Molecular Marker Development and Gene Cloning for Diverse Disease Resistance in Pepper (Capsicum annuum L.): Current Status and Prospects
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Plant Breed. Biotech. 2020 (June) 8(2):89~113 Online ISSN: 2287-9366
https://doi.org/10.9787/PBB.2020.8.2.89 Print ISSN: 2287-9358
REVIEW ARTICLE
Molecular Marker Development and Gene Cloning for Diverse
Disease Resistance in Pepper (Capsicum annuum L.):
Current Status and Prospects
Geleta Dugassa Barka, Jundae Lee*
Department of Horticulture, Institute of Agricultural Science & Technology, Jeonbuk National University, Jeonju 54896, Korea
ABSTRACT The production of chili pepper (Capsicum annuum L.) is hindered by several biotic factors even though striding
progresses were made in genetic improvement in the last two decades. Among the advancements were the fast-track genetic
improvement of disease-resistant varieties by the use of marker-assisted selection (MAS) and the conventional breeding-based intro-
gression of major resistance genes. Marker development, marker-based identification and fine mapping have revealed a large number
of resistance genes, from which cloning of some candidate genes demonstrated the applicability and versatility of map-based cloning
for disease resistance. In some of the recent fine mapping of disease resistance QTLs, closely linked DNA markers were identified,
which in turn resulted in the rapid introgression of target gene(s) into breeding lines. Also, progresses were made on the characterization
and map-based cloning of resistance genes conferring broad-spectrum resistance. As the number of identified and characterized
resistance genes and the DNA markers linked to resistance genes are steadily generated, the development of multiple/durable resistance
to major chili pepper diseases is accelerated by MAS. In the present review, the development of molecular markers, marker-based
mapping of genes conferring resistance to ten major chili pepper diseases were discussed, focusing on the recent advancements in major
and QTL-spanning resistance gene mapping. The review provides up-to-date insights into the development of DNA markers linked to
disease resistance genes and the cloning of resistance genes, which are all so crucial in pepper breeding for disease resistance.
Keywords Disease resistance, Fine mapping, Introgression, Map-based cloning, QTL, Resistance gene
INTRODUCTION diverse diseases is highly required (Wiesner-Hanks and
Nelson 2016). It can be achieved by the fast-track ac-
Chili pepper (Capsicum annuum L.) is among the top cumulation of disease resistance genes through the use of
economically valuable vegetable crops mainly due to the marker-assisted selection (MAS) (Ribaut and Hoisington
high demand and popularity of spicy foods in many parts of 1998; Cobb et al. 2019). Recently, the rapid detection of
the world (Pinto et al. 2016). However, the production of single nucleotide polymorphism (SNP) markers associated
pepper is hindered by diverse diseases including fungal with disease resistance genes by the high-throughput
(anthracnose and powdery mildew), oomycete (phytophthora genotyping methods combined with the next-generation
root rot), viral (Cucumber mosaic virus [CMV], tobamo- sequencing (NGS) technologies has substantially shorten-
viruses, potyviruses, Tomato spotted wilt virus [TSWV], ed the time required for genetic map construction, quan-
etc.), bacterial (bacterial spot and bacterial wilt) and titative trait loci (QTL) analysis and candidate gene iden-
nematode (root-knot nematodes) (Barchenger et al. 2019). tification in plant molecular breeding (Rafalski 2002;
Therefore, pepper breeding for multiple resistances to Varshney et al. 2009; Kumar et al. 2012; Mammadov et al.
Received March 31, 2020; Revised May 15, 2020; Accepted May 15, 2020; Published June 1, 2020
*Corresponding author Jundae Lee, ajfall@jbnu.ac.kr, Tel: +82-63-270-2560, Fax: +82-63-270-2581
Copyright ⓒ 2020 by the Korean Society of Breeding Science
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.90 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
2012; Thomson 2014; Huq et al. 2016; Phan and Sim 2017; through inheritance analysis (Kim et al. 2007, 2008d). In
Xu et al. 2017). The frequently adopted methods for another study, the resistance of C. baccatum ‘PBC80’ to C.
high-throughput SNP genotyping include genotyping-by- scovillei was controlled by two genes, co4 and Co5, based
sequencing (GBS) (Deschamps et al. 2012; Poland and on phenotypic data (Mahasuk et al. 2009b). Two QTLs,
Rife 2012; Kim et al. 2016), double-digest restriction An8.1 and An9.1, for resistance to C. scovillei were de-
association DNA sequencing (ddRAD-seq) (Peterson et al. tected in an F2 population derived from a cross between C.
2012), and specific-locus amplified fragment sequencing baccatum var. pendulum (‘Cpb’) (resistant) and C. baccatum
(SLAF-seq) (Sun et al. 2013). Molecular marker develop- ‘Golden-aji’ (susceptible) (Kim et al. 2010). A major QTL
ment and fine mapping for disease resistance genes and the CaR12.2 for the resistance was found in an introgressed
eventual identification and characterization of such genes BC1F2 population by interspecific crosses between C. annuum
by map-based cloning are believed to bring a paradigm ‘SP26’ (susceptible) and C. baccatum ‘PBC81’ (resistant),
shift in the speed of disease-resistant pepper variety de- and so was the development of CaR12.2M1-CAPS marker
velopment. closely linked to the major QTL CaR12.2 (Lee et al. 2010,
The aim of the present review was to discuss some of the 2011). Another QTL analysis revealed that the resistance
latest advancements in marker development and gene of C. chinense ‘PBC932’ to C. scovillei is controlled by a
cloning for major disease resistances in chili pepper. The major dominant QTL on chromosome P5 (Sun et al. 2015).
review could also be used as an important input for such Recently, three major (RA80rP2, RA80rP3.1, and RA80rHP1)
molecular breeding programs involving MAS, as it high- and two minor (RA80rP3.2 and PA80rHP2) QTLs for
lighted some of the latest reports on the identification of resistance to C. scovillei in the ripe fruit stage were iden-
candidate disease resistance genes and associated/tightly- tified in an F2 population derived from an intraspecific
linked DNA markers. cross between C. baccatum ‘PBC80’ and ‘CA1316’ (Mahasuk
et al. 2016). Two markers, SCAR-Indel and SSR-HpmsE032,
associated with resistance to C. scovillei were validated in
MARKER DEVELOPMENT FOR PEPPER two C. annuum anthracnose resistant introgression lines,
DISEASE RESISTANCE PR1 derived from ‘PBC932’ and PR2 derived from ‘PBC80’,
resulted in the selection efficiency of 77% when both
Anthracnose markers were used together (Suwor et al. 2017).
Pepper anthracnose is characterized by water-soaked QTL analysis for resistance to C. siamense and C.
and sunken circular lesions on mature/immature fruits truncatum in a cross between C. annuum ‘Jatilaba’ (sus-
caused by Colletotrichum species including C. scovillei ceptible) and C. chinense ‘PRI95030’ (resistant) revealed
(formerly C. acutatum), C. truncatum (formerly C. capsici), one main QTL (B1) and three other QTLs (B2, H1, and D1)
and C. siamense (formerly C. gloeosporioides) (Mongkolporn for the resistance (Voorrips et al. 2004). Inheritance
and Taylor 2018). It has been reported that some genetic analysis indicated that the resistance of ‘PBC932’ to C.
resources belonging to two Capsicum species, C. baccatum truncatum was responsible by a single recessive gene
(‘PBC80’, ‘PBC81’, ‘PI594137’, and ‘Cbp’) and C. chinense (Pakdeevaraporn et al. 2005; Kim et al. 2008d). Three
Jacq. (‘PBC932’), have resistance to anthracnose (AVRDC different recessive genes, co1, co2, and co3, were re-
2003; Yoon et al. 2004; Kim et al. 2008e; Park et al. 2009). sponsible for the resistance to C. truncatum of green fruit,
The DNA markers linked to anthracnose resistance in red fruit, and seedling, respectively, from a cross between
Capsicum species were summarized in Table 1. C. annuum ‘Bangchang’ and C. chinense ‘PBC932’, and
The resistances of C. annuum ‘AR’ derived from C. two QTLs RA932g (co1) and RA932r (co2) were detected
chinense ‘PBC932’ and C. baccatum ‘PI594137’ to C. in the same population (Mahasuk et al. 2009a, 2016). A
scovillei were reported to be controlled by a single re- major QTL CcR9 for the resistance of ‘PBC81’ to C.
cessive gene and a single dominant gene, respectively, truncatum was identified, and the CcR9M1-SCAR markerTable 1. Molecular markers linked to the genes or QTLs resistant to fungal diseases in pepper.
