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Application of genetics and genomics towards Capsicum translational research

November 2013
Plant Biotechnology Reports

November 2013

DOI:10.1007/s11816-013-0306-z

Authors:

Nirala Ramchiary

Mechuselie Kehie

Sustainable Development Research Centre

Vijaya Brahma

Jawaharlal Nehru University

Suman Kumaria

North - Eastern Hill University, India, Shillong

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Capsicum species commonly known as Chili peppers are economically important group of plants belonging to the Solanaceae family. Of the 38 species reported, only six species namely, Capsicum annuum, C. assamicum, C. baccatum, C. frutescence, C. chinense and C. pubescens are cultivated. They are very important component of the human being as peppers are used as vegetables, spices, and a coloring agent and for medicinal purposes. Based on pungency trait which is due to the presence of a group of compounds known as capsaicinoids, cultivated capsicums are classified into sweet peppers and hot peppers. Although conventional breeding and classical genetic analysis were successful in estimating the number of genes for economically important traits governed by few major genes and their incorporation in the breeding pro-gramme, the advent of molecular markers and recently developed next generation sequencing technologies sup-plemented greatly in dissecting the genetic and molecular basis of economically important traits in the capsicum genome for applied research. Here in this review, we tried to highlight the use of molecular markers, comparative mapping and advanced genomics technologies and their integrated use in the translational research of cultivated Capsicums.

ariability in the fruit morphology of different Capsicum species

…

dentified QTL/genes which could be used in Capsicum translational research

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Development of an efficient protocol for regeneration of C. chinense that could be used in translational research. a Seed germination of C. chinense in MS medium (Bar 1 cm) producing explants. b Multiple shoots induction from nodal segments of C. chinense in MS medium containing 18.16 lM TDZ after 5 weeks culture (Bar 1 cm). c Elongation and root induction of regenerated shoots in MS medium containing 5.70 lM indole-3-acetic acid (IAA) (Bar 1 cm). d Regenerated hardened plantlet transferred to earthen pot (Bar 10 cm). e Hardened plantlet bearing normal fruit in green house (Bar 10 cm). f Field-grown plantlets bearing fruits

…

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REVIEW ARTICLE

Application of genetics and genomics towards Capsicum

translational research

Nirala Ramchiary •Mechuselie Kehie •

Vijaya Brahma •Suman Kumaria •

Pramod Tandon

Received: 10 September 2013 / Accepted: 21 October 2013

ÓKorean Society for Plant Biotechnology and Springer Japan 2013

Abstract Capsicum species commonly known as Chili

peppers are economically important group of plants

belonging to the Solanaceae family. Of the 38 species

reported, only six species namely, Capsicum annuum,C.

assamicum,C. baccatum,C. frutescence,C. chinense and

C. pubescens are cultivated. They are very important

component of the human being as peppers are used as

vegetables, spices, and a coloring agent and for medicinal

purposes. Based on pungency trait which is due to the

presence of a group of compounds known as capsaicinoids,

cultivated capsicums are classiﬁed into sweet peppers and

hot peppers. Although conventional breeding and classical

genetic analysis were successful in estimating the number

of genes for economically important traits governed by few

major genes and their incorporation in the breeding pro-

gramme, the advent of molecular markers and recently

developed next generation sequencing technologies sup-

plemented greatly in dissecting the genetic and molecular

basis of economically important traits in the capsicum

genome for applied research. Here in this review, we tried

to highlight the use of molecular markers, comparative

mapping and advanced genomics technologies and their

integrated use in the translational research of cultivated

Capsicums.

Keywords Capsaicinoids Transcriptome 

Molecular markers Quantitative traits loci

Introduction

Chili peppers belonging to the Capsicum genus and family

Solanaceae have been a part of the human diet in the

Americas since 7500 BC. The archaeological evidence at

sites located in southwestern Ecuador showed that chili

pepper was domesticated more than 6,000 years ago, and it

is one of the ﬁrst cultivated crops in the Central and South

Americas (Bosland 1996; Perry et al. 2007). The genus

Capsicum comprises about 38 species (USDA-ARS 2011)

of which only six species (namely Capsicum annuum,C.

baccatum,C. frutescence,C. chinense,C. pubescens and C.

assamicum) are cultivated. The last one was recently

identiﬁed as a distinct domesticated species from the North

Eastern part of India (Purkayastha et al. 2012). These

domesticated species are derived from different ancestral

stocks found in three distinct centers of origin: Mexico

being the primary center of origin for C. annuum, the most

widely cultivated species, with Guatemala as a secondary

center; Amazonia for C. chinense and C. frutescens; and

Peru and Bolivia for C. baccatum and C. pubescens.C.

annuum and C. frutescens are widely distributed from

Mexico through Central America and throughout the

Caribbean region (Greenleaf 1986). Of the domesticated

species, C. chinense is the most pungent fruit type. Chili

fruits contain rich metabolites that are beneﬁcial for human

health, such as carotenoids (provitamin A), vitamins (C and

E), ﬂavonoids and capsaicinoids (Maga 1975). These

N. Ramchiary (&)M. Kehie

Translational and Evolutionary Genomics Lab, School of Life

Sciences, Jawaharlal Nehru University, New Delhi 110067, India

e-mail: nrudsc@gmail.com

V. Brahma

Laboratory of Comparative and Evolutionary Genomics,

Department of Cell and Systems Biology, University of Toronto,

25 Willcocks St., Toronto, ON M5S3B2, Canada

S. Kumaria P. Tandon

Department of Botany, North Eastern Hill University,

Shillong 793022, India

123

Plant Biotechnol Rep

DOI 10.1007/s11816-013-0306-z

compounds (apart from functioning as antioxidants and

nutrients) are used in traditional medicine due to their

enormous medicinal properties. Capsaicinoids cause the

spicy ﬂavor (pungency) of chili pepper fruit. Kosuge et al.

(1965) established that the pungent principle of red pepper

consists not only of one chemical but actually of the

unsaturated and saturated amides capsaicin and dihydro-

capsaicin, which were named in combination as capsaici-

noids (Pruthi 1980). Capsicum species are classiﬁed as hot

peppers and sweet peppers based on the presence or

absence of pungency traits in the fruits which are due to

capsaicinoids.

Although many classical studies reported genetics of

economically important traits in Capsicum species, the

advent of molecular markers and recently developed next-

generation sequencing (NGS) technologies supplemented

greatly in translating those studies into the applied breeding

of Capsicum species. Here in this review, we tried to

highlight the resources, current status in genetic and

genomics studies and their prospects in translational

research.

Genetic and genomic resources: foundation

for translational research

Germplasm resources

Genetic resources are important for ﬁnding beneﬁcial

alleles present in a large number of germplasm and for their

use in breeding programs for improving yield, quality and

biotic and abiotic stress resistances/tolerances. Although

many institutes/organizations are collecting and maintain-

ing important genetic resources in the form of germplasm

of the different Capsicum species for their use in applied

and basic research, few institutes listed in http://www.

thechileman.org/ have a comparatively large collection of

the different capsicum germplasm collections. The Asian

Vegetable Research and Development Center (AVRDC,

Taiwan) has the largest collection with a total of 8,170

accessions belonging to wild and domesticated Capsicum

species from around the world, followed by the United

States Department of Agriculture (USDA) with 6,067

collections. In India, The National Bureau of Plant Genetic

Resources, New Delhi has 2,774 accessions of indigenous

and exotic collections. The other prominent institutes

involved in collection and maintenance of capsicum spe-

cies are: the Chile Pepper Institute (New Mexican State

University), the Centre for Genetic Resources (CGN), the

Netherlands, Banco de Germoplasma de Hortalic¸as (BGH),

Brazil, The German Research Centre for Biotechnology

(CAP), Germany. The capsicum germplasms are being

continuously evaluated for fruit morphology, plant type,

resistance to diseases and pests and abiotic stresses. Many

capsicum germplasms showing resistance against arthro-

pods, nematodes and pathogens are recently reviewed by

Babu et al. (2011).

Molecular markers and genomic resources

Among many, the most important use of molecular markers

in translational research is genome mapping and ﬁnding the

genetic loci/quantitative trait loci (QTL) for economically

important traits. Although tomato markers were used in

Capsicum at the beginning, several researchers developed

molecular markers, especially simple sequence repeats

(SSRs) from Capsicum species (Sanwen et al. 2001; Lee

et al. 2004). Apart from the SSRs developed from genomic

sequences, the EST databases provide a new source for

developing SSR markers in a rapid and cost-effective

manner since the number of expressed sequence tags

(ESTs) is increasing considerably in public databases with

the ongoing multinational efforts of transcriptome

sequencing. Yi et al. (2006) by in silico analysis of 10,232

non-redundant ESTs of pepper identiﬁed a total of 1,201

SSRs, which corresponded to one SSR in every 3.8 kb of

the ESTs. Of these, 812 primer pairs satisfying all condi-

tions were designed, of which 513 SSRs (63.1 %) were

successfully ampliﬁed and 150 of them (29.2 %) showed

polymorphism between C. annuum ‘TF68’ and C. chinense

‘Habanero’. Portis et al. (2007) reported the development

of 783 SSRs form 576 non-redundant EST sequences, of

which 348 SSRs primer pairs were designed. Wu et al.

(2006) developed a set of 2,869 conserved single-copy

orthologolous set of markers (COSII) by aligning ortholo-

gous sequences of multiple plant species, including model

Arabidopsis species which could be used in a broad range

of plant taxa, including those plants lacking any sequence

databases. These markers were widely used to develop

genetic maps and comparative genome analysis between

many species, including peppers, tomato and eggplants

belonging to the Solanaceae family (Wu et al. 2009a,b).

The SOL Genomics Network (SGN; http://sgn.cornell.

edu), a genomics information resource for the Solanaceae

and related families in the Asterid Clade, was developed as

a part of International Solanaceae Project (SOL) formed by

researchers working on Solanaceae plants in many coun-

tries. The aims of SGN are cataloging and maintaining

genetic maps and markers, disseminating sequence infor-

mation especially ESTs as unigenes, cataloging and pub-

lishing phenotypic information of Solanaceae plants and

assembling, analyzing and publishing sequencing data of

Solanaceae plant species such as tomato, potato, peppers

and eggplant (Mueller et al. 2005).

Kim et al. (2008a) ﬁrst developed chili pepper EST

databases with a total of 122,582 sequenced ESTs from 21

Plant Biotechnol Rep

123

pepper EST libraries. This pepper EST database provided

functional genomic resources which could be used for

identifying unigenes, analyzing tissue speciﬁc expressed

genes in different developmental tissues and under condi-

tions of stress, with a comparative analysis of pepper ESTs

with those of other members of the Solanaceae family.

Tecle et al. (2010) developed a new tool, solQTL, which

employs a user-friendly web interface for uploading raw

phenotype and genotype data to the database, with R/QTL

mapping software for QTL analysis. This module contains

algorithms for online visualization and cross-referencing of

QTL to other relevant datasets and tools such as the SGN

Comparative Map Viewer and Genome Browser.

Huan-huan et al. (2011) developed 755 SSR markers

from EST sequences, 210 of which were tested in eight

pepper cultivars; and the PCR result revealed the existence

of polymorphism for 127 SSR primer pairs. Shirasawa

et al. (2013) developed a total of 5,751 SSR primer pairs

from 118,060 publicly available EST sequences of C.

annuum. Nucleotide sequences spanning 2,245 SSRs of C.

annuum markers were successfully mapped onto the

tomato genome, and 96 of these, which spanned the entire

tomato genome, were selected for further analysis. Out of

77 SSRs, 60 produced speciﬁc DNA amplicons and were

polymorphic among the 192 Capsicum lines examined.

Sugita et al. (2005) developed non-redundant 2- or 3-base

SSR markers from enriched C. annuum genomic libraries

and from C. annuum cDNA sequences in public databases.

The group obtained 1,736 genomic SSR markers and 1,344

cDNA-derived SSR markers from 6,528 clones and 13,003

sequences, respectively.

Recent advances in high-throughput genomics technol-

ogies such as NGS technologies have opened up new

opportunities to combine conventional breeding through

marker trait association/QTL mapping with the develop-

ment of large-scale genome and transcriptome sequences.

In Capsicum, recently, a few studies adopted transcriptome

sequencing to identify genes that are involved in different

developmental and metabolic biosynthesis pathways.

´ngora-Castillo et al. (2012) sequenced C. annuum

transcriptomes from different tissues and assembled a total

of 32,314 high-quality contigs from 1,324,516 raw reads

and developed a Capsicum transcriptome database (DB,

http://www.bioingenios.ira.cinvestav.mx:81/Joomla/). Nic-

olaı

¨et al. (2012) sequenced the C. annuum cv. ‘Yolo

Wonder’ transcriptome using Roche 454 pyrosequencing

and assembled de novo 23,748 contigs and 60,370 single-

tons. Further they mapped 10,886,425 sequence reads

obtained by the Illumina GA II sequencing of C. annuum

cv. ‘Criollo de Morelos 334’ to the ‘Yolo Wonder’ tran-

scriptome and identiﬁed a total of 11,849 reliable SNPs

spread across 5,919 contigs besides detecting a total of 853

single sequence repeats.

