Phytophthora root rot is a highly damaging disease of chile peppers present throughout the globe and is very difficult to manage [2]. With modern molecular plant breeding tools, disease resistance QTL and genes can be detected, which ultimately contributes to the development of resistant chile pepper varieties. GWAS is a tool used in plant breeding which provides higher mapping resolution than traditional biparental mapping approaches [47]. This study employed multi-locus GWAS approaches to better understand the genetics of resistance to P. capsici root rot in chile pepper. The virulence of P. capsici isolates used for screening exhibited variation, where some genotypes displayed resistance to specific races. A complex genetic relatedness was observed within the Capsicum population used for screening P. capsici root rot. Further, the complex genetic basis of disease resistance is reflected by the diversity of candidate genes identified in this study.
Variation in virulence for the P. capsici isolates and race-specific resistance
The genotypes that are virulent to the same host are referred to as a physiological race of a pathogen [48]. This study involved three isolates of P. capsici, each of which belonged to different physiological races with different levels of virulence. Around 82.8% of the accessions screened with isolate ‘PWB-186’ showed no significant difference in the average disease ratings compared to the resistant control (‘CM-334’). For isolates ‘PWB-185’ and ‘6347’, 22.3% and 17.2% of the accessions, respectively, did not exhibit significant differences from the resistant control. The susceptible checks for ‘PWB-186’ were not dead at 14-DPI, whereas for ‘PWB-185’ and ‘6347’, the susceptible checks were completely dead at 14-DPI and 10-DPI, respectively. Thus, the isolate ‘PWB-186’ was the least virulent among the three; however, isolates ‘PWB-185’ and ‘6347’ were highly virulent, with ‘6347’ being more virulent than ‘PWB-185’. Isolate ‘6347’ was also described as the most virulent isolate (designated as ‘PWB-175’) among 13 isolates used in a previous study [49].
Breeding peppers for resistance against P. capsici is a challenging task due to the constant emergence of new races that can overcome the existing host resistance [50, 51]. Different resistance genes are required for different physiological races within each disease phase [10]. Race-specific resistance was observed for some of the accessions in this study. For example, ‘Floral Gem’ was resistant to isolates ‘PWB-185’ and ‘PWB-186’ but was susceptible to isolate ‘6347’. In a previous study, this cultivar was screened against 10 isolates of P. capsici and was found to be resistant to eight isolates and susceptible to isolates ‘PWB-24’ and ‘PWB-73’ [52]. ‘Paladin’ was susceptible to all three isolates in this study, but it was resistant to three (‘PWB-53’, ‘PWB-75’, and ‘PWB-54’) out of eight isolates in a previous study [52]. ‘Paladin’ was also found to be resistant to isolates from New York and North Carolina, USA [53, 54]. In this study, ‘NuMex Vaquero’ was susceptible to all three isolates, but a previous study reported that this variety had resistance against Phytophthora root rot races 2 and 3 [55]. Five accessions, namely ‘Chilhuacle Orange’, ‘Tipo Ancho’, ‘NMCA10237’, ‘13C905-6’, and ‘Tipo Pasilla’ exhibited broad-spectrum resistance and were found to be completely resistant to the three isolates used in this study. These accessions can serve as potential resistant sources for future genomic breeding aimed at developing chile peppers with Phytophthora root rot resistance.
Population structure of the Capsicum spp. accessions
Genetic subpopulations for the genotypes were derived using Bayesian iterative algorithm approach used in STRUCTURE software. Based on the ad hoc Evanno criterion, the optimal number of clusters, K, was determined to be 7 (Fig. 3D), indicating that the population could be divided into seven subpopulations. This finding was also supported by the results of the phylogenetic analysis (Fig. 3C). About 97.45% of the panel used in this study consisted of C. annuum accessions and only four accessions were interspecific hybrids between C. annuum and C. frutescens. These four interspecific accessions formed a separate cluster based on the results from STRUCTURE, PCA, and Neighbour-joining analysis (Group E; Figs. 3B and 3C). The rest of the six clusters consisted of the C. annuum accessions. In previous studies, complex genetic relatedness has been found within the C. annuum group [32, 56], where accessions clustered into different groups. Taranto et al. [57] found clustering in C. annum based on geographical locations and fruit-related traits. Similar to previous studies [58, 59], clusters of C. annuum accessions were detected on the basis of fruit or pod type in this study. The PCA revealed five groups whereas the STRUCTURE analysis resulted in K = 7 optimum clusters; this disparity between PCA and STRUCTURE results has been previously noted in other studies [32, 58]. The optimum K derived in performing STRUCTURE analysis may not necessarily represent the ideal number of clusters since it is determined based on a pre-determined sampling method. Therefore, when interpreting the results of STRUCTURE, it is crucial to consider the biological significance of the optimum number of clusters [60].