Population
Resistance Type of Inheritance Status of
Group Disease Pathogen Chr. Marker or gene Number Reference
locus marker Parents Generation pattern research
of plants
Fungi Anthracnose Colletotrichum CaR12.2 12 CaR12.2M1-CAPS CAPS ‘SP26’ × BC1F2 87 QTL Genetic Lee et al.
scovillei ‘PBC81’ mapping 2011
(formerly C. Co5 4 BACSNP-4-63, -60 SNP ‘PBC80’ × F2 146 QTL Genetic Mahasuk
acutatum) ‘CA1316’ mapping et al. 2016
AnR5 5 InDel, HpmsE116 InDel, ‘77013’ × BC1 186 QTL Genetic Sun et al.
SSR ‘PBC932’ mapping 2015
CaR12.2 12 SCAR-Indel, SCAR, ‘PS’ × ‘PR1’, F2, BC1 468 Single Marker Suwor
HpmsE032 SSR ‘PS’ × ‘PR2’ dominant analysis et al. 2017
Colletotrichum CcR9 9 CcR9M1-SCAR SCAR ‘SP26’ × BC1F2 87 QTL Genetic Lee et al.
truncatum ‘PBC81’ mapping 2011
(formerly C. co1, co2 2 CAP_T22290_0_1_429, SNP ‘Bangchang’ × F2 126 QTL Genetic Mahasuk
capsici) CAP_T39318_0_1_1042 ‘PBC932’ mapping et al. 2016
RCt1 11 CtR-431, CtR-594 STS ‘Punjab Lal’ × F2, BC1 354 Single Genetic Mishra
‘Arka Lohit’ dominant mapping et al. 2019
Powdery Leveillula Lt_6.1 6 E36/M59-380h AFLP ‘H3’ × DH 101 QTL Genetic Lefebvre
mildew taurica ‘Vania’ mapping et al. 2003
Lt_9.1 9 D11_0.8h RAPD ‘H3’ × DH 101 QTL Genetic Lefebvre
‘Vania’ mapping et al. 2003
LtR4.2 4 Ltr4.1-40344, SNP ‘SP26’ × BC1F2 87 QTL Marker Kim et al.
Ltr4.2-56301, ‘PBC81’ analysis 2017a
Ltr4.2-585119
PMR1 4 ZL1_1826, HPGV_1313, SCAR, ‘VK515R’ × F2:3 102 Single Candidate Jo et al.
HPGV_1344, SNP ‘VK515S’ F2 80 dominant gene 2017
HPGV_1412, Cultivar ‘PM identifi-
KS16052G01 Singang’ cation
Marker Development and Gene Cloning for Pepper Disease Resistance ∙ 9192 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
closely linked to the QTL CcR9 was developed (Lee et al. (Lefebvre et al. 2003). Recently, a novel powdery mildew
2010, 2011). An SSR marker HpmsE032 was associated resistance locus, PMR1, and cosegregating markers, one
with resistance in progressive lines derived from ‘PBC80’ sequence characterized amplified region (SCAR) marker
to C. truncatum at green fruit stages and could be con- (ZL1_1826) and five high-resolution melting (HRM) (Liew
sidered useful in the selection of resistance derived from et al. 2004) markers (HPGV_1313, HPGV_1344, HPGV_
‘PBC80’ (Suwor et al. 2015). Recently, reference genome 1412, KS16052G01, and HRM2_A4), were identified on
sequences of C. baccatum and QTL information for re- pepper chromosome 4 using two populations, 102 ‘VK515’
sistance to C. truncatum revealed 64 nucleotide-binding F2:3 families and 80 ‘PM Singang’ F2 plants (Jo et al. 2017).
and leucine-rich-repeat proteins (NLRs) from a 3.8 Mb In addition to that, the report indicated that PMR1 locus
region of chromosome 3 as candidate resistance genes for might have been introgressed from C. baccatum.
C. truncatum (Kim et al. 2017b). Bulked segregant an-
alysis (BSA) combined with inter-simple sequence repeat Phytophthora root rot
(ISSR) and amplified fragment length polymorphism Phytophthora capsici is one of the destructive pathogens
(AFLP) markers has resulted in the development of two posing a serious threat to vegetables and fruits including
sequence-tagged site (STS) markers (CtR-431 and CtR-594) chili pepper (C. annuum). Several resistant resources to
linked to the resistance (RCt1) locus against C. truncatum Phytophthora root rot, including C. annuum ‘Vania’,
in C. annuum (Mishra et al. 2019). ‘Perennial’, ‘Criollo de Morelos 334 (CM334)’, ‘CM331’,
‘AC2258’, ‘YCM334’, ‘PI201234’, ‘PBC280’, ‘PBC495’,
Powdery mildew and ‘PBC602’, have been reported (Lee et al. 2012b). In
Powdery mildew, caused by Leveillula taurica, anamorph pepper, resistance to P. capsici is attributed to single
Oidiopsis taurica, is a serious disease of pepper (C. dominant gene (Monroy-Barbosa and Bosland 2008) and
annuum) grown in greenhouses (de Souza and Café-Filho the joint action of hundreds of the most diversified partial
2003). The symptoms are characterized by a powdery- resistance QTLs (Truong et al. 2012). The list of QTLs and
white fungal growth on the undersides of leaves and light- DNA markers associated with the resistance is shown in
green to yellow blotches on the upper leaf surfaces (de Table 2.
Souza and Café-Filho 2003). Nine resistant resources to Comparative QTL analysis for resistance to Phytophthora
powdery mildew, including three C. annuum accessions capsici was performed in three intraspecific pepper po-
(H3, H-V-12 and 4648) and six C. baccatum accessions pulations derived from three different resistant accessions,
(CNPH 36, 38, 50, 52, 279 and 288), were identified after C. annuum ‘Vania’, ‘Perennial’, and ‘CM334’, and the
evaluating a total of 162 Capsicum genotypes (Daubeze et major resistance factor on chromosome P5 was found to be
al. 1995; de Souza and Café-Filho 2003). There are several common to the populations (Thabuis et al. 2003). More-
reports on the development of molecular markers for over, resistance alleles to P. capsici at four QTLs were
powdery mildew resistance in Capsicum species (Table 1). transferred from a small-fruited pepper into a bell pepper
The resistance to powdery mildew from an African using four markers, ASC031 (P2), ASC037 (P5), E43M53-
pepper line ‘H3’ (C. annuum) was controlled by two or 159y (P5), and E35M61-114y (P10) (Thabuis et al. 2004).
three genetic factors with additive and partial dominance The D4 SCAR marker for the detection of Phyto.5.2, a
effects (Daubeze et al. 1995). Two common QTLs, Lt_6.1 major QTL for resistance to P. capsici, was developed
(a closely linked AFLP maker E36/M59-380h) and Lt_9.1 (Quirin et al. 2005). In another study, two intraspecific
(a closely linked random amplified polymorphic DNA linkage maps, ‘PSP-11’ × ‘PI201234’ and ‘Joe E. Parker’ ×
[RAPD] marker D11_0.8h), for resistance to powdery ‘CM334’, were constructed to identify QTLs conferring
mildew under natural and artificial infections were de- resistance to P. capsici root-rot and foliar-blight diseases
tected in the doubled haploid (DH) progeny from the cross (Ogundiwin et al. 2005). Three QTLs, Phyt-1, Phyt-2, and
between ‘H3’ (highly resistant) and ‘Vania’ (susceptible) Phyt-3, for resistance to Phytophthora blight, were de-Table 2. Molecular markers linked to the genes or QTLs resistant to Phytophthora capsici in pepper.
