Identification The Resistance Gene Of Soybean Cultivar Qichadou 1 To Phytophthora Sojae And Analysis The Candidate Gene

Phytophthora root rot?PRR?,caused by Phytophthora sojae M.J.Kaufmann&J.W.Gerdemann,is one of the most economically destructive diseases of soybean.Phytophthora sojae is a soil-borne oomycete that is capable of infecting soybean plants at all developmental stages,resulting in symptoms including damping-off,root and stem rot,leaf yellowing,and wilting.Globally,the annual economic losses resulting from PRR-infected soybean plants is about$1-2billion.Using Phytophthora-resistant soybean plants harboring dominant Rps genes is the most economical,effective and environmentally safe method to prevent this disease.The study were well studied the resistance spectrum,phenotypic and genetic analysis,and resistance gene of Phytophthora-resistance cultivar Qichadou 1.The PRR resistant gene RpsQ of Qichadou 1 were finely mapped by using the segregatant population constructed by susceptible parent Jikedou 2 and resistant parent Qichadou 1.The candidate gene of RpsQ was predicted according to the reference genome sequence information and the two parents sequence information in the candidate region of RpsQ,and the genomic sequence of the candidate gene was analyzed by using bioinformatics technology.The expression pattern of the candidate gene of RpsQ was also detected by using RT-PCR technique.The candidate gene of RpsQ in related soybean cultivars?lines?with a kinship of Qichadou 1were amplified and sequenced,then the haplotype diversity of the candidate gene were characterized.This study also invented a new,simple,fast and reliable detached petiole inoculation technique which suitable for population phenotypic identification of PRR resistance.The main results are as follow:1.The Chinese soybean cultivar Qichadou 1 exhibited a broad spectrum resistance,and has a distinct resistance phenotype by compared with known P.sojae-resistant differentials,following inoculation with 36 Chinese P.sojae isolates.Genetic analyses indicated that the disease resistance in Qichadou 1 is controlled by a single dominant gene.This gene locus was designated as RpsQ and mapped to a 118 kb region between BARCSOYSSR₀3₀165 and InDel281 on soybean chromosome3,and co-segregated with Insert11,Insert144 and SNP276.Within this region,the gene Glyma.03g27200 encoding a protein with serine/threonine protein kinase structure is a typical resistant gene,and RT-PCR analysis showed that this gene induced by P.sojae infection,which was suggested as a best candidate gene of RpsQ.2.Based on candidate gene sequence differences between the resistant and susceptible parents,a InDel marker Insert144 was developed.To verify the effectiveness of the mark,the InDel marker Insert144 was used to distinguish RpsQ from the other known Rps genes on chromosome 3.Identical polymerase chain reaction amplification products were produced for cultivars Qichadou 1?RpsQ?and Ludou 4?Rps9?.All other cultivars carrying Rps genes on chromosome 3 produced different PCR products,which all lacked a 144-bp fragment present in Qichadou 1.The phenotypes of the analyzed cultivar Qichadou 1 and Ludou 4 combined with the physical position of the PRR resistance locus,candidate gene analyses,and the candidate gene marker test revealed RpsQ is Rps9 or an allele gene of Rps9,and confer resistance to P.sojae.3.The sescupibile parent Jikedou 2,the resistant parent Qichadou 1 harboring RpsQ and 26related soybean cultivars?lines?with a kinship of Qichadou 1 were inoculated with 8 different virulent P.sojae isolates and obtained the phenotypic reactions of all cultivars?lines?.The results showed that Qichaodu1 resistant to 4 isolates and had a distinct reaction type.The referenced candidate gene of RpsQ in different cultivars?lines?were cloned and sequenced,and haplotypes and polymorphism analysis were also performed.There were multiple diversity in gDNA sequences of the 28 cultivars?lines?and the 28 cultivers?lines?formed 26 haplotypes,of which Qichadou 1 represent a kind of haplotype.Baded on the reaction types of all cultivars?lines?and the gDNA sequence difference of all cultivars?lines?,it implies that the other 27 cultivars?lines?doesnot harbor Phytophthora–resistant gene RpsQ of Qichadou 1,or harbor the allele genes of RpsQ.4.This study also developed a rapid,effective and reliable in vitro PRR inoculation method,namely the detached petiole inoculation technique.In this study,the reliability of the detached petiole was verified in soybean cultivars and segregate populations.The detached petiole and hypocotyl inoculation techniques were both used to evaluate resistance of 13 soybean cultivars and the F₂ and F_2:3populations of Zhonghuang47×Xiu94-11.The results showed that all reactions of the thirteen cultivars to three P.sojae isolates were respectively consistent between the two inoculation techniques.The reaction comparison of the F₂ and F_2:3 populations to isolate PsMC1 had a 95.20%similarity using the two methods and the segregant ratios of resistance and susceptible were all fit 3:1.The results reveals that the PRR resistance in Xiu94-11 is controlled by a single dominant gene.The phenotypic ratios of Jikedou 2×Qichadou 1 F₂ population tested using the detached petiole inoculation technique fitted well3:1?R:S?,verifying that Qichadou1 contain a single dominant gene resistance to P.sojae.This detached petiole inoculation technique is effective and reliable and can save seeds and non-destructive to plant.This is suitable for multiple repeated,especially for the genetic populations with a few seeds.It also can carry out for large-scaled resistance screening using multiple P.sojae isolates in soybean.This technology is an effective and reliable evaluation method for PRR resistance in soybean...