Functional Characterization and Molecular Marker Development of the Wheat Transcription Factor Gene TaWRKY65-3A

WRKY transcription factors are the plant-specific transcription factor families, and play a crucial role in plant growth, development, nutrient uptake, and response to biotic and abiotic stresses. Characterized the function of the wheat transcription factor gene TaWRKY65-3A and developed the molecular markers, providing genetic resources for wheat germplasm improvement through molecular breeding in this research.

The sequence of TaWRKY65-3A was cloned from the cDNA of wheat variety Hanxuan 10. The domains were analyzed via the SMART website. The expression patterns of TaWRKY65-3A gene were analyzed by qRT-PCR under phytohormone ABA treatments, PEG and NaCl stresses. Predicted the polymorphisms of TaWRKY65-3A through the Wheat Genome Variation Joint Database, and validated them by using 21 wheat germplasms with rich genetic diversity and further developed molecular markers. Association analysis between TaWRKY65-3A haplotypes and phenotypic traits was carried out in a natural population consisting of 323 wheat accessions. The haplotype of 189 modern wheat cultivars from different released years were analyzed, aiming to clarify the selection trends of superior haplotypes during wheat breeding in China.

TaWRKY65-3A is 1998 bp in full length, comprising 2 exons and 1 intron, and harbors the characteristic WRKY domain and a C₂H₂-type zinc finger motif typical of the WRKY transcription factor family. The promoter region of TaWRKY65-3A contains a variety of cis-acting elements, including hormone-responsive elements, (e.g., to abscisic acid and methyl jasmonate) and stress-responsive elements (e.g., to drought and low temperature). Moreover, the expressions of TaWRKY65-3A was influenced by ABA, PEG and NaCl stresses. Six SNPs were identified in the TaWRKY65-3A region, forming three haplotypes: Hap-3A-1, Hap-3A-2, and Hap-3A-3. dCAPS markers were developed at positions of 1265 bp (G/C) in the coding sequence and 1658 bp (T/C) in the non-coding sequence, which were significantly associated with thousand-kernel weight, yield per plant, spikelet number per spike, and kernel number per spike across multiple environments. Notably, the haplotype Hap-3A-2 was demonstrated to be significantly correlated with higher spikelet number per spike, a great kernel number per spike, a higher thousand-kernel weight, and a higher yield per plant in multiple environments, including drought, high temperature, and the combined stress of drought and high temperature, and it was positively selected during wheat breeding in China.

TaWRKY65-3A gene responded to phytohormones ABA, PEG and NaCl stress, and were significantly correlated with spikelet number per spike, kernel number per spike, thousand-kernel weight and yield per plant under various environments such as drought, high temperature, and the combined stress of drought and high temperature. Hap-3A-2 is the favorable haplotype with higher spikelet number per spike, more kernel number per spike, higher thousand-kernel weight, and higher yield per plant. The molecular markers of dCAPS developed in this study provide valuable tools for marker-assisted breeding, facilitating the selection of germplasm with superior yield-related traits.