Identification Of Candidate Genes Behind The Wheat Flag Leaf Width QTL TaFLW1

Leaves are important organs for crop photosynthesis,providing assimilates for crop growth and development.Flag leave of wheat stay green longest after anthesis,and are considered a key source of nutrients and energy for grain filling and formation of yield.In our previous study,a major QTL Qflw.nau-5A controlling the flag leaf width was identified on chromosome 5A using a recombinant inbred line population derived from the cross between Nanda 2419 and Wangshuibai.With the help of the secondary populations derived from the cross of Mianyang 99-323 and PH691 with their respective near-isogenic lines,Qflw.nau-5A was fine mapped to an interval of 0.000061-0.000116 cM flanked by Xwgrb1356 and Xwgrb1619,and named TaFLW1.However,the physical distance spanning TaFLW1 is as large as 32.9 Mb,indicating a low-recombinogenic pericentromeric region.This makes it arduous to identify the candidate gene behind TaFLWl through a traditional map-based cloning approach.In this study,the genomical interval of TaFLW1 was analyzed using the Chinese Spring reference sequence and the resequencing data of Wangshuibai,Nanda 2419,Mianyang 99-323 and PH691 produced in our laboratory.The RNA_seq database shows that there are 56 expressed genes in the candidate interval of TaFLW1.The haplotype analysis showed that Chinese Spring was a narrow-leaf genotype in the candidate interval.Among the 56 expressed genes,26(named Gene1 to Gene26)have sequence variations between the wide-and narrow-leaf materials.In addition,12 genes have variations in the promoter regions,8 have variations in the coding regions,and 9 genes have variation in the introns.The results of mRNA sequence splicing and CDS prediction showed that 2 genes with the variations in the promoter regions and 2 genes with the variations in the coding regions,were not to splice complete mRNA sequences.Only Gene6 of the 9 genes with the intron variations caused alternative splicy.Gene1 displays variation in the 5’UTR;Gene5,Gene7 and Gene8 display variations in the CDS regions.Gene7 and Gene8 cause amino acid changes while Genes does not.Gene 18 and Gene 19 show the whole gene deletion in the wide leaf materials.Function predictions revealed that Gene7,Gene 10 and Gene23 with the promoter variations are related to the development of leaves.It is also true for Gene7,Gene8 and Gene18 with the amino acid variations.qPCR analysis showed that the expression level of each of Gene8 and Gene18 was different between the wide and narrow leaf materials.PROVEAN analysis showed that Gene8 was greatly affected by the functional redundancy of Gene8-5B and Gene8-5D.Gene8 encodes a cell division inhibitor PTEN protein,and its expression level in wide leaf materials is higher than in narrow leaf materials.Therefore,Gene8 may not be the candidate gene of TaFLW1.PROVEAN analysis showed that Gene18 was less affected by its 5B and 5D homologs,and it had a larger space for its function.qPCR showed that the expression level of Gene18 in narrow-leaf materials was higher than that in wide leaf materials,and the expression level of homologous genes Gene18-5B and Genel8-5D was much lower than that of Gene 18.In summary,Gene18 was identified as the candidate gene of wheat flag leaf width gene TaFLW1.