Development and validation of KASP markers for resistance to Phytophthora capsici in Capsicum annuum L

Abstract

Resistance of Capsicum annuum to Phytophthora blight is dependent on the genetic background of the resistance source and the Phytophthora capsici isolate, which poses challenges for development of generally applicable molecular markers for marker-assisted selection. In this study, the resistance to P. capsici of C. annuum was genetically mapped to chromosome 5 within a 1.68-Mb interval by genome-wide association study analysis of 237 accessions. In this candidate region, 30 KASP markers were developed using genome resequencing data for a P. capsici-resistant line (0601 M) and a susceptible line (77,013). Seven of these KASP markers, located in the coding region of a probable leucine-rich repeats receptor-like serine/threonine-protein kinase gene (Capana05g000704), were validated in the 237 accessions, which showed an average accuracy of 82.7%. The genotyping of the seven KASP markers strongly corresponded with the phenotype of 42 individual plants in a pedigree family (PC83-163) developed from the P. capsici-resistant line CM334. This research provides a set of efficient and high-throughput KASP markers for marker-assisted selection of resistance to P. capsici in C. annuum.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11032-023-01367-3.

Introduction

Phytophthora blight, including foliar blight as well as root-, stem-, and fruit-rot, caused by Phytophthora capsici is a serious disease of pepper (Capsicum annuum) worldwide. The disease causes an annual global loss of more than US$100 million (Bosland 2008). The pathogen may spread rapidly throughout a field within days resulting in 100% loss (Barchenger et al. 2018). Different pepper blight syndromes reflect different sites of infection, and a specific resistance gene is required for control of each disease syndrome and of individual physiological races of P. capsici within each syndrome (Király et al. 2007; Monroy-Barbosa and Bosland 2010). Therefore, independent resistance genes are needed to control each disease syndrome (Walker and Bosland 1999; Sy et al. 2005), which increases the complexity of resistance breeding.

The inheritance of pepper resistance to P. capsici is considered to be a qualitative or quantitative trait among the different resistance sources. Studies reporting inheritance models of P. capsici resistance include at least two genes in CM334 (Guerrero-Moreno and Laborde 1980; Reifschneider et al. 1992; Thabuis et al. 2003), one dominant gene in PI201234 (Kim and Hur 1990; Saini and Sharma 1978; Wang et al. 2016), and multiple genes with additive or epistatic effects in the chili pepper Perennial (Lefebvre and Palloix 1996). Qualitative control of P. capsici resistance is race-specific (Sy et al. 2008; Foster and Hausbeck 2010; Jo et al. 2014) and dependent on the evaluation method for disease resistance used (Liu et al. 2014; Wang et al. 2016). Researchers have predominantly studied P. capsici resistance as a quantitative trait, and a number of major and minor resistance-associated quantitative trait loci (QTLs) have been mapped (Barchenger et al. 2018).

Phytophthora capsici resistance genes have been localized on all 12 chromosomes of Capsicum using different resistance sources and P. capsici isolates (Lefebvre and Palloix 1996; Thabuis et al. 2003; Quirin et al. 2005; Bonnet et al. 2007; Kim et al. 2008; Truong et al. 2012; Liu et al. 2014; Rehrig et al. 2014; Wang et al. 2016; Xu et al. 2016; Siddique et al. 2019; Lozada et al. 2021; Ro et al. 2022). However, in numerous studies, the predominant QTLs associated with resistance to P. capsici are consistently located in close physical proximity on chromosome P5 regardless of the resistance source or P. capsici isolate (Lefebvre and Palloix 1996; Thabuis et al. 2003, 2004a, b; Ogundiwin et al. 2005; Sugita et al. 2006; Bonnet et al. 2007; Minamiyama et al. 2007; Kim et al. 2008; Truong et al. 2012; Mallard et al. 2013; Liu et al. 2014; Rehrig et al. 2014; Wang et al. 2016; Siddique et al. 2019; Du et al. 2021b, a; Li et al. 2021; Lozada et al. 2021; Ro et al. 2022). Mallard et al. (2013) detected three clustered QTLs (Pc5.1, Pc5.2, and Pc5.3) on chromosome P5 involved in resistance to P. capsici by developing anchor markers for three published maps. A homoserine kinase gene, DOWNY MILDEW RESISTANT 1 (DMR1; CA00g97170 of CM334 v1.55 and Capana05g000668 of Zunla-1 v2.0 reference genomes), which cosegregates with Pc5.1 was considered to be a strong candidate gene for P. capsici resistance (Rehrig et al. 2014). In addition, Du et al. (2021b, a) proposed Snakin-1 (SN1) gene (CA05g05250) account for Pc5.1 (Mallard et al. 2013) which control the wide-spectrum resistance. Wang et al. (2016) mapped the race-specific resistance locus CaPhyto to a 3.3 cM interval on chromosome P5, and two genes (Capana05g000764 and Capana05g000769) were confirmed as candidate genes. Siddique et al. (2019) detected three major-effect loci (QTL5.1, QTL5.2, and QTL5.3) that confer resistance to a broad spectrum of P. capsici isolates by combining traditional QTL mapping and genome-wide association study (GWAS). The QTL5.2 locus was also detected using GWAS in combination with single-nucleotide polymorphisms (SNPs) and coincided with the previously identified QTLs Pc5.1 (CaDMR1) (Mallard et al. 2013; Rehrig et al. 2014), the dominant gene CaPhyto (Wang et al. 2016), and the tightly linked marker Phyto5NBS1 (Liu et al. 2014).

