Molecular identification of Phytophthora resistant genes in soybean

Phytophthora root and stem rot (PRSR), caused by oomycete Phytophthora sojae, is the most severe soil-borne disease of soybean (Glycine max (L.) Merr.) worldwide. The disease can be effectively managed by introducing resistance to P. sojae (Rps) genes into soybean cultivars by breeding, which requires continuous efforts on identification of resistance resources from soybean germplasm. Previously, two resistance genes, Rps2-cas (former name Rps2-das) and Rps14 (former name Rps1-f), were mapped by linkage analysis from soybean landraces, PI 594549 C and PI 340029, respectively. The resistance underlying PI 594592 also need further characterization given its broad resistance spectrum. In this study, Rps-2cas and Rps14 were further mapped, and Rps2-b, was identified and initial mapped from PI 594592. Thus, this thesis research was divided into three parts for three Rps genes.

The first part mainly focuses advances on Rps2-cas. Marker-assisted spectrum analysis was performed for Rps-2cas to confirm its potential in disease management. A high-quality genome assembly of PI 594549 C was generated, and KASP markers were developed based on comparison between new reference and Williams 82 reference genome. The gene was further mapped to a 32.67-kb region on PI 594549 C reference genome harboring three expressed NLRs by 24 recombinants screened from a large F₄ population. Comparative genomics analysis suggests the only intact NBS-LRR gene in the fine mapping region is the best candidate gene for Rps2cas, and its function was validated by stable transformation. Evidences from other high-quality assembly genomes suggest Rps2-cas originated from an ancient unequal crossing over event.

In the second part, Rps14 was further mapped using 21 recombinants identified from a F₃ population consisting of 473 plants. In commonly used Williams 82 reference genome, the assembly of fine mapping region was incomplete, and Rps14 region showed drastic variation in size and copy number of NLRs in 23 high-quality genome assemblies, suggesting the complexity of Rps14 region and high-quality reference sequence of donor line is required for isolation of Rps14 candidate genes. Marker assisted resistance test showed Rps14 had wider resistance spectrum to different P. sojae isolates comparing to other Rps genes on chromosome 3, and phylogenic analysis further supported the potential of Rps14 to be a novel resistance gene.

For the third part, an F₂ population derived from a cross between PI 594592 and Williams was tested by P. sojae race 1. The 3:1 and 1:2:1 Mendelian segregation ratios were observed in F₂ individuals and F_2:3 families, respectively, suggesting a single dominant Rps gene in PI 594592. The gene was initially mapped to the distal end chromosome 16 overlapped with Rps2, and the gene was tentatively named as Rps2-b. Polymorphic SSR markers and InDel markers designed based on re-sequencing data of PI 594592 and Williams was used to genotyping all the F_2:3 families, and a linkage map was constructed for Rps2-b. Rps2-b was mapped to a 461.8-kb region flanked by SSR marker Satt431 and InDel marker InDel3668 according to the reference genome (Wm82. a2). Marker-assisted resistance test showed Rps2-b hold a wide resistance spectrum.