Population
Resistance Marker or Type of Inheritance Status of
Group Disease Pathogen Chr. Number Reference
locus gene marker Parents Generation pattern research
of plants
Oomycetes Phytophthora Phytophthora Phyto5.2 5 D04.717-SCAR SCAR ‘CM334’ × F3 9 Single Marker Quirin et al.
root rot capsici ‘Yolo B’ families families dominant development 2005
CAMS420 SSR ‘Manganji’ × DH 96 QTL Genetic Minamiyama
‘CM334’ mapping et al. 2007
P5-SNAP SNAP ‘CM334’ × F2 100 QTL Marker Kim et al.
‘Chilsungcho’ development 2008b
M3-CAPS CAPS ‘Subicho’ × F2 96 Single Marker Lee et al.
‘CM334’ dominant development 2012b
SA133_4, SCAR, ‘YCM334’ × RIL 126 Single Marker Truong
UBC553 RAPD ‘Tean’ dominant development et al. 2013
Phyto5NBS1 SNP ‘YCM334’ × RIL 128 Single Candidate gene Liu et al.
‘Tean’ dominant identification 2014
Pc5.1 5 CA036100, SNP ‘H3’ × ’Vania’ DH 101 QTL Candidate gene Mallard
CA004482 ‘Perennial’ × DH 114 identification et al. 2013,
‘Yolo Wonder’ Rehrig
‘Yolo Wonder’ × RIL 297 et al. 2014
‘CM334’
CaPhyto 5 ZL6726, SSR ‘Shanghaiyuan’ F2 794 QTL Candidate gene Wang et al.
ZL6970 × ‘PI201234’ identification 2016
Phyt-1 5 M10E3-6 AFLP ‘K9-11’ × DH 176 QTL Genetic Sugita et al.
‘AC2258’ mapping 2006
Phyt-2 1 RP13-1 RAPD ‘K9-11’ × DH 176 QTL Genetic Sugita et al.
‘AC2258’ mapping 2006
Phyt-3 11 M9E3-11 AFLP ‘K9-11’ × DH 176 QTL Genetic Sugita et al.
‘AC2258’ mapping 2006
PhR10 10 P52-11-21, SSR ‘CM334’ × F2, BC1 853 Single Candidate gene Xu et al.
P52-11-41 ‘NMCA10399’ dominant identification 2016
QTL5.1, 5 EC5-bin27 SNP ‘CM334’ × RIL 188 QTL Genetic Siddique
QTL5.2, S05_27703815 ‘ECW30R’ mapping et al. 2019
QTL5.3 EC5-bin51
Marker Development and Gene Cloning for Pepper Disease Resistance ∙ 9394 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
tected using an intraspecific DH population derived from a (race 3) of P. capsici using BSA and SLAF-seq, and two
cross between C. annuum ‘K9-11’ (susceptible) and ‘AC2258’ flanking SSR markers, P52-11-21 and P52-11-41, of the
(resistant), and three markers, M10E3-6 AFLP, RP13-1 PhR10 locus were also identified (Xu et al. 2016). Two
RAPD, and M9E3-11, for the QTLs, respectively, were candidate genes, Capana05g000764 and Capana05g000769,
identified (Sugita et al. 2006). Two markers, CAMS420 for a dominant gene CaPhyto controlling the resistance of
SSR (linked to a major QTL on LG15) and CTT/ACT3M ‘PI201234’ to P. capsici race 2, were identified, and one
AFLP (a minor QTL on LG3), for resistance to P. capsici SSR marker, ZL6726, most closely linked to CaPhyto at a
were identified in a segregating DH population developed distance of 1.5 cM, was developed (Wang et al. 2016).
by anther culture of an F1 plant crossed between C. annuum Through GBS-based QTL mapping and GWAS analysis,
‘Manganji’ (susceptible) and ‘CM334’ (resistant) (Minamiyama three major QTLs (5.1, 5.2, and 5.3) conferring broad-
et al. 2007). Two bacterial artificial chromosome (BAC)- spectrum resistance to P. capsici were identified (Siddique
derived markers, P5-SNAP and SSR-9, were developed et al. 2019).
from two RFLP markers, CDI25 (P5) and CT211 (P9),
linked to P. capsici resistance which were detected in an F2 Cucumber mosaic virus
population from a cross between C. annuum ‘CM334’ (re- Cucumber mosaic virus (CMV), a member of the
sistant) and ‘Chilsungcho’ (susceptible) (Kim et al. 2008b). Cucumovirus genus in the family Bromoviridae, is a plant
The M3-CAPS marker tightly linked to the major QTL RNA virus which often causes significant losses in dicots
Phyto.5.2 for resistance to Phytophthora root rot, was including pepper and some monocot crops due to the rapid
developed using two segregating F2 populations from a spread of the disease by aphids, and other vectors
cross of ‘Subicho’ × ‘CM334’ and self-pollination of a com- (Roossinck 2001). Up to date, CMV resistance has been
mercial cultivar ‘Dokyacheongcheong’ (Lee et al. 2012b). identified in various genetic sources of pepper (Capsicum
One common QTL (P5) and four isolate-specific QTLs spp.) including C. annuum ‘Perennial’ (Lapidot et al.
(P10, P11, Pb, and Pc) for resistance to Phytophthora root 1997), ‘Vania’ (Caranta et al. 2002), ‘Sapporo-oonaga’
rot were detected using two P. capsici isolates (09-051 and and ‘Nanbu-oonaga’ (Suzuki et al. 2003), ‘Bukang’ (Kang
07-127) and an intraspecific recombinant inbred line (RIL) et al. 2010), ‘BJ0747-1-3-1-1’ (Yao et al. 2013), ‘CA23’
population from a cross between ‘YCM334’ (resistant) and (Rahman et al. 2016), C. frutescens ‘BG2814-6’ (Grube et
‘Tean’ (susceptible) (Truong et al. 2012). Subsequently, a al. 2000b), ‘Tabasco’, ‘LS1839-2-4’ (Suzuki et al. 2003),
codominant SCAR marker SA133_4 and a RAPD marker ‘PBC688’ (Guo et al. 2017a), and C. baccatum ‘PI439381-
UBC553, linked to the QTL P5, were developed (Truong et 1-3’ (Suzuki et al. 2003).
al. 2013). By means of meta-analyses, a key QTL, Pc5.1, Several researches on CMV resistance in Capsicum
conferring broad-spectrum resistance to P. capsici was species have been reported (Table 3). The resistance of
identified (Mallard et al. 2013). A resistance gene, C. these resources was reported to be quantitatively controlled.
annuum DOWNY MILDEW RESISTANT 1 (CaDMR1), as Two additive QTLs on LG3 and Noir and one epistatic
a candidate gene responsible for the major QTL on ch- QTL between TG124 (positioned on Noir) and TG66
romosome P5 for resistance to P. capsici was identified by (positioned on Pourpre) were identified using 94 DH lines
generating a high-density map with 3887 markers in a set of obtained from the F1 of the ‘Perennial’ and ‘Yolo Wonder’
RIL derived from the highly resistant C. annuum ‘CM334’ parental varieties (Caranta et al. 1997b). In the same popu-
and the susceptible ‘Early Jalapeno’ (Rehrig et al. 2014). lation, the major QTL for CMV resistance was positioned
2
The Phyto5NBS1, a reliable marker for P. capsici re- on chromosome 12, with an R (coefficient of determina-
sistance, was developed using BSA and Affymetrix tion) of 19% and a strong linkage with the A5.1 marker
GeneChips (Liu et al. 2014). A single dominant gene, (Pflieger et al. 1999). Also, four QTLs, cmv4.1, cmv6.1,
PhR10, mapped on chromosome 10, was identified to be cmv11.1, and cmv13.1, were detected using 180 F3 families
responsible for the resistance of ‘CM334’ to an isolate Byl4 derived from a cross between C. annuum ‘Maor’ andTable 3. Molecular markers linked to the genes or QTLs resistant to viruses in pepper.