Several types of molecular markers for P. capsici resistance in chili pepper have been developed, such as single-nucleotide amplified polymorphisms (Kim et al. 2008), simple sequence repeats (SSRs) (Kim et al. 2008; Wang et al. 2016; Xu et al. 2016), cleaved amplified polymorphic sequences (Kim et al. 2008), high-resolution melting markers (HRM) (Liu et al. 2014; Kim et al. 2019; Ro et al. 2022), and sequence-characterized amplified regions (SCARs) (Quirin et al. 2005; Truong et al. 2013). Several of these markers show a high accuracy for given host materials and P. capsici isolates. The genotype of the HRM marker Phyto5NBS1 showed 91% agreement with the resistance phenotype to the P. capsici isolate MY-1 in 100 F₁ hybrids (Liu et al. 2014). The SSR marker ZL6726 showed 100% accuracy in 20 pepper lines (Wang et al. 2016). The HRM marker CaNB-5480 showed the highest cosegregation percentage of 86.9% with 61 Capsicum accessions among 11 HRM markers (Kim et al. 2019). The accuracy of Chr02-1126 marker in predicting pepper resistance in 64 resistant plants and 32 susceptible plants was 78.5% (Ro et al. 2022). However, the accuracy of P. capsici resistance-linked markers varies greatly when used with diverse genotypes with variable disease phenotypes (Liu et al. 2014; Siddique et al. 2019). Therefore, from a breeding perspective, resistance resource- and isolate-specific markers are more reliable and effective for marker-assisted selection in the transfer of P. capsici resistance.

The objective of this study was to map a candidate interval for resistance to P. capsici in C. annuum accessions by means of GWAS analysis and to develop KASP markers tightly linked to the candidate gene for P. capsici resistance for use in molecular marker-assisted selection.

Materials and methods

Plant materials

Two hundred and thirty-seven Capsicum annuum accessions provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF-CAAS; Beijing, China), were used for GWAS analysis and marker validation (Cao et al. 2022). There were three replications of each accession with 10 plants in each replication. Germinated seeds were sown in seedling trays in a greenhouse under natural lighting. Five F₁ hybrids were raised by crossing the P. capsici susceptible line 77,013 with three resistant lines (CM334, Perennial, and 0601 M) and two susceptible lines (IVF2014032 and H3) for polymorphic marker screening in Kompetitive Allele-specific PCR (KASP™) assays. Phytophthora capsici resistance-associated candidate markers were also tested in a pedigree family (PC83-163) developed by crossing the P. capsici susceptible line 83–163 with a resistant individual from among the F₂ progeny of the cross 83–58 × CM334.

Phytophthora capsici inoculation

The P. capsici isolate Jo-1 was kindly provided by Dr B. Xie (Vegetables Disease Group, Institute of Vegetables and Flowers, CAAS). A 1-cm² square of the source isolate was transferred to a plate containing V8 medium (100 ml V8 juice, 0.2 g calcium carbonate, and 20 g agar) and incubated at 25 °C for 7 days. To assess foliar blight resistance, the V8 plate was cut into 1-cm² squares, and the plate was one-quarter filled with ddH₂O. The plate was incubated in the dark at 25–27 °C for 3–4 days, followed by 1–2 days in the light. The plate was placed in a refrigerator at 4 °C for 30 min to release Phytophthora zoospores. The number of Phytophthora zoospores was counted using a hemacytometer, and then individual plants were inoculated with 3 ml of the zoospore suspension (1 × 10³ zoospores per milliliter).

