Mapping Of Early Leaf Senescence Gene Els1 And Cloning Of Powdery Mildew Resistance Gene Pm41 In Wheat Based On BSR-Seq

Wheat is an important food crop in the world.Early senescence and powdery mildew can directly threaten the wheat production worldwide.Early senescence of functional leaves significantly reduces the photosynthetic time and efficiency,seriously affecting grain yield and quality in wheat.Discovering genes responsible for early leaf senescence are necessary for developing novel germplasms and cultivars with delayed leaf-senescence through molecular manipulation and marker assisted selection.Powdery mildew is one of the severest wheat diseases worldwide.Mapping and cloning of powdery mildew genes are necessary for molecular breeding by design and undersatnding the mechanism of disease resistance.In present study,early leaf senescence gene elsl and powdery mildew resistance gene Pm41 were fine mapped rapidly by applying bulked segregant analysis plus RNA-Seq（BSR-Seq）.High-density genetic linkage maps of els1 and Pm41 were constructed.Physical map of Pm41 was also constructed by screening genomic BAC library of the Pm41 donor wild emmer accession IW2.Combining BAC clone sequencing,expression analysis and genetic transformation,the candidate gene of Pm41 was identified.The main results are as following:1.An early leaf senescence segregating F3 family,La,that segregated in a 3 normal:1 early leaf senescent ratio was identified in a breeding population.The F3:4 families segregated in a 1 normal:2 segregation:1 early leaf senescent ratio.Homozygous early leaf senescent line M114 was crossed with homozygous normal line W301 to develop a F2 population that segregated in a 3 normal:1 early leaf senescent ratio.The F2:3 families segregated in a 1 normal:2 segregation:1 early leaf senescent ratio These results suggested that early leaf senescence of M114 was controlled by a single recessive gene,provisionally designated els1.2.BSR-Seq approach was applied to map the els1 gene.els1 associated SNPs were found on chromosome 2BS and 4 polymorphic SNP markers（WGGB302,WGGB303,WGGB304 and WGGB305）and 2 polymorphic SSR markers（WGGB306 and WGGB307）were developed and linked with els1.els1 was mapped in a 1.5 cM genetic interval flanked by SNP markers WGGB305 and WGGB303,and co-segregated with SNP marker WGGB302 on wheat chromosome 2BS,which corresponds to 9.2 Mb genomic region in Chinese Spring.Sixty-nine putative genes were annotated in this genomic region.Among them,NB-ARC,cytochrome P450,receptor-like protein kinase family protein and WRKY transcription factor were identified that may be associated with early leaf senescence or cell death.3.In order to construct a fine genetic linkage map of Pm41,BSR-Seq approach was applied to find polymorphic SNPs in the Pm41 genomic region on chromosome 3BL.Over all,7 polymorphic SNP markers（B3-6,B3-14,B3-78,B3-97,B3-104,B3-112 and B293）tightly linked with Pm41 were developed.Pm41 was mapped in a 0.2 cM genetic interval flanked by SNP markers B3-14 and B3-104,and co-segregated with markers B3-78 and B3-97 on wheat chromosome 3BL.4.BAC library of Pm41 donor wild emmer accession IW2 was screened to construct a physical map of Pm41.Altogether,14 positive BAC clones were identified and sequenced.An 850 kb contig sequence was assembled to cover the physical interval of Pm41 locus.The genomic region of Pm41 corresponds to a 733 kb genomic region in Chinese Spring 3BL and a 901 kb genomic region in wild emmer accession Zavitan,respectively.Seven putative genes were annotated in the corresponding genomic regions of IW2 and Zavitan,while only 6 genes in were annotated in the corresponding genomic region of Chinese Spring.A CC-NBS-LRR（CNL）gene was identified in wild emmer accessions IW2 and Zavitan,but not in Chinese Spring,and was considered to be the candidate of Pm41.5.The genomic DNA sequences of the CNL gene were cloned from IW2 and Langdon.There are 14 SNPs in the 3,370 bp sequences between IW2 and Langdon from the start codon ATG to the stop codon TGA.The full-length CDS of CNL in IW2 is 2,955 bp encoding 985 amino acids with RX-CC_like,NB-ARC and LRR₃ conserved domains.The CNL was strongly induced by Bgt infection in resistance wild emmer accession IW2,however the CNL allele was not expressed in susceptible durum wheat Langdon.Four types of alternative splicing located in the untranslated region of CNL were detected in IW2.Over-expression vector pTPCK303-Hyg-CNL and complementary vector pCAMBIA1300-Hyg-CNL were constructed and used for transformation of highly susceptible powdery mildew wheat variety Fielder by agrobacterium-mediated method.6.Functional marker of Pm41,Pm41-427,was developed and can be used in molecular marker assisted selection in wheat breeding program.Pm41 was only present in wild emmer and not found in 262 tested Chinese wheat microcore collection germplasm,providing valuable disease resistance resource for wheat improvement.