Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper

Abstract

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum. Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated. Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F2 population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10(3)/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F1 commercial cultivars. Among the markers, Phyto5NBS1 showed about 90% accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.

Identification and Expression Analysis of Candidate Genes Associated with Defense Responses to Phytophthora capsici in Pepper Line “PI 201234”

“…Recent reports have suggested that the major resistance QTL is located on chromosome 5 [22,23] and that CaDMR1 is highly associated with this QTL, but functional validation has not been reported. Among the 12 examined DEGs (Figure 7), six DEGs ( XLOC_021142 , XLOC_021386 , XLOC_021757 , XLOC_021821 , XLOC_021928 and XLOC_022272 ) were on chromosome 5.…”

“…Recently, CaDMR1 , a candidate gene that encodes a homoserine kinase, was reported to be highly associated with the major QTL Pc5.1 [22]. Additionally, a single nucleotide polymorphism marker (SNP) on chromosome 5, Phyto5NBS1, was reported to be highly associated with resistant/susceptible traits against low virulence P. capsici strains [23]. …”

Bulked sample analysis in genetics, genomics and crop improvement

“…Tissues sampled from the phenotypic extremes are pooled first, and then, a single DNA/RNA/protein isolation is performed; or DNA/RNA/protein is isolated first from each extreme individual, and then, an equal amount of the extraction from each individual is bulked. As the two bulking methods for DNA analysis do not give significantly different results (Liu et al ., ), bulking before extraction is more cost‐effective.…”

“…The examples of BSA based on DNA/RNA/chip are summarized in Table . The method has been used to study a dozen of recessive mutants in maize (Liu et al ., ), leaf rust in wheat (Forrest et al ., ), sulphur and selenium contents in Arabidopsis thaliana (Becker et al ., ), salt resistance in cotton (Rodriguez‐Uribe et al ., ), bean common mosaic virus in common bean (Bello et al ., ), Phytophthora root rot in pepper (Liu et al ., ) and photosynthetic traits in poplar (Wang et al ., ).…”

QTL Mapping of Kernel Traits and Validation of a Major QTL for Kernel Length-Width Ratio Using SNP and Bulked Segregant Analysis in Wheat

“…SNPs are thus rapidly replacing traditional markers such as RFLP and SSR, and SNPs are now widely used in the construction of animal and plant genetic linkage maps. The BSA + gene chip method can quickly detect the genetic differences between two different phenotypes 22,23 . The SNP mutation and its chromosomal segment are more accurate than genome-wide gene mapping.…”