The knowledge of physical distance of LD decay across the population of interest facilitates the selection of the number of markers for association studies. If LD decays over long distances, fewer markers are necessary for association mapping, resulting in lower resolution. Conversely, if LD decays rapidly within a short distance, a larger number of markers is needed for association mapping and the resolution is higher [61]. Rapid LD decay (~ 0.10 Mb) was observed for the population in this study. The marker density of one single nucleotide polymorphism (SNP) per 48.04 kb, on average, was thus sufficient for detecting the genomic loci associated with P. capsici root rot resistance. Our results were consistent with previous studies which detected rapid LD decay for C. annuum at 0.07 Mb [58] and 0.01 Mb [57]. While rapid LD decay in chile peppers would require an increased number of markers, it would also provide advantages to breeders in performing fine mapping of the genes of interest identified from association studies.
Significant markers associated with P. capsici root rot resistance
Performing multi-locus GWAS resulted in the detection of a total of 330 SNP markers with the highest being detected using the isolate ‘6347’ (114 SNPs). Fifty-six markers distributed across all 12 chromosomes were detected which were common in two or more isolates. Previous studies have also mapped the resistance loci on chromosomes 1–12 of chile pepper [8, 9, 27, 28, 62–70]. Chromosome 5 has been previously identified as a prominent region for the occurrence of resistance genes against P. capsici in Capsicum spp. [8, 9, 28, 71]. In this study, seven significant SNPs were identified on chromosome 5 with S5_14665044 explaining the maximum phenotypic variation of up to 29.6%. This marker was located ~ 0.2Mb upstream of a major QTL peak detected using bulk segregant analysis that covered 18.8 cM and was delimited by two markers, CONTIG6473 and CONTIG1896 [72]. Phyto5SAR marker showing highest LOD at the major QTL detected by Liu et al. [72], was located ~ 1.4 Mb downstream to the S5_25470050 SNP identified in this study. GBS-derived-SNPs on chromosome 5 detected by Siddique et al. [9] at 27.0 to 29.5 Mb, were also located ~ 1.4 Mb downstream to the S5_25470050. Three SNP markers (S5_9345710, S5_14665044, and S5_25470050) detected using GWAS in this study coincide with the extended Pc5.1 (Ext-Pc5.1) region on chromosome 5 that is located between 8.35 Mb and 38.13 Mb [73]. These SNPs also coincide with the MQTL5.1 identified from meta-QTL analysis in chile pepper [71]. SNP S5_207353938 detected in this study was located ~ 0.19 Mb upstream to significant SNP S05_207549766 detected for isolate ‘KPC-7’ in another study with phenotypic variation ranging from 3.5–29.5% [9]. The findings of this study along with previous research provide further evidence supporting the significance of chromosome 5 for Phytophthora root rot resistance.
The SNP markers S6_170978694 and S12_225476747 identified in this study was ~ 0.53 Mb upstream and ~ 1.28 Mb downstream to significant SNP markers, S06_171517874 and S12_224191357 detected on chromosomes 6 and 12, respectively [9]. Another SNP marker, S10_197712360 identified in this study was ~ 1.69 Mb downstream to the SNP, S10_196014162 on chromosome 10 detected in a previous GWAS [9]. The significant marker, S8_133105638 identified in the current GWAS on chromosome 8 was ~ 0.45Mb downstream to QTL.Pc8.1 detected using a RIL population obtained by crossing ‘CM-334’ (resistant accession) and ‘Early Jalapeno’ (susceptible accession) [8]. The previously identified QTL.Pc9 was also ~ 1.91 Mb upstream to the significant SNP, S9_237909149 detected on chromosome 9 in the present work [8]. In another GWAS, significant SNPs were identified at 9.14 Mb and 208.4 Mb on chromosomes 7 and 12 respectively, which were ~ 0.11 Mb and ~ 0.91 Mb upstream to the SNPs, S7_9249493 and S12_209274913 detected in this study [30]. A novel major SNP marker, S6_705421 was discovered in this study which accounted for up to 54.4% of the phenotypic variation. Most of the significant SNPs identified in this research represent potential novel resistance loci which can be targeted in breeding toward resistance. Though chromosome 5 has been known to be the major region with disease resistance loci against P. capsici, this study as well as previous studies have detected genomic regions on other chromosomes which demonstrates the complexity of Phytophthora root rot resistance in chile peppers [8, 9, 27, 30, 68, 74].
