Genome-Wide Correlation of 36 Agronomic Traits in the 287 Pepper (Capsicum) Accessions Obtained from the SLAF-seq-Based GWAS

Abstract

There are many agronomic traits of pepper (Capsicum L.) with abundant phenotypes that can benefit pepper growth. Using specific-locus amplified fragment sequencing (SLAF-seq), a genome-wide association study (GWAS) of 36 agronomic traits was carried out for 287 representative pepper accessions. To ensure the accuracy and reliability of the GWAS results, we analyzed the genetic diversity, distribution of labels (SLAF tags and single nucleotide polymorphisms (SNPs)) and population differentiation and determined the optimal statistical model. In our study, 1487 SNPs were highly significantly associated with 26 agronomic traits, and 2126 candidate genes were detected in the 100-kb region up- and down-stream near these SNPs. Furthermore, 13 major association peaks were identified for 11 key agronomic traits. Then we examined the correlations among the 36 agronomic traits and analyzed SNP distribution and found 37 SNP polymerization regions (total size: 264.69 Mbp) that could be selected areas in pepper breeding. We found that the stronger the correlation between the two traits, the greater the possibility of them being in more than one polymerization region, suggesting that they may be linked or that one pleiotropic gene controls them. These results provide a theoretical foundation for future multi-trait pyramid breeding of pepper. Finally, we found that the GWAS signals were highly consistent with those from the nuclear restorer-of-fertility (Rf) gene for cytoplasmic male sterility (CMS), verifying their reliability. We further identified Capana06g002967 and Capana06g002969 as Rf candidate genes by functional annotation and expression analysis, which provided a reference for the study of cytoplasmic male sterility in Capsicum.

Keywords: agronomic traits; genome-wide association study (GWAS); multi-trait pyramid breeding; pepper; restorer-of-fertility (Rf) gene; specific-locus amplified fragment sequencing (SLAF-seq).

Figure 1

Density thermal map of specific-locus amplified fragment (SLAF), polymorphic SLAF (ploy-SLAF) and single nucleotide polymorphisms (SNPs) on chromosomes. (a) The number of SLAF within 1 Mb window size. (b) The number of poly-SLAF within 1 Mb window size. (c) The number of SNPs within 1 Mb window size. Color index indicates the number of labels.

Figure 2

Population structure analysis for 287 pepper accessions. (a) NJ tree of 287 pepper accessions constructed from simple matching distance of genome SNPs. The Z1, Z2, Z3, Z4, Z5, and Z6 represent fruit shape is lantern, cons, horns (cattle and sheep horns), fingers, linear and the other, respectively. (b) Plots of the first three principal components of 287 pepper accessions. The Z1, Z2, Z3, Z4, Z5, and Z6 represent fruit shape is lantern, cons, horns (cattle and sheep horns), fingers, linear and the other, respectively. (c) Admixture in sub-populations (K = 13) resolved using 9,557,790 SNPs. The accessions were divided into 13 subgroups: Q1–Q13. The values represent accession number and percentage for each group. (d) Relatedness value thermal map of 287 pepper accessions. The color bar represents relatedness value.

Figure 3

The position of 37 polymerization region for 23 agronomic traits on each chromosome. On the left aside of the chromosomes, the starting position of polymerization region was indicated by the black triangle, and the end position of that was indicated by the red triangle. Different colors and shapes represent the trait in various categories.

Figure 4

Phenotype correlation network of agronomic traits in pepper. Red and green lines represent negative and positive correlations, respectively. Line width is proportional to the strength of the correlation. Different shape nodes represent stem-related, leaf-related, flower-related and fruit-related traits, respectively. Different color frames of nodes represent plant structure-related, color-related, shape-related, size-related, pubescence-related and other traits, respectively.

Figure 5

Significant SNPs and potential candidate gene numbers in pairwise traits. The upper triangle is the significant SNPs numbers in pairwise trits; The lower triangle is the potential candidate gene numbers in pairwise traits. The color bar represents the number of SNPs or candidate genes in pairwise traits.

Figure 6

Comparison of molecular mapping with GWAS signals for Rf gene. (a) the molecular mapping result of Rf. (b) Comparison of the results of molecular mapping and GWAS mapping of Rf (factored spectrally transformed linear mixed (FaST-LMM) model). The red arrow represents the major peaks associated with male-sterility (Rf gene). The highly significant threshold is shown as a dash blue line (p < 1.707 × 10⁻⁸) and the significant threshold is shown as a dash red line (p < 1.707 × 10⁻⁷).