Population
Resistance Type of Inheritance Status of
Group Disease Pathogen Chr. Marker or gene Number Reference
locus marker Parents Generation pattern research
of plants
Viruses CMV Cucumber CMV 12 A5.1 RAPD ‘Perennial’ × DH 94 Single Genetic Pflieger et al.
mosaic ‘Yolo Wonder’ dominant mapping 1999
virus cmv11.1 11 E35/M48-101 AFLP ‘Maor’ × ‘Perennial’ F3 families 180 QTL Genetic Ben Chaim
mapping et al. 2001
cmv12.1 12 E33/M48-132, AFLP ‘H3’ × ‘Vania’ DH 101 QTL Genetic Caranta et al.
E40/M47-262 mapping 2002
Cmr1 2 CaTm-int3-HRM, SNP Cultivar ‘Bukang’ F2 309 Single Marker Kang et al.
CaT1616BAC, dominant development 2010
240H02sp6
qcmv.hb-8.2 11 UBC829 RAPD ‘BJ0747’ × ‘XJ0630’ F2, BC1 334 QTL Genetic Yao et al. 2013
mapping
qCmr11.1 11 Indel-11-64 InDel ‘PBC688’ × ‘G29’ F2 289 QTL Candidate gene Guo et al.
identification 2017a
qcmv11.1 11 Marker6201026 SNP ‘BJ0747’ × ‘XJ0630’ F2 195 QTL Genetic Li et al. 2018
qcmv11.2 Marker5409028 mapping
qcmv12.1 Marker17652010
cmr2 8 Affy4, IBP160, SNP ‘Lan32’ × ‘Jeju’ F2 129 Single Genetic Choi et al.
cmvAFLP recessive mapping 2018
Potyvirus Pepper pvr1 4 Pvr1-S, pvr1-R1, CAPS R and S accessions Line 23 Single Marker Yeam et al.
mottle virus (= pvr2) pvr1-R2 recessive development 2005
(PepMoV) eIF4E-A614G, ARMS-PCR ‘Yolo Wonder’ × F2 - Single Marker Rubio et al.
-G325A, -T236G, ‘CM334’, ‘Perennial’ × recessive development 2008
-T200A ‘Yolo Y’, ‘Perennial’ ×
‘Florida VR2’
KASP_pvr1 KASP ‘Habanero’ × F2 56 Single Marker Holdsworth and
‘PI159234’ recessive development Mazourek
2015
Marker Development and Gene Cloning for Pepper Disease Resistance ∙ 95Table 3. Continued.
Population
Resistance Type of Inheritance Status of
Group Disease Pathogen Chr. Marker or gene Number Reference
locus marker Parents Generation pattern research
of plants
Pvr4 10 Pvr4-CAPS CAPS ‘Yolo Wonder’ × F2 151 Single Marker Caranta et al.
(= Pvr7) ‘CM334’ dominant development 1999
SCUBC19 SCAR ‘SCM334’ × F2 110 Single Marker Arnedo-Andrés
‘Yolo Wonder’ dominant development et al. 2002
HpmsE031 SSR ‘CM334’ × F2 100 Single Genetic Kim et al.
‘Chilsungcho’ dominant mapping 2011
MY1421 SNP ‘SR-231’ × ‘CM334’ F2 204 Single Genetic Devran et al.
dominant mapping 2015
SNP-H2.4, SNP ‘9093’ × ‘Jeju’ 916 Single Candidate gene Venkatesh
96 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
F2
SNP-H1.5, dominant identification et al. 2018
SNP-H1.6
(Pvr4 = Pvr7)
Pepper pvr6 3 eIF(iso)4E InDel ‘DH218’ × ‘F’ F2 182 Single Gene cloned Ruffel et al.
veinal gene-based recessive 2006
mottle marker
virus
(PVMV)
Chilli veinal Pvr6-SCAR SCAR ‘Dempsey’ × F2 187 Single Marker Hwang et al.
mottle ‘Perennial’ recessive development 2009
virus
(ChiVMV)
Tobamov Pepper mild L3 11 21L24M, A339, SCAR, ‘KOS’ × ‘NDN’ F2 3,391 Single Define of Tomita et al.
irus mottle 197AD5R, SNP ‘PI159236’ × F2 2,016 dominant cosegregating 2008
virus 253A1R ‘LS1839-2-4’ region
(PMMoV)
L3, L4 11 L3-SCAR, SCAR Cultivars F1 53 Single Marker Lee et al.
L4-SCAR dominant development 2012a
L3-HRM, SNP Cultivars ‘Special’, F2 631, Single Marker Yang et al.
L4-HRM ‘Myoung-sung’ 858 dominant development 2012
TSWV Tomato Tsw 10 SCAC568 CAPS ‘Cupra’ × ‘Baltasar’ BC1-like 92 Single Marker Kim et al.
spotted dominant development 2008c
wilt virusMarker Development and Gene Cloning for Pepper Disease Resistance ∙ 97
‘Perennial’ (Ben Chaim et al. 2001). Among them, QTL 2006). A total of 10 CMVP1-resistant peppers were identi-
cmv11.1 was detected in all the experiments (Volcani 97, fied by evaluating 199 pepper genetic resources using
2
98, and Cornell 97) and had the largest R values (16-33%) enzyme-linked immunosorbent assays (ELISA) (Shin et al.
(Ben Chaim et al. 2001). Four QTLs, cmv5.1, cmv11.1, 2013). The CMVP1 resistance of C. annuum ‘I7339’ was
cmv11.2, and cmv12.1, which involved the partial restri- controlled by two different recessive genes, cmr3E and
ction of long-distance CMV movement, were mapped in a cmr3L, which were linked with one RAPD marker OPAT16
DH population derived from the F1 hybrid between C. on pepper chromosome 6 (Min et al. 2014). Recently, two
annuum ‘H3’ and ‘Vania’ (Caranta et al. 2002). The major- QTLs cmvP1-5.1 and cmvP1-10.1, conferring CMVP1 re-
effect QTL cmv12.1, detected in two separate experiments sistance were identified with trait variation of 17.81% and
using the CMVMES and CMVN strains, was positioned 22.78%, respectively (Eun et al. 2016). Furthermore, a
between two AFLP markers, E33/M48-132 and E40/ single recessive gene, cmr2, conferring a broad-spectrum
M47-262, on pepper chromosome 12 and explained 45.0- type of resistance to CMVP1 in C. annuum ‘Lam32’ was
63.6% of the phenotypic variation (Caranta et al. 2002). identified by inheritance analysis, and a SNP marker,
CMV resistance in C. annuum ‘BJ0747-1-3-1-1’ was con- Affy4, positioned 2.3 cM from the gene on chromosome 8
trolled by six QTLs, qcmv.hb-4.1, -7.1, -8.1, -8.2, -8.3, and (Choi et al. 2018).
-16.1, derived from experiments conducted over two
growing seasons (summer and autumn) (Yao et al. 2013). Tobamoviruses
Two stable and major QTLs, qcmv.hb-8.2 and -4.1, were Capsicum plants have genes, designated L genes, con-
found on linkage groups 8 and 4, and explained 37.7-43.5% ferring resistance to Tobamovirus spp. which generate di-
and 10.7-11.2% of the trait variation, respectively (Yao et verse symptoms including the chlorosis of leaves, stunting,
al. 2013). In the same population, three QTLs, qcmv11.1, and distorted and lumpy fruiting structures (Boukema
1
qcmv11.2, and qcmv12.1, conferring CMV resistance were 1980). There are four resistant alleles for L locus: L
additionally detected using SLAF-seq with trait variation (derived from C. annuum accessions) confers resistance to
of 10.2%, 19.2%, and 7.3%, respectively (Li et al. 2018). P0 pathotype viruses such as Tomato mosaic virus
2
Recently, two QTLs, qCmr2.1 and qCmr11.1, were iden- (ToMV); L (C. frutescens) confers resistance to P0 and P1
tified through genome-wide comparison of SNP profiles pathotype Paprika mild mottle virus (PaMMV) that over-
1 3
between the CMV-resistant and CMV-susceptible bulks comes L resistance; L (C. chinense) confers resistance to
constructed from an F2 population of C. frutescens ‘PBC688’ P0, P1, and P1,2 pathotype Pepper mild mottle virus
2 4
(resistant) and C. annuum ‘G29’ (susceptible), and the gene (PMMoV) that overcomes L resistance; L (C. chacoense)
CA02g19570 was identified as a possible candidate gene of confers resistance to P0, P1, P1,2, and P1,2,3 pathotype
3
qCmr2.1 (Guo et al. 2017a). PMMoV that overcomes L resistance (Boukema 1980,
Only in C. annuum ‘Bukang’, the CMV resistance was 1982, 1984; Tsuda et al. 1998; Tomita et al. 2008, 2011).
controlled by single dominant gene Cmr1 (Kang et al. The genes conferring resistance to various tobamoviruses
2010). Three CMVKorean and CMVFNY resistance SNP and their markers were listed in Table 3.
