1. Introduction
Pepper, which belongs to the
Capsicum genus, holds a significant position as one of the world’s most important vegetable crops, including in South Korea [
1]. The
Capsicum genus encompasses around 35 species [
2], with 5 of them being cultivated and of economic importance:
Capsicum annuum L.,
Capsicum chinense Jacq.,
Capsicum frutescens L.,
Capsicum baccatum L., and
Capsicum pubescens Ruiz and Pav. [
3]. However, other species of
Capsicum species (
Capsicum chacoense and
Capsicum galapagoense) have also important traits. According to the data from FAOSTAT [
4] spanning from 2010 to 2021, the overall production of pepper has experienced a noteworthy growth of roughly 20.28%. During this period, the production of green peppers increased by 18.12%, while the production of dried peppers exhibited substantial growth at 36.43%. In 2021, global pepper production reached a total of 41.13 million tons, with 36.89 million tons of fresh pepper and 4.84 million tons of dried pepper [
4]. In the same year, China led the world in fresh pepper production, producing 16.72 million tons, followed by Turkey with 3.09 million tons and Indonesia with 2.75 million tons. In terms of dried pepper production, India emerged as the foremost producer, contributing 2.05 million tons to the worldwide market [
4].
However, the annual production and cultivation of pepper have been impacted by prevalent pepper diseases, resulting in a significant decrease in yield. Notable among these ailments are
Phytophthora root rot (
Phytophthora capsici) [
5], anthracnose (in the forms of
Colletotrichum scovillei and
C. truncatum, previously known as
C. acutatum and
C. capsici, respectively [
6]), powdery mildew (
Leveillula taurica) [
7], bacterial wilt (
Ralstonia solanacearum) [
8], bacterial spot (
Xanthomonas campestris pv. vesicatora) [
9], cucumber mosaic virus (
CMV) [
10], pepper mild mottle virus (
PMMoV) [
11], tomato spotted wilt virus (
TSWV) [
12], and pepper mottle virus (
PepMoV) [
12,
13]. These diseases pose a formidable challenge to control, even with the application of agricultural chemicals.
Molecular markers are widely employed to enhance the effectiveness of plant breeding initiatives, create genetic linkage maps, and identify genes or the quantitative trait locus (QTL) responsible for specific characteristics [
14,
15,
16]. Marker-assisted selection (MAS) and marker-assisted backcrossing (MABC) are key methods in plant breeding, streamlining trait selection. MAS targets specific traits, while MABC hones in on genomic regions in backcross generations [
15]. This approach expedites breeding by leveraging codominant markers to detect traits early, eliminating the need for full plant maturity or inoculation, reducing the timeline and generations required compared to traditional phenotypic selection [
12,
15].Various molecular markers have been developed for selecting resistant pepper varieties against prevalent diseases. Pepper’s bacterial spot resistance genes (Bs2 and Bs3) were cloned [
17,
18], followed by the development of gene-based codominant markers: 14F/14R for Bs2 and PR-Bs3 for Bs3 [
9,
18]. Additionally, two dominant markers were reported for detecting a major QTL, Phyto.5.2, associated with resistance to
P. capsici [
19]. Furthermore, codominant markers M3-CAPS and Phyto5NBS1-HRM for the same trait have been developed [
5,
20]. Two CAPS markers, pvr1-R1 and pvr1-R2, were devised to detect pvr1 and pvr12 alleles for potyvirus resistance in
C. chinense accessions [
21]. Moreover, Pvr4 and Tsw genes were cloned: Pvr4 is a potyvirus resistance gene, originated from
C. annuum ‘CM334’, while Tsw is a TSWV resistance gene found in
C. chinense accessions ‘PI159236’ and ‘PI152225’ [
22]. Additionally, three SNP markers were developed from the single dominant gene Cmr1, which is associated with CMV resistance [
23].
