification, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp. Theoretical and Applied Genetics 115:907-916

Han K, Jeong H-J, Sung J, Keum YS, Cho M-C, Kim J-H, Kwon J-K, Kim B-D, Kang B-C (2012) Biosynthesis of capsinoid is controlled by the Pun1 locus in pepper. Molecular Breeding 31:537-548

Hill TA, Ashrafi H, Reyes-Chin-Wo S, Yao J, Stoffel K, Truco MJ, Kozik A, Michelmore RW, Van Deynze A (2013) Characterization of Capsicum annuum

genetic diversity and population structure based on parallel polymorphism discovery with a 30K unigene Pepper GeneChip. PLoS One 8:e56200

Iwai K, Suzukl T, Fujiwake H (1979) Simultaneous microetermination of capsaicin and its four analogues by using high-performance liquid chromatography and gas chromatography-mass spectrometry. Journal of Chromatography 172:303-311

Jeong H-J, Hwang D-Y, Ahn J-T, Chun J-Y, Han K-E, Lee W-M, Kwon J-K, Lee Y-J, Kang B-C (2012) Development of a simple method for detecting capsaicinoids using Gibb's reagent in pepper. Korean Journal of Horticultural Science and Technology 30:294-300

Joo JI, Kim DH, Choi J-W, Yun JW (2010) Proteomic analysis for antiobesity potential of capsaicin on white adipose tissue in rats fed with a high fat diet. Journal of Proteome Research 9:2977-2987

Kang BC, Nahm SH, Huh JH, Yoo HS, Yu JW, Lee MH, Kim B-D (2001) An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers. Theoretical and Applied Genetics 102:531-539 Kao C-H, Zeng Z-B, Teasdale RD (1999) Multiple interval mapping for

quantitative trait loci. Genetics 152:1203-1216

Kearsey MJ (1998) The principles of QTL analysis (a minimal mathematics approach). Journal of Experimental Botany 49:1619-1623

Kearsey MJ, Farquhar AGL (1998) QTL analysis in plants; where are we now? Heredity 80:137-142

Kozukue N, Han J-S, Kozukue E, Lee S-J, Kim J-A, Lee K-R, Levin CE, Friedman M (2005) Analysis of eight capsaicinoids in peppers and pepper-containing foods by high-performance liquid chromatography and liquid chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry 53:9172-9181

Lang Y, Yanagawa S, Sasanuma T, Sasakuma T (2006) A gene encoding a putative acyl-transferase involved in pungency of Capsicum. Breeding Science 56:55-62

Lee C-J, Yoo E, Shin JH, Lee J, Hwang H-S, Kim B-D (2005) Non-pungent Capsicum contains a deletion in the capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers. Molecules and Cells 19:262-267

Luo XJ, Peng J, Li YJ (2011) Recent advances in the study on capsaicinoids and capsinoids. European Journal of Pharmacology 650:1-7

Mazourek M, Pujar A, Borovsky Y, Paran I, Mueller L, Jahn MM (2009) A dynamic interface for capsaicinoid systems biology. Plant Physiology 150:1806-1821

Mori A, Lehmann S, O'Kelly J, Kumagai T, Desmond JC, Pervan M, McBride WH, Kizaki M, Koeffler HP (2006) Capsaicin, a component of red peppers, inhibits the growth of androgen-independent, p53 mutant prostate cancer cells. Cancer Research 66:3222-3229

plant domestication traits in tomato and pepper. Journal of Experimental Botany 58:3841-3852

Peng J, Li YJ (2010) The vanilloid receptor TRPV1: role in cardiovascular and gastrointestinal protection. European Journal of Pharmacology 627:1-7

Portepr AM (1937) Effect of light intensity on the photosynthetic efficiency of tomato plants. Plant Physiology 12:225-252

Rao GU, Chaim AB, Borovsky Y, Paran I (2003) Mapping of yield-related QTLs in pepper in an interspecific cross of Capsicum annuum and C. frutescens. Theoretical and Applied Genetics 106:1457-1466