Phenotype of Capsicum annuum infected by Phytophthora capsici

The resistance to P. capsici foliar blight of the 237 accessions was determined following the method of Wang et al. (2016). Resistance was assessed in accordance with Chinese standard NY/T 2060.1–2011 (Ministry of Agriculture of the People’s Republic of China 2011) scored on a 0–5 scale (0 = no visible symptoms; 1 = brownish lesions had begun to appear on stems, leaves not wilted or slightly wilted; 2 = stem lesions extended to 1–2 cm in length, leaves wilted irrecoverably, and lower leaves had begun to abscise; 3 = stem lesions extended to be longer than 2 cm, leaves wilted or abscised; 4 = long, brownish lesions on stems extended and dehydrated, all leaves except for the uppermost leaves abscised, plant almost dead; 5 = plant dead). The disease index (DI) was calculated as follows:

$$DI=\sum (s\times n)/(N\times S)\times 100$$

where s is the disease level, n is the number of plants for each disease level, N is the total number of surveyed plants, and S is the representative value of the highest disease level. The resistant grades were categorized as I = immunity (DI = 0), HR = highly resistant (0 < DI ≤ 10), R = resistant (10 < DI ≤ 30), MR = moderately resistant (30 < DI ≤ 50), S = susceptible (50 < DI ≤ 70), and HS = highly susceptible (DI > 70). The inoculation treatment was designed with three replicates, each containing eight plants. The disease index and the resistant grade of each replicate were obtained from the eight plants. The average of the replicates, which are at the same resistant grade and the difference is ≤ 20, was used for GWAS.

Inoculation of stem cuttings was performed in accordance with the method of Lefebvre and Palloix (1996). The stem was excised at the first flower bud node with a sterilized blade; a small fungus cake was pasted on the wound, wrapped with plastic film for 24 h, and then the film was removed. The disease severity was evaluated based on the lesion length, which was measured after 7 days.

Genome-wide association analysis of Phytophthora capsici resistance

Whole-genome resequencing of the 237 accessions was performed by Berry Genomics (Beijing, China) on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA) and has been published (Cao et al. 2022). The GWAS was performed by Allwegene (Beijing, China) in accordance with the procedure of Zhang et al. (2020). The raw data were subjected to quality control using the NGSQC toolkit (Dai et al. 2010). Burrows-Wheeler Aligner (BWA) software (Li and Durbin 2010) was used to align the clean reads to the Zunla-1 v2.0 reference genome (Qin et al. 2014). The GWAS analysis was performed using a general linear model with a threshold of − log(P) ≥ 4.0 using homozygous SNPs with a read depth of not less than 4 and an average quality score ≥ 20. Quantile–quantile plots and Manhattan plots were drawn using the CMplot and qqman R packages, respectively (Turner 2014).

Molecular marker development and detection

Three hundred and eighty-seven SNPs between the sweet pepper lines 77,013 and 0601 M in the candidate interval of Phytophthora capsici resistance were selected based on the results of previous studies (Zhang et al. 2020). In addition, 398 SNPs between the chili peppers Perennial and Zunla-1 in the candidate region were selected from alignment of the Perennial whole-genome sequence (NCBI SRA accession no. SRX1362495) and the Zunla-1 v2.0 reference genome (Qin et al. 2014). The SNP discovery for Perennial was performed by Allwegene (Beijing, China) as described by Zhang et al. (2020). Primer design and the KASP assay were performed in accordance with the procedures of Zhang et al. (2020).

Results

GWAS for Phytophthora capsici resistance

A total of 53 P. capsici-resistant and 188 susceptible accessions were used for the GWAS analysis, for which the DI ranged from 1.48 to 49.52 and 50.36 to 95.48, respectively (Table 1). The average sequencing depth was approximately 8 × for the 237 accessions. A total of 3,329,373 SNPs were used for the GWAS analysis. A general linear model was used to analyze the observed phenotype corresponding to P. capsici resistance. The P. capsici resistance locus was localized on chromosome 5 within a same 1.68 Mb interval from 23,844,243 to 25,526,786 bp based on the phenotypes of disease index and resistant grade. Three hundred and ninety-eight SNPs were found to be associated with P. capsici resistance, with P-value between 1.17 × 10⁻¹⁸ and 1.17 × 10⁻¹³ (Fig. 1). Nine genes were annotated in the candidate region (Table 2), in which the Capana05g000704 encoding a leucine-rich repeats receptor-like serine/threonine-protein kinase (LRR-RLK) was considered as the most likely candidate gene for P. capsici resistance of C. annuum.

