Genome-wide linkage and association mapping identified a novel candidate gene, TaGLK-A1, underlying fructan content...

A nuclear-localized G2-like transcription factor, TaGLK-A1 (TraesCS7A02G539600), was identified as a strong candidate gene for grain fructan content in wheat using GWAS, linkage mapping and transcriptome analysis. Fructans, a type of natural polysaccharides or oligosaccharides polymerized from fructose molecules, play important physiological roles in crops and confer great health benefits to humans. In this study, we detected 78 stable loci associated with fructan content across all 21 wheat chromosomes across three environments and in the BLUE, explaining 4.4-10.0% of the phenotypic variance by a genome-wide association study (GWAS) using 243 wheat cultivars and advanced lines genotyped with the Axiom wheat 660 K single-nucleotide polymorphism (SNP) array. Among these, a significant locus of fructan content was localized in a 4.02 Mb interval (713.87-717.89 Mb) on chromosome 7AL, designated QGfc.hkd-7A. QGfc.hkd-7A was validated in 164 recombinant inbred lines (RILs) from a cross between Avocet and Chilero in five environments using diversity array technology (DArT), which was tightly linked to DArT markers SNP2260399, 3575400, SNP3028492, SNP100415949, 3533474 and 100091413, accounting for 16.4-24.6% of the total phenotypic variance in the RIL population. Favorable allele of QGfc.hkd-7A significantly increased grain fructan content by 10.5% (P < 0.01) compared with lines carrying unfavorable alleles. To further validate QGfc.hkd-7A, we generated differential gene expression profiles using RNA-seq for high-fructan (AC6012) and low-fructan (AC6361) lines from the Avocet/Chilero population. TaGLK-A1 (TraesCS7A02G539600), a differential expressed gene (DEG), was subsequently cloned and sequenced from both genotypes. Sequence comparison of the full-length TaGLK-A1 cDNA (1545 bp) revealed a single SNP at position 109 causing a proline (CCA) to threonine substitution between Avocet and Chilero. Subcellular localization analysis confirmed that the G2-like transcription factor encoded by TaGLK-A1 localizes to the nucleus. Transactivation assays revealed that it lacks intrinsic transcriptional activation activity, suggesting it may function as a transcriptional repressor or require cofactors for regulatory function. These results provide a novel and strong candidate gene for fructan content and a forward genetics strategy for gene discovery in molecular breeding.