Identifying Wild Versus Cultivated Gene-Alleles Conferring Seed Coat Color and Days to Flowering in Soybean

Abstract

Annual wild soybean (G. soja) is the ancestor of the cultivated soybean (G. max). To reveal the genetic changes from soja to max, an improved wild soybean chromosome segment substitution line (CSSL) population, SojaCSSLP5, composed of 177 CSSLs with 182 SSR markers (SSR-map), was developed based on SojaCSSLP1 generated from NN1138-2(max)×N24852(soja). The SojaCSSLP5 was genotyped further through whole-genome resequencing, resulting in a physical map with 1366 SNPLDBs (SNP linkage-disequilibrium blocks), which are composed of more markers/segments, shorter marker length and more recombination breakpoints than the SSR-map and caused 721 new wild substituted segments. Using the SNPLDB-map, two loci co-segregating with seed-coat color (SCC) and six loci for days to flowering (DTF) with 88.02% phenotypic contribution were identified. Integrated with parental RNA-seq and DNA-resequencing, two SCC and six DTF candidate genes, including three previously cloned (G, E2 and GmPRR3B) and five newly detected ones, were predicted and verified at nucleotide mutant level, and then demonstrated with the consistency between gene-alleles and their phenotypes in SojaCSSLP5. In total, six of the eight genes were identified with the parental allele-pairs coincided to those in 303 germplasm accessions, then were further demonstrated by the consistency between gene-alleles and germplasm phenotypes. Accordingly, the CSSL population integrated with parental DNA and RNA sequencing data was demonstrated to be an efficient platform in identifying candidate wild vs. cultivated gene-alleles.

Keywords: SNP linkage disequilibrium block (SNPLDB); annual wild soybean (G. soja Sieb. and Zucc.); chromosome segment substitution line (CSSL); cultivated soybean (G. max (L.) Merr.); days to flowering; seed coat color; whole-genome re-sequencing.

Figure 1

Molecular characteristics of SojaCSSLP5 derived from NN1138-2 and N24852. (A) The physical map of SojaCSSLP5 genotyped by 182 SSR markers (SSR-map). (B) The physical map of SojaCSSLP5 genotyped by 1366 SNPLDB markers (SNPLDB-map). (C) The frequency distribution of NN1138-2-recovery rate in SojaCSSLP5. (D) The frequency distribution of wild segment number per line in SojaCSSLP5. (E) The frequency distribution of wild segment length in SojaCSSLP5. (F) The SSR-segment broken into new SNPLDB segments. In (A,B,F), the light turquoise and red bars denote the NN1138-2 and N24852 segments, respectively.

Figure 2

Identification of wild alleles for seed coat color using bulk segregant analysis. SSR-map, the physical map constructed with SSR markers; SNPLDB-map, the physical map constructed with SNPLDB markers. The seed coat color (SCC) was classified as yellow, green and black in SojaCSSLP5, including 147, 7 and 23 in a total of 177 CSSLs, respectively. 0(144) or 0(147) represent the number of lines with genotype 0 (homozygous NN1138-2) are 144 or 147, while 2(3) or 2(0) represent the number of lines with genotype 2 (homozygous N24852) are 3 or 0.

Figure 3

Joint comparisons of CSSLs significantly different from NN1138-2 in DTF. DTF, days to flowering. SSR-map, the physical map constructed with SSR markers; SNPLDB-map, the physical map constructed with SNPLDB markers. The wild segments on the markers in gray cells are of negative additive effects; the others are of positive additive effects. In 0(143), 0 represents NN1138-2 genotype while (143) represents 143 CSSLs with genotype 0 in intermediate flowering group. Similarly, 2(3) represents 3 CSSLs with genotype 2 (N24852) in intermediate flowering group.

Figure 4

Sequence analysis of SCC and DTF candidate genes between N24852 and NN1138-2. (A) and (B), are candidate genes of SCC, respectively; (C–H) are respective DTF candidate genes. The black boxes represent exons; the lines between boxes represent introns; red arrowheads and broken lines represent the variant position. Red nucleotides represent SNP between N24852 and NN1138-2, which are nonsynonymous mutations and marked with red arrowheads.