Molecular identification of Phytophthora resistant genes in soybean

<p>Phytophthora root and stem rot (PRSR), caused by oomycete <i>Phytophthora sojae</i>, is the most severe soil-borne disease of soybean (<i>Glycine max</i> (L.) Merr.) worldwide. The disease can be effectively managed by introducing resistance to <i>P. sojae</i> (<i>Rps</i>) genes into soybean cultivars by breeding, which requires continuous efforts on identification of resistance resources from soybean germplasm. Previously, two resistance genes, <i>Rps2-cas</i> (former name <i>Rps2-das</i>) and <i>Rps14 </i>(former name<i> Rps1-f</i>), 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, <i>Rps-2cas</i> and <i>Rps14</i> were further mapped, and <i>Rps2-b</i>, was identified and initial mapped from PI 594592. Thus, this thesis research was divided into three parts for three <i>Rps</i> genes.</p><p>The first part mainly focuses advances on <i>Rps2-cas</i>. Marker-assisted spectrum analysis was performed for <i>Rps-2cas</i> 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 <i>Rps2cas</i>, and its function was validated by stable transformation. Evidences from other high-quality assembly genomes suggest <i>Rps2-cas</i> originated from an ancient unequal crossing over event.</p> <p>In the second part, <i>Rps14</i> was further mapped using 21 recombinants identified from a F₃population consisting of 473 plants. In commonly used Williams 82 reference genome, the assembly of fine mapping region was incomplete, and <i>Rps14</i> region showed drastic variation in size and copy number of NLRs in 23 high-quality genome assemblies, suggesting the complexity of <i>Rps14</i> region and high-quality reference sequence of donor line is required for isolation of <i>Rps14</i> candidate genes. Marker assisted resistance test showed <i>Rps14</i> had wider resistance spectrum to different <i>P. sojae </i>isolates comparing to other <i>Rps</i> genes on chromosome 3, and phylogenic analysis further supported the potential of <i>Rps14</i> to be a novel resistance gene. </p> <p>For the third part, an F₂population derived from a cross between PI 594592 and Williams was tested by <i>P. sojae</i> race 1. The 3:1 and 1:2:1 Mendelian segregation ratios were observed in F₂individuals and F_2:3families, respectively, suggesting a single dominant <i>Rps</i> gene in PI 594592. The gene was initially mapped to the distal end chromosome 16 overlapped with <i>Rps2</i>, and the gene was tentatively named as <i>Rps2-b</i>. Polymorphic SSR markers and InDel markers designed based on re-sequencing data of PI 594592 and Williams was used to genotyping all the F_2:3families, and a linkage map was constructed for <i>Rps2-b</i>. <i>Rps2-b</i> 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 <i>Rps2-b</i> hold a wide resistance spectrum. </p>