Lu et al. (2012) recently reported the transcriptome

analysis of red pepper (Capsicum annuum L. TF68) using

454 GS-FLX pyrosequencing. Approximately 30.63

megabases (Mb) of EST data with a total of 9,818 contigs

and 23,712 singletons with an average length of 375 base

pairs (bp) were obtained. All unigenes showed approxi-

mately equal distribution on chromosomes when aligned

with tomato (Solanum lycopersicum) pseudomolecules.

Furthermore, 1,536 high quality single nucleotide dis-

crepancies were discovered using the Bukang mature fruit

cDNA collection as a reference. A total of 758 SSR motifs

were mined from 614 contigs, from which 572 primer sets

were designed. The SSR motifs corresponded to di- and tri-

nucleotide motifs (27.03 and 61.92 %, respectively). De

novo transcriptome assembly in C. frutescens has been

reported by Liu et al. (2013). The group obtained a total

54,045 high-quality unigenes. Ashraﬁ et al. (2012)

sequenced the transcriptome of three pepper genotypes,

namely Maor, Early Jalapen

˜o and Criollo de Morelos-334

(CM334) using NGS technology. They assembled two

reference pepper transcriptomes separately. The ﬁrst

assembly of 31,196 contigs was made using 125,000 San-

ger-EST sequences mainly derived from a Korean F1-

hybrid line, Bukang, wherefrom overlapping probes were

designed to construct a pepper Affymetrix GeneChip

microarray. They identiﬁed a total of 4,236 SNPs and 2,489

SSRs from the assembly. The second transcriptome

assembly was constructed based on more than 200 million

Illumina Genome Analyzer II reads and about 22,000 high-

quality putative SNPs, and 10,398 SSR markers were

identiﬁed within the Illumina transcriptome assembly.

Hill et al. (2013) developed the Pepper GeneChip array

from Affymetrix for polymorphism detection and expres-

sion analysis in Capsicum. Probes on the array were

designed from 30,815 unigenes assembled from ESTs.

Hybridization of genomic DNA from 40 diverse C. annu-

um lines and few lines from other cultivated species (C.

frutescens,C. chinense and C. pubescens) detected 33,401

SPP markers within 13,323 unigenes. Among the C. ann-

uum lines, 6,426 SPPs covering 3,818 unigenes were

identiﬁed. An estimated threefold reduction in diversity

was detected in non-pungent lines to the pungent lines

besides an observation of 8.7 cM region without poly-

morphism around the Pun1 in non- pungent C. annuum.

This suggests that non pungent traits in different pepper

genotypes might have originated from the same source.

Genetic maps: dissecting Capsicum genome

for translational research

The development of molecular markers in the late 1980s

helped to identify the genomic region harboring important

Plant Biotechnol Rep

123

traits loci and thereby created a foundation for transla-

tional research in crop plants (Tanksley et al. 1988). As a

result, in Capsicum species also several genetic maps

have been constructed and published in the past 20 years

(reviewed by Kumar et al. 2011). The ﬁrst genetic map in

pepper with 85 RFLP markers was constructed using an

inter-speciﬁc cross by Tanksley et al. (1988). Shortly,

thereafter using anonymous markers such as genomic

DNA based restriction fragment length polymorphic

(RFLPs), Ampliﬁed fragment length polymorphism (AF-

LPs), Random ampliﬁed length polymorphic DNA

(RAPD) and isozyme markers, many intra-speciﬁc or

inter-speciﬁc pepper genetic maps based on F2, RIL,

doubled haploid or backcross mapping populations were

developed (Livingstone et al. 1999; Kang et al. 2001; Rao

et al. 2003; Lee et al. 2004; Ben-Chaim et al. 2006). The

most comprehensive pepper–tomato comparative map

using AFLP, RFLP and RAPD markers was constructed

by Livingstone et al. (1999). The group reported a genetic

map of Capsicum (pepper) from an interspeciﬁc F

pop-

ulation consisting of 11 large (76.2–192.3 cM) and two

small (19.1 and 12.5 cM) linkage groups (LGs) that

covered a total of 1,245.7 cM. Ben-Chaim et al. (2006)

used a total of 728 molecular markers including 489

SSRs, 195 AFLP, eight speciﬁc PCR-based and 36 RFLP

markers to construct a genetic map in an F

population of

C. annuum (NuMex RNaky) 9C. frutescens cross. This

linkage maps includes candidate genes, pAMT,COMT

and Bcat genes that are involved in a proposed model of

capsaicinoid biosynthesis pathway (Blum et al. 2003;

Stewart et al. 2005). The map consists of 12 major and

four small LGs with a total length of 1,358.7 cM. In

addition to these inter-speciﬁc maps, several intra-speciﬁc

C. annuum maps have been reported.

An integrated molecular linkage map of pepper (C.

annuum L.) was constructed by aligning three intra-

speciﬁc linkage maps of C. annuum L. (Lefebvre et al.

2002). The individual maps varied from 685 to

1,668 cM with 16 to 20 LGs. Lee et al. (2009)con-

structed an integrated pepper map using RFLP, SSR,

CAPS, AFLP, WRKY, rRAMP and BAC end sequence-

derived markers. They developed 132 new PCR-based

markers (57 STS from BAC end sequences, 13 STS

from RFLP, and 62 SSRs). This integrated map con-

tained 169 SSR, 354 RFLP, 23 STS from BAC end

sequences, six STS from RFLP, 152 AFLP, 51 WRKY

and 99 rRAMP markers on 12 chromosomes. This

constructed integrated map consisted of 805 markers

(map distance of 1,858 cM) in interspeciﬁc populations

and 745 markers (map distance of 1,892 cM) in intra-

speciﬁc populations. Lee et al. (2004) developed a

linkage map which contains 46 SSR markers. An inte-

grated genetic map of pepper by combining six indi-

vidual maps was constructed with a total of 2262

marker loci (1528 AFLP, 440 RFLP, 288 RAPD and

known gene sequences, isozymes and morphological

markers). The map covered a total distance of 1832 cM

of capsicum genome (Paran et al. 2004). Minamiyama

et al. (2005) developed 626 unique SSR markers for

Capsicum species by constructing four SSR-enriched

genomic DNA libraries from C. annuum.Theydevel-

oped a pepper genetic linkage map containing 374

markers, including 106 new SSR markers distributed in

13 LGs which covered 1,042 cM of the pepper genome.

Yi et al. (2006) mapped 150 EST SSRs in a population

derived from C. annuum ‘TF68’ and C. chinense ‘Haban-

ero’ in the previously generated pepper linkage map of

inter-speciﬁc cross TF68 9Habanero. Some 139 ESTS-

SRs were located on the linkage map, in addition to 41

previous SSRs and 63 RFLP markers, forming 14 LGs and

spanning 2,201.5 cM. Portis et al. (2007) screened a total

of total of 204 EST SSRs for polymorphism between four

C. annuum inbred lines and one C. frutescens accession

parental lines used for developing mapping population. Wu

et al. (2009a) developed ﬁrst comprehensive pepper genetic

map that contain a total of 587 conserved orthologous set II

markers (COSII) of which 299 were orthologous markers

between the pepper and tomato genomes in 12 LGs of

pepper. Based on synteny analysis between tomato and

pepper, they found that the two genomes shared 35 con-

served syntenic segments (CSSs) within which gene/mar-

ker order is well preserved.

Lu et al. (2012) through comparative transcriptomics

developed 41 SNP markers and integrated those markers

by mapping in previous genetic map of red pepper C.

annuum. Their new map contained a total of 458 molec-

ular markers. Sugita et al. (2013) mapped 597 markers,

including 265 of the newly developed EST SSR markers

by using doubled haploid (DH) lines derived from cross-

ing between two contrasting C. annuum lines. The map,

designated as the KL-DH map, covered a total distance of

2,028 cM pepper genome in 12 LGs. A linkage map of

pepper using a DH population derived from a cross

between two C. annuum genotypes, a bell-type cultivar

‘California Wonder’ and a Malaysian small-fruited culti-

var ‘LS2341’ has been reported by Mimura et al. (2012).

A total of 253 markers (151 SSRs, 90 AFLPs, 10 CAPSs

and two sequence-tagged sites) were mapped, spanning

1,336 cM of the Capsicum genome. This is the ﬁrst SSR-

based map to consist of 12 LGs, corresponding to the

haploid chromosome number in an intraspeciﬁc cross of

C. annuum.

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123

Candidate genes and QTL mapping: ﬁnding genetic loci

governing economically important traits

for translational research

The development of molecular markers and genetic maps

help to dissect capsicum genome into many parts and

associate with the economically important traits by QTL

mapping or using the Candidate gene approach. Several

researchers reported mapping and identiﬁcation of many

QTL/candidate genes in peppers for fruit size, shape and

color, male sterility system and fertility restorer genes and

metabolites such as pungency and disease resistances. Of

the many reported, only few listed in Table 1could be

applied in pepper translational research as they were shown

directly associating with the trait of interest.

Fruit traits

Capsicum species show a large variation in fruit traits such

as fruit size and shape, texture and chemical composition

(Fig. 1). QTL-mapping studies have identiﬁed several loci

that control fruit size and shape in pepper (Ben-Chaim

et al. 2001; Rao et al. 2003; Zygier et al. 2005; Barchi et al.

2009; Borovsky and Paran 2011; Tsaballa et al. 2011).

Ben-Chaim et al. (2001) did QTL analysis for 14 traits

including fruit traits in an F

population derived from a

cross between two C. annuum genotypes, the bell-type

cultivar Maor and the Indian small-fruited line Perennial. A

total of 55 QTL were detected which included 5, 4, 4, 3, 4,

4 and 6 QTL for fruit weight, fruit diameter, fruit length,

fruit shape, pericarp thickness and 6 for soluble solid

concentration, respectively. The fruit shape QTL detected

on chromosome 3 was a major QTL that explained around

60 % of phenotypic variation. Ben-Chaim et al. (2003a)

mapped an anthocyanin QTL and a major fruit shape QTL

fs10.1on chromosome 10, using a population derived from

C. annum and C. chinense. Rao et al. (2003) studied 10

yield-related traits that included fruit weight, size, shape

and seed weight in BC

and BC

generations derived by

a crossing between the cultivated species C. annuum cv.

Maor and the wild C. frutescens BG 2816 accession. A

total of 58 QTLs were detected. Two QTL for yield were

detected, of which one (yld8.1) was also associated with

fruit weight and the other (yld1.1) with ﬂowering and fruit

setting. The major QTL affecting fruit shape was fs3.1. The

same QTL was previously found to be the major QTL

affecting fruit shape in C. annuum which accounted for

more than 60 % of the phenotypic variation (Ben-Chaim

et al. 2001). Zygier et al. (2005) mapped fruit size and

shape QTL using two populations derived from interspe-

ciﬁc cross between C. chinense and C. frutescence on

chromosome 2 and chromosome 4. They detected a major

QTL fw2.1 for fruit weight that explained 62 % of phe-

notypic variation on chromosome 2 which was co-mapped

with a minor fruit-shape QTL, fs2.1, and two fruit weight

QTL fw4.1 and fw4.2 on chromosome 4. Major QTL fw2.1

and fw4.2 for fruit-weight QTL were also detected in other

Capsicum mapping populations for fruit-size variations.

Barchi et al. (2009) mapped a total of 76 QTL for 13

traits that included plant morphology and fruit traits. Of the

many QTL reported, only fs10.1 for fruit elongation loca-

ted on chromosome 10 was characterized, which can be

used in breeding programs. Lee et al. (2008) developed

CAPs markers tightly linked to the pendant and erect

fruiting habit of Capsicum. Through a genetics study in F

and BC

of a cross between erect and pendant types, they

showed that the erect fruiting habit was recessive to the

pendant habit. Further analysis in natural germplasm with

erect versus pendant gave the expected result suggesting

that these markers could be used for marker-assisted

breeding of this trait.