Significant advancements in the field of DNA sequencing and SNP (single-nucleotide polymorphism) genotyping have been achieved in recent decades, including next-generation sequencing (NGS) and high-throughput SNP genotyping [
24,
25,
26,
27]. High-throughput SNP genotyping specifically holds substantial promise in the realm of crop breeding [
27]. Remarkably, molecular markers can be rapidly developed for SNPs, which are the most common types of genetic variations, exhibiting extensive nucleotide diversity among individual organisms, even within the same species [
28]. Presently, a wide array of automated platforms designed for high-throughput analysis have made it possible to process substantial volumes of data rapidly [
29,
30]. As an illustration, the Fluidigm dynamic arrays employ automated PCR techniques in conjunction with nanofluidic integrated fluid circuits (IFCs) [
31]. The Fluidigm platform has found extensive application in the realm of SNP genotyping and the development of SNP markers for distinguishing cultivars in various plant species [
32,
33,
34,
35].
Fluidigm SNP-type genotyping markers have been developed for various diseases of pepper, including bacterial spot, anthracnose,
Phytophthora root rot, powdery mildew, potyviruses, CMV, TMV (tobamovirus), and TSWV [
12]. These Fluidigm SNP markers were utilized in the current study. In this research, a large collection of Capsicum germplasm (5658 accessions) from diverse species and geographical locations, preserved within the genebank of the National Agrobiodiversity Center (NAC), Rural Development Administration, was subjected to assessment. We utilized 19 SNP markers through the Fluidigm genotyping system to identify disease-resistant germplasm against eight important diseases of pepper. This geographically and genetically diverse dataset serves as a valuable resource, not only shedding light on the genetic basis of disease resistance in different
Capsicum species but also offering a nuanced understanding of their distribution and prevalence, thereby informing future breeding strategies and enhancing global crop improvement efforts.
3. Discussion
Peppers (
Capsicum spp.) are vulnerable to many diseases, which can significantly reduce their yield and quality. To find pepper accessions that are resistant to these diseases, it is crucial to use efficient screening methods in breeding programs. In our study, we used SNP markers with the Fluidigm genotyping system to screen for disease resistance in a large group of pepper plants from the National Agrobiodiversity Center genebank. In this study, various markers from known resistance genes were used to screen for disease resistance. Three dominant resistance genes, Bs1, Bs2, and Bs3, which are not different forms of the same gene (alleles), have been identified as conferring resistance against
X. campestris pv. vesicatora (Xcv) [
36]. The avirulence genes avrBs1, avrBs2, and avrBs3, obtained from Xcv, have been isolated and demonstrated to induce resistances specific to particular races. Notably, the Bs2 gene in pepper (
Capsicum spp.) exhibits resistance against the most prevalent Xcv races [
9]. This study utilized markers designed from the resistance loci of the Bs2 and Bs3 genes to identify bacterial spot-resistant genotypes in pepper. The Bs2 and Bs3 genes are known for conferring resistance to bacterial spot disease caused by
X. campestris pv. vesicatoria [
17,
18]. These genes have been identified in certain wild pepper species, such as
C. chacoense (Bs2) and
C. annuum (Bs3), but their resistance mechanisms can also be effective in other
Capsicum species and related plant species. In the previous study, which included a small sample of accessions from various
Capsicum species, the unique marker BS2 was found to identify resistant accessions exclusively from
C. chacoense [
12]. Our current research expands on this by confirming resistance in the majority of
C. chacoense accessions (10 out of a total of 11) and also identifying a few resistant accessions from
C. annuum (8 accessions) and
C. baccatum (3 accessions) when utilizing markers derived from the Bs2 gene. The larger sample size in our study allowed us to identify resistant accessions not only in
C. chacoense but also in other species. On the other hand, when employing markers (Bs3-1 and Bs3-3) derived from the resistance locus of the Bs3 gene, a large number of
C. annuum accessions were identified as resistant to bacterial spot. The variation in resistant accession distributions for bacterial spot using three markers (Bs2, Bs3-1, and Bs3-2) might be due to genetic diversity within the plant population, differences in pathogen strains, and the specific resistance genes from which the markers are derived. According to gene-for-gene interactions between resistance (R) genes and their corresponding avirulence genes, bacterial spot caused by
Xe has been classified into eleven races (P0–P10) [
37]. Bs1 confers resistance against races P0, P2, and P5; Bs2 against races P0, P1, P2, P3, P7, and P8; and Bs3 against races P0, P1, P4, P7, and P9 [
37]. Therefore, resistant genes originating from different sources (species) exhibit varied interactions with different pathogen races.