4
markers, CaTm-int3HRM, CaT1616BAC, and 240H02sp6 A RAPD marker WA31-1500, linked to the L allele that
associated with Cmr1 gene, were developed through the confers resistance to PMMoV, was identified using an F2
comparative genetic mapping between pepper and tomato population derived from a cross between ‘AP-PM04’ (re-
(Kang et al. 2010). Besides, a total of 1,941 Capsicum sistant; derived from ‘PI260429’) and ‘Mie-midori’ (sus-
accessions were evaluated using the 240H02sp6 marker, of ceptible) (Matsunaga et al. 2003). Three SCAR markers;
which 89 and 162 were homozygously and heterozygously PMFR11269, PMFR11283 and PMFR21200, positioned at a
3
resistant, respectively (Ro et al. 2012). distance of 4.0 cM from the L locus, were developed from
In Korea, CMVP1 strain breaking the CMVP0 resistance two RAPD markers, E18272 and E18286, which were de-
of pepper in the field was first reported in 2006 (Lee et al. veloped by applying the BSA method to two DH popu-98 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
lations, K9-DH and K9/AC-DH, derived from F1 hybrid aceous crops such as tomato, pepper, potato, and tobacco
3
‘K9’ that harbors the L gene derived from ‘PI159236’ (Caranta et al. 1997a; Kyle and Palloix 1997). Capsicum
(Sugita et al. 2004). A SCAR marker L4SC340, which was species have various potyvirus resistance genes such as;
4 1
mapped 1.8 cM from the L locus, was developed from an pvr1 (C. chinense ‘PI159236’ and ‘PI152225’), pvr2 (C.
2
AFLP marker L4-c, which was identified by applying annuum ‘Yolo RP10’ and ‘Yolo Y’), pvr2 (C. annuum
BSA-AFLP method to a near-isogenic BC4F2 population ‘PI264281’, ‘SC46252’, and ‘Florida VR2’), pvr3 (C.
generated by using C. chacoense ‘PI 260429’ (carrying the annuum ‘Avelar’), Pvr4 (C. annuum ‘CM334’ and ‘Serrano
4
L allele) as a resistant parent (Kim et al. 2008a). A SNP Criollo de Morelos’), pvr5 (C. annuum ‘CM334’ and
4
marker 087H03T7 with a distance of 1.5 cM from the L ‘Serrano Criollo de Morelos’), pvr6 (C. annuum ‘Perennial’),
locus was developed by sequencing a BAC clone 082F03 and Pvr7 (C. chinense ‘PI159236’) (Caranta et al. 1996;
that harbors the tomato I2 and potato R3 homologs (Yang et Kyle and Palloix 1997; Grube et al. 2000a). These genes
al. 2009). were mapped with molecular markers and cloned by
3
To clone the L gene, fine mapping and BAC library map-based cloning or candidate gene approach (Tables 3
3
analysis were performed (Tomita et al. 2008). The L gene and 5).
was mapped between I2 homolog marker IH1-04 and A recessive resistance pvr1 gene against PepMoV and
BAC-end marker 189D23M by using an intraspecific F2 TEV was mapped to a small linkage group, containing
population (2,016 individuals) of C. annuum (introduced TG56, A313, TG135, and CT128b markers, with synteny
from C. chinense ‘PI152225’) and an interspecific F2 popu- to the short arm of tomato chromosome 3 (Murphy et al.
lation (3,391 individuals) between C. chinense ‘PI159236’ 1998). The pvr1 gene encodes a translation initiation factor
3 3 2 2 1 2
(L /L ) and C. frutescence ‘LS1839-2-4’ (L /L ) (Tomita et eIF4E and is allelic with pvr2 and pvr2 , previously known
al. 2008). to be eIF4E with narrower resistance spectra (Kang et al.
1 2
The L4segF&R marker was developed based on the 2005). Two additional resistant alleles, pvr1 and pvr1 ,
4
LRR region of the L candidate gene identified in previous were identified (Kang et al. 2005), and three CAPS
4
study and applied to two L -segregating F2 populations markers, Pvr1-S, pvr1-R1, and pvr1-R2, were developed to
+ 1 2
derived from commercial cultivars ‘Special’ and ‘Myoung- discriminate between Pvr1 , pvr1, pvr1 , and pvr1 alleles
sung’ (Yang et al. 2012). The L4segF&R marker, however, (Yeam et al. 2005). Four functional markers, eIF4E-T200A,
4
did not completely cosegregate with the L gene, sug- eIF4E-T236G, eIF4E-G325A, and eIF4E-A614G, were
4 + 1 2
gesting that the candidate is not an actual L gene (Yang et designed for distinguishing between pvr2 , pvr2 , pvr2 ,
4 3
al. 2012). An L -specific HRM marker L4RP-3F/L4-RP3R and pvr2 alleles using the tetra-primer amplification
4
precisely detected the L allele in 90 out of 91 lines (Yang refractory mutation system (ARMS)-PCR method (Rubio
et al. 2012). Furthermore, a set of allele-specific markers of et al. 2008). Polymorphism analysis of the pvr2-eIF4E
L locus, including L1-SCAR, L2-CAPS, L3-SCAR, L4- coding sequence in 25 C. annuum accessions revealed 10
1 2 3 4 5 6
SCAR, L0c-SCAR, and L0nu-CAPS markers, was develop- allelic variants; pvr2 , pvr2 , pvr2 , pvr2 , pvr2 , pver2 ,
7 8 9
ed using five pepper differential hosts including C. annuum pver2 , pver2 , pvr2 , and pvr1 (Charron et al. 2008).
0 0 1 1
‘ECW’ (L /L ), C. annuum ‘Tisana’ (L /L ), C. annuum User-friendly markers for the pvr1 gene were also develop-
2 2 3 3
‘CM334’ (L /L ), C. chinense ‘PI159236’ (L /L ), and C. ed using the Kompetitive Allele-Specific PCR (KASP)
4 4
chacoense ‘PI260429’ (L /L ) (Lee et al. 2012a). genotyping system (Holdsworth and Mazourek 2015).
A recessive pvr3 gene for PepMoV resistance from C.
Potyviruses annuum ‘Avelar’ was reported to be different from the pvr1
The genus Potyvirus contains over 180 distinct viruses gene for PepMoV and TEV resistance from C. chinense
including Potato virus Y (PVY), Tobacco etch virus (TEV), ‘PI159236’ and ‘PI152225’ (Murphy et al. 1998).
and Pepper mottle virus (PepMoV), most of which cause A dominant Pvr4 gene for PVY and PepMoV resistance
significant losses in many agriculturally important Solan- from C. annuum ‘CM334’ was located on pepper chromo-Marker Development and Gene Cloning for Pepper Disease Resistance ∙ 99
some 10 (Dogimont et al. 1996; Grube et al. 2000a). The transient expression system in N. benthamiana (Tran et al.
Pvr4 gene was mapped on a linkage group containing eight 2015).
AFLP markers; E33/M54-126, E41/M49-645, E38/M61- In addition, a dominant Cvr1 (C. annuum ‘CV3’) and a
403, -414, -460, E41/M55-102, E41/M49-296, and E41/ recessive cvr4 (C. annuum ‘CV9’) genes were reported to
M54-138, and one of them, E41/M49-645 was converted confer ChiVMV resistance (Lee et al. 2017). Recently, a
into a CAPS marker (Caranta et al. 1999). RAPD and new resistance gene to Pepper yellow mosaic virus
SCAR markers, UBC191432 and SCUBC191432, linked to the (PepYMV), that is different from Pvr4 was reported in C.
