Cloning Of TaSST Genes Associated With The Synthesis Of Water Soluble Carbohydrate,Development Of Functional Marker And Association Mapping

Water soluble carbohydrates（WSC） accumulated in wheat stems is an important carbon source for grain weight and yield. The main storage form of WSC in wheat stems is fructans.Sucrose:sucrose-1-fructosyltransferase（1-SST） is the key enzyme for graminan biosynthesis. Characterization of 1-SST gene and development of functional markers are of high importance for marker-assisted selection in wheat breeding. In the present study, TaSST genes were cloned by homologous cloning in combination with PCR（Polymerase chain reaction） amplification, and functional markers were developed and validated with166 cultivars. Meantime, quantitative trait loci（QTL） for WSC content in common wheat were mapped using recombinant inbred lines（RILs） derived from the Shi 4185/Doumai cross grown in two environments.Genome-wide association study（GWAS） for WSC was conducted using the 166 cultivars. Both RIL population and 166 cultivars were genotyped with wheat 90 K iSelect chip. The main results are summarized as follows:1. Fructan biosynthetic enzymes genes TaSST in common wheat were cloned. The full-length genomic DNA sequence of TaSST-A1, TaSST-A2 and TaSST-D1 on chromosomes 4A, 7A and 7D, respectively, were characterized by homologous cloning and PCR verification. The coding sequences（CDS） were 1,992, 1,989,and 1,989 bp for TaSST-A1, TaSST-A2, and TaSST-D1, respectively. Similar to barley 1-SST gene, four exons interspaced by three introns were present in the TaSST genes. Sequence analysis of TaSST-A2 revealed a single nucleotide polymorphism（SNP） in the first exon, four SNPs, a one-bp Insertion/Deletion（InDel） in the first intron, 8 SNPs in the third exon and a 13 bp InDel in the third intron. Sequence analysis of TaSST-D1 revealed15 SNPs in the third exon. No sequence diversity was found at TaSST-A1 locus.2. A cleaved amplification polymorphism sequence（CAPS） marker WSC7 D was developed based on the SNP at the position 1,216 bp in TaSST-D1 gene. Two fragments of 633 and 137 bp were generated in the genotype TaSST-D1 a, whereas a fragment of 770 bp was produced in TaSST-D1 b. The specific bands of 633 and 770 bp can be used to discriminate TaSST-D1 a and TaSST-D1 b. WSC7 D was located on chromosome7 DS using a set of Chinese Spring（CS） nulli-tetrasomics and ditelosomic 7DS, consistent with the linkage analysis based on SNP markers and RILs. Consequently, WSC7 D was validated on 166 wheat cultivars and advanced lines, showing highly significant（P < 0.01） association with wheat stem WSC content. Moreover,the same results were also detected in Shi 4185/Doumai RIL population. Therefore, WSC7 D could serve as a gene-specific marker for improving WSC content in wheat breeding programs.3. QTL for WSC content in common wheat were mapped using the RIL population grown in two environments and the RIL population was finally genotyped with 7,412 SNP markers and a new CAPS marker developed in this study. Three QTL for WSC content were detected in the RIL population across two environments and the averaged data, designated QWSC.caas-4BS, QWSC.caas-7AS, and QWSC.caas-7DS, on chromosomes 4BS, 7AS, and 7DS, respectively. TaSST-D1 is within one of the three QTL. QWSC.caas-4BS was spanned by Tdurum_contig31139₁43 and Excalibur_c36630₂194, with a genetic distance of 8.6 cM;QWSC.caas-7AS was flanked by Kukri_c107638₂53 and Excalibur_c7538₂718, with a genetic distance of10.0 cM; QWSC.caas-7DS was flanked by WSC7 D and BS00108793₅1 with a genetic distance of 6.1 cM.QWSC.caas-4BS, QWSC.caas-7AS, and QWSC.caas-7DS explained 7.1-9.3%, 4.6-5.4%, and 8.8-11.3% of phenotypic variances, respectively.4. A GWAS for WSC was performed with a panel of 166 Chinese wheat（Triticum aestivum L.） cultivars across four environments. Genotyping data consisted of 18,207 mapped SNP markers derived from the 90 K iSelect array. Considering associations with a-log10（P-value） ≥ 3.0, a total of 52 marker-trait associations（MTAs） distributed on 23 chromosome regions were linked with WSC content, with the R2 values ranged from6.8% to 15.2%. Eight candidate genes were detected based on the significant MTAs. They were SDP6, Hgsnat,CBL7, PPR-repeat, RPD8L3, RPM1, TaMPK21-1, and WAK3.This study first reported the allelic variations of TaSST-D1, and the functional marker WSC7 D was of high value in wheat breeding.