Two important markers (M3-2 and M3-3) derived from a known locus (
Phyto.5.2) associated with resistance against
Phytophthora root rot were utilized to screen resistant accessions. The
Phyto.5.2 locus, renowned for its significant impact on
P. capsici resistance and its ability to confer broad resistance against multiple isolates [
38], proved to be a valuable genetic marker for identifying and selecting resistant pepper varieties. These two markers identified resistant accessions from all
Capsicum species used in this study but predominantly from
C. chinense and
C. annuum. A similar report indicated that the dominant OpD04.717 allele, linked to that locus, was present for all
C. chinense accessions in the authors’ study and for a few
C. annuum individuals. Accessions of
C. chacoense were identified as resistant material, which is in line with [
39], a study that identified resistant
C. chacoense accessions. However, the authors of another study [
40] reported that they did not identify any resistant genotypes within the
C. chacoense species. These
C. chacoense accessions originated from Bolivia, Argentina, the United Kingdom, and Germany, and two accessions were of unknown origin. Regardless of their origin, they were persistently identified as resistant accessions for several diseases, including
Phytophthora root rot. Resistant accessions of
C. chinense primarily originated from Hungary, Bolivia, Brazil, Colombia, Costa Rica, Ecuador, and Peru. In contrast, resistant accessions of
C. annuum mainly came from the United States, Vietnam, South Korea, India, and China. In a previous report, both
C. annuum and
C. chinense resistant accessions predominantly originated from Central America and the Caribbean region, where the most durable source of resistance to
P. capsici, ‘CM334’, was identified [
41].
Capsicum annuum,
C. chinense, and to a lesser extent
C. baccatum have been recognized as sources of resistance against various races of
P. capsici [
42]. In this study, resistant accessions from
C. baccatum were also identified using both markers.
Anthracnose resistance is a primary target in chili pepper breeding endeavors. In this study, three SNP markers (
CA09g12180,
CA09g19170, and
CcR9) were employed to screen for anthracnose resistance, yielding consistent results. Resistance was observed across various species of
Capsicum, suggesting that resistance is not species-specific. However, a notable proportion of resistant accessions were identified from
C. baccatum compared to other species. This finding aligns with previous research, which has highlighted
C. baccatum as having higher levels of resistance to anthracnose compared to other
Capsicum species, making it an essential genetic resource for anthracnose resistance [
43,
44,
45]. Multiple studies have reported sources of anthracnose resistance in pepper from different countries, with
C. baccatum and
C. chinense being commonly identified as reservoirs of resistance [
46,
47,
48,
49]. However, according to [
47], resistant accessions of
C. chinense are frequently utilized in breeding programs targeting anthracnose resistance due to their genetic proximity to
C. annuum, facilitating the transfer of resistance genes between species. Notably, no resistant accessions to anthracnose were identified from
