Identification of Loci and Candidate Genes Responsible for Fiber Length in Upland Cotton (Gossypium hirsutum L.) via Association Mapping and Linkage Analyses

Abstract

Fiber length (FL) is an important fiber quality trait in cotton. Although many fiber quality quantitative trait loci (QTL) responsible for FL have been identified, most cannot be applied to breeding programs, mainly due to unstable environments or large confidence intervals. In this study, we combined a genome-wide association study (GWAS) and linkage mapping to identify and validate high-quality QTLs responsible for FL. For the GWAS, we developed 93,250 high-quality single-nucleotide polymorphism (SNP) markers based on 355 accessions, and the FL was measured in eight different environments. For the linkage mapping, we constructed an F ₂ population from two extreme accessions. The high-density linkage maps spanned 3,848.29 cM, with an average marker interval of 1.41 cM. In total, 14 and 13 QTLs were identified in the association and linkage mapping analyses, respectively. Most importantly, a major QTL on chromosome D03 identified in both populations explained more than 10% of the phenotypic variation (PV). Furthermore, we found that a sucrose synthesis-related gene (Gh_D03G1338) was associated with FL in this QTL region. The RNA-seq data showed that Gh_D03G1338 was highly expressed during the fiber development stage, and the qRT-PCR analysis showed significant expression differences between the long fiber and short fiber varieties. These results suggest that Gh_D03G1338 may determine cotton fiber elongation by regulating the synthesis of sucrose. Favorable QTLs and candidate genes should be useful for increasing fiber quality in cotton breeding.

Keywords: GWAS; QTL; fiber length; sucrose synthesis; upland cotton.

FIGURE 1

Correlation analyses of fiber length (FL) based on eight environments among 355 accessions. ^∗∗Indicates that the correlation reached significance at 0.001.

FIGURE 2

Genome-wide association scan of FL. (A) Manhattan plot of FL using the best linear unbiased prediction of FL value. The red dashed horizontal line represents the significance threshold (P < 2.5 × 10^-5). (B) Quantile–quantile plots of FL.

FIGURE 3

Box plots of FL based on the haplotypes of two SNPs (AA, n = 52 and TT, n = 262) on chromosome D03 in eight environments (E1: AY-2014, E2: AY-2015, E3: AY-2016, E4: SHZ-2014, E5: SHZ-2015, E6: SHZ-2016, E7: HG-2016, and E8: SY-2016). The significance of the difference was analyzed with two-tailed Student’s t-tests.

FIGURE 4

Genome-wide distribution of SNPs throughout the physical and genetic maps. (A) Genome-wide distribution of SNPs and genetic variants throughout the CRI50 and CRI60 genomes. The outermost box with a scale represents the 26 cotton chromosomes. The blue histogram represents the density of SNPs that are polymorphic between CRI50 and CRI60; the purple histogram indicates the density of insertions or deletions (Indels) between CRI50 and CRI60; and the orange histogram represents the density of aa × bb markers between CRI50 and CRI60. (B) Genetic map constructed by SNP markers.

FIGURE 5

Combined results of the association mapping and linkage analysis of chromosome D03. (A) Peak region (41.03–42.21 Mb) on chromosome D03. (B) Pairwise LD between the SNP markers is indicated as D′ values, where dark red indicates a value of 1, and gray indicates a value of 0. The blue dotted line shown in (A) indicates the LD blocks that contain two significant SNPs (red dots). (C) The right part is the linkage map and the results of the linkage mapping, and the green bar indicates overlapping regions across the GWAS and linkage mapping results.

FIGURE 6

Identification of the FL causal gene Gh_D03G1338 on chromosome D03. (A) Exon–intron structure of Gh_D03G1338 and the polymorphism in two haplotypes with the ‘C’ and ‘T’ alleles. (B) Box plot of FL based on the two haplotypes mentioned above (n = 39 versus 36). Differences between the haplotypes were analyzed by two-tailed Student’s t-tests. (C) Comparison of expression levels of Gh_D03G1338 during fiber development stages by qRT-PCR (^∗∗indicates significance at the 0.001 probability level).