Mining And Utilization Of Late Blight Resistance Genes In Potato SD20

Potato is the fourth largest food crop behind rice,wheat and corn,and plays an important role in ensuring China’s food security.Potato late blight,caused by Phytophthora infestans(Pi),is the destructive disease of potato production in the world.This disease caused annual economic losses of～$6.7 billion in the world and～$1 billion in China.The main method for late blight control is chemical pesticides spraying,which raised serious environmental and human health concerns.Moreover,P.infestans has overcome resistance to some of the pesticides in use,which brings new problems to late blight control.Hence,planting resistant varieties is the most economic and effective method to control this disease.An important prerequisite for late blight resistance breeding is to find the excellent potato germplasm with high resistance and even immune to late blight,explore resistant genes and analysis the resistance mechanism.In this study,we found that the tetraploid potato cultivar SD20(Jiaxiang 1)was highly resistant to P.infestans.Using the methods of Bulked Segregation Analysis(BSA),2brestriction site-associated DNA(2b-RAD)sequencing,Resistance gene enrichment sequencing(RenSeq),BAC library construction and screening,the genetic segments and resistance genes to potato late blight in SD20 were identified,and the disease resistance regulation mechanism in response to exogenous methyl jasmonate(MeJA)was analyzed.The main conclusions are as follows:1.Identification of late blight resistance in potato tetraploid genotype SD20Forty potato materials were inoculated with six Pi strains(CN152,90128,T30-4,F80029,428-2,89148),which exhibited different virulence levels.The results showed that potato SD20,a tetraploid genotype,can resist the infection of all six Pi strains,implying harboring the unknown new resistance genes in it.2.Two candidate disease resistance genetic segments were mined based on isolated population evaluation and simplified genome sequencingThe F1 segregating population(containing 262 individual plants)was obtained by crossing with potato SD20 and Desiree.And the resistance of each individual plant in F1 was identified by spray inoculation and in-vitro inoculation of detached leaflets.Then based on the phenotypic data,30 extremely resistant(ER)and 30 extremely susceptible(ES)genotypes were screened to generate the ER-pool and ES-pool,respectively.The 2b-RAD simplified genome sequencing was carried in these two DNA pools.The results showed that several differently genetic regions were identified on chromosome 4,9,10,and 11.Combined with the analysis of the significance of the difference between resistant and susceptible pools,two genetic regions of chromosome 4 and 10 were selected as candidates with resistance to late blight in SD20.3.Two positive BAC clones containing the resistance candidate genes were obtained using BAC library screening combined with RenSeq analysis.Renseq was applied to the potato resistant genotypes SD20,314(MaR9),and 321(an advanced clone Xiong 2-27 with certain resistance to late blight)and NBS-LRR genes were enriched.The gene specific primers were designed to amplify genome DNA of potato Desiree,314,SD20 and 321.We found that two pairs of primers,PR125 and PR640,were specifically amplified in SD20.The corresponding genes of the two pairs of primers were located at 46 Mb and 42 Mb of chromosome 4,respectively,which was consistent with the candidate resistant segments excavated by 2b-RAD sequencing.The genomic library(BAC Library)of SD20 with an average insertion fragment of 90kb containing 200,000 clones was constructed by using CopyControlTMPCC1BACTM(Hind Ⅲ)vector.The BAC library was screened with specific primers PR125 and PR640 respectively,and two BAC clones of BA29-E31 and BA191-J20,which contained disease resistance candidate genes,were obtained.4.Exogenously applied methyl jasmonate induces early defense related genes in response to P.infestans infection in potato SD20Potato genotype SD20 exhibits strong resistance against the highly virulent Pi isolate CN152,while infected by the super virulent isolate 2013-18-306.While exogenously applied MeJA can significantly delayed the onset and alleviated the symptoms of late blight,indicating exogenous MeJA could induce resistance to Pi in the early biotrophic stage of infection in SD20 plants.To further identify key genes involved in JA signal transduction,gene expression profiling via RNA sequencing(RNA-seq)in SD20 plants treated with exogenously applied MeJA was performed.A total of 2,927 differentially expressed genes were specifically induced,the majority encoded transcription factors,protein kinases,defense enzymes and disease resistance related proteins.GO functional annotation and KEGG metabolic pathway analysis showed that exogenously applied MeJA rapidly induced the expression of genes related to immune response regulation,pathogen defense,and other biological processes,and stimulated endogenous JA synthesis and signal transduction,and the overall early pathogen defense response in SD20.Four candidate disease resistance genes were obtained by RNA-Seq and 2b-RAD genetic segment analysis,which may play an important role in SD20 disease resistance defense.5.A high resistant late blight advanced clone WS29 was developed with SD20 as parent.Using SD20 as parent,a total of 746 seeds were obtained from nine cross combinations,Three excellent potato families were selected,SD20 ×Xisen 6,SD20 × Zhongshu 3,and SD20×V7.Through field evaluation and CN152 inoculation identification,a new clone WS29 with high resistance to late blight was screened from the progeny of SD20×Zhongshu 3.WS29 was medium-late maturing,resistant to PVY and PLRV in field,and has a high vitamin C content.