Annotation of Cultivar Variations at the Multigenic Rhg1/Rfs2 Locus of Soybean

Soybean Glycine max (L.) Merr. suffers major seed yield losses due to root pathogens. The major pathogens included  Heterodera glycine I. (agent of soybean cyst nematode SCN or moonlight disease) and Fusarium virguliforme (Snyder, Haas, Aoki;  agent of sudden death syndrome (SDS)). The major resistance locus to both pathogens was the Rhg1/Rfs2 locus located at a sub-telomeric region of the soybean chromosome 18 (molecular linkage group G). The locus underlies half of the coinheritance of resistance to SDS and SCN and  a reduction in root growth and plant seed yield. One gene (Glyma18g02690; the receptor like kinase (GmRLK18-1) at Rhg1/Rfs2 has been shown to confer partial resistance in stable transgenic plants. A bacterial artificial chromosome (BAC) clone, (B73P06) of 82,157 bp, in insert size that contained the Rfs2/Rhg1 locus was sequenced. Analysis of sequence identified 800 SNPs (1bp) and  57 indels (of 2-56 bp). Polymorphism was clustered and encompassed 59 kbp of the insert, in three sections that had probably resulted from 3 recombination events (743 SNPs in the 59 Kbp; from 1,500–60,500 bp). There were 5 recombinationt breakpoints that were identified at 733; 6,601; 15,035; 44,740 and 79,211 bp. The third and fourth breakpoints were located before gene 3 and after gene 5 (Glyma18g02690; the RLK at Rhg1/Rfs2) which is therefore inferred to be Peking derived within Rhg1/Rfs2 region.  The highly polymorphic DNA found in a resistant cultivar, Forrest, was inferred to be from the resistance donor Peking. Compared to two susceptible cultivars, Williams 82 and Asgrow 3244 that had just 11 SNPs and no indels between them in the same region the DNA sequence was very different. Comparisons  to single nucleotide polymorphisms (SNPs) from among 32 semi-domesticated and domesticated genomes showed 80% of the total possible SNPs were found in B73P06. In this region Forrest’s DNA was most homologous to ‘Beijing No.4’ a black seeded semi-domesticated line. Annotation of the BAC insert predicted it encoded 9-10 genes with genes 6 and 7 being one fused gene in Forrest but not Williams 82. These genes were comprised of 57 exons (2–8 per gene). Gene 1 (Glyma18g02650) was an arogenate dehydrogenase (AgDH; EC 1.3.1.43, NCBI Reference Sequence: XP_003552195.1); gene 2 (Glyma18g02660) had three conserved domains (Pfam 11891; Pfam 04187; CG 3016) with unknown functions; gene 3 (Glyma18g02670) was a biotin binding like protein (NCBI: NP_567035.1);  gene 4 was a hypothetical protein (NCBI: AET79249.1) conserved across the viridiplantae; the GmRLK18-1 proven in stable transgenic soybean plants to underlie part  of the Rhg1/Rfs2 locus was gene 5 (Glyma18g02680) (NCBI: ACI05083.1, Gene ID, symbol LOC547641); the laccase (GenBank AET79244.1.) was coded by a fusion of genes 6 and 7 (Glyma18g02690); sodium-hydrogen ion antiporters (pfam00999) were coded by gene 8 and 9 (Glyma18g02700) and gene 10 (Glyma18g02620) was predicted to be a helicase (DEXDc ,cd00046; HELICc, cd00079). There were only 31 SNPs within genes. Just 11 SNPs caused amino acid changes. SNPs from 32 genomes indicated the regions introgressed from Peking and the sites where recombination(s) occurred. Therefore, the locus was multigenic, pleiotropic and epistatic for 3 traits.