Table 1.

Resistant grades and KASP marker genotyping of 237 accessions of Capsicum annuum

Resistance grades	Number of accession	Disease index	No. of correctly genotyped accessions	Marker accuracy
HR	3	1.48–3.33	2	66.7%
R	15	10.19–28.83	6	40.0%
MR	35	31.22–49.52	6	17.1%
S	71	50.36–70.00	70	98.6%
HS	113	70.45–95.48	112	99.1%
Total	237	–	196	82.7%

HR highly resistant, R resistant, MR moderately resistant, S susceptible, HS highly susceptible

Fig. 1.

Genome-wide association analysis of Phytophthora capsici resistance in Capsicum annuum. a Manhattan plots of disease index; b quantile–quantile plots of disease index; c Manhattan plots for resistant grades; d quantile–quantile plots for resistant grades. The highly significant threshold is indicated by a red line, and the significant threshold is indicated by a blue line

Table 2.

Candidate genes for Phytophthora capsici resistance within the candidate interval on chromosome 5 of Capsicum annuum “Zunla-1”

Name	Position	Description
Capana05g000692	23711423:23711824: −
Capana05g000694	23907550:23907969: −	BURP domain-containing protein
Capana05g000695	24067120:24067753: +
Capana05g000696	24069652:24071682: +	Histone acetyltransferase HAC1
Capana05g000700	24588673:24589874: +
Capana05g000701	24681455:24685093: +	Protein AIG1
Capana05g000702	25000520:25001096: +
Capana05g000704	25470196:25473671: +	Probable LRR receptor-like serine/threonine-protein kinase
Capana05g000705	25510280:25517420: −	Extended synaptotagmin-2

Polymorphic KASP marker screening

In total, 785 SNPs were chosen for KASP marker development in the candidate interval identified by the GWAS. Of these SNPs, 387 were detected between 77,013 and 0601 M by comparison with the CM334 reference genome, and 398 SNPs were detected between Perennial and the Zunla-1 (Not resistant to P. capsici) reference genome. Sequences of CM334 and Zunla-1 flanking these SNPs were utilized to develop the KASP markers. In total, 165 KASP marker primers were successfully designed from the 785 SNPs, and 47 high-quality primers were sampled for the KASP assay. Ultimately, 30 KASP markers that showed polymorphism among the P. capsici-resistant and susceptible lines were considered to be associated with P. capsici resistance (Table 3; Table S1).

Table 3.

Information on SNPs for KASP markers located in the candidate interval of the Capsicum annuum genome associated with resistance to Phytophthora capsici identified by genome-wide association study

Markers	SNPs	Variation type	Amino acid changes	Physical location*		CDS
				Zunla-1	CM334	Zunla-1	CM334
5–128	G/A	Intergenic		23,861,414	28,330,327
5–130	G/C	Upstream		23,907,998	28,277,706	Capana05g000694	CA05g06460
5–131	G/T	Intergenic		23,969,648	28,229,668
5–133	G/A	Intergenic		24,019,537	28,179,669
5–134	C/T	Intergenic		24,105,234	28,084,195
5–136	A/T	Intergenic		24,194,881	28,696,830
5–138	G/A	Intergenic		24,362,884	28,823,093
5–139	G/T	Intergenic		24,435,615	28,902,368
5–141	T/C	Intergenic		24,666,761	29,013,377
5–142	C/T	Intronic		24,684,561	29,032,212
5–145	G/A	Intergenic		25,228,029	29,218,639
5–146	A/G	Intergenic		25,397,519	28,427,721
5–148	A/T	Intergenic		25,482,127	28,499,658
5-156a	T/A	Upstream		25,468,479	28,486,079	Capana05g000704	CA05g06470
5-157a	A/G	Missense	Leu/Pro	25,471,408	28,489,059	Capana05g000704	CA05g06470
5-158a	C/G	Missense	His/Asp	25,471,602	28,489,253	Capana05g000704	CA05g06470
5–159	C/A	Missense	Ser/Tyr	25,471,889	28,489,542	Capana05g000704	CA05g06470
5-160a	G/T	Missense	Ala/Ser	25,472,161	28,489,814	Capana05g000704	CA05g06470
5-153a	A/G	Exonic	Ser/Ser	25,472,265	28,489,918	Capana05g000704	CA05g06470
5-161a	C/A	Missense	Leu/Ile	25,472,626	28,490,279	Capana05g000704	CA05g06470
5–154	T/C	Exonic	Leu/Leu	25,472,832	28,490,485	Capana05g000704	CA05g06470
5-162a	G/A	Synonymous	Leu/Leu	25,472,832	28,490,485	Capana05g000704	CA05g06470
5–164	C/G	Downstream		25,478,721	28,496,255	Capana05g000704	CA05g06470
5–34	C/T			25,515,132	28,036,997	Capana05g000705	CA05g06430
5–35	C/A	Synonymous		25,513,237	28,038,829	Capana05g000705	CA05g06430
5–37	A/G	Downstream		25,510,219	28,041,969	Capana05g000705	CA05g06430
5–38	G/A	Downstream		25,505,350	28,042,837	Capana05g000705	CA05g06430
5–41	G/A	Downstream		25,506,413	28,046,085	Capana05g000705	CA05g06430
5–39	C/T	Downstream		25,508,010	28,044,519	Capana05g000705	CA05g06430
5–150	C/T	Intronic		25,515,991	28,036,138	Capana05g000705	CA05g06430