Tsaballa et al. (2011) successfully cloned and charac-

terized Ovate-like genes (designated as CaOvate) from two

pepper cultivars of different fruit shape, cv. ‘Mytilini

Round’ and cv. ‘Piperaki Long’. The CaOvate encodes a

335 amino-acid polypeptide, shares 63 % identity with the

tomato OVATE protein encoding gene and exhibits high

similarity with OVATE sequences from other Solanaceae

species. Analysis showed no signiﬁcant structural differ-

ences but observed that expression of CaOvate followed a

different developmental proﬁle between the two cultivars,

being higher in cv. ‘Round’. Furthermore, down-regulation

of CaOvate through VIGS in cv. ‘Round’ showed its fruit

had a more oblong form, indicating that CaOvate is

involved in determining fruit shape in pepper. Borovsky

and Paran (2011) constructed near-isogenic lines for fs10.1

and mapped it in a BC

population. In this population,

fs10.1 segregated as a Mendelian locus and mapped 0.3 cM

away from the closest molecular marker. They further

veriﬁed the effect of fs10.1 in an F

population from an

independent cross between elongated and conical-fruited

parents. Fruit shape mutation was mapped to the fs10.1

region and was determined to be allelic to the QTL. By

measuring the fruit shape of near-isogenic lines for fs10.1

during fruit development, they found that the shape of the

fruit was determined primarily in the ﬁrst 2 weeks after

anthesis. Lu et al. (2012) detected 23 QTL for 11 traits

which included QTL for Phytophthora resistances in C.

annum. Recently, QTL analyses for plant architectural,

phenological, and fruit traits in a pepper mapping popula-

tion consisting of 92 recombinant inbred lines derived from

a cross between C. frutescens acc. 2814-6 and C. annuum

var. NuMexRNAKY were done and identiﬁed a total of 96

QTL for 38 traits, including 12 QTL associated with

capsaicinoid levels (Yarnes et al. 2013).

Plant Biotechnol Rep

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Table 1 Identiﬁed QTL/genes which could be used in Capsicum translational research

Cross/species QTL/gene Trait References

Capsicum sp. fs10.1 Fruit elongation Barchi et al. (2009)

C. annuum CaOvate Fruit shape Tsaballa et al. (2011)

PI 152225 (C. chinense)x

Kelvin, 4751 (C. annuum)

cl,c2,yFruit color Popovsky and Paran (2000)

Capsicum sp. Phytoene synthase (PSy), phytoene

desaturase and capsanthin,

capsorubin synthase (Ccs) genes,

LcyB,CrtZ-2

Fruit color Ha et al. (2007), Rodriguez-Uribe

et al. (2012)

Capsicum sp. Pal,Ca4h,Comt, 3-ketoacyl-ACP

synthase (Kas), putative

aminotransferase (pAmt), Acl,Fat,

SB2-149 and SB1-158 clones

(similarity to pAmt and Kas). SB2-

66, SB2-115, SB2-115

Capsaicinoid biosynthesis Curry et al. (1999), Kim et al.

(2001), Aluru et al. (2003)

NMCA 30036 (C. chinense) Pun 1 (locus C), At3 Capsaicinoid biosynthesis Blum et al. (2002)

Capsicum sp. pun1

Capsaicinoid biosynthesis Stewart et al. (2007)

C. annuum CaMF2 Pollen development Chen et al. (2011)

C. annuum Methionine sulfoxide reductase (B2) Plant defense Oh et al. (2010)

C. annuum Ipcr Resistance to P. capsici Reeves et al. (2013)

C. annuum cv ‘Bukang’ Cmr1 (cucumber mosaic resistance 1) Resistance to CMV Kang et al. (2010)

C. annuum CaBtf3 Defense-related genes Huh et al. (2012b)

C. annuum CaWRKYb Resistance gene Lim et al. (2011)

C. annuum and C. chinense Phyto.5.2 P. capsici resistance Quirin et al. (2005)

C. annuum L. CaMi Nematode-resistant gene Chen et al. (2007)

Capsicum sp. Pcpme 1–9 Pme genes Li et al. (2011)

CM33 (C. annuum) CaRGA2 P. capsici resistance Zhang et al. (2013)

Capsicum sp. caWRKY40 Resistance to R. solanacearum Dang et al. (2013)

Capsicum sp. Bs1,Bs4,bs5,bs6,gdr Resistance to Xanthomonas

campestris pv. vesicatoria

Jones et al. (2002,2004), Csillery

et al. (2004)

C. annuum CaPO2 Defense against Xanthomonas

campestris pv. vesicatoria

Choi et al. (2007)

C. annuum CaMLO2 Mildew resistance locus O gene Kim and Hwang (2012)

Capsicum sp. pvr1,pvr21,pvr22,pvr3,pvr4,pvr5,

pvr6,Pvr7

Resistance genes Caranta et al. (1996), Kyle and

Palloix (1997), Grube et al.

(2000)

C. annuum cv ‘Bukang’ Cmr1 Resistance gene Kang et al. (2010)

C. chinense Tsw. Resistance gene Moury et al. (2000)

Capsicum sp. L1,L3,L4 Resistance gene Lefebvre et al. (1995), Ben-Chaim

et al. (2001), Matsunaga et al.

(2003), Tomita et al. (2008)

C. annuum CaWRKYb,CaPR-10,CaPR-1,

CaPR-5

Reduce resistance pathogenesis-

related gene

Lim et al. (2011)

C. annuum Me4,Mech1,Mech2 Root-knot nematode resistance

gene

Djian-Caporalino et al. (2001,

2007)

C. annuum CaSD1 Senescence-delaying Seo et al. (2013)

C. annuum CaMsrB2 Methionine sulfoxide reductase

B2 gene

Oh et al. (2010)

C. annuum orf456 Cytoplasmic male sterility Kim et al. (2007)

C. annuum PmsM1-CAPS Genetic male sterility Lee et al. (2010a)

C. annuum ms1,ms3 Genetic male sterility Lee et al. (2010b,c)

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Fruit colors

The white, yellow, orange, red and chocolate color of

Capsicum fruits depends on the intensity and amount of

accumulation of carotenoids pigments and xanthophylls

produced during ripening. More than 30 different pigments

have been identiﬁed in the pepper fruits (Bosland and

Votava 2000). The red chili and paprika carotenoids used

by the food industry as natural red colorants also have

immense nutritional value as it contains provitamin A and

antioxidant properties. Improvement of Capsicum for

carotenoid and nutrient content is the goal of several

breeding. Three independent loci have been reported to

control the mature pepper fruit (cl,c2 and y) in different

allelic combination, that is, dominant alleles at these three

loci results in red compared to white color when all alleles

are recessive (Hurtado-Hernandez and Smith 1985).

Genetic linkage analyses showed that c2 and yloci corre-

spond to genes that encode phytoene synthase (Psy) and

capsanthin–capsorubin synthase (Ccs), respectively (Le-

febvre et al. 1998; Popovsky and Paran 2000; Thorup et al.

2000; Huh et al. 2001). The deletion of the Ccs gene is

reported to cause yellow and orange fruit color (Lefebvre

et al. 1998; Lang et al. 2004). Kim et al. (2010b) reported

that a splicing mutation in the gene encoding Psy causes

orange coloration in Habanero pepper fruits.

To further understand the pigmentation process and the

expression of those three genes in different fruit color

capsicum genotypes, Ha et al. (2007) analyzed the levels

and composition of carotenoid accumulation in seven dif-

ferently colored (fully ripe) capsicum varieties at different

stages of ripening and correlated with the expression pat-

terns of the carotenoid biosynthetic genes. They observed

that red peppers accumulate increasing levels of total

carotenoids during ripening compared to non-red peppers.

The gene involved in the carotenoid biosynthesis pathway,

PSy, phytoene desaturase, and Ccs were highly expressed

in peppers with high levels of total carotenoids, whereas

one or two of these genes were not expressed in peppers

with lower levels of total carotenoids. Two structural

mutations in the Ccs gene were observed in two yellow

pepper varieties expressing this gene, suggesting that either

a premature stop codon or a frame-shift mutation might be

the cause for creating yellow phenotypes rather than

deletion of the gene itself.

Rodriguez-Uribe et al. (2012) again did a correlation

analysis of the carotenoid composition and transcript

abundances of four carotenogenic enzymes, Psy,LcyB,

CrtZ-2and Ccs, in seven orange-colored C. annuum cul-

tivars. In the cultivar ‘Fogo’, with premature termination

mutation in the ccs-3 allele, they could not observe the Ccs

protein although the transcript of this gene was found,

Fig. 1 Variability in the fruit morphology of different Capsicum species

Plant Biotechnol Rep

123

thereby preventing the synthesis of capsanthin and cap-

sorubin forms of carotenoids. In ‘Orange Grande’ and

‘Oriole’, both orange-colored cultivars, no red pigments

and Ccs transcript was detected although wild-type alleles

of all four carotenoids biosynthesis genes were there.

While the Ccs protein was not observed in ‘Canary’ cul-

tivar, the transcripts for all four of the wild-type alleles

were detected due to which no red carotenoids were syn-

thesized. This observation suggests that there might be a

non-structural gene which controls color development in

Capsicum fruits.

Apart from carotenoids, anthocyanin also controls pep-

per color and is found to be governed by the incompletely

dominant gene A. Ben-Chaim et al. (2003b) mapped the A

locus onto pepper chromosome 10 in a C. annuum

(5226) 9C. chinense (PI 159234) F

population. An

additional locus Fc, for the purple anther ﬁlament in an F

population from a cross of IL 579, a C. chinense intro-

gression line and its recurrent parent 100/63, was at the

same position as A. These two anthocyanin loci were found

to be linked to a major quantitative trait locus, fs10.1, for

fruit-shape index.

Pungency trait

Pungency, an economically important quality trait in pep-

per fruits, have been studied by many researchers. This trait

is due to the presence of capsaicinoid complex of which

capsaicin followed by dihydrocapsaicin are two major

components. Capsaicin is an amide derivative of vanillyl-

amine and 8-methylnon-trans-6-enoic acid (Bennett and

Kirby 1968). The capsaicin biosynthetic pathway is rela-

tively well characterized. Capsaicin biosynthesis in plants

is deﬁned by two pathways: phenylpropanoid (which

determines phenolic structure) and fatty acid metabolism

(which determines the molecule’s fatty acids; Ochoa-Alejo

and Go

´mez-Peralta 1993). The vanillylamine moiety of

capsaicinoids is derived from phenylalanine and synthe-

sized by the shikimate/arogenate pathway; whereas the

branched fatty acid moiety is derived from valine (Dı

´az

et al. 2004; Fig. 1). It is usually assumed that the vanil-

lylamine moiety is synthesized via the phenylpropanoid

pathway. The phenylpropanoid origin of the vanillylamine

moiety has been supported in the case of Capsicum species,

wherein transcript accumulation has been reported for the

genes Pal,Ca4h and Comt, corresponding to the phenyl-

propanoid pathway (Curry et al. 1999). So far, the most

elusive point in the pathway is capsaicinoid synthase,

which catalyzes the ﬁnal condensation of vanillylamine

and the branched-chain fatty acid. Curry et al. (1999) iso-

lated Pal,Ca4hand Comt genes from a cDNA library of

Habanero (C.chinense) placenta. The transcript levels of

these three genes were studied throughout development in

six cultivars differing in pungency in fruit tissues and

found that the abundance of transcripts were positively

correlated with the degree of pungency in placental tissues.

Habanero, the most pungent chile fruit, had the highest

transcript levels, CalWonder, a non-pungent fruit, had the

lowest levels. By using tissue-speciﬁc expression analysis

in fruits, it was also found that both the 3-ketoacyl-ACP

synthase (Kas) and the putative aminotransferase (pAmt)

sequences were only expressed at signiﬁcant levels in

placental tissues, where the capsaicinoids were synthe-

sized. Kim et al. (2001) generated a cDNA subtractive

library from C. chinense cv. Habanero utilizing placental

tissues. The group reported that SB2-149 and SB1-158

clones showed a high similarity to the pAmt and Kas genes.

Similarly, the SB2-66 and SB2-115 clones were also

thought to be involved in capsaicinoid biosynthesis.

Aluru et al. (2003) carried out a differential screen of a

Habanero (C. chinense) placenta cDNA library and char-

acterized three candidate genes: Kas,Acl and Fat. Tran-

scription of these three genes was found to be placental-

speciﬁc and RNA abundance was positively correlated with

degree of pungency. Kas and Acl were mapped to linkage

group 1 and Fat to linkage group 6. None of the genes was

linked to the pungency locus, C, on linkage group 2. Kas

accumulation was positively correlated with pungency.

Soon after, Stewart et al. (2005) found that the SB2-66

clone, previously isolated by Kim et al. (2001), co-segre-

gated with the pungency trait, and it was mapped to a locus

in close proximity to Pun 1 (locus C), which modiﬁes the

pungency level (Blum et al. 2002). Using genome walking,

the SB2-66 genomic DNA was isolated and compared with

certain sequences from pungent and non-pungent chili

peppers, showing that sequences from non pungent fruits

have a 2.5-kb deletion, encompassing part of the putative

promoter and the ﬁrst exon. That allele was named pun1,

and because the SB2-66 clone has acyltransferase domains

and labeled as At3. In order to demonstrate that At3 was

related to capsaicinoid production, virus-induced gene

silencing (VIGS) was done to silence the At3 gene. Lee

et al. (2005) also observed perfect co-segregation of

capsaicinoid synthetase (CS) gene with the C locus. The

sequence analysis from four pungent and four non-pungent

pepper lines revealed that the non-pungent peppers had a

2529 bp-deletion in the 50upstream region of CS gene.