C. galapagoense and
C. pubescens species.
Powdery mildew resistance has been identified in various species, primarily
Capsicum annuum,
Capsicum baccatum,
Capsicum chacoense,
Capsicum chinense, and
Capsicum frutescens (
Table 4). Several studies have pinpointed pepper genotypes with varying degrees of resistance against powdery mildew in these species [
50,
51,
52,
53,
54]. In this investigation, a substantial number of
C. baccatum accessions are identified as resistant to powdery mildew using three markers: Ltr4.1-40344248 (248 accessions), Ltr4.2-56301 (243 accessions), and Ltr4.2-585119 (244 accessions). These markers yield similar results, indicating the presence of the target resistance gene in
C. baccatum compared to other species. Notable resistant pepper genotypes include ‘H-V-12’ and ‘4638’ (
C. annuum), ‘IHR 703’ (
C. frutescens), and CNPH 36, 38, 50, 52, 279, and 288 (
C. baccatum) against
L. taurica [
50,
51]. However, the number of
C. annuum accessions resistant to powdery mildew is relatively low (
Table 4). Supporting reports suggest that while most
C. annuum species are susceptible to powdery mildew,
C. baccatum,
C. chinense, and
C. frutescens species often exhibit resistance. This indicates that resistance to powdery mildew is primarily found in
Capsicum species other than
C. annuum [
51]. Moreover, the dominant pattern of inheritance of powdery mildew resistance in ‘VK515R’, similar to
C. baccatum, suggests that resistance in ‘VK515R’ may have been introgressed from
C. baccatum, possibly facilitated by
C. chinense as a bridge species, given the lack of cross-compatibility between
C. annuum and
C. baccatum [
55,
56,
57].
Plant viruses are responsible for considerable reductions in both crop yield and quality on a global scale [
58]. Pepper cultivation faces considerable challenges due to the presence of numerous plant pathogens, with over 60 viruses identified as significant threats [
59]. Managing these viral pathogens presents difficulties because of their wide range of hosts and the multitude of insect vectors involved. Utilizing resistant cultivars remains the most effective and often the sole approach to mitigating plant viral diseases [
60]. The screening of
Capsicum accessions for resistance to various viral diseases (potyvirus, CMV, TMV, and TSWV) was conducted using SNP markers. Resistance to potyviruses was investigated by using different markers linked to
pvr1,
pvr2, and
pvr4 resistant genes [
21,
61,
62]. In this work, we utilized different markers including ones from the resistant genes
pvr1,
pvr2, and
Pvr4. These markers identified resistant accessions predominantly from
C. chinense (
Table 5 and
Table 6).
C. chinense lines emerge as the most promising resource against potyviruses in previous work [
39] since the predominant
pvr1 allele protects pepper plants against TEV, PVY (0), and PepMoV [
21]. Markers made from the resistant genes
pvr2 and
pvr4 identified resistant accessions primarily from
C. annuum. Additionally, a few resistant accessions for potyvirus were identified from
C. chinense and
C. frutescens using the markers from resistant genes (
pvr2 and
Pvr4) (
Table 5 and
Table 6). The dominant locus
Pvr4, which controls the complete inhibition of viral replication and accumulation, was investigated, and the resistant allele was observed in all but three
C. baccatum, 56.1% of
C. chinense, and 12.1% of
C. annuum, suggesting that those lines carry potential resistance to PVY (0, 1, 2) and PepMoV [
39]. On the other hand, accessions from the wild
Capsicum species (
C. chacoense), which exhibited resistance to other diseases, were predicted to be susceptible to all viral diseases evaluated in this study. As reported in previous studies [
12,
58,
63], the distribution of resistant accessions across markers derived from the resistant genes pvr1 and pvr2 differed, a trend also observed in our findings. The
pvr1 assay primarily detected resistance alleles in
C. chinense accessions, while the
pvr2-123457 assay, originating from
C. annuum and
C. frutescens, showed a higher frequency of resistant reactions in
C. annuum. This pattern illustrates the genetic diversity among
Capsicum species and supports our findings, wherein markers derived from the
Pvr4 gene, originating from
C. annuum, detected a greater number of resistant accessions compared to markers derived from the
pvr1 gene, which is associated with
C. chinense. These observations highlight the importance of considering genetic diversity and marker specificity in virus resistance screening within pepper populations. Furthermore, the race specificity of these resistance genes may also contribute to the observed patterns of resistance, underscoring the complex interplay between genetic factors and virus strain specificity in pepper virus resistance.
