Map-based Cloning Of Leaf Senescence Gene TaLS-2D In Hexaploid Wheat(Triticum Aestivum L.)

Senescence is an important agronomic trait that affects both crops yield and quality.Effective senescence can increase crop yield and enhance the adaptability of crop to environment.Therefore,the identification and function analysis of senescence related genes could reveal the regulation mechanism of wheat senescence and provide new genetic resources for the breeding of new wheat varieties.Wheat senescence mutant m68 was screened from the elite wheat cultivar Yan Zhan 4110?YZ4110?EMS mutant library and the senescence of the mutant leaves started earlier than that of wild type YZ4110.In order to find the casual genes affecting senescence,both wild-type YZ4110 and a diverse parent 188?NX188?were crossed with mutant m68.Two populations of YZ4110 x m68 BC₂F_2:3?763individuals?and NX188 x m68 F_2:3?10133 individuals?were generated.Genetic analysis showed that the senescence trait of m68 was controlled by a recessive gene.By screening and mapping 713 public SSR markers,the results showed that the target gene was flanked by two markers cfd116 and gwm157located on wheat chromosome 2D.This mapping result was consistent with previous results obtained from using wheat 660K SNP analysis.This novel gene controlling wheat senescence trait was named TaLS-2D.One thousand one hundred and nine new SSR markers and forty six new KASP markers were developed based on comparative genomics using the reference genome sequences of the D genome progenitor Aegilops tauschii and hexaploid wheat.Finally,the gene TaLS-2D was further mapped between newly developed SSR markers M270 and M1065 with only one recombinant plant found between these two markers.Newly developed SNP marker S3 co-segregated with the TaLS-2D gene.The corresponding physical distances at this region were approximately 3Mb on Ae.tauschii and hexaploid wheat reference genome sequences,while it was 93.8 Kb,116 Kb and 107 Kb on Brachypodium,rice and sorghum genome sequences,respectively.By using the TriAnnot tool,19,20,and 23 genes were predicted in the candidate gene region on the AK58,CS and Ae.tauschii genome sequences,respectively.In combination with transcriptome expression profiles of YZ4110 and m68 flag leaves at different senescence stages,there were three genes related to the senescence process in the candidate gene region,in which the expression levels of gene2 and gene4 gradually increased with the progress of leaf senescence,while the expression level of gene6 gradually decreased with the progress of leaf senescence.Although a SNP?C>T?mutation was found in the coding region of gene2 between YZ4110 and m68,it was not the casual mutation as it was not co-segregating with the senescence phenotype according to the results from sequencing the recombinants from the cross of NX188 x m68 and individuals with the senescence phenotype from BC₂F₂ plants of YZ4110 x m68.Other predicted genes in the candidate region were also sequenced.The results showed that a SNP was detected in the first exon of gene13 between YZ4110 and m68.This mutation has lead to the amino acid change from proline to serine.Further analysis confirmed that this SNP was the casual mutation,and gene13 was the candidate gene for further study.Transcriptome sequencing was performed on the flag leaves at four different senescence stages of WT and m68.Altogether 2396,8383,13355 and 16130 differentially expressed genes?DEGs?were detected at S1,S2,S3 and S4 stages,respectively.Among the 366,3142,6452 and 8761 were down-regulated genes and 2030,5241,6903,and 7369 were up-regulated genes,respectively.GO enrichment analysis of DEGs showed that the down-regulated expression genes were enriched for 141GO terms and the up-regulated expression genes were enriched for 143 GO terms.WRKY transcription factors and kinase genes were involved in the initiation of m68 leaf senescence.Senescence affects chlorophyll,photosynthesis and glucose metabolism,etc.The KEGG pathway analysis of DEGs showed that plant hormones were also involved in regulation of m68 leaf senescence.Among them,abscisic acid,salicylic acid and jasmonic acid were positively regulating m68 leaf senescence;while cytokinin was negatively regulating m68 leaf senescence.Cluster analysis of DEGs showed that 1012genes in m68 were associated with leaf senescence,including transcription factors,transporter genes,kinase genes,cytochrome C P450 family genes,etc.These genes provide a theoretical basis for studying and analyzing the mechanism of the wheat leaf senescence.