They developed four SCAR markers which could be used

for early detection of non-pungent individuals in breeding

programs.

Quantitative trait loci analysis in a F

population derived

from an inter-speciﬁc cross between a mildly pungent C.

annuum ‘NuMex RNaky’ and the wild, highly pungent C.

frutescens accession BG2814-6 detected a total of six QTL

for three capsaicinoids: capsaicin, dihydrocapsaicin and

Plant Biotechnol Rep

123

nordihydrocapsaicin (Blum et al. 2003). Co-localization of

capsaicinoid biosynthetic pathway genes involved in valine

catabolism, BCAT and 3A2 were found with the detected

QTL for capsaicinoid on chromosomes 3 (cap3.1) and 4

(cap4.1), respectively. Stewart et al. (2007) analyzed the

At3 gene in a non-pungent C. chinense NMCA 30036 chili

pepper. The At3 gene sequence revealed a 4-bp deletion in

the ﬁrst exon, and this allele was named pun

. Due to that

deletion, the AT3 protein was not detected in NMCA 30036

fruits, but low levels of the transcript were detected in 20

and 50 DPA chili pepper fruits.

The more illustrated and updated capsaicinoid bio-

synthesis pathway has been described, where more than

30 putative genes have been placed in the capsaicinoid

biosynthetic pathway (Mazourek et al. 2009;Aza-Gon-

´lez et al. 2011). Mazourek et al. (2009) developed a

comprehensive model indicating speciﬁc genes in the

capsaicinoid biosynthesis pathway, which is made pub-

licly available within the SolCyc database at the SGN

(http://www.sgn.cornell.edu). Recently, Yarnes et al.

(2013) mapped 12 QTL for capsaicinoids content in

recombinant inbred lines derived from a cross between

Capsicum frutescens acc. 2814-6 and C. annuum var.

NuMexRNAKY.

Male sterility

Male sterility is an important trait required for pepper

hybrid breeding. Identiﬁcation of stable sterile lines and

fertility restorer genes is important requisite for hybrid

breeding. Wang et al. (2004) performed QTL analysis of

fertility restoration based on the test-cross progeny of

77013A (cytoplasmic-genetic male sterile line) and a

doubled haploid population derived from Yolo wonder

(sterility maintainer line) and Perennial line (a fertility-

restorer line). One major QTL, accounting for 20–69 % of

the phenotypic variation for fertility restoration was map-

ped to chromosome P6. Four additional minor QTL were

also detected on chromosomes P5 and P2 and LGs PY3 and

PY1, explaining about 7–17 % of the phenotypic variation.

SCAR markers for early identiﬁcation of cytoplasmic male

sterility (CMS) genotype in C. annuum L. were reported by

Kim and Kim (2005). Two CMS-speciﬁc SCAR markers

were developed to distinguish N-cytoplasm from S-cyto-

plasm. A novel ORF, termed orf456, was found at the 30

end of the coxll gene which might represent a strong can-

didate gene from among the many CMS-associated mito-

chondrial genes for determining the male-sterile phenotype

of CMS in chili pepper (Kim et al. 2007).

CMS in chili pepper is restored by one major dominant

nuclear gene, restorer of fertility (Rf), together with some

modiﬁer genes and is also affected by temperature (Lee

et al. 2008). Lee et al. (2010a) developed a co dominant

cleaved ampliﬁed polymorphic sequence (CAPS) marker

named PmsM1-CAPS, tightly linked to genetic male ste-

rility (GMS) in paprika and colored pepper, using bulked

segregant analysis (BSA) which could be applied in

Paprika breeding for a GMS system. Further, they devel-

oped a co-dominant SCAR marker and three AFLP

markers which were later converted to CAPS markers

linked to the genic male sterility genes ms1 and ms3,

respectively, in peppers (Lee et al. 2010b,c). Chen et al.

(2011), through a cDNA-AFLP analysis in the genic male

sterile–fertile line 114AB of C. annuum L., identiﬁed a

transcript-derived fragment (TDF) which speciﬁcally

accumulated in the ﬂower buds of the fertile line but not in

the sterile line. The identiﬁed anther-speciﬁc gene named

as capsicum male fertile 2 (CaMF2) encodes a lipid

transfer protein with 94 amino acids and VIGS of this gene

showed low pollen germination ability and shrivelled pol-

len grains, suggesting a vital role of CaMF2 in the pollen

development of C. annuum. Ma et al. (2013) studied the

efﬁciency of maintaining and restoring CMS systems in

pepper (C. annuum L.). An Rf-linked molecular marker

was employed to analyze the interaction between six CMS

lines (A), ﬁve maintainers (B) and six restorers (C). They

observed that the six restorers had diverse restoring abili-

ties in individual CMS lines and concluded that the

restorers 208C and 207C can transmit the Rf gene or the Rf-

linked marker to F

hybrids.

Trichomes

Trichomes, which are found in most land plants, are known

for protecting against insect herbivores, drought and UV

radiation. The trichome-bearing phenotype is conferred by

the dominant allele of the pepper trichome locus 1 (Ptl1)in

C. annuum. Kim et al. (2010a) ﬁne-mapped the trichomes

trait in Capsicum annuum in an F

population derived from

a cross involving CM334 (with trichome) and Chilsungcho

varieties. The trichome locus Ptl1 region screened in BAC

libraries and found in one 80 kb BAC clone, TT1B7, which

further was found to co-localize in chromosome 10 by

ﬂuorescence in situ hybridization (FISH). Two closely

linked markers, one co-dominant (Tco) and one dominant

(Tsca), were successfully developed from the TT1B7 BAC

sequence that could be used for screening the presence of

this trait in segregating population or for markers assisted

breeding. Further study using F

population derived from

the parental lines, that is, C. annuum CM334 (trichome-

bearing and PepMoV resistant) and Chilsungcho (glabrous

and Pep-MoV susceptible) detected two major QTL each

for trichome enhancing (Ptel1 and Ptel2) and pepper mottle

virus resistance QTL (pep1 and Pep2 on LG 24 or

Plant Biotechnol Rep

123

chromosome 10; Kim et al. 2011). While Ptel1 on chro-

mosome 10 (LG24) contributed for stem trichome, Ptel2 on

LG 2 showed major QTL for calyx trichome. Pep1 was co-

mapped within the same genetic interval of Ptel1 in the

R-gene cluster (RGC) for potyvirus resistance including

Pvr4, a locus showing broad spectrum resistance to poty-

viruses, thereby suggesting the high-density trichome on

the pepper main stem can be used as a morphological

marker for potyvirus resistance breeding in peppers. Seo

et al. (2013) recently isolated the secreted protein gene

Capsicum annuum senescence-delaying 1 (CaSD1), the

gene family of which is known for multiple roles in plant

development, metabolism and stress response, and trans-

formed in Nicotiana benthamiana. The transformed plants

leaves resulted in delayed senescence of leaves, increasing

the number of trichomes and enlarging the epidermal cells.

Disease resistance

Phytophthora resistance

Phytophthora capsici Leonian is one of the most destruc-

tive pathogen of peppers which attacks all parts of the

plant, causing symptoms such as root rot, crown rot, stem

lesion, fruit rot, damping-off of seedlings and so on.

(Ristaino 1990; Quirin et al. 2005). Resistant sources for

this pathogen have been identiﬁed by many researchers,

such as ‘CM334’ from Mexico, ‘AC2258’, ‘PI201232’ and

‘PI201234’ from Central America, among which ‘CM334’

has found to get the highest resistance level (Bosland and

Lindsey 1991; Ortega et al. 1992; Quirin et al. 2005).

Thabuis et al. (2003) mapped QTL for P. capsici resistance

in three C.annuum populations derived from independent

resistance and susceptible parental lines. Of the 18 QTL

detected, one major loci on chromosome 5 was common in

all the three populations, while another locus was common

to two populations and the remaining were cross-speciﬁc.

Transfer of the resistance factors from CM334 into a bell

pepper genetic background was reported by Thabuis et al.

(2003). Quirin et al. (2005) screened a large number of C.

annuum and C. chinense with RAPD markers and found

one RAPD marker OpD04 to be tightly linked to highly

resistant lines. After converting this RAPD marker into

SCAR markers, they mapped into capsicum genome and

observed mapping of these markers to previously reported

P. capsici resistant locus Phyto.5.2 on chromosome 5,

suggesting that the Phyto.5.2 QTL was widely distributed

in highly resistant germplasm. The markers developed

could be used for the rapid selection of highly resistant

genotypes in peppers. Ogundiwin et al. (2005) mapped P.

capsici resistance QTL in two populations, one RIL and

another F

, derived from resistant and susceptible parental

crosses. A total of 16 chromosomal intervals containing

single or clusters of resistance QTL for root rot and (or)

foliar blight were detected, thereby indicating a complex

set of genetics involved in resistance to P. capsici. Another

ﬁve QTL were detected in the F

population that conferred

resistant to root rot.

Kim et al. (2008b) mapped four QTL for root rot and

three QTL for damping off in an intraspeciﬁc F

population

derived from a cross between Mexican resistant landrace C.

annuum ‘CM334’ and a susceptible Korean landrace C.

annuum ‘Chilsungcho’. They developed one single-nucle-

otide ampliﬁed polymorphic marker (SNAP) and two

SSRs, and CAPS markers from BAC clones which could

be used for the selection of genotypes resistant to P. cap-

sici. A pepper (C. annuum) methionine sulfoxide reductase

B2 gene (CaMsrB2) was isolated, and its roles in plant

defense were studied (Oh et al. 2010). Measurement of

ROS levels in CaMsrB2-silenced pepper plants revealed

that suppression of CaMsrB2 increased the production of

ROS, which in turn resulted in the acceleration of cell

death via the accumulation of ROS.

Li et al. (2011) identiﬁed nine Pme genes (Pcpme 1–9)

from a genomic library of highly virulent P. capsici strain

SD33 and analyzed the expression pattern of nine genes on

three hosts: pepper, tomato and cucumber using qRT-PCR

during all stages of infection. All nine genes were found to

be differentially expressed in three host species in the

course of P. capsici interaction. The expression levels of

the respective genes increased from 1 to 7 dpi in pepper.

Thus, evidence of Pcpme gene expression in different

plants at a range of time points suggests that the late stages

of infection may represent the most critical time for P.

capsici to successfully express or/and secret PMEs. Reeves

et al. (2013) reported a novel disease resistance inhibitor

gene [inhibitor of P. capsici resistance (Ipcr)], found in the

chile pepper (C. annuum) variety ‘New Mexico Capsicum

Accession 10399’ (NMCA10399), which inhibits resis-

tance to Phytophthora capsici but not to other species of

Phytophthora.

Wang et al. (2013) reported the cloning and isolation of

three polygalacturonase-inhibiting proteins (PGIPs) from

peppers which play the role of inhibiting fungal endo-

polygalacturonases (PGs). The three PGIPs were up-regu-

lated at different times following stimulation of the pepper

leaves by P. capsici and abiotic stresses. Silencing of these

genes in peppers showed enhanced activity of susceptibil-

ity to P. capsici while the application of puriﬁed recom-

binant proteins individually inhibited the activity of PGs

produced by Alternaria alternata and Colletotrichum ni-

cotianae, respectively. The crude protein from the CaP-

GIP1 transgenic tobacco enhanced resistance to the PG

activity of both fungi and a signiﬁcant reduction in the

number of infection sites, number and average size of

Plant Biotechnol Rep

123

lesions in the leaves, suggesting that CaPGIPs conferred

defense response and play an important role in a plant’s

resistance to diseases. Zhang et al. (2013) isolated and

characterized a P. capsici resistance gene, CaRGA2, from a

high-resistance pepper (C. annuum, CM334). Quantitative

real-time PCR analysis indicated that CaRGA2 was rapidly

induced by P. capsici evidenced by showing very high and

rapid expression in the resistant line compared to the sus-

ceptible cultivars, and when the gene was silenced, the

resistance level was clearly suppressed, suggesting

involvement of CaRGA2 gene in conferring resistance

against P. capsici.

WRKY proteins are a class of zinc ﬁnger-containing

transcription factors (TFs) which are involved in regulating

defence against biotic stresses or tolerance of abiotic

stresses in plants (Rushton et al. 2010). WRKY TFs from

Capsicum species are found to positively and negatively

regulate resistance to bacterial disease caused by Ralstonia

solanacearum, a devastating soil-borne bacterium causing

wilting disease in pepper. Overexpression of C. annum

WRKY 40 (caWRKY40) enhanced resistance to R. solan-

acearum and tolerance to heat shock in tobacco, while

silencing of this gene enhanced susceptibility to R. solan-

acearum and impaired thermotolerance in pepper (Dang

et al. 2013). Further analysis found that CaWRKY40 tran-

scripts was induced by signalling mechanisms mediated by

the stress hormones salicylic acid (SA), jasmonic acid (JA)

and ethylene (ET). Another gene, CaWRKY58, was shown

to negatively regulate the resistance to R. solanacearum

(Wang et al. 2013). The CaWRKY58-overexpressed

tobacco plants showed enhanced susceptibility coupled

with the decreased expression of hypersensitive response

(HR) and defence-associated genes. In contrast, CaW-

RKY58 silenced pepper plants displayed enhanced resis-

tance to the highly virulent R. solanacearum strain

FJC100301 coupled with the enhanced transcripts of

defence-related pepper genes.