Candidate genes for Phytophthora root rot resistance
This study identified candidate genes linked to disease resistance. Nucleotide-binding site leucine-rich repeat (NBS-LRR) class resistance genes are widely recognized for conferring disease resistance. The LRR regions of these genes interact with extracellular ligands while cytoplasmic kinase domains facilitate signal transduction through phosphorylation [75]. These are essential for the proper function of pathogen recognition as they are responsible for identifying effectors that pathogens deliver to host cells during infection which leads to the activation of a strong resistance response known as effector-triggered immunity [76, 77]. A total of nine genes that encode LRR receptor-like serine/threonine-protein kinase protein was detected near SNP S2_137881264 on chromosome 2, two genes near S12_5016287, and one gene near S3_38944749, S4_212420204, S10_122768586, and S10_189438330. Most resistance (R) proteins that play a role in detecting pathogens and triggering innate immune responses possess a central nucleotide-binding domain (NBS found in NBS-LRR proteins) [78]. The NBS domain, also known as the NB-ARC domain [79] is comprised of three subdomains, NB, ARC1, and ARC2. The NB-ARC domain performs as a functional ATPase domain, and the activity of the R protein is regulated by its nucleotide-binding state [78]. A total of six genes that code for the NB-ARC domain were detected near SNP S6_705421, S6_6636269, S10_201485448, and S11_44892.
Receptor-like proteins and receptor-like kinases are extracellular surface receptors that play role as pattern recognition receptors in plants. They detect the molecular patterns derived from both microbes and the host, initiating the first stage of inducible defense for plant immunity, growth, and development [9, 80]. The genes encoding these proteins were detected in this study near SNPs S5_207353938, S8_141949349, S10_201485448, and S12_37348368. These candidate genes were also previously detected and are good candidates for Phytophthora root rot resistance in chile peppers [9]. SAR8.2 gene, also designated as CASAR82A, is a systemic acquired resistance (SAR)-related gene which plays role in pathogen infection, environmental stresses, and abiotic elicitors [81]. This gene was also identified previously in other P. capsici resistance study [9] and was also detected near SNP, S5_14665044 in this study suggesting its involvement in Phytophthora root rot resistance in chile peppers.
Ethylene plays a vital role in numerous developmental processes and is known to be a critical mediator of abiotic and biotic stress responses in plants [82]. Previous studies have documented the involvement of ethylene responsive transcription factors in pathogen attack [83–86]. These factors were recently identified to be upregulated in plants infected with P. capsici [87]. In this study, the ethylene responsive transcription factor genes were identified near S4_206485683, S4_212420204, and S12_5016287 and might be important candidate genes involved in P. capsici resistance. Glucan endo-1,3-beta-glucosidases, also known as β-1,3-glucanases, are essential hydrolytic enzymes in pathogenesis-related groups of proteins and abundant in various plant species following pathogen infection [88]. These enzymes contribute significantly to the defense response by degrading the β-1,3-glucans that are present in the cell wall of microbes, especially fungus and generating signaling glucans which trigger the activation of global responses [89]. The gene coding for this enzyme detected in the proximity of SNPs S7_9249493, S12_1728396, S12_1902119, S12_5016287, and S12_209274913 might have a crucial role in degrading the β-1,3-glucans that are present in the cell wall of P. capsici and can be a strong candidate gene for resistance in chile peppers. A few late blight resistance genes were found to be associated with SNPs S2_137881264 (1), S6_705421 (4), S6_6636269 (1), S11_44892 (3), and S12_225476747 (1). These genes also belong to NB-ARC-domains containing resistance genes and encode a specific domain of potato resistance genes [90]. The plant cell wall serves as the initial physical barrier against pathogen intrusion and plays role in detecting external signals and stimulating defense response [91, 92]. The candidate genes associated with cell wall biosynthesis, organization, and modification like hydroxyproline-rich glycoproteins (S2_137881264, S8_140731657), UDP-glucuronic acid decarboxylase (S3_29930687, S5_9345710), and β-D-xylosidase (S1_65866380, S4_212420204), were identified which can play role in defense response against P. capsici. Previous reports have also identified the involvement of epigenetic mechanisms in the defense response to P. capsici infection [8, 71, 73]. In this study, candidate genes for DNA methylation, histone methylation, acetylation, and ubiquitination were detected which support the argument for the involvement of the epigenome in defense response. Overall, many candidate genes involved in different mechanisms were detected reflecting the complex genetic architecture of resistance to P. capsici root rot in chile peppers. Further molecular analysis needs to be conducted to better understand and confirm their roles in conferring root rot disease resistance in Capsicum.