Genomic heterogeneity in soybean Williams 82

Soybean is a self-pollinating species that has relatively low nucleotide polymorphism rates compared to 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 intra-cultivar 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 intra-cultivar 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. www.plantphysiol.org on July 19, 2017 Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved. INTRODUCTION Intra-cultivar genetic heterogeneity refers to the genetic variation present from plant to plant within a named cultivar or variety. Although the phenomenon of intra-cultivar heterogeneity has long been recognized in crop species (Byth and Weber, 1968) it is oftentimes ignored, as most researchers assume that elite cultivars are composed of relatively homogenous genetic pools (Fasoula and Boerma, 2007). However, a small number of studies have documented the phenotypic consequences of intra-cultivar genetic heterogeneity in inbred crop accessions, including studies in tobacco (Gordon and Byth, 1972), maize (Higgs and Russell, 1968; Tokatlidis, 2000), wheat (Tokatlidis et al., 2004) and cotton (Tokatlidis et al., 2008). The segregation of parental loci during the breeding process is one source of intracultivar heterogeneity. For self-pollinating species, new cultivars are typically derived from either intermating elite lines or backcrossing traits into elite lines, followed by several rounds of single-seed descent via self-mating and subsequent seed increase generations. At the termination of the single-seed descent generations, any remaining heterozygous loci will segregate in subsequent generations. Assuming that the population remains intact, each plant lineage will eventually fix almost all of the segregating loci in the homozygous state of either parent. Therefore, the population will maintain some degree of plant to plant variation due to the heterogeneity at these loci. Genetic heterogeneity may also be generated de novo by spontaneous mutation (Shaw et al., 2000; Ossowski et al., 2010), novel recombination events, DNA transposition or epigenetic processes (Rasmusson and Phillips, 1997). Recent studies in yeast and fungi have reported striking genomic structural variation, such as large-scale duplications, deletions and rearrangements, induced de novo in response to drug treatments or nutrient-stressed conditions (Gresham et al., 2008; Selmecki et al., 2009). Furthermore, a recent study has reported striking genomic structural variation derived de novo in Arabidopsis thaliana lineages within five or fewer generations when individuals are grown in stressful conditions (Debolt, 2010). Studies that have investigated the genetic basis of intra-cultivar heterogeneity have primarily reported on the rates of molecular marker polymorphisms within cultivars and inbred lines of crops such as barley, maize, rice, sunflower and wheat (Zhang et al., 1995; Olufowote et al., 1997; Gethi et al., 2002; Roder et al., 2002; Sjakste et al., 2003; Soleimani et al., 2005; Giarrocco et al., 2007). However, the number and types of markers applied in these studies www.plantphysiol.org on July 19, 2017 Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved. limited their throughput and ability to resolve major features of structural variation, including large-scale deletions, duplications and more complicated genomic rearrangements. Consequently, little is known about the origins and mechanisms of intra-cultivar heterogeneity. In soybean (Glycine max), Fasoula and Boerma have reported on the impact of intracultivar variation on several traits, including seed composition, seed weight, maturity, plant height and lodging (Fasoula and Boerma, 2005, 2007). They noted that this remnant variation could be used to select elite individuals from within existing cultivars, and recently registered a total of 18 lines directly selected from within the cultivars ‘Benning,’ ‘Cook’ and ‘Haskell’ (Fasoula et al., 2007a, 2007b, 2007c). The recent sequencing of the soybean genome (Schmutz et al., 2010) has enabled the development of genomic tools and methodologies that can address the question of soybean intracultivar heterogeneity in great detail. Williams 82, the sequenced accession, was derived from a composite of four individual plants selected from a Williams × Kingwa BC6F3 generation (Bernard and Cremeens, 1988). This implies that Williams 82 experienced two rounds of singleseed descent following the six backcross generations. Residual heterozygous loci in the BC6F2 generation may have differentially segregated among the four BC6F3 individuals, and in subsequent generations. In theory, this process would fix genetic heterogeneity into the Williams 82 population after several rounds of self-pollination. Furthermore, as seed is propagated and distributed throughout the scientific community, founder effects from geneticbottleneck events may give rise to distinct Williams 82 sub-populations at different locations, resulting in disparate Williams 82 lines among researchers. Detailed genomic comparisons of different Williams 82 individuals should resolve the regions of intra-cultivar genomic heterogeneity. Further comparisons of each Williams 82 individual to the Williams and Kingwa parents would then trace the ancestry of the heterogeneous regions to either parent; different Williams 82 individuals would match different parents within these regions. Additionally, genomic variation derived de novo after the split of the Williams 82 lineages may also contribute novel heterogeneous loci. In this case, the genomic comparisons of different Williams 82 individuals would still resolve the regions of intra-cultivar genomic heterogeneity. However, the ancestry of these loci would not specifically trace back to either parent. We would expect to observe novel genomic compositions at such loci. www.plantphysiol.org on July 19, 2017 Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved. In this study, we have utilized high density single nucleotide polymorphism (SNP) genotyping, comparative genomic hybridization (CGH) and exome resequencing data to obtain an unprecedented resolution of the genetic heterogeneity that is extant in Williams 82. The SNP genotyping resolved the parental origins of Williams 82 genetic heterogeneity. Furthermore, the CGH and exon resequencing analyses from more than 203,000 loci revealed the consequences of this heterogeneity in terms of structural and gene content variants between the Williams 82 individuals. Collectively, these findings demonstrate that intra-cultivar genetic heterogeneity can be pervasive in soybean. Implications on the interpretation of the Williams 82 reference genome and the potential of applying similar approaches to inter-specific comparative genomics are discussed.