CMV is one of the most persistent viruses affecting peppers in South Korea [
64]. We employed a marker (Cmr1-2) associated with the CMV-resistant gene Cmr1. Over recent decades, diverse sources of resistance to CMV have been uncovered in
Capsicum. Most of these sources exhibit polygenic resistance controlled by multiple genes. Notable examples include
C. annuum varieties such as “Perennial” [
65,
66,
67], “Vania” [
68], “Sapporo-oonaga”, and “Nanbu-oonaga” [
69], as well as
C. frutescens “BG2814-6” [
66],
C. frutescens “LS1839-2-4”, and
C. baccatum “PI439381-1-3” [
23,
69]. In our investigation, resistant accessions were identified from
Capsicum species other than
C. chacoense utilized in the study, showing significant variability in terms of accessions. A substantial number of accessions were predominantly identified from
C. annuum (25%),
C. chinense (59%), and
C. frutescens (88%). A probable source of resistance to CMV was discovered in 94.1% of
C. frutescens and 90.2% of
C. chinense accessions but not in any genotype from other domesticated or wild species [
39]. Additionally,
Capsicum chinense and
C. frutescens were also highlighted as good sources of resistance against CMV by [
40] Di Dato et al., using the same CAPS marker, as well as through phenotypic assays [
66].
We utilized a single marker (TSW1-4) to identify potential resistance among accessions to TSWV, focusing on the Tsw dominant resistant allele. The resistant accessions were predominantly discovered within
C. chinense, with subsequent findings in
C. annuum,
C. frutescens, and
C. baccatum. These results echo those of a prior study by [
39], which documented resistant accessions across a wide spectrum of
Capsicum species, including
C. chinense,
C. frutescens,
C. baccatum,
C. chacoense,
C. eximium, and
C. cardenasii, alongside a limited number from
C. annuum. Moreover, previous studies have highlighted the potential for exploiting resistance in
C. annuum populations from Mexico, Peru, and Spain, which harbor additional alleles that are candidates for resistance [
40,
70].
Among the recognized genetic factors providing resistance to TMV, the L4 allele at the L locus is known for its wide resistance spectrum against various pathotypes [
71]. In our research, we utilized two markers linked to resistance genes, L1 (L1-3K) and L4 (L4). Based on the L1-3k marker, resistant accessions were found in
C. annuum,
C. chinense,
C. frutescens, and
C. baccatum. However, when considering marker L4, no homozygous accessions were identified; instead, one heterozygous accession was discovered, which we anticipated to be resistant given its association with the resistance gene. In contrast to our findings, a prior study revealed that the dominant resistant allele for the marker (060I2END), linked to L4, was present in nearly all accessions from both domesticated and wild species, except for
C. annuum, where potential resistance to TMV was observed only in three landraces [
39,
40]. Moreover, reports have suggested the identification of resistant sources from
C. chacoense genotypes carrying the L4 allele [
11,
39].
A comparison of the disease evaluation results and Flufigm SNP genotyping results for CMV and TSWV is essential for validating the efficacy of marker-assisted selection (MAS) in plant breeding programs. Assessing the agreement between traditional disease phenotyping techniques and molecular marker data allows breeders to verify the reliability of SNP markers in identifying resistant and susceptible plant materials. The observed discrepancies between the two methods underscore the intricate nature of disease resistance mechanisms and emphasize the importance of employing complementary approaches in breeding for disease resistance. The high accuracy (98.68%) of the TSWV SNP markers in predicting resistance or susceptibility indicates their potential for expediting breeding efforts and hastening the development of resilient crop varieties. While the accuracy for CMV SNP markers was 70.22%, this analysis provides valuable insights for refining breeding strategies and optimizing marker selection to enhance crop resilience against these devastating viral pathogens.