Pvr4 locus were developed using segregating progenies ob- annuum ‘PIM-025’ (Rezende et al. 2019).
tained by crossing a homozygous resistant variety (‘Serrano
Criollo de Morelos-334’) with a homozygous susceptible Tomato spotted wilt virus
variety (‘Yolo Wonder’) (Arnedo-Andrés et al. 2002). Tomato spotted wilt virus (TSWV) disease is identified
Interestingly, trichome density of pepper main stem was by various symptoms including ringspots (yellow or brown
tightly linked to the Pvr4 gene resistant to PepMoV (Kim et rings) or other line patterns, black streaks on petioles or
al. 2011). A cosegregating marker, MY1421, with Pvr4 stems, necrotic leaf spots, or tip dieback (Boiteux et al.
gene was identified using an NGS method for which a total 1993). The resistance was found to be determined by a
of 204 F2 individuals derived from a cross between C. single dominant gene, Tsw, in three C. chinense accessions
annuum ‘SR-231’ (susceptible) and ‘CM334’ (resistant) (‘PI152225’, ‘PI159236’, and ‘7204’) (Moury et al. 1997).
were used (Devran et al. 2015). There are a few reports on the development of DNA
The complementation between recessive pvr6 (‘Perennial’) markers for TSWV resistance in Capsicum spp. (Table 3).
2
and pvr2 (‘Florida VR2’) genes confers complete resist- A CAPS marker SCAC568 was developed from the
ance to PVMV (Caranta et al. 1996). The pvr6 gene was OPAC10593 RAPD marker linked to Tsw gene to assist
positioned on linkage group 4 (LG4) of a pepper map selection of TSWV resistance in pepper (Moury et al.
generated by using a DH population from the hybrid 2000), and it was applied to paprika cultivars, suggesting
between ‘Perennial’ and ‘Yolo Wonder’ (Caranta et al. that SCAC568 can be deployed in pepper breeding programs
1997a). The pvr6 gene was identified to correspond to an in combination with TSWV-resistant cultivars from ‘Zeraim’
eIF(iso)4E gene which encodes the second cap-binding (Kim et al. 2008c). The Tsw gene was mapped on pepper
isoform identified in plants (Ruffel et al. 2006). Two chromosome 10 and a RAPD marker Q-06270 was identi-
simultaneous recessive alleles at pvr2 (eIF4E) and pvr6 fied using the segregating BC4F1 plants developed by
(eIFiso4E) loci confer resistance to PVMV as well as Chili backcrossing C. chinense ‘PI152225’ with C. annuum
veinal mottle virus (ChiVMV) in pepper (Ruffel et al. ‘Cuby’ and ‘Spartacus’ (Jahn et al. 2000). Recently, a
2006; Hwang et al. 2009). genome-based approach cloning revealed that Tsw
A dominant Pvr7 gene confers resistance to the PepMoV (CcNBARC575) gene encodes typical NLR proteins (Kim
Florida (V1182) strain and is tightly linked to the Pvr4 gene et al. 2017c). However, TSWV isolates breaking the Tsw
with a genetic distance of 0.012 to 0.016 cM and to Tsw resistance gene were reported (Hobbs et al. 1994; Moury et
gene on pepper chromosome 10 (Grube et al. 2000a). al. 1997; Jiang et al. 2017). Moreover, Tsw resistance was
Recently, Pvr7 (C. chinense ‘PI159236’ and C. annuum overcome by some TSWV isolates from paprika at high
‘9093’) and Pvr4 (C. annuum ‘CM334’) were revealed to temperatures (30 ± 2℃) (Chung et al. 2018). Therefore, a
be the same dominant resistant gene through sequence research for the identification of novel and stable TSWV-
analysis of the Pvr7 flanking markers and the Pvr4-specific resistant resources will be necessary.
gene (Venkatesh et al. 2018).
The resistance of Pvr9 gene, which is an ortholog of Bacterial spot
Rpi-blb2 conferring a hypersensitive response (HR) to Bacterial spot of pepper causes leaf and fruit spots,
PepMoV in Nicotiana benthamiana, was characterized in a which lead to defoliation, sun-scalded fruit, and yield loss100 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113 (Scherer, https://content.ces.ncsu.edu/bacterial-spot-of- due to a 13-bp insertion) promoters. A codominant SCAR pepper-and-tomato). It is caused by Xanthomonas campestris marker PR-Bs3 was developed by designing primers to pv. vesicatoria, which includes race 0 to 10 (Stall et al. amplify the indel region of Bs3 and bs3 promoters (Römer 2009). Capsicum species are known to have five dominant et al. 2010). Furthermore, user-friendly markers for the Bs3 and two recessive genes resistant to bacterial spot including gene were developed using the KASP genotyping system Bs1 (C. annuum ‘PI163192’), Bs2 (C. chacoense ‘PI260435’), (Holdsworth and Mazourek 2015). Bs3 (C. annuum ‘PI271322’), Bs4 (C. pubescens ‘PI235047’), Two recessive genes, bs5 and bs6, resistant to a pepper BsT (C. annuum commercial cultivars), bs5 (C. annuum xcv race 6 strain, were identified by using an F2 and two BC ‘PI163192’ and ‘PI271322’), and bs6 (C. annuum ‘PI163192’ populations derived from a cross between ECW12346 (re- and ‘PI271322’) (Hibberd et al. 1987; Stall et al. 2009). sistant) and ECW123 (susceptible) (Jones et al. 2002). Pepper resistance genes differentially interacts with races According to Vallejos et al. (2010), these two recessive of xanthomonads: Bs1 gene confers resistance to races 0, 2, genes when combined confer full resistance to race 6 and and 5; Bs2 gene confers resistance to races 0, 1, 2, 3, 7, and five AFLP markers (PepA2, PepC2, PepF4, PepB7, and 8; Bs3 gene confers resistance to races 0, 1, 4, 7, and 9; Bs4 PepG4) were linked to the bs5 gene. gene confers resistance to races 0, 1, 3, 4, and 6 (Stall et al. 2009). The DNA markers linked to the Bs2, Bs3, and bs5 Bacterial wilt genes have been reported (Table 4). Bacterial wilt, caused by a soil-borne pathogen Ralstonia The Bs2 resistance gene of pepper was positioned on a solanacearum, is a serious disease in a wide range of crops high-resolution genetic map constructed by RAPD and including pepper and tomato (Peeters et al. 2013). Five AFLP markers and was found to cosegregate with one Capsicum accessions, including ‘MC-4’, ‘PBC631’, AFLP marker A2 (Tai et al. 1999a). Three yeast artificial ‘PBC066’, ‘PBC1347’, and ‘PBC473’, were selected for chromosome (YAC) clones, YCA22D8, YCA80H11, and pepper breeding with bacterial wilt resistance (Lopes and YCA164C12, containing the Bs2 gene, were selected using Boiteux 2004). A Malaysian pepper accession ‘LS2341’ two probes from the A2 and B3 markers previously de- (C. annuum) was identified to be highly resistant to R. veloped (Tai and Staskawicz 2000). The Bs2 gene, which solanacearum strains from Japan (Mimura and Yoshikawa encodes a tripartite NBS and an LRR motif, was identified 2009). In addition, six inbred lines (‘KC350-3-4-2’, by coexpression with avrBs2 in an Agrobacterium-medi- ‘KC351-2-2-2-4’, ‘KC980-3-1’, ‘KC995-2-1’, ‘KC999-3-1’, ated transient assay (Tai et al. 1999b). Two tetra-primer and ‘KC1009-3-2’) resistant to bacterial wilt were reported ARMS-PCR markers, 25-1 and 25-2, were developed for (Tran and Kim 2010). Inheritance analysis and marker marker-assisted selection of the Bs2 gene in pepper development for the resistance to bacterial wilt were poorly (Truong et al. 2011). studied (Table 4). The Bs3 gene governing recognition of the Xanthomonas A major QTL Bw1 for bacterial wilt resistance, ex- campestris pv. vesicatoria AvrBs3 protein was mapped plaining 33% of genetic variance, was detected on pepper using AFLP markers and was delimited within two chromosome 1 using a DH population derived from a cross flanking markers, P23-70 and P22-3, with a genetic dis- between ‘California Wonder’ (susceptible) and ‘LS2341’ tance of 0.13 cM (Pierre et al. 2000). The Bs3 gene was (resistant) and an SSR marker CAMS451 was identified to physically delimited within two BAC clones, BAC128 and be closely linked to Bw1 (Mimura et al. 2009). A total of six BAC104, and was located between two markers, B104SP6 polymorphic AFLP bands, three bands (103, 117, and 161 and B103T7 (Jordan et al. 2006). Bs3 gene encodes flavin bp) linked with the resistant recessive allele and three monooxygenases with a previously unknown structure bands (183, 296, and 319 bp) linked with the dominant (Römer et al. 2007). The report indicated that recognition susceptible allele of the bacterial wilt resistance gene, were specificity between Bs3 and AvrBs3 resides in Bs3 (re- detected using C. annuum ‘Pusa Jwala’ (highly suscep- cognized by AvrBs3) and bs3 (not recognized by AvrBs3 tible), ‘Ujwala’ (highly resistant), and ‘Anugraha’ (a resist-
Table 4. Molecular markers linked to the genes or QTLs resistant to bacterial and nematode diseases in pepper.
