Rapid Identification of Known Alleles and Discovery of Novel Alleles at the Soybean E3 Locus via Genotyping-by-Sequencing

In Eastern Canada, earliness is an important trait given the short growing season. The aim of this work was to develop markers/haplotypes for breeders to rapidly assess the alleles present in their germplasm at the four recently cloned maturity genes (E1 to E4). The tremendous throughput of modern DNA sequencing technology, has allowed the use of genotyping by sequencing (GBS) approaches to identify and genotype thousands of single nucleotide polymorphisms (SNP) across the entire genome. We have developed a GBS protocol and SNP-calling pipeline for soybean and used these to characterize 144 lines relevant to the study of E genes, including near isogenic lines, as well as a set of 305 lines representative of the Eastern Canadian genepool. Five distinct haplotypes in and around the E3 locus were observed: one corresponded to the wild-type Harosoy-E3 allele and another to the mutant e3T allele that differs by a 13.3 kb deletion in the 3’ end of the GmPhyA3 gene. Sequencing of the GmPhyA3 gene in lines presenting the third and fourth haplotypes revealed two novel alleles both characterized by a premature stop codon. As expected, lines carrying these haplotypes were early-maturing. Molecular characterizations of genotypes that share the fifth haplotype lead to the discovery of another allele defined by an amino acid substitution. However, the impact of this mutation on the phenotype remains to be explored. These results suggest that this approach is a powerful method for rapid allelic characterization, and it application to other maturity genes will be useful for breeding purposes.