Molecular Mapping Of Late Blight Resistance Gene From S.pinnatisectum And Transcriptional Analysis Of PME/PMEI Genes

Potato?Solanum tuberosum L.?belongs to Petota Dumortier section,Potatoe?G.Don?A'Arcy subgenus,Solanum L.genus,Solanaceae family.Potato,originated and domesticated from the Andes mountains of South America,has been widely planted in Europe since the 18th century and grown in about 160 countries and regions so far.As eaten by more than a billion people worldwide,it has been the fourth most important food crop in the world after rice,wheat and maize.China is the largest potato producer in the world,and its annual planting area and production rank the top of other countries and regions.Potato late blight,a cosmopolitan disease caused by Phytophthora infestans,is regarded as the first killer of potato production on account of its strong epidemic,rapid spread and serious destruction.The pathogenic oomycetes have both sexual and asexual reproduction modes.The frequent variation of race virulence brings risks to resistance genes which may be overcome.Therefore,it is significant for potato resistance breeding against late blight to exploring new disease-resistant germplasm,researching new resistance genes and expanding the resistance-breeding resource library.High level and broad spectrum late blight resistance has also been observed in the Mexican diploid wild species Solanum pinnatisectum.In this study,the genetic mechanism of resistance gene from a S.pinnatisectum clone PI275233 was researched by means of building mapping population,screening linkage molecular markers and collinearity analysis.The response to late blight of disease-resistance related genes,PME/PMEI,in S.pinnatisectum was investigated by transcriptome sequencing induced by P.infestans.The main results are summarized as follow:1.A backcross population including 931 progenies was developed by crossing the susceptible diploid S.cardiophyllum?PI186548?as the male parent with the resistant diploid S.pinnatisectum?PI275233?.The result of detached leaf identification reflected that the BC₁ generation produced 440 resistant plants and 491susceptible plants.The segregation ratio fit a monogenic Mendelian inheritance model of 1:1?resistant:susceptible?in the population??²=2.794,P=0.095?,which suggested that a single dominant locus,named as Rpi2,controlled the late blight resistance in S.pinnatisectum?PI275233?.2.A total of 324 EcoRI+3/MseI+3?196 E-A/M-C and 128 E-A/M-A?AFLP primer combinations were screened by BSA strategy and a subpopulation of BC₁population?164 susceptible and 101 resistant plants with extreme phenotypes?.The two closest markers,EAGCMCGA-450 and EACAMAGG-330,were determined to be linked to the resistance locus at distances of 1.2 and 0.8 cM,respectively.Based on their sequence information,EAGCMCGA-450 and EAACMATC-330 was respectively converted into the CAPS markers SpAFLP1 and SpAFLP2.The sequence of EAGCMCGA-450 was used as query to BLAST analysis.A contig,assembled with 23ESTs and named as cEST1,was obtained.Fortunately,cEST1 contained a previous reported RFLP marker TG572,which was co-segregated with SpAFLP1.Adopting the similar strategy,molecular markers SpAL21?SpCT226?SpGot2 and SpT1756 were discovered to exhibit linkage relationship with Rpi2.SpGrol-1 was a RGA molecular marker linked with Rpi2 at distance of 2.8cM.Eventually,the genetic linkage map,including 11 molecular markers,was established,and the target gene Rpi2 was flanked by SpT1756?1.6cM?and SpAFLP2?0.8cM?.3.The molecular marker sequences were used as queries to search for homologous loci in the genome sequence databases of potato and tomato.Because cloning and sequencing failed,homologous loci of SpGot2-1 and SpTG20 were not found.The other 9 molecular markers mapped in the Rpi2 genetic linkage map hit their homologous loci and located a 2.9Mb orthologous region on potato genome and a 2.4Mb orthologous region on tomato genome.Scanning potato genome suggested the physical distance between StAFLP2 and StT1756 was about 84 kbp.Unfortunately,there was a 50-kbp gap in this potato genome region that potentially contained the homologous gene of Rpi2.However,the homologous segment in the tomato genome was assembled completely,in which 3 of 10 loci were annotated as NBS-LRR class disease resistance proteins?Accession nos.Solyc07g056180.1,Solyc07g056190.2,and Solyc07g056200.2?.4.A total of 135 PME/PMEI related genes were identified in potato genome,including 35 StproPMEs,34 StPMEs and 66StPMEIs.The physical and chemical characteristics of proteins encoded by these genes were analyzed and summarized,involving protein length,molecular weight,isoelectric point,signal peptide and transmembrane domain.Subcellular location analysis indicated that the entire StproPMEs,StPMEs and StPMEIs were located in extracellular matrix.5.The Phylogenetic and conserved motifs analysis results indicated that StproPMEs could be divided into nine subfamilies,named as Group?^?,and identified 28 conserved motifs;StPMEs could be divided into three subfamilies,named as Group?,Group?and Group?;StPMEIs included nine subfamilies,and 26conserved motifs were identified in the 66 StPMEIs.6.Analysis of the gene expression patterns in different tissues showed that some StproPME,StPME and StPMEI genes were specifically expressed in the reproductive tissues?mature flowers and stamens?and almost not expressed in other tissues,including 8 StproPME genes,4 StPME genes and 9 StPMEI genes.In consideration of the previous research on Arabidopsis and the role of stamen forming pollen,we hypothesized that these genes were involved in the potato pollen formation and pollen tube elongation.7.The result of transcriptome sequencing induced by P.infestans reflected that partial potato PME/PMEI genes were regulated to expression changes after infection and differently expressed between resistant and susceptible materials.Among the potato PME genes,the expression patterns presented two types.The first type was that gene expression was positively associated with potato resistance.For instance,the expression level of StproPME22 significantly increased after inoculation in resistance material and gradually decreased in susceptible material,while the initial expression levels between the resistant and susceptible were identical.The other type was that gene expression was positively associated with potato resistance.For instance,the expression levels of StproPME16,StproPME28 and StPME28 gradually decreased after inoculation.Thereinto,StPME28 was the specific expression gene in resistant material.The potato PMEI genes,such as StPMEI23,StPMEI25,StPMEI29,StPMEI40,StPMEI45,StPMEI52,StPMEI60 and StPMEI62,were specifically expressed in resistant material and up-regulated after inoculation.