Bacterial and fungal spot diseases

Xanthomonas campestris pv. vesicatoria (Xcv) causes the

bacterial spot disease in peppers. A total of six genes, four

dominant (Bs1 to Bs4) and three recessive genes (bs5 and

bs6, gdr) were reported conferring resistance to Xcv (Jones

et al. 2002,2004; Csillery et al. 2004). AFLP markers

tightly linked to Bs2 gene conferring resistance to Xan-

thomonas campestris pv vesicatoria was detected and then

subsequently cloned and expressed in tomato (Tai et al.

1999a,b). The identiﬁed Bs2 gene encodes motifs char-

acteristic of the nucleotide binding site leucine-rich repeat

class of resistance genes which controls the hypersensitive

response when transiently expressed in susceptible pepper

and tomato lines, and in a non-host species, Nicotiana

benthamiana. Pierre et al. (2000), using BSA of resistant

and susceptible plants, identiﬁed AFLP markers linked to

the Bs3 gene conferring resistance to Xanthomonas cam-

pestris pv. vesicatoria which were converted into locus-

speciﬁc PCR-based markers and mapped in a linkage map

with a genetic resolution of 0.13 cM. Vallejos et al. (2010)

identiﬁed bs5 and bs6 resistance genes in breeding line

ECW12346 showing resistance to race P6 in C. annuum.

The combined effect of these two genes gave full resistance

to race P6 (indicating a positive epistatic interaction) and

allele-speciﬁc PCR primers for bs5 were developed to

facilitate the genetic manipulation of this gene.

C. annuum peroxidase (CaPO2), an extracellular gene,

was isolated from pepper (C. annuum) and their role in

defence against Xanthomonas campestris pv. vesicatoria

was studied (Choi et al. 2007). CaPO2-silenced pepper

plants were highly susceptible to Xcv infection and showed

compromised hydrogen peroxide (H

) accumulation and

hypersensitive cell death in leaves. The overexpression of

CaPO2 in Arabidopsis conferred enhanced resistance to

Xcv accompanied by cell death, H

accumulation and

PR gene induction, suggesting involvement of this gene in

ROS generation, both locally and systemically, activation

of cell death and PR gene induction during the defense

response to pathogen invasion.

Another pathogen causing bacterial spot is Colletotri-

chum gloeosporioides. Voorrips et al. (2004) mapped QTL

for resistance to this pathogen using F

population derived

from a cross involving resistant parent C. chinense and a

susceptible line C. annuum. One major resistant QTL was

identiﬁed for the infection frequency, the true lesion

diameter and the overall lesion diameter for C. gloeospo-

rioides apart from detecting three other small effect QTL

for overall lesion and true lesion diameter. Kim and Hwang

(2012) isolated a pepper (C. annuum) mildew resistance

locus O gene (CaMLO2) which is transcriptionally unreg-

ulated by Xanthomonas campestris pv. vesicatoria infec-

tion, salicylic acid and abiotic stresses. Silencing of

CaMLO2 in pepper plants confers enhanced resistance

against virulent Xcv accompanied by a compromised sus-

ceptibility cell-death response and reduced bacterial

growth, as well as an accelerated reactive oxygen species

burst. Pathogen infection induced expression of the sali-

cylic acid-dependent defense marker gene CaPR1 in

CaMLO2-silenced leaves while CaMLO2 over-expression

in Arabidopsis enhances susceptibility to Pseudomonas

syringae pv. Tomato (Pst) and Hyaloperonospora arabid-

opsidis. In contrast, leaves of plants over-expressing

CaMLO2 exhibit a susceptibility cell-death response and

high bacterial growth during virulent Pst infection. This

suggests that CaMLO2 is involved in the susceptibility

cell-death response and bacterial and oomycete prolifera-

tion in pepper and Arabidopsis.

Plant Biotechnol Rep

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Powdery mildew

This disease caused by Leveillula taurica is important

pathogen in pepper. Lefebvre et al. (2003) mapped pow-

dery mildew resistance QTL in a doubled-haploid popu-

lation derived from crossing ‘H3’ (resistant) and ‘Vania’

(susceptible) by phenotyping in both natural ﬁeld infection

and artiﬁcial inoculation tests. A total of seven genomic

regions including additive QTL and epistatic interactions

were detected, of which two genomic regions were com-

monly detected by both the evaluation methods.

Virus diseases

Viruses, the intracellular obligate pathogens, are important

to study as they damage many cultivated Capsicum species

causing huge loss to the crops. Several resistance gene

sources have been identiﬁed against tobacco mosaic virus

(TMV), potyviruses, cucumber mosaic viruses (CMVs) and

so on. Some of the resistances are conferred by the plant’s

HR reaction resulting in cell death of the invading cells,

thereby blocking further multiplication and movement of

the viruses in the resistant phenotypes.

Potyviruses

Peppers are found to be infected by potyviruses such as

potato virus Y (PVY), tobacco etch virus (TEV), pepper

mottle virus, pepper veinal mottle virus and chili veinal

mottle virus (Green and Kim 1991). Although many

resistance genes have been reported, Kyle and Palloix

(1997) re-designated them into six resistance genes,

namely pvr1,pvr2

,pvr22,pvr3,pvr4 and pvr5, based on

their chronological order of identiﬁcation. The ﬁrst iden-

tiﬁed and cloned resistance gene against potyvirus was

pvr2 which was analyzed and mapped on chromosome 4

(Murphy et al. 1998; Kang et al. 2005). This recessive

resistance gene against PVY was later found to be

eukaryotic translation initiation factor 4E (eIF4E; Ruffel

et al. 2002). This gene codes for 228 amino acid and

sequences of resistant lines was found to differ for two

amino acids from the susceptible lines. Further, they sug-

gested that the interaction between the potyvirus genome-

linked protein (VPg) and eIF4E are important for virus

infectivity, and resistance could be due to incompatibility

between the VPg and eIF4E. Another recessive resistance

gene, pvr6, against potyviruses was mapped on chromo-

some 3 (Caranta et al. 1996). Pvr7, a dominant resistant

gene against the pepper mottle potyvirus (PepMoV) from

C. chinense Jacq. ‘PI159236’ was reported to be tightly

linked to the dominant potyvirus resistance gene Pvr4

(Grube et al. 2000). This gene was mapped in pepper

chromosome 10 using an interspeciﬁc pepper map. They

reported the ﬁrst identiﬁed dominant resistance gene clus-

ter of Pvr4,Pvr7 and Tsw [a gene conferring resistance to

tomato spotted wilt virus (TSWV)] in Capsicum species.

Caranta et al. (1997b) mapped 11 resistant QTL for two

PVY isolates and two potyvirus E in a doubled-haploid

progeny from the hybrid between the Perennial resistance

line and the susceptible Yolo Wonder line. Upon com-

parison, few QTLs were detected in the vicinity of the pvr2

and pvr6 loci. Caranta et al. (1999) developed CAPS

marker tightly linked to the Pvr4 resistance gene in pepper

which confers a complete resistance to the three pathotypes

of PVY and pepper mottle virus (PepMoV). Arnedo-An-

dres et al. (2002) reported the development of SCAR

markers for Pvr4 from a RAPD marker against resistance

to PVY in pepper (C. annuum L.). Rubio et al. (2008)

developed functional markers based on single nucleotide

polymorphisms (SNPs) at the pvr2-eIF4E locus that confer

resistance to several potyviruses. They claimed that the

developed SNP based markers could be used for marker-

assisted selection of virus-resistant paper genotypes in

breeding programme. It has been reported that double and

simultaneous mutations in translation initiation factors

eIF4E (commonly known as pvr2) and eIF (iso) 4E9

(known as pvr6) are required to prevent pepper veinal

mottle virus infection in pepper (Ruffel et al. 2006; Hwang

et al. 2009; Rubio et al. 2009).

Cucumber mosaic virus (CMV) is one of the most

destructive viruses in the Solanaceae family. Caranta et al.

(1997b) ﬁrst reported mapping three QTL for resistance to

CMV using a DH lines derived from crossing between a

partially CMV-resistant C. annuum perennial line and Yolo

Wonder, a CMV-susceptible C.annuum line. The three

QTL were found explaining together a total of 57 % phe-

notypic variation. They also identiﬁed a digenic epistasis

interaction between a locus controlling signiﬁcant trait

variation and a second locus that by itself had no demon-

strable effect on the trait. Ben-Chaim et al. (2001) mapped

four main effect QTL for resistance to CMV using an

intraspeciﬁc C. annuum F

mapping population from a

cross involving a susceptible bell type cultivar Maor and

the resistant small-fruited Indian perennial line. Two di-

genic interactions were observed and the QTL controlling

the largest percentage (16–33 %) of the observed pheno-

typic variation (cmv11.1) was found linked with the Llocus

conferring resistance to TMV which was reported by

Pochard et al. (1983). Caranta et al. (2002) mapped QTL

using doubled haploid mapping populations derived by

crossing a partial resistance parent ‘Vania’ and a suscep-

tible line ‘H3’. One major and six minor effects QTL were

found to be associated with the partial resistance to long

distance movement of the CMV viruses. Kang et al. (2010)

showed that the C. annuum cultivar ‘Bukang’ contains a

Plant Biotechnol Rep

123

single dominant resistance gene Cucumber mosaic resis-

tance 1 (Cmr1) against CMV (Korean) and CMV (FNY)

strains. Genetic mapping and FISH analysis revealed that

the Cmr1 gene is located at the centromeric region of LG2,

syntenic to the ToMV resistance locus (Tm-1) in tomatoes.

Three SNP markers, one intron-based marker based on

pepper homolog of Tm-1, and two SNP markers using

tomato and pepper BAC sequences mapped near Cmr1.

Tomato Spotted Wilt Virus

Resistance source of TSWV has been reported in C.

chinense (Moury et al. 2000). The resistance to this virus is

govern by single dominant gene, Tsw. Bulk segregant

analysis using RAPD markers in 153 F

individual derived

from crossing between a resistant C. chinense and a sus-

ceptible cultivar detected 4 linked RAPD markers. One

tightly linked RAPD marker was converted into a co-

dominant CAPS. The developed tightly linked CAPS

marker to Tsw was used for marker-assisted selection for

resistance traits. Jahn et al. (2000) also mapped Tsw gene in

distal portion of chromosome 10 in C. annuum. However,

recently naturally occurring resistance-breaking (RB)

TSWV strains have been identiﬁed. Analysis conﬁrmed

that S RNA, which encodes both the nucleocapsid protein

(N) and a nonstructural protein (NSs), carries the genetic

determinant responsible for Tsw resistance breakdown

(Margaria et al. 2007). Comparative sequence analysis of

full-length S RNA segments or proteins between the wild

type and RB strains gave indirect evidence that the NSs

protein is the avirulence factor in question. Characteriza-

tion of two RB strains carrying deletions in the NSs protein

showed that NSs is important in maintaining TSWV

infection in newly emerging leaves over time, thereby

giving evidence that local necrotic response is not sufﬁ-

cient for resistance in Capsicum spp. carrying the Tsw

gene. Lovato et al. (2008) showed through over-expression

analysis that N protein of TSWV is associated with the

induction of programmed cell death (PCD) in C.chinense

plants by hypersensitive reaction.

