Fine Mapping And Candidates Mining Of A Novel Gene Pm6Sl Conferring Resistance To Wheat Powdery Mildew

Powdery mildew is a foliar disease caused by the fungus Blumeria graminis f.sp.tritici（Bgt）.Screening of new sources resistant to wheat powdery mildew for the development and cultivation of resistant varieties is currently the most effective and economical friendly approach to control this disease.Our laboratory previously found that the 6S^l#3 chromosome of the Chinese Spring（CS）-Aegilops longissima addition line TA7548 harbored a gene conferring powdery mildew resistance,and designated the gene as Pm6Sl.In this study,66 specific molecular markers and 136 plants with 6S^l#3 chromosome structural variation were developed,the Pm6Sl gene was successfully mapped to a 0.94 Mb interval on the proximal end of 6S^l#3L by means of an approach named by “three-step fine mapping of alien genes”.By combining disease-resistance gene enrichment sequencing（Mut Ren Seq）of Pm6Sl resistance loss mutants,transcriptome sequencing and chromosomal collinearity analysis,5 Pm6Sl candidate genes were mined in the interval.In addition,4 new germplasms with tiny fragments harboring Pm6Sl and 2 diagnostic markers for Pm6Sl were also identified.The results not only lay a foundation for further cloning of Pm6Sl and understanding of disease resistance mechanism,but also provides new disease resistance genes and molecular marker-assisted selection technology for wheat disease resistance breeding.The main contents and results are as follows:1、Fine mapping of Pm6SlThe Pm6Sl gene was finely mapped to a 0.94 Mb interval between markers EMO03991 and EMO04335 closing the end of the long arm of chromosome 6S^l#3 by the “three-step fine mapping of alien genes”.Step 1: Chromosome arm mapping of Pm6Sl.A double-monosomic 6S^l#3/6A segregating population was constructed using a CS-Ae.longissima 6S^l#3[6A] substitution line TA7548 S,and then 608 6S^l#3 individuals from the population were screened with four 6S^l#3-specific molecular markers.It identified 1 translocation of 6S^l#3 short-arm,6 short-arm telos,and 10 long-arm telos.After powdery mildew resistance assay by inoculation of Bgt-isolate E26,the Pm6Sl gene was mapped to the long arm of 6S^l#3.Step 2: Chromosome segment mapping of Pm6Sl.The 6S^l#3 segregating population with homozygous gene ph1 b which induced homoeologous recombination was constructed by6S^l#3[6A] substitution line TA7548 S.Fourteen 6S^l#3 long-arm recombinants were identified from795 individual plants using four 6S^l#3 long-arm specific markers.Afterwards,24 6S^l#3 specific markers were analyzed,and mapped the Pm6Sl gene in the proximal segment of 6.43% of the6S^l#3 long arm,about 42.60 Mb range.Step 3: Fine mapping of Pm6Sl.The Pm6Sl secondary segregation population was constructed by self-pollinated heterozygous 6S^l#3 long-arm recombinants of powdery mildew resistance.Using 2 markers flanking the initial mapping interval,105 new individuals with recombined points in the interval,which includes 38 resistant and 67 susceptible plant,were identified from 6000 plants in the secondary segregation population.Furthermore,34 molecular markers were used to fine-mapped Pm6Sl to an interval of about 0.94 Mb between markers EMO03991（654.11 Mb）and EMO04335（655.04 Mb）at the proximal end of 6S^l#3L.2、Pm6Sl candidate gene mining,cloning and functional analysisBased on Ae.longissima accession reference genome sequences,combined analyses of the transcriptome sequences of 6S^l#3 addition line TA7548,the full-length transcriptome sequences of TA1910,and chromosome collinearity analysis,13 NLR class,9 Kinase class and 1 STP class candidates were mined in Pm6Sl fine-mapped interval.The mutant candidate gene sequence amplification and BSMV-VIGS gene silencing of the NLR gene EMO16698 confirmed that the gene is not a candidate for Pm6Sl.3、Development of Pm6Sl mutants with loss of resistance to powdery mildew and MutRenSeq analysisIn order to develop Pm6Sl mutants with loss of resistance to powdery mildew for further gene isolation by susceptible mutant-based cloning approach,seeds of 6S^l#3/6A substitution line TA7548 S were treated with ethyl methanesulfonate（EMS）.Based on powdery mildew resistance identification,mutant authenticity by 6S^l#3 specific markers,genetic background consistent identification by SSR marker and 55 K gene chips,55 authentic loss-of-resistance mutants from37 M₂ lines were identified from 466 M₂ lines.5 of the independent mutants were selected to perform Mut Ren Seq analysis,giving rise to a total of 301 Contigs with single base variation（SNV）compared with the wild-type sequence,of which,1 contig had SNV in 3 mutants,and 9 contigs with SNV in 2 mutants.However,none of the Contigs with SNVs were in Pm6Sl fine mapped region.Since Mut Ren Seq can only clone NLR-type resistance genes,and the NLR-type gene EMO16698 had already been verified as a non-candidate gene,so Pm6Sl was excluded as an NLR-type gene,thus reducing the candidate genes to 4 Kinase-type and one STP-type genes.4、Pm6Sl tiny recombinants and diagnostic molecular markersResistance assay by inoculations of more than 30 Bgt isolates displayed that Pm6Sl conferred broad-spectrum resistance to powdery mildew.4 resistant recombinants were identified having tiny fragment harboring Pm6Sl accounting for 1.22%,1.59%,3.31% and 3.52% of the total length of 6S^l#3,respectively.In addition,the markers EMO39960 and EMO09126 in the fine mapping interval were not amplified in 20 wheat varieties and advanced breeding lines,indicating those markers were diagnostic for Pm6Sl.