Underlined numbers indicate that the physical location retrieved by BLAST searches

^aMarkers selected for further validation in the GWAS study population

Validation of KASP markers

Seven KASP markers (Table 3) from the coding sequence of Capana05g000704 were selected for further validation in the GWAS study population. The genotypes of these seven KASP markers were identical in the 237 accessions. The average accuracy of the molecular markers was 82.7%; however, the accuracy differed substantially between the P. capsici HR-resistant (66.7%) and HS-susceptible (99.1%) groups of accessions (Table 1).

Forty-two plants of the pedigree family PC83-163 were genotyped with four KASP markers (5–130, 5–141, 5–148, and 5–150) scattered in the candidate gene (Capana05g000704) region, using CM334 as a disease-resistant control and Qiemen as a disease-susceptible control (Fig. 2). CM334 showed strong disease resistance, with an average lesion length of 0.43 cm compared with that of 8.38 cm for Qiemen (Figs. 3a and 4). The majority of plants of the PC83-163 population showed stronger disease resistance than Qiemen; the lesion length ranged from 1.4 to 10.0 cm with an average of 5.14 cm (Figs. 3b and 4). The genotypes of the five KASP markers were identical in CM334, Qiemen, and the PC83-163 population. CM334 and Qiemen exhibited homozygous disease-resistant and -susceptible genotypes, respectively. In addition to disease-resistant and -susceptible genotypes, heterozygous genotypes were detected in the PC83-163 population.

Fig. 2.

Genotypes of KASP markers a 5–130, b 5–141, c 5–148, and d 5–150 in the PC83-163 pedigree population. Blue and green triangles indicate individuals homozygous for the FAM-labeled recessive susceptible allele and HEX-labeled dominant resistant allele, respectively. Red triangles correspond to heterozygous individuals, and black dots represent the non-template control

Fig. 3.

Phenotype of resistance to Phytophthora capsici in the PC83-163 pedigree family assessed using stem-cutting inoculation. a Resistance control CM334 and infection control Qiemen; b phenotype of the PC83-163 population. Red arrows indicate relatively susceptible plants

Fig. 4.

Distribution of stem lesion length and KASP marker genotypes of the Phytophthora capsici resistant line CM334, the susceptible line Qiemen, and the PC83-163 pedigree population