Population
Resistance Type of Inheritance Status of
Group Disease Pathogen Chr. Marker or gene Number Reference
locus marker Parents Generation pattern research
of plants
Bacteria Bacterial Xanthomonas Bs2 9 A2-SCAR, S19-SCAR SCAR ‘ECW’ × F2, BC1 1577 Single Genetic Tai et al.
spot campestris ‘ECW-123R’ dominant mapping 1999a
pv. vesicatora Bs3 2 P23-70, P22-3 AFLP Cultivars Line 17 Single Fine mapping Pierre
dominant et al. 2000
B104SP6, B103T7 STS Cultivars Line 17 Single Define of cose- Jordan et al.
dominant gregating region 2006
PR-Bs3 InDel Accessions Line 19 Single Marker Römer et al.
dominant development 2010
KASP_Bs3 KASP Accessions Line 25 Single Marker Holdsworth and
dominant development Mazourek 2015
bs5 6 PepA2, PepC2, PepF4 AFLP‘NuMex R F2 100 Two Genetic Vallejos et al.
Naky’ × recessive mapping 2010
‘PI159234’ (bs5 and bs6)
Bacterial Ralstonia Bw1 8 CAMS451 SSR ‘LS2341’ × DH 94 QTL Genetic Mimura et al.
wilt solanacearum ‘CW’ mapping 2009
qRRs-10.1 10 ID10-194305124 SNP ‘BVRC25’ × F2, BC1 504 QTL Candidate gene Du et al. 2019
‘BVRC1’ identification
Nematodes Root- Meloidogyne Me3, 9 HM1, HM2, SSCP_B322 AFLP, ‘PM687’ × DH 103 Single Genetic Djian-Caporalino
knot spp.(M. Me4 SSCP ‘Yolo Wonder’ dominant mapping et al. 2001
nematode incognita, Me1, 9 SCAR_CD (PM54), SCAR ‘DH330’ × F2 373 Single Genetic Djian-Caporalino
M. javanica, Mech2 SCAR_HM60, SCAR_PM54 ‘DLL’ dominant mapping et al. 2007
M. arenaria) Me7, 9 CAPS_F4R4 (HM58), CAPS, ‘DLL’ × F2 301 Single Genetic Djian-Caporalino
Mech1 Q04_0.3, SSCP_B322 RAPD, ‘PM702’ dominant mapping et al. 2007
(PM6) SSCP
N 9 SCAR_PM6a, SCAR_PM6b, SCAR, ‘CW’ × F2 132 Single Genetic Fazari et al.
SSCP_PM5, SCAR_N SSCP ‘20080-5-29’ dominant mapping 2012
CA_CAPS_2, CA_SSR37 CAPS, ‘CW’ × F2 256 Single Candidate gene Celik et al.
SSR ‘AZN-1’ dominant identification 2016
Me1 9 CL000081-05555, CAPS, ‘AZN-1’ × F2 100 Single Marker Uncu et al.
C2At2g06530, CL001943-1222 COSII ‘PM217’ dominant development 2015
16830-H-V2, 16830-CAPS HRM, ‘DH330’ × BC1 1,598 Single Fine mapping Wang et al.
CAPS ‘0516’ dominant 2018
Me loci 9 SCAR_PM54 SCAR Accessions Line 14 Single Validity test of Pinar et al.
dominant marker 2016
Me3 9 11F6F, 43N9R, Me3-F/R, STS HDA149 Line 1 Single Physical Guo et al.
242G21R, 25F15F dominant mapping 2017b
Me7 9 G24U5, SF164076, CA1-1b, SNP, ‘ECW30R’ × F2 714 Single Candidate gene Changkwian
611109646, SCAR_PM6a, SCAR ‘CM334’ dominant identification et al. 2019
SCAR_PM6b, SF164024,
SF16406, 2111b1
Marker Development and Gene Cloning for Pepper Disease Resistance ∙ 101102 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
ant near isogenic line to ‘Pusa Jwala’) through a BSA- ‘AZN-1’ (susceptible line) and ‘PM217’ (resistant inbred
AFLP approach (Thakur et al. 2014). Recently, a major line derived from ‘PI201234’) (Uncu et al. 2015). An SSR
QTL, qRRs-10.1, conferring bacterial wilt resistance was marker (0.8 cM away) tightly linked to the N gene was
identified in an F2 population of BVRC25 (susceptible) × developed through fine mapping of NBS-coding resistance
BVRC1 (resistant) using SLAF-BSA analysis, and the genes to the Me-gene cluster on pepper chromosome 9
SNP marker ID10-194305124 tightly linked to the QTL (Celik et al. 2016). The SCAR_PM54 marker was identi-
peak was developed (Du et al. 2019). fied to be fully consistent with artificial nematode (M.
incognita race 2) testing, correctly predicting resistant
Root-knot nematode (‘PM687’, ‘PM217’, and ‘Carolina Cayenne’) and suscep-
The root-knot nematode (Meloidogyne spp.), which tible (‘Yolo Wonder B’, ‘California Wonder 300’, and
often shows symptoms of stunting, wilting or chlorosis ‘CM331’) genotypes (Pinar et al. 2016). Two BAC clones,
(yellowing), is a major plant pathogen, diseasing several PE25F15 and PE11F6, containing the Me3 gene, were
solanaceous crops including pepper (Djian-Caporalino et identified using BAC library and physical mapping an-
al. 2007). At least 10 dominant Me genes (N, Me1, Me2, alysis (Guo et al. 2017b). Recently, two markers, an HRM
Me3, Me4, Me5, Me6, Me7, Mech1, and Mech2) were marker 16830-H-V2 and a CAPS marker 16830-CAPS,
reported to be resistant to the nematode (Djian-Caporalino tightly linked to the Me1 gene, were developed through a
et al. 1999, 2001, 2007). Accordingly, Me4, Me5, Mech1, fine mapping approach and the CA09g16830 gene was
and Mech2 are specific to certain Meloidogyne species or identified as a candidate gene for Me1 (Wang et al. 2018).
populations and N, Me1, Me3, and Me7 are effective In addition, a recent development was the identification of
against a wide range of Meloidogyne species including M. nine SNP markers cosegregating with RKN resistance gene
arenaria, M. javanica, and M. incognita. In C. annuum ac- (Me7) for the utilization in MAS and the characterization of
cessions, ‘PI201234’ has Me1 and Mech2 genes, ‘PI322719’ 25 NLR class candidate resistance genes spanning the Me7
has Me3 and Me4 genes, and ‘CM334’ has Me7 and Mech1 region (Changkwian et al. 2019).
genes (Djian-Caporalino et al. 2007). These genes are
clustered on pepper chromosome 9 (Fig. 1, Table 4).
