Results and discussion

The Ve gene is a cell surface-localized receptor that confers resistance to race 1 of the vascular wilt fungus Verticillium dahliae through (Fradin et al. 2009; Kawchuk et al. 2001) recognition of the Ave1 effector (Song et al. 2017). Homologous Ve genes have been identified in many land plants from diverse plant families (Song et al. 2017). These homologs have been found to mediate resistance in other solanaceous species like diploid tomato (Solanum lycopersicoides) (Chai et al. 2003), polyploidy potato (S. tuberosum) (Bae et al. 2008; Uribe et al. 2014), eggplant (S. melongena) (Liu et al. 2015), wild eggplant (S. torvum), tobacco (Nicotiana glutinosa) (Fei et al. 2004; Song et al. 2017), and in a non-solanaceous species such as Mentha longifolia (Vining and Davis 2009), grape (Vitis vinifera) (Tang et al. 2016), lettuce (Lactuca sativa) (Hayes et al. 2011), and cotton (Gossypium barbadense) (Chen et al. 2016). Additionally, transformation of individual tomato Ve genes into susceptible potato plants conferred resistance against Verticillium, demonstrating that the tomato Ve-resistant genes are effective in other Solanaceae species (Kawchuk et al. 2001).

The five previously reported primer sets for Ve-associated markers based on homology to tomato and potato were evaluated with chile pepper genomic DNA (Bae et al. 2008; Park et al. 2008; Uribe et al. 2014). None of these Ve primer sets amplified PCR products of the expected size. However, the primers amplified expected amplicons from control tomato or potato DNA, confirming that the primers and reaction conditions were suitable for amplification of Ve homologs. Based on these results, it appeared that the chile pepper Ve orthologs are sufficiently divergent from the tomato and potato genes to preclude amplification using these primers.

Using the tomato Ve1 (accession: FJ464557.1) tomato Ve2 (accession FJ464562.1), we queried the draft chile pepper genome (GCA_000710875.1 v1.0) and identified a 3423 bp ORF that was 97 and 88% identical to the tomato Ve1 and Ve2 genes, respectively. Similar to Ve in tomato, the chile pepper Ve homolog was located on chromosome 9 between 110,090,054 and 110,087,826 bp. Based on the sequence of this ORF, primers were designed to amplify the putative Ve homologs from VW resistant and susceptible accessions of C. annuum. These primers produced amplicons from both VW susceptible and VW resistant accessions that were cloned and sequenced. These sequences were submitted to the National Center for Biotechnology Information (accessions MG062689-MG062698). The CaVe ORF encodes a putative polypeptide of 1140 amino acids. The Ve genes of tomato encode extracellular leucine-rich repeat receptor-like proteins (eLRR-RLPs) (Kawchuk et al. 2001; Wang et al. 2008, 2010). Similarly, the predicted CaVe is leucine-rich (15.5%) as is expected for a resistance gene with many of the L residues occurring in canonical L rich repeats (LRR) that align well with tomato Ve genes. Between the known resistant and susceptible plants, 16 nucleotide polymorphisms were identified, 13 of which were nonsynonymous mutations (Table 1).

Table 1 Single nucleotide polymorphism designation, location, nucleotide, and predicted amino acid of the putative CaVe gene identified between resistant and susceptible chile pepper accessions as well as the four TaqαI restriction sites used for CAPS marker development
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For CAPS marker development, the CaVe primers (CaVeFw19: 5′-CTATGGCTTTTCTTGATACCCTT-3′ CaVeRv2223: 5′-GTTGACAAGAGA TTTTGGCAGCT -3′) were developed and the restriction enzyme Taqα1 was used for digestion. There were four recognition sites in the resistant accessions but only two in the susceptible accessions (Table 1 and Fig. 1). The resistant lines had digestion bands at ~ 530, 570, and 1000 bp, while the susceptible lines had digested bands at ~ 1000 and 1200 bp (Fig. 1). However, despite using reported resistant and susceptible lines for co-dominant marker development, when screened using diverse Capsicum germplasm, the CaVe marker had 48% selection accuracy (Table 2). There are several possible reasons for the low level of matching between the marker and the phenotype. Firstly, the number of resistance sources in Capsicum is limited (Gurung et al. 2015), which is due in no small part to the overall lack of an unequivocal disease screen. It is also possible that the V. dahliae isolate collected in New Mexico used in this study is different from other isolates use in previous studies. These two points are highlighted by the difference in phenotypic results in our study and those reported by Gurung et al. (2015).

Fig. 1
figure 1

Segregation of co-dominant CAPS marker in resistant and susceptible chile pepper accessions. Total DNA was isolated from individual chile plants and amplified with CaVe primers. Amplicons were digested with TaqαI, run on a 1.5% gel, and visualized with EtBr staining. Lane 1 is a size standard 100 bp ladder marker (GeneRuler 100 bp plus), lanes M1–M6 are resistant accession samples, lanes M7 and M8 are susceptible accession samples, and lanes M9 and M10 are heterozygotes

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Table 2 Genotypic and phenotypic disease rating of diverse germplasm evaluated in testing the efficacy of the CaVe-CAPS marker developed in known resistant and susceptible lines
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This molecular marker is a promising tool that can be used in marker-assisted select for Verticillium wilt resistance. The marker developed here has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Therefore, we suggest that this work be used to provide a basis for future research in plant breeding and plant pathology. Research needs to be conducted to better understand the epidemiology of V. dahliae and to characterize the isolates collected from diverse production regions. Furthermore, the identification of highly resistant accessions of chile pepper is necessary before we can better understand the function of Ve homologs in chile pepper.