Sanatombi K, Sharma GJ (2008) Capsaicin content and pungency of different

Capsicum spp. cultivars. Notulae Botanicae Horti Agrobotanici Cluj 36:89-90

Singh S, Asad SF, Ahmad A, Khan NU, Hadi SM (2001) Oxidative DNA damage by capsaicin and dihydrocapsaicin in the presence of Cu(II). Cancer Letters 169:139-146

Spanyar P, Blazovich M (1969) A thin-layer chromatographic method for the determination of capsaicin in ground paprika. Analyst 94:1084-1089

Stellari GM, Mazourek M, Jahn MM (2010) Contrasting modes for loss of pungency between cultivated and wild species of Capsicum. Heredity 104:460-471

Stewart C, Jr., Kang BC, Liu K, Mazourek M, Moore SL, Yoo EY, Kim BD, Paran I, Jahn MM (2005) The Pun1 gene for pungency in pepper encodes a putative acyltransferase. The Plant Journal 42:675-688

Stewart C, Jr., Mazourek M, Stellari GM, O'Connell M, Jahn M (2007) Genetic control of pungency in C. chinense via the Pun1 locus. Journal of Experimental Botany 58:979-991

Sudré CP, Gonçalves LSA, Rodrigues R, Júnior ATdA, Riva-Souza EM, Bento CdS (2010) Genetic variability in domesticated Capsicum spp as assessed by morphological and agronomic data in mixed statistical analysis. Genetics and Molecular Research 9:283-294

Sukrasno N, Yeoman MM (1993) Phenylpropanoid metabolism during growth and development of Capsicum frutescens fruits. Phytochemistry 32:839-844 Tewksbury JJ, Nabhan GP (2001) Directed deterrence by capsaicin in chillies.

Nature 412:403-404

Tewksbury JJ, Reagan KM, Machnicki NJ, Carlo TA, Haak DC, Penaloza AL, Levey DJ (2008) Evolutionary ecology of pungency in wild chilies. Proceedings of the National Academy of Sciences 105:11808-11811

Thoennissen NH, O'Kelly J, Lu D, Iwanski GB, La DT, Abbassi S, Leiter A, Karlan B, Mehta R, Koeffler HP (2010) Capsaicin causes cell-cycle arrest and apoptosis in ER-positive and -negative breast cancer cells by modulating the EGFR/HER-2 pathway. Oncogene 29:285-296

Thul ST, Lal RK, Shasany AK, Darokar MP, Gupta AK, Gupta MM, Verma RK, Khanuja SPS (2009) Estimation of phenotypic divergence in a collection of

Capsicum species for yield-related traits. Euphytica 168:189-196

van Eeuwijk F, Glasbey C (2012) SPICY: towards automated phenotyping of large pepper plants in the greenhouse. Functional Plant Biology 39:870 Voorrips RE (2002) MapChart: software for the graphical presentation of linkage

maps and QTLs. The Journal of Heredity 93:77-78

Walsh BM, Hoot SB (2001) Phylogenetic relationships of Capsicum (Solanaceae) using DNA sequences from two noncoding regions: the chloroplast atpb-rbcl spacer region and nuclear waxy introns. International Journal of Plant Sciences 162:1409-1418

Wang S, Basten CJ, Zeng Z-B (2012) Windows QTL Cartographer 2.5. Department of Statistics. North Carolina State University

Yarnes SC, Ashrafi H, Reyes-Chin-Wo S, Hill TA, Stoffel KM, Van Deynze A (2013) Identification of QTLs for capsaicinoids, fruit quality, and plant architecture-related traits in an interspecific Capsicum RIL population. Genome 56:61-74

Zygier S, Chaim AB, Efrati A, Kaluzky G, Borovsky Y, Paran I (2005) QTLs mapping for fruit size and shape in chromosomes 2 and 4 in pepper and a comparison of the pepper QTL map with that of tomato. Theoretical and Applied Genetics 111:437-445