Tobacco Mosaic Virus

Genetic analysis of resistance to TMV was reported to be

governed by several alleles of the L gene. Subsequently

L1 gene showing TMV resistant was mapped in chro-

mosome 11 of C.annuum genome (Lefebvre et al. 1995;

Ben-Chaim et al. 2001). Matsunaga et al. (2003) detected

a RAPD marker linked to the pepper mild mottle virus

(PMMoV) resistant locus (L4) and developed a tightly

linked SCAR marker ‘WA31-1500S’ in Capsicum spe-

cies. Ampliﬁcation of this SCAR marker in a few geno-

types showed speciﬁc bands for both the resistant and

susceptible genotypes which they suggested, could be

used for breeding resistant cultivar for PMMoV. Tomita

et al. (2008) ﬁne-mapped the tobamovirus resistance gene

L3 of C. chinense in a 400-kb region of R-like genes

cluster embedded in highly repetitive sequences. Lim

et al. (2011) isolated C. annuum WRKYb (CaWRKYb)

gene, which is rapidly induced during TMV pathotype P

infection in hot pepper. They observed that a CaWRKYb-

knockdown plant showed reduce resistance level by

reduction of the number of hypersensitive response local

lesions upon TMV-P

infection and exhibited an accu-

mulation of more TMV through decreased expression of

the C. annum pathogenesis-related genes CaPR-10,

CaPR-1and CaPR-5, suggesting that CaWRKYb plays as

a positive transcription factor in defense-related signal

transduction pathways in hot pepper. Huh et al. (2012b)

analyzed the function of C. annuum basic transcription

factor 3 (CaBtf3) through VIGS and found that CaBtf3

was involved in HR cell death against (TMV-P

) infection

by inducing a transcriptional change in defense-related

genes. Huh et al. (2012a) characterized C. annuum

WRKY transcription factor d (CaWRKYd) isolated from a

microarray analysis in TMV-P

-infected hot pepper

plants. In their experiment they found that CaWRKYd

transcripts were induced by various hormone treatments

and (TMV-P

) inoculation. Silencing of this gene affected

TMV-P

-mediated HR cell death and the accumulation of

TMV-P

coat protein in local and systemic leaves, which

showed a reduction of expression of some pathogenesis-

related (PR) and HR-related genes, suggesting that this

gene regulates HR cell death. Another gene, WRKY

transcription factor b (CaWRKYb) was found to bind the

CaPR-10 promoter and functions as a positive regulator in

innate immunity upon TMV infection by acting as a

positive transcription factor in defense-related signal

transduction pathways in hot pepper (Lim et al. 2011).

Virus-induced gene silencing is based on the sequence-

speciﬁc degradation of RNA. Hernan et al. (2013) reported

a gene silencing vector derived from EuMV-YP, named

pEuMV-YP:DAV1, to silence NPR1 genes in both N.

benthamiana and C. annuum. After silencing, the plants’

viral symptoms increased to levels similar to those seen in

wild-type plants. These results suggest that NPR1 plays a

role in the compatible interactions of EuMV-YP N.

benthamiana and EuMV-C. annum var. anaheim. Bento

et al. (2013) investigated inheritance of resistance to Pep-

per yellow mosaic virus (PepYMV) in progenies derived

from a cross involving C. baccatum var. pendulum acces-

sions UENF 1616 (susceptible) and UENF 1732 (resistant)

lines. Genetic analysis showed that the minimum number

of genes that control resistance was seven, thereby sug-

gesting polygenic and complex inheritance of resistance to

this disease.

Plant Biotechnol Rep

123

Root-knot nematode disease

Meloidogyne species, causing root knot diseases are

important pathogens in many crop plants including Cap-

sicum species. Many genes showing resistance to root-knot

nematodes are reported mainly in C. annuum. Thies and

Fery (2000,2002) reported characterization of nematode

resistance gene N. Genes that were stable at high temper-

ature were reported in three distinct genotypes showing

highly resistant phenotypes plants, namely PI 322719, PI

201234 and CM334. The genes Me4,Mech1 and Mech2

were shown to be speciﬁc to certain Meloidogyne species

or populations, whereas Me1,Me3 and Me7 were non-

speciﬁc and effective against wide range of root knot

pathogen species (Djian-Caporalino et al. 2001,2007).

Djian-Caporalino et al. (2001) reported mapping of Me

and Me

resistance genes in a segregating population

derived from the cross of resistant parental line PM687

with a susceptible line and found both genes to be linked

together. Recently the six genes mentioned above were

found to be clustered in the P9 chromosome of pepper.

Through allelism tests and ﬁne mapping using the BSA-

AFLP approach, these genes were reported to be different

but linked to form a resistant gene cluster within a 28 cM

interval of P9 chromosome containing other disease resis-

tance genes. Comparative analysis of this P9 region was

found to be co-linear with chromosome T12 of tomato and

chromosome XII of potato containing four other nematode

resistances, suggesting that these nematode resistance

genes are located in orthologous genomic regions in

Solanaceae (Djian-Caporalino et al. 2007). Chen et al.

(2007) isolated a nematode resistant gene CaMi and

expressed in transgenic tomato plants. Transformed plants

carrying CaMi showed signiﬁcantly improved resistance

against the root-knot nematodes compared to untrans-

formed susceptible plants by triggering a hypersensitive

response around nematodes infection. Recently, Fazari

et al. (2012) found co-localization of N-gene in the Me-

genes cluster on the P9 chromosome on pepper. Based on

their study, they reported that Nwas mapped at 7 cM from

Me1 and 2 cM from Me3 and N gene was found to be

allelic to Me7 through allelism tests. They developed PCR

based markers tightly linked to Me- and N-genes which

could be very useful for the marker-assisted breeding of

nematode resistance in pepper.

Tissue culture and genetic transformation in Capsicum

translational research

In vitro plant regeneration from cells, tissues and organ

cultures is a fundamental process for plant propagation,

plant breeding and the genetic improvement of plants.

Gunay and Rao (1978) for the ﬁrst time tested different

hormonal regimes for organogenesis in Capsicum. Coty-

ledon and hypocotyl explants of two C. annuum cultivars

(Pimento and California Wonder) and a hybrid of C.

frutescens (Bharath) were cultured on Murashige and

Skoog (1962) medium containing the auxins, indole-3-

acetic acid (IAA), naphthaleneacetic acid (NAA), 2,4-

dichlorophenoxyacetic acid (2,4-D) and the cytokinins,

benzyladenine (BA), kinetin (KN), Zeatin (Zea), Zeatin

(Zea), 6-benzyl 9-tetra hydropyrane adenine (SD 8339),

adenine and coconut milk. Several other attempts have

been made on in vitro plant regeneration of the genus

Capsicum through different protocols and morphogenic

pathways (Reddy et al. 2002; Venkataiah et al. 2003;

Anilkumar and Nair 2004; Peddaboina et al. 2006; Khan

et al. 2006; Mezghani et al. 2007). An attempt was made by

us to develop an efﬁcient, simple and promising protocol

for in vitro plantlet regeneration of Naga King Chili

belonging to C. chinense from nodal segments and shoots

tips in MS medium containing 18.16 lM thidiazuron

(TDZ; Kehie et al. 2012a). The number of shoots devel-

oped was nearly twofold more than reported earlier (San-

atombi and Sharma 2008; Fig. 2). This protocol may be

applied for a large-scale propagation of individual geno-

types of this species of chili besides using in conservation.

The production of capsaicinoids, the hot principle of

chili pepper fruits, by cells and callus tissues has been

another area of intense research. The biosynthetic capacity

of in vitro cultured cells and tissues to produce capsaici-

noids has been investigated by different researchers

(Lindsey et al. 1983; Mavituna and Park 1985; Ravishankar

et al. 1998; Salgado-Garciglia and Ochoa-Alejo 1990;

Ochoa-Alejo and Salgado-Garciglia 1992; Sudha and

Ravishankar 2002; Nunez-Palenius and Ochoa-Alejo 2005;

Gutie

´rrez-Carbajal et al. 2010; Kehie et al. 2012b). Cell

suspension cultures and immobilized cells can be adopted

for the production of food additives or pharmaceuticals by

biotransformation processes.

Genetic manipulation is an attractive proposition where

it involves recombination of an efﬁcient cell or tissue

culture regeneration system with recombinant DNA tech-

nology, which would transfer speciﬁc genes from other

taxa, or the modiﬁed expression of speciﬁc native genes

(Kothari et al. 2010). In chili pepper, application of genetic

transformation has been limited due to lack of efﬁcient

transformation. Genetic transformation in chili pepper was

ﬁrst reported by Liu et al. (1990) employing in vitro

seedling explants co-cultured with the wild tumorigenic

strains A281 and C58 of Agrobacterium tumefaciens and

with a disarmed strain bearing the plasmid pGV 3850. Zhu

et al. (1996) obtained transgenic sweet pepper from

Agrobacterium-mediated transformation using Agrobacte-

rium strain GV311-SE harboring cucumber mosaic

Plant Biotechnol Rep

123

cucumovirus coat protein (cms-cp) gene. Kim et al. (1997)

investigated RNA-mediated resistance to cucumber mosaic

virus in progeny of transgenic plants of hot pepper that

expresses RNA. Several workers have attempted to

transform Capsicum species but with less than 0.1 %

transformation efﬁciency were obtained (Jayashankar

et al. 1997). A highly efﬁcient genetic transformation

system using pepper cotyledons as explants was estab-

lished by Li et al. (2003). A. tumefaciens strain LBA 4404

with the plasmid pBI 121 was employed for transforma-

tion of pepper. Solı

´s-Ramos et al. (2009) described the

Agrobacterium tumefaciens-mediated transformation of

Habanero chili pepper (C. chinense) with the gene

WUSCHEL from Arabidobsis thaliana under the control

of an estradiol inducible promoter as a means of over-

coming the somatic embryogenesis formation recalcitrance

of this species. Several attempts have been made for

regeneration and genetic transformation of Capsicum

species (Liu et al. 1990; Engler et al. 1993; Lee et al.

1993; Zhu et al. 1996; Kim et al. 1997; Jayashankar et al.

1997; Manoharan et al. 1998; Phillips et al. 2000; Delis

et al. 2005; Sharma et al. 2008). Recently, Gururaj et al.

Fig. 2 Development of an

efﬁcient protocol for

regeneration of C. chinense that

could be used in translational

research. aSeed germination of

C. chinense in MS medium (Bar

1 cm) producing explants.

bMultiple shoots induction

from nodal segments of C.

chinense in MS medium

containing 18.16 lM TDZ after

5 weeks culture (Bar 1 cm).

cElongation and root induction

of regenerated shoots in MS

medium containing 5.70 lM

indole-3-acetic acid (IAA) (Bar

1 cm). dRegenerated hardened

plantlet transferred to earthen

pot (Bar 10 cm). eHardened

plantlet bearing normal fruit in

green house (Bar 10 cm).

fField-grown plantlets bearing

fruits

Plant Biotechnol Rep

123

(2012) reported the functional validation of putative ami-

notransferase gene for vanillylamine biosynthesis in het-

erologous system using Agrobacterium mediated genetic

transformation studies in C. frutescens calli cultures. At

present, apart from Agrobacterium tumefaciens mediated

genetic transformations, other methods such as particle

gun bombardment, electroporation, etc. are emerging as

the methods of choice for introduction of agronomically

important genes for quality improvement, regulation of

secondary metabolites, and for engineering biotic and

abiotic stress resistances. Increased production of capsa-

icins, carotenoids and vitamins content through genetic

engineering would help in the maximum use in pharma-

ceutical, nutraceutical and food industry.

Comparative mapping: identifying genes/QTL using

information from related species

To date, the most characterized and well studied species

belonging to the Solanaceae family is tomato. In the early

1990s tomato RFLPs markers were used for the develop-

ment of linkage maps in peppers (Tanksley et al. 1988).

Comparative mapping in several studies reported that,

among plant species belonging to the same family, there is

a macro- and micro-level conservation of gene order and

chromosomal structure, although divergence and fractio-

nations of genomes are observed (Livingstone et al. 1999).

Through comparative mapping of pepper genome with

tomato, several syntenic regions were found apart from

observation of 30 breakpoints between the genome of two

species. Thorup et al. (2000) mapped 10 structural genes

from the pepper carotenoid biosynthetic pathway in C.

annuum 9C. chinense F

genetic map anchored in Lyc-

opersicon (tomato). The position of these genes in the

pepper map was compared with the position of those genes

in tomato. Mapping results showed that the color-produc-

ing genes C2 and capsanthin–capsorubin synthase (Ccs)

co-segregated with phytoene synthase and Y, respectively,

on chromosome 6. A comparison of map position between

species showed that Ccs in pepper mapped to a homolo-

gous region containing the Blocus for hyperaccumulation

of b-carotene in tomato; the lycopene b-cyclase gene in

pepper mapped to the corresponding region containing

gene lutescent-2mutation in tomato; and the lycopene

cyclase locus in pepper corresponded to the lycopene

cyclase locus y Del mutation for hyperaccumulation of D-

carotene in tomato. Further, co-mapping of structural genes

and quantitative loci for pepper and tomato fruit color were

observed. Their observations suggested that comparative

analyses using candidate genes from one species could be

used to isolate homologs in related species.

A comparative analysis of 13 genes involved in antho-

cyanin pigmentation was done in pepper, tomato, eggplants

and potato, and found conservation of the anthocyanin

producing gene, an2, among those species, suggesting that

a conserved orthologous regulatory locus might have been

subjected to parallel selection in the domestication of many

solanaceous crops. Zygier et al. (2005) using a pepper

population of introgressed lines from C. chinense and C.

frutescence for chromosomes 2 and 4, mapped QTL for

fruit weight and shape, and compared with the tomato QTL

for those traits. A major fruit-weight QTL fw2.1 in chro-

mosome 2 explaining about 62 % phenotypic variation and

a minor fruit shape QTL fs.1 were syntenic to the tomato

chromosomal region containing fruit-shape gene ovate.In

chromosome 4, two fruit-weight QTL (fw.1 and fw4.2), one

of which co-mapped with a fruit-shape QTL were detected,

suggesting the pleiotropy or close linkage of the genes

controlling fruit size and shape. The mapping of QTL for

traits in the homologous region of pepper and tomato

suggests that the pepper and tomato QTL are orthologous.