The Me3 and Me4 genes conferring heat-stable resist- GENE CLONING FOR PEPPER DISEASE
ance to root-knot nematodes were mapped using DH lines RESISTANCE
and F2 progeny from a cross between ‘Yolo Wonder’ (sus-
ceptible) and ‘PM687’ (resistant) by using RAPD and To date, many disease resistance (R) genes have been
AFLP analyses combined with BSA (Djian-Caporalino et identified and characterized in diverse plants (Dangl and
al. 2001). A RAPD marker Q04_0.3 (10.1 cM) and an Jones 2001; Gururani et al. 2012; Kourelis and van der
RFLP marker CT135 (2.7 cM) were linked to the Me3 Hoom 2018). The plant R genes can specifically detect a
gene, which was positioned at 10 cM of genetic distance pathogen attack and promote a counter-attack system
from Me4 gene (Djian-Caporalino et al. 2001). The six- against the pathogen (van der Biezen and Jones 1998;
dominant root-knot nematode resistance genes (Me1, Me3, Shehzadi et al. 2017). These R genes encode NLR and
Me4, Me7, Mech1, and Mech2) were found to be clustered non-NLR type proteins, which play important roles in
in a single genomic region within 28 cM on the pepper effector-triggered immunity (ETI) plant defense (Jacob et
chromosome 9 (Djian-Caporalino et al. 2007). Another al. 2013). In pepper, a total of 755 NLR-encoding genes,
root-knot nematode resistance gene, N-gene, was co-localized including 27 TNL, 236 CNL, 159 NL, 15 TN, 143 CN, and
in the Me-genes cluster on pepper chromosome 9 (Fazari et 175 N type genes, were identified through genome-wide
al. 2012). A codominant CAPS marker, CL000081-0555, analysis (Seo et al. 2016). In addition, 25 Pto-like protein
located 1.13 cM away from the Me1 gene, was developed kinases (PLPKs), which were non-NLR type proteins,
using an F2 population of a cross between C. annuum were identified in pepper genome (Venkatesh et al. 2016).Marker Development and Gene Cloning for Pepper Disease Resistance ∙ 103
Fig. 1. Position of disease resistance genes or QTLs on the pepper reference genome (Capsicum annuum cv. CM334 v1.55;
http://peppergenome.snu.ac.kr; Kim et al. 2014). Bs3, Bacterial spot 3 (Römer et al. 2007); Cmr1, Cucumber mosaic
resistance 1 (Kang et al. 2010); qCmr2.1, QTL Cucumber mosaic resistance 2.1 (Guo et al. 2017a); pvr6, potyvirus
resistance 6 (Ruffel et al. 2006); PMR1, Powdery Mildew Resistance 1 (Jo et al. 2017); pvr1, potyvirus resistance
1 (Kang et al. 2005); pvr2, potyvirus resistance 2 (Kang et al. 2005); LtR4.2, Leveillula taurica resistance 4.2 (Kim
et al. 2017a); Phyto5.2, QTL phytophthora resistance 5.2 (Quirin et al. 2005); Pc5.1, QTL Phytophthora capsici
5.1 (Mallard et al. 2013); CaPhyto, QTL Capsicum Phytophthora (Wang et al. 2016); AnR5, QTL Anthracnose Resistance
5 (Sun et al. 2015); Pvr9, Potyvirus resistance 9 (Tran et al. 2015); bs5, bacterial spot 5 (Vallejos et al. 2010);
Lt_6.1, QTL Leveillula taurica 6.1 (Lefebvre et al. 2003); cmr2, cucumber mosaic resistance 2 (Choi et al. 2018);
Bw1, Bacterial wilt 1 (Mimura et al. 2009); Bs2, Bacterial spot 2 (Tai et al. 1999b); Me gene cluster, Me1, Me3,
Me4, Me7, Mech1, Mech2, and N genes (Fazari et al. 2012); CcR9, QTL Colletotrichum capsici Resistance 9 (Lee
et al. 2011); qRRs-10.1, QTL Resistance Ralstonia solanacearum 10.1 (Du et al. 2019); PhR10, QTL Phytophthora
Resistance 10 (Xu et al. 2016); Tsw, Tomato spotted wilt resistance (Kim et al. 2017c); Pvr4, Potyvirus resistance
4 (Kim et al. 2017c); Pvr7, Potyvirus resistance 7 (Venkatesh et al. 2018); RCt1, QTL Resistance Colletotrichum
truncatum 1 (Mishra et al. 2019); Phyt-3, QTL Phytophthora 3 (Sugita et al. 2006); qcmv11.1 and qcmv11.2, QTL
cucumber mosaic virus 11.1 and 11.2 (Li et al. 2018); L, resistance locus to tobamoviruses (Tomita et al. 2008);
qcmv12.1, QTL cucumber mosaic virus 12.1 (Li et al. 2018); CaR12.2, QTL Colletotrichum acutatum Resistance
12.2 (Lee et al. 2011).
In light of the rapidly evolving molecular markers and Xanthomonas campestris pv. vesicatoria (Xcv) was the
map-based cloning, identification and characterization of first cloned disease resistance gene in pepper (Tai et al.
disease resistance genes in Capsicum species have ad- 1999b). Two recessive genes, pvr1 and pvr6, which encode
vanced the pace of introgression of resistance genes into eIF4E and eIF(iso)4E proteins, respectively, were cloned
elite varieties (Srivastava and Mangal 2019). Fine mapping and reported to confer resistance to potyviruses such as
and identification of resistance genes and QTLs have PepMoV, PVMV, and ChiVMV (Kang et al. 2005; Ruffel
prompted the discovery of several resistance genes in et al. 2006). The Bs3 gene resistant to the Xcv with AvrBs3
pepper (Table 5). The Bs2 gene, which encodes an NLR was identified to encode flavin monooxygenase, which is
protein that interacts with the corresponding bacterial an unusual protein encoded by plant disease resistance
avirulence protein avrBs2, conferring resistance to genes (Römer et al. 2007). According to the report, AvrBs3Table 5. Cloned and candidate genes for pepper disease resistance.
Locus Chr. Encoding protein Gene name Resistance resource Reference
Bs2 9 nucleotide binding site–leucine-rich repeat (NLR) protein Bs2 C. chacoense ‘PI260435’ Tai et al. 1999b
C. annuum ‘ECW-20R’
pvr1 4 Eukaryotic translation initiation factor 4E (eIF4E) eIF4E C. chinense ‘PI152225’, Kang et al. 2005
‘PI159234’, ‘PI159236’
104 ∙ Plant Breed. Biotech. 2020 (June) 8(2):89~113
pvr6 3 Eukaryotic translation initiation factor iso 4E (eIF(iso)4E) eIF(iso)4E C. annuum ‘Perennial’ Ruffel et al. 2006
Bs3 2 Flavin-dependent monooxygenase (FMOs) Bs3 C. annuum ‘PI271322’ Römer et al. 2007
C. annuum ‘ECW-30R’
3
L 11 coiled-coil, nucleotide-binding, leucine-rich repeat protein (CC-NB-LRR) L C. chinense ‘PI152225’ Tomita et al. 2011
Pc5.1 5 Homoserine kinase (HSK) CaDMR1 C. annuum ‘CM334’ Rehrig et al. 2014
Pvr9 6 CC-NB-ARC-LRR protein Pvr9 C. annuum ‘CM334’ Tran et al. 2015
CaPhyto 5 Leucine rich repeat receptor-like serine/threonine-protein kinase BRI1-like 2 (BRL2) Capana05g000764 C. annuum ‘PI201234’ Wang et al. 2016
Disease resistance protein RPP13 Capnan05g000769
Tsw 10 Nucleotide-binding and leucine-rich domain protein (NLR) CcNBARC575 C. chinense ‘PI159236’ Kim et al. 2017c
Pvr4 10 Nucleotide-binding and leucine-rich domain protein (NLR) CaNBARC322 C. annuum ‘CM334’ Kim et al. 2017c
qCmr2.1 2 N-like protein (TMV resistance protein) (TIR-NBS-ACR-LRR) CA02g19570 C. frutescens ‘PBC688’ Guo et al. 2017a
PMR1 4 NLR domain-containing R protein 408 and 556 C. annuum ‘VK515R’, Jo et al. 2017
C. annuum ‘PM Singang’
Me1 9 Putative late blight resistance protein (homolog with R1A-3 gene) CA09g16830 C. annuum ‘PI201234’ Wang et al. 2018You can also read