Discussion

Loci for resistance to P. capsici are located on all 12 chromosomes of C. annuum as detected using genetically diverse populations and multiple types of molecular markers (Lefebvre and Palloix 1996; Thabuis et al. 2003; Quirin et al. 2005; Bonnet et al. 2007; Kim et al. 2008; Truong et al. 2012; Liu et al. 2014; Rehrig et al. 2014; Wang et al. 2016; Xu et al. 2016; Siddique et al. 2019; Du et al. 2021b, a; Li et al. 2021; Lozada et al. 2021; Ro et al. 2022). The candidate genes CaDMR1 (Rehrig et al. 2014), Capana05g000764 and Capana05g000769 (Wang et al. 2016), and Snakin-1 (CA05g05250) (Du et al. 2021b, a) were predicted in CM334, PI201234, and 305R, respectively. However, a majority of studies have shown that the candidate interval is relatively large, ranging from less than 1 Mb to more than 100 Mb, which includes three to tens of candidate genes associated with disease resistance (Mallard et al. 2013; Xu et al. 2016; Siddique et al. 2019; Du et al. 2021b, a). In the present study, a GWAS-derived candidate region for P. capsici resistance loci was located at 23,844,243 to 25,526,786 bp (Fig. 1) on chromosome 5 of the Zunla-1 reference genome (Qin et al. 2014). This candidate interval overlapped with the location of the P. capsici resistance loci Pc5.1 (CaDMR1) (Mallard et al. 2013; Rehrig et al. 2014) and QTL5.2 (Siddique et al. 2019) and the closely associated marker Phyto5NBS1 (Liu et al. 2014). According to previous studies, three well-known resistance source lines of C. annuum (CM334, Perennial, and PI201234) each harbor major resistance sites on chromosome 5, thus indicating that major P. capsici resistance genes or gene clusters are located on chromosome 5 (Lefebvre and Palloix 1996; Thabuis et al. 2003; Bonnet et al. 2007; Truong et al. 2012; Mallard et al. 2013; Rehrig et al. 2014; Wang et al. 2016; Siddique et al. 2019).

Plant disease-resistance genes or QTLs are often located in clusters on specific chromosomes. In C. annuum, clusters of genes for resistance to bacterial wilt (Kang et al. 2016), viruses (Kim et al. 2017; Naresh et al. 2017), powdery mildew (Jo et al. 2017), and aphids (Sun et al. 2020) were predicted to encode LRR-RLKs or nucleotide-binding site leucine-rich repeat (NBS-LRR) domain-containing proteins. A cluster of multiple NBS-LRR genes is located in the candidate interval for resistance to P. capsici on pepper chromosome 5 (Liu et al. 2014; Siddique et al. 2019). In the present study, a predicted LRR-RLK-encoding gene, Capana05g000704, located in the candidate interval for P. capsici resistance was revealed by GWAS (Fig. 1; Table 2). Fine mapping of these major QTLs and GWAS-identified loci will provide useful information for resistance of pepper to P. capsici and will contribute to marker-assisted selection in pepper breeding.

A number of markers linked to P. capsici resistance loci have been developed, and the applicability of some of these has been evaluated. Troung et al. (2013) reported that one SCAR marker (SA133_4) and one RAPD marker (UBC553) correctly identified resistance or susceptibility to P. capsici in nine commercial pepper cultivars. Liu et al. (2014) developed the HRM marker Phyto5NBS1, which showed 91% accuracy in phenotype prediction among 100 pepper lines susceptible or resistant to the low-virulence P. capsici isolate MY-1. Wang et al. (2016) mapped the race-specific resistance gene CaPhyto and reported that the accuracy of the genotype detected by the markers ZL6726 and ZL6970 was 100% and 70%, respectively, in 20 pepper lines. Ro et al. (2022) obtained SNPs related to P. capsici resistance through GWAS analysis and developed an HRM marker Chr02-1126, with an accuracy of 78.5%. In the present study, seven KASP markers located in the coding region of the candidate gene Capana05g000704 showed 82.7% accuracy in phenotype prediction for 237 accessions (Table 1). The locus will be useful for molecular marker-assisted selection in breeding for P. capsici resistance (Figs. 2, 3, and 4). The genotypes of the reported markers perform differently when matched with resistance phenotypes tested with different degrees of virulence (Liu et al. 2014), and when applied to relatively small groups of pepper lines and commercial cultivars (Troung et al. 2013; Wang et al. 2016). The present study also showed that the matching of marker genotypes to disease-resistance phenotypes was superior in the disease-resistant accessions compared with that of the disease-susceptible accessions. These results suggested that the resistance of pepper accessions to Phytophthora blight was determined by the interaction between the resistance source and the P. capsici isolate. Development of isolate- and resistance source-specific molecular markers will be useful for molecular marker-assisted selection.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

ZHZ participated in the design of the study and developed the KASP primers. YCC, HLY, and HMW performed the GWAS analysis. LJ and XGF performed the phenotypic assessment. YFW, DLA, and YSZ conducted the KASP genotyping assay. BXZ and LHW designed the study and revised the manuscript. All the authors read and approved the final version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2016YFD0100200), the China Agricultural Research System (CARS-23-A15), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Science (CAAS-ASTIP-IVFCAAS).

Data availability

Not applicable.

Code availability

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Declarations

Ethics approval

The experiments in this study comply with the current laws of China.

Consent to participate

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Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.