Ben-Chaim et al. (2006) by comparative mapping between

tomato and pepper showed the conservation of QTL for

fruit weight, pericarp thickness and fruit shape. One major

fruit weight QTL, fw2.1 in chromosome 2 of pepper, was

syntenic to fw2.1 of tomato and another minor QTL fw2.2

of pepper; and these were syntenic to the major QTL fw2.2

of tomato. For fruit shape, fs8.1 was detected in the

orthologous region in tomato and peppers. Their opinion is

that the difference in the degree of QTL conservation

between pepper and tomato for fruit weight might be due to

convergent domestication which resulted into mutations

and selection at common loci in different species (Zygier

et al. 2005; Ben-Chaim et al. 2006).

Comparative mapping studies using common markers

mapped both in tomato and pepper showed that chromo-

somal region containing disease resistance gene clusters

were syntenic to both the species (Thabuis et al. 2003).

Several disease resistance loci have been found to co-map

in the same pepper genomic region. The resistance locus

for P. capsici, a generalist resistance factor, and the Llocus

conferring resistance to TMV, resistance to CMV, pot-

yviruses and TSWV were reported mapping in the same

chromosomal region of the P11 pepper chromosome

((Lefebvre et al. 1995; Caranta et al. 1997a,b; Moury et al.

2007). This chromosomal region which also contains the

‘generalist’ QTL was claimed to be conserved with the R

gene Pto containing the chromosomal region in tomato

chromosome 5. The synteny between the major P. capsici

resistance loci on pepper chromosome P5 and the QTL

involved in partial resistance to Phytophthora infestans in

potato was reported (Leonards-Schippers et al. 1994;

Sandbrik et al. 2000; Thabuis et al. 2003). Pﬂieger et al.

Plant Biotechnol Rep

123

(2001) reported, in pepper, a co-localization of this con-

served resistance factor with a defense gene (class III

chitinase). The common generalist resistance on P10 was

mapped in the vicinity of R gene ‘cluster’ where two

potyvirus R genes (Pvr4 and Pvr7) and a TSWV resistance

Tsw gene were mapped (Caranta et al. 1999; Grube et al.

2000; Moury et al. 2000; Lefebvre et al. 2002; Thabuis

et al. 2003). This region is shown to be orthologous to a

region of tomato chromosome 1 containing two R genes

(Cf-1 and Cf-4) conferring speciﬁc resistance to Clado-

sporium fulvum (Jones et al. 2002). The Phytophthora

resistance locus was mapped in P2 close to an R gene for

nematode resistance (Me3 linked to CT135; Lefebvre et al.

2002), a putative orthologous region in tomato and potato

harboring R genes conferring resistance to nematodes, Mi3

and Gpa2, respectively (Djian-Caporalino et al. 2001).

The recessive resistance locus pvr2 conferred by the

translation initiation factor 4E (eIF4E) against the PVY in

pepper (C. annuum) was found to be in syntenic region

with the pot-1 region of tomato that conferred recessive

resistance to PVY and TEV. The eIF4E in tomato co-

mapped in the same genomic region as pot-1 and few

polymorphism between the resistance and susceptible line

gene sequences were observed suggesting that the eIF4E

gene is a key component of recessive resistance to pot-

yviruses which is conserved among Solanaceae species

(Ruffel et al. 2006). The observation of co-localization of

resistance gene clusters suggests that there is conservation

at an intraspeciﬁc, interspeciﬁc and inter-generic level. It is

believed that the gene clusters observed might have

evolved differently to engender various resistance factors

to unrelated pathogens and within the Solanaceae family to

maintain resistance factors to related pathogens such as

Phytophthora (Thabuis et al. 2003).

The observation of conserved synteny of gene/QTL for

similar traits in Solanaceae species gives an ample

opportunity to use sequence information of extensively

characterized species such as tomato for ﬁnding the

orthologous genes from other species. Furthermore, the

recently published draft genome sequences of tomato and

potato (The Potato Genome Sequencing Consortium 2011;

The Tomato Genome Consortium 2012) would be very

helpful for ﬁnding orthologous genes governing econom-

ically important traits in pepper, such as fruit traits, disease

resistances and other biochemical traits, as orthologous

genes are found to govern the same traits in those species

(Paran and Van Der Knaap 2007). Comparative mapping

and use of sequence information from related species

would accelerate the development of translational

genomics tools in peppers as none of the genome sequence

of Capsicum species is available at present although the

release of draft genome sequence of C. annuum is expected

soon.

Conclusion: integrated use of conventional breeding

and omics technologies should be adopted

for accelerating pepper translational research

As pepper is an important crop species with various uses

from vegetables to medicine, several advances were made

in ﬁnding the genetic loci responsible for important traits

such as pungency, fruit size and shape, resistance to dis-

eases and pests. However, only handfuls of gene/QTL loci

(listed in Table 1) were amenable for incorporation in the

translational research of capsicum species.

Out of approximately 30 or more genes reported by

Mazourek et al. (2009) that are involved in the capsaicinoid

biosynthetic pathway, only Pun1 is found to be directly

associated, suggesting that many more genes need to be

discovered and validated in a large number of genotypes

through association mapping and transformation. Similarly,

only Ovate was shown to be directly involved in fruit shape

recently although many QTL were detected (Tsaballa et al.

2011). The conventional QTL mapping and ﬁnding the

gene of interest is time-consuming. Therefore, combination

with modern technologies such as high-throughput low-

cost NGS technologies would be a great help towards

accelerating the development of breeding tools that could

be used in pepper translational genomics (Li et al. 2013).

The soon to be released whole genome sequence of C.

annuum would be helpful in ﬁnding genes of interest and

their manipulation in breeding programs (Choi et al. 2013).

The genes/QTL identiﬁed are mostly for traits which are

govern by few major genes. Therefore, sequencing of a

large-scale number of transcriptomes from various tissues

and developmental stages of phenotypically distinct

germplasm and their association with the phenotypes to

genotypes would help to develop genome-wide SNP

markers from functional genes. Linkage disequilibrium and

haplotype maps could be created by developing high-den-

sity SSR/SNP linkage maps using already developed

genomics resources and additional transcriptomic

sequencing from a large number of diverse germplasm that

would help in association mapping, besides the identiﬁca-

tion of germplasm containing noble beneﬁcial alleles.

Although pungency and carotenoids are the two exten-

sively studied traits, other metabolites and bioactive com-

pounds including whole metabolomics could be studied

and translational genomics tools could be developed.

Genes and genetic network analysis by combining large-

scale phenotyping and expression analysis coupled with

mapping studies would complement and accelerate the

development of pepper translational research tools. Since

many comparative mapping studies reported the conser-

vation of functional QTL/genes governing similar traits

among Solanaceae, candidate gene information from the

sequenced tomato and potato genomes could be used for

Plant Biotechnol Rep

123

the isolation and manipulation of homologous genes in

pepper.

Acknowledgments This work was supported by the Department of

Biotechnology, Ministry of Science and Technology, Goverment of

India, in the form of prestigious Ramalingaswami Fellowship cum

Project Grant (No. BT/RLF/Re-entry/46/2011) to Nirala Ramchiary.

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Functional genomics to understand the tolerance mechanism against biotic and abiotic stresses in Capsicum species

Chapter

Full-text available

Oct 2022

The Capsicum L. belonging to the family Solanaceae is an economically important genus of flowering plants. This genus comprises more than 30 species with its unique characteristic feature of pungency in its fruits, which are used as spices, in raw salads and pickles. People also use the species of Capsicum for ethnomedicinal and ethnoveterinary purposes. The fruits of Capsicum contain several metabolites including capsaicinoids (alkaloids responsible for the unique pungent taste), vitamins, flavonoids, carotenoids, mineral elements, and other health beneficial compounds, which have medicinal potential. Pharmacological researches have also demonstrated the role of Capsicum in the treatment of many diseases. However, the cultivated species of Capsicum face biotic and abiotic stresses that affect the normal growth and development of the plant and fruits. These stresses negatively affect yields and incur high economic losses to the farmers and the exchequer. Therefore breeding varieties resistant to these stresses is a desirable strategy to avoid/reduce the loss. The development of improved stress-resistant cultivars of Capsicum requires the knowledge of the genetic, genomic, proteomic, and metabolomic basis of stress responses active inside the plants. The recent advancements in high-throughput genomics, proteomics, and metabolomics technologies enable the understanding, quantification, and analysis of genes, proteomes, and metabolomes in various tissues and their response to different stresses at substantial speed and precision. These tools, en masse, known as omics tools allow comparative investigation of wild and cultivated species of plants; thereby in many of the cases could lead to the identification of the genes or proteins and the elucidation of their functions and assisting in the development of multiple stress-tolerant crop designs. In this chapter an attempt has been made to give an overview of the studies currently available toward understating genomic, proteomic, and metabolomic responses to biotic and abiotic stresses in Capsicum species. It further provides insights into designing resistant cultivars using genome editing and other biotechnological approaches.

Genome-wide association study and candidate gene identification for agronomic traits in 182 upward-growing fruits of C. frutescens and C. annuum

Article

Full-text available

Jun 2024

Pepper agronomic traits serve as pivotal indicators for characterizing germplasm attributes and correlations. It is important to study differential genotypic variation through phenotypic differences of target traits. Whole genome resequencing was used to sequence the whole genome among different individuals of species with known reference genomes and annotations, and based on this, differential analyses of individuals or populations were carried out to identify SNPs for agronomic traits related to pepper. This study conducted a genome-wide association study encompassing 26 key agronomic traits in 182 upward-growing fruits of C. frutescens and C. annuum. The population structure (phylogenetics, population structure, population principal component analysis, genetic relationship) and linkage disequilibrium analysis were realized to ensure the accuracy and reliability of GWAS results, and the optimal statistical model was determined. A total of 929 SNPs significantly associated with 26 agronomic traits, were identified, alongside the detection of 519 candidate genes within 100 kb region adjacent to these SNPs. Additionally, through gene annotation and expression pattern scrutiny, genes such as GAUT1, COP10, and DDB1 correlated with fruit traits in Capsicum frutescens and Capsicum annuum were validated via qRT-PCR. In the CH20 (Capsicum annuum) and YB-4 (Capsicum frutescens) cultivars, GAUT1 and COP10 were cloned with cDNA lengths of 1065 bp and 561 bp, respectively, exhibiting only a small number of single nucleotide variations and nucleotide deletions. This validation provides a robust reference for molecular marker-assisted breeding of pepper agronomic traits, offering both genetic resources and theoretical foundations for future endeavors in molecular marker-assisted breeding for pepper.

Transcriptome Analyses Reveal Distinct Defense Strategies in Chili Plants under Soilborne Disease Intervention

Article

Full-text available

Nov 2023

Chili (Capsicum annuum L.) is highly susceptible to soilborne diseases, thereby presenting a significant threat that results in considerable yield losses in chili production. The exploration of genes conferring resistance and the underlying defense mechanisms presents a promising strategy for bolstering plant disease control. In this study, we selected two distinct cultivars, the disease-sensitive ‘Hailan 99’ and the disease-tolerant ‘Sanxiaqing’, to elucidate the molecular basis of their responses to soilborne disease intervention. We conducted a comprehensive analysis of root morphological characteristics and transcriptome profiles under stress conditions. Our findings revealed that, when subjected to soilborne disease intervention, these two cultivars exhibited contrasting root system characteristics and responses, reflecting diverse defense strategies. The disease-resistant cultivar demonstrated superior adaptability, possibly owing to its capacity for swift recognition of pathogen effectors, activation of defense responses, and effective containment of infection at localized sites, thus impeding disease progression. Noteworthy genes such as T459_04053, implicated in effector recognition; MSTRG.26158, MSTRG.30886, and T459_22510, associated with secondary metabolite biosynthesis; and T459_05615, partaking in the autophagy pathway, along with other differentially expressed genes linked to effector recognition, immune activation, and modulation of cell death processes, offer valuable insights into enhancing soilborne disease resistance in chili. Furthermore, these findings contribute to an enhanced understanding of the molecular mechanisms underlying soilborne disease resistance in diverse plant crops.

