The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82

Abstract

Soybean (Glycine max) is a self-pollinating species that has relatively low nucleotide polymorphism rates compared with other crop species. Despite the low rate of nucleotide polymorphisms, a wide range of heritable phenotypic variation exists. There is even evidence for heritable phenotypic variation among individuals within some cultivars. Williams 82, the soybean cultivar used to produce the reference genome sequence, was derived from backcrossing a Phytophthora root rot resistance locus from the donor parent Kingwa into the recurrent parent Williams. To explore the genetic basis of intracultivar variation, we investigated the nucleotide, structural, and gene content variation of different Williams 82 individuals. Williams 82 individuals exhibited variation in the number and size of introgressed Kingwa loci. In these regions of genomic heterogeneity, the reference Williams 82 genome sequence consists of a mosaic of Williams and Kingwa haplotypes. Genomic structural variation between Williams and Kingwa was maintained between the Williams 82 individuals within the regions of heterogeneity. Additionally, the regions of heterogeneity exhibited gene content differences between Williams 82 individuals. These findings show that genetic heterogeneity in Williams 82 primarily originated from the differential segregation of polymorphic chromosomal regions following the backcross and single-seed descent generations of the breeding process. We conclude that soybean haplotypes can possess a high rate of structural and gene content variation, and the impact of intracultivar genetic heterogeneity may be significant. This detailed characterization will be useful for interpreting soybean genomic data sets and highlights important considerations for research communities that are developing or utilizing a reference genome sequence.

Figure 1.

SNP genotyping reveals the parental origins of Williams 82 genetic heterogeneity. The Infinium SNP genotypes of the Wm82-SGC-01 and Wm82-ISU-01 individuals are shown in A and B, respectively. Blue spots indicate SNP positions that match the Williams genotype. Red spots indicate SNP positions that match the Kingwa genotype. Green spots indicate SNP positions that match neither Williams nor Kingwa. Gray X indicates SNPs that were nonpolymorphic between Wm82, Williams, and Kingwa. Data were jittered along the x axis of each chromosome to better resolve individual data points.

Figure 2.

SNP genotyping reveals the parental origins of the Williams 82 reference sequence. The Infinium genotypes of Williams and Kingwa were compared with the Williams 82 reference sequence to identify which haplotypes are represented in the reference sequence within regions of Wm82 heterogeneity. The genotype of the Williams 82 reference sequence is shown for chromosomes 3, 7, 14, 15, and 20. Blue spots indicate SNP positions that match the Williams genotype. Red spots indicate SNP positions that match the Kingwa genotype. Green spots indicate SNP positions that match neither Williams nor Kingwa. Gray X indicates SNPs that were nonpolymorphic between Wm82, Williams, and Kingwa. Regions of heterogeneity appear to be mosaics of Williams and Kingwa sequences in the Williams 82 reference sequence, as evidenced by the interspersion of blue and red spots throughout the regions of heterogeneity. Data were jittered along the x axis of each chromosome to better resolve individual data points.

Figure 3.

Structural variation within regions of heterogeneity between Wm82-ISU-01 and Wm82-SGC-01. A detailed view of CNV on chromosomes 3, 7, 15, and 20 reveals major structural polymorphism within known regions of heterogeneity (Fig. 1; Table I). Each data point represents the log₂ ratio of the hybridization for a given microarray probe. Colored data points represent probes within significant CNV segments that exceeded the significance threshold value. Red data points are CNV located within known regions of heterogeneity based on SNP genotyping. Blue data points are CNV outside of known regions of heterogeneity. Gray data points indicate probes that are not located in significant segments. All significant CNV are located within known regions of heterogeneity between the genotypes, except for the left-most feature on chromosome 7.

Figure 4.

A detailed view of CNV between Wm82 individuals reveals three distinct structural compositions for chromosome 3 based on differential introgressions from Kingwa. Each data point represents the log₂ ratio of the hybridization for a given microarray probe for each genotype versus the Williams reference. In the top panel, CNV were compared between Kingwa and Williams as a reference for differences between the Wm82 parents. Red data points represent probes within significant CNV segments that exceeded the significance threshold value. The other panels display the CNV patterns of Wm82-ISU-01, Wm82-SGC-01, and Wm82-MN-01 versus the Williams reference. For all panels, gray data points indicate probes that are not located in significant segments. (Wm82-PU-01 exhibited a similar chromosome 3 structure to Wm82-SGC-01 and thus is not included here.)

Figure 5.

Exome resequencing reveals gene content variation between two Williams 82 lines. Genomic DNA for Wm82-ISU-01 and Wm82-SGC-01 was captured on a soybean exome microarray and then sequenced via the Illumina II_X system. The relative frequency of reads matching the soybean Glyma gene models is shown for the two Williams 82 lines; 134 gene models are shown. Colored triangles indicate gene models that exhibited presence in one line and absence (no captured exon reads) in the other line. Nearly 90% of the presence-absence gene content variants identified between Wm82-ISU-01 and Wm82-SGC-01 reside within the 10-Mb region of chromosome 3 shown here.

Figure 6.

A model for the origin of genomic heterogeneity in two Williams 82 lines. A, The Williams × Kingwa BC₆ generation, in which contributions from Williams are shown in blue and contributions from Kingwa are shown in red. In this example, 10 loci are heterozygous; the Kingwa Rps₁^k locus has been selected near the top of chromosome 3. B, The BC₆F₂ plant after one generation of selfing. In this example, loci that fix the Williams type are shown in red triangles, loci that fix the Kingwa type are shown in red rectangles, and loci that remain heterozygous are shown in red circles. C, The heterozygous loci from the BC₆F₂ have segregated and fixed homozygosity within each individual plant after several rounds of selfing. The resulting individuals, Wm82-SGC-01 and Wm82-ISU-01, fix heterogeneous types for four of these loci. On chromosome 3, the Rps₁^k locus is fixed for the Kingwa type in both individuals; however, differential recombination below this locus fixes heterogeneous types for much of the chromosome.