Construction and Evaluation of Pepper Core Collection Based on Phenotypic Traits and SSR Markers

Preprint

Full-text available

Oct 2023

155 pepper germplasm from different region are used to conduct diversity analysis on 24 phenotypic traits, aiming to reduce the resource conservation workload, improve resource utilization and breeding efficiency. The best core collection was selected from three levels: the proportion of samples within the group, the overall sample size, and the gradual clustering of samples within the group. Representative evaluation of the phenotypic trait data of the core collection was conducted using indicators such as mean, variance, range, and coefficient of variation, and the core collection was confirmed by principal component analysis. At the same time, the Shannon Weaver diversity index and phenotype Shannon index were used to evaluate the representativeness of SSR molecular marker data of germplasm. The results showed that the pepper core collection constructed based on phenotype data contained 41 materials, which were found to perfectly preserved the genetic diversity of the original pepper germplasm using basic genetic parameters and principal component analysis. 54 polymorphic loci were obtained from 155 germplasm using SSR molecular markers, and core collection containing 32 materials was screened. Integrate phenotype and molecular core collection, the core collection composed of 57 varieties with the 92.55% phenotype retention ratio and 100% polymorphism site retention ratio were determined, and had the most genetic diversity according to a 90% compression ratio, which can fully represent the genetic diversity of the original germplasm.

Resistance to Anthracnose Rot Disease in Capsicum

Article

Full-text available

May 2023

Pepper (Capsicum spp.) is an important vegetable crop worldwide with high economic and nutritional value. The Capsicum genus comprises more than 30 species, of which C. annuum, C. chinense, C. baccatum, C. frutescens, and C. pubescens are the five domesticated ones. Anthracnose fruit rot, caused by Colletotrichum spp., is one of the most destructive fungal diseases of pepper. In this review, we compiled up-to-date information from 40 publications on anthracnose resistance in Capsicum species. In total, 375 accessions were described as showing different levels of resistance against Colletotrichum spp. These accessions belonged to different species, including C. annuum (160), C. baccatum (86), C. chacoense (4), C. chinense (90), and C. frutescens (16), as well as 19 accessions of which the species were not reported. High levels of resistance were mainly present in C. baccatum and C. chinense. For some of the resistant accessions, resistance genes or quantitative trait loci (QTL) were reported. Using associated molecular markers, we located 31 QTLs and 17 resistance-related genes in the recently published Capsicum genomes, including C. annuum CM334 version 1.6, C. chinense version 1.2, and C. baccatum version 1.2. Our results could be helpful for making use of some reported accessions in the breeding of pepper cultivars with resistance to anthracnose rot disease.

Analysis of Volatile Aroma Components and Regulatory Genes in Different Kinds and Development Stages of Pepper Fruits Based on Non-Targeted Metabolome Combined with Transcriptome

Article

Full-text available

Apr 2023
INT J MOL SCI

Aroma is a crucial attribute affecting the quality of pepper and its processed products, which has significant commercial value. However, little is known about the composition of volatile aroma compounds (VACs) in pepper fruits and their potential molecular regulatory mechanisms. In this study, HS-SPME-GC-MS combined with transcriptome sequencing is used to analyze the composition and formation mechanism of VACs in different kinds and development stages of pepper fruits. The results showed that 149 VACs, such as esters, alcohols, aldehydes, and terpenoids, were identified from 4 varieties and 3 development stages, and there were significant quantitative differences among different samples. Volatile esters were the most important aroma components in pepper fruits. PCA analysis showed that pepper fruits of different developmental stages had significantly different marker aroma compounds, which may be an important provider of pepper’s characteristic aroma. Transcriptome analysis showed that many differential genes (DEGs) were enriched in the metabolic pathways related to the synthesis of VACs, such as fatty acids, amino acids, MVA, and MEP in pepper fruits. In addition, we identified a large number of differential transcription factors (TFs) that may regulate the synthesis of VACs. Combined analysis of differential aroma metabolites and DEGs identified two co-expression network modules highly correlated with the relative content of VACs in pepper fruit. This study confirmed the basic information on the changes of VACs in the fruits of several Chinese spicy peppers at different stages of development, screened out the characteristic aroma components of different varieties, and revealed the molecular mechanism of aroma formation, providing a valuable reference for the quality breeding of pepper.

Diversity and expression analysis of ZIP transporters and associated metabolites under zinc and iron stress in Capsicum

Article

Feb 2023
PLANT PHYSIOL BIOCH

The members of ZRT, IRT-like protein (ZIP) family are involved in the uptake and transportation of several metal ions. Here, we report a comprehensive identification of ZIP transporter genes from Capsicum annuum, C. chinense, and C. baccatum, and their expression analysis under Zn and Fe stress. Changes in root morphology and differential accumulation of several metabolites from sugars, amino acids, carboxylic acids, and fatty acids in root and leaf tissues of plants in the absence of Zn and Fe were observed. Further, metabolites such as L-aspartic acid, 2-ketoglutaric acids, β-L-fucopyranose, quininic acid, chlorogenic acid, and aucubin were significantly upregulated in root and leaf tissues under Zn/Fe deprived conditions. qRT-PCR analysis of 17 CaZIPs in different tissues revealed tissue-specific expression of CaZIP1-2, CaZIP4-8, CaZIP13, and CaZIP16-17 under normal conditions. However, the absence of Zn and Fe significantly induced the expression of CaZIP4-5, CaZIP7-9, and CaZIP14 genes in root and leaf tissues. Additionally, in the absence of Fe, upregulation of CaZIP4-5 and CaZIP8 and increased uptake of mineral elements Cu, Zn, Mg, P, and S were observed in roots, suggesting their potential role in metal-ion uptake in Capsicum. The identified genes provide the basis for future studies of mineral uptake and their biofortification to increase the nutritional values in Capsicum.

Capsaicin-Producing Plants: Species Diversity, Distribution and Botanical Details

Chapter

Jan 2024

The species of Capsicum are known to synthesise capsaicin. It has a molecular weight of 305.5 g/mol and the chemical formula C18H27NO3. The IUPAC name of capsaicin is (E)-N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methylnon-6-enamide. It is responsible for the unique pungent taste of the Capsicum. It is crystalline, colourless, odourless and lipophilic. This molecule belongs to a special type of alkaloids called capsaicinoids. Capsicum spp. are known to produce several types of capsaicinoids such as capsaicin, homocapsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and norcapsaicin. Capsaicin and dihydrocapsaicin constitute about 80–90% of capsaicinoids and hence are the major capsaicinoids of the Capsicum. The only genus that has been found to produce and accumulate capsaicinoids is Capsicum. Capsaicin concentration in plants varies and is generally affected by genetic, environmental and various other factors including biotic stresses. The capsaicin finds applications in diverse sectors including flavouring, food, cosmetics, defence sprays, medicine and pharmacy. This book chapter provides a brief overview of the diversity of various capsaicin-producing plants along with their botanical, cytological and distribution details. This chapter is important for those who are interested in botany, taxonomy and diversity of the capsaicin-producing species.

Genome-wide association study and candidate gene identification for agronomic traits in 182 upward-growing fruits of C. frutescens and C. annuum

Preprint

Full-text available

Feb 2024

Pepper agronomic traits serve as pivotal indicators for characterizing germplasm attributes and correlations. Investigating genotypic disparities through phenotypic variations holds significant scientific merit Whole genome resequencing facilitates comprehensive examination of diverse individuals with known references, enabling subsequent differential analyses to pinpoint single nucleotide polymorphisms(SNPs) linked to pepper agrnomic. This study ,conducted a genome-wide association study(GWAS) encompassing 26 agronomic traits in 182 pepper specimens. Rigorous measures, including phylogenetic analysis, population structure analysis, population principal component analysis, kinship analysis, and linkage disequilibrium analysis, were employed to ensure the precision and reliability of GWAS results. The optimal statistical model. Was determined through these analyses. A total of 929 SNPs significantly associated with 26 agronomic traits, were identified, alongside the detection of 519 candidate genes within 100kb region adjacent to these SNPs. Additionally, through gene annotation and expression pattern scrutiny, genes such as SCPL13, extensin-1-like, and DDB1 correlated with fruit traits in Capsicum frutescens and Capsicum annuum were validated via qRT-PCR. This validation provides a robust reference for molecular marker-assisted breeding of pepper agronomic traits, offering both genetic resources and theoretical foundations for future endeavors in molecular marker-assisted breeding for pepper.

Integrated Use of Molecular and Omics Approaches for Breeding High Yield and Stress Resistance Chili Peppers

Chapter

Full-text available

Jan 2023

In a rapidly changing environment along with the growing population, crop production and improvement are the most sought-after sectors in the world. Pepper (Capsicum spp.) is a highly consumed plant species in the form of spice or whole fruit and is widely popular for its pungent flavour and vibrant colours worldwide. Like other crop plants, pepper is also severely affected by environmental and genetic constraints and reflects a massive loss in yield, quality and production rate. Therefore, it is imperative to immediately change our focus towards the development of stress-resilient and high-yielding Capsicum varieties. This chapter discusses the integrated use of conventional, molecular and multi-omics tools towards meeting the end goal of Capsicum trait improvement. Conventional breeding programmes have utilized natural sources of genes and QTLs attributed to biotic and abiotic stress tolerance and other important horticultural traits in pepper improvement which involve time-consuming multiple and tedious crossing cycles followed by selection. The development of reliable molecular markers like SSRs, SNPs, InDels etc. have enabled genetic and biparental QTL mapping, and genome-wide association studies which all have not only helped in the rapid identification of significant QTLs governing important traits but also marker-assisted selection of desired phenotypes. Several candidate genes for Capsicum plant and fruit morphology, metabolite content, disease resistance and stress tolerance have been identified and are available for introgression via marker-assisted breeding and genetic engineering into elite genetic backgrounds. Further, genome and transcriptome sequencing projects have changed the tides for advancement in Capsicum research and provided new insights into Capsicum genome structure, size, features and function. Other omics approaches like epigenomics, proteomics and metabolomics are rapidly evolving to reveal transcriptional, post-transcriptional and post-translational modifications in Capsicum genes and proteins. In brief, established breeding strategies combined with new technological hues can be successfully exploited for breeding high-quality, better performing, stress-resistant and sturdy Capsicum varieties to meet the needs of the growing human population.Keywords Capsicum Stress resistantGenomicsTranscriptomicsMetabolomicsProteomicsEpigenomics

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Sweet Pepper (Capsicum annuum) Transformation using Agrobacterium rhizogenes

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Jun 1997

In vitro genetic transformation of chile pepper, Capsicum annuum var. New Mexico 6-4, was achieved. Seeds of `New Mexico 6-4' were grown aseptically on Murashige and Skoog medium. Seedlings 22 days old were wounded on the hypocotyl region using a sterile hypodermic needle. A. rhizogenes strain K 599 harboring the plasmid p35S GUS Intron was inoculated on the wound site. Three days later the seedlings were transferred onto MS media with antibiotics (Cefotaxime, Carbenicillin, Amoxicillin, Clavulanic acid, and Kanamycin). New roots were seen to initiate from the wound site 15 to 20 days after inoculation. The roots were morphologically identified as “hairy roots.” Glucuronidase ( Gus ) assay performed 40 days after inoculation on randomly chosen roots that had grown into the selection medium, showed that 6/25 (24%) of the inoculated seedlings had roots that showed intense blue coloration. Presence of an intron makes it impossible for the bacteria to express the reporter gene. The seedlings that had transformed roots had a different morphology with wrinkled leaves and short internodes. The pattern of expression of the introduced gene varied greatly. Some positive tissues had the root tips alone being blue; a few had the vascular tissues and the root tips blue; and others had the vascular tissues, the surrounding parenchyma cells, root tips, and the root hairs turn very dark blue. The transformed roots did not need to grow into the selection media to be Gus positive. Isolated roots cultured on MS media supplemented with 0.2 mg/L IAA were maintained for 120 days and continued to express the reporter gene. Currently, methods to regenerate transformed shoots from roots are being tested. The “hairy root” transformation system in pepper could have application in the testing of root-expressible constructs for transgene expression assays.

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Sep 2004

A rapid and reproducible regeneration protocol has been established in Bell pepper (Capsicum annuum L. cv. Early California wonder) via in vitro shoot multiplication. Multiple shoots were induced from shoot tip explants cultured on MS medium fortified with 1 mg/l Kin and 7 mg/l BA. Addition of 20% coconut milk enhanced the vigour of shoot growth. An optimum of 8.5 shoots was obtained per shoot tip explants after 45 days of culture. Three repeated subcultures up to 120 days produced a maximum of 22 shoots per explant. Rooting was achieved from the shoots of 2-3cm long placed on MS basal medium augmented with 2% sucrose. The in vitro regenerated plantlets were hardened in vermiculite and subsequently planted in the soil and 90% survival rate was observed.

A revised medium for rapid growth and bioassays with tobaco tissue cultures

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Capsicum

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Historically, capsicum has provided potent and characteristic sensory properties to foods. This review summarizes the progress made to date in understanding this unique product. Areas included are horticultural considerations, physiological effects, sensory properties, methods of analysis, and chemical properties.

Application of genetics and genomics towards Capsicum translational research

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