The TIFY family, a plant-specific group of transcription factors, plays critical roles in regulating growth, development, and stress responses. This study aimed to clone TaTIFY11c-4A in wheat, validate its genetic effects, and provide a theoretical basis for high-yield molecular breeding of wheat.

The wheat cultivar Hanxuan 10 was used to clone TaTIFY11c-4A and allelic variations were detected in germplasms. The tissue-specific expression patterns of TaTIFY11c-4A and its responses to various hormones and stresses were analyzed via quantitative real-time PCR (qRT-PCR). The subcellular localization of TaTIFY11c-4A was determined through transient expression in tobacco. A molecular marker targeting the polymorphic site in TaTIFY11c-4A was developed to assess the genotypes in the natural population, and association analysis was performed to evaluate the correlations between the genotypes and phenotypes. Additionally, the spatial and temporal distribution of different genotypes were analyzed. Synergistic effects of TaTIFY11c-4A and TaSRL1-4A haplotypes were explored to identify superior genotype.

TaTIFY11c-4A was successfully cloned, comprising three exons and two introns, encoding a 198-amino acid protein with conserved TIFY and Jas domains. TaTIFY11c-4A is expressed in roots, root bases and leaves at the seedling stage, and highly expressed in roots and leaves at the booting stage. There are multiple cis-acting elements related to hormone responses, stress adaptation, and endosperm development in the promoter of TaTIFY11c-4A. Its expression responds to plant hormones (ABA, IAA, MeJA) and abiotic stresses (drought, high salinity, low and high temperature). A SNP (G/A) was identified in its promoter at -405 bp. A molecular marker was developed based on the SNP and association analysis revealed significant correlations between TaTIFY11c-4A alleles and plant height, thousand grain weight under multiple environments such as drought and high temperature, and root depth at tillering stage. Compared with genotype SNP-G, wheat germplasms carrying the SNP-A allele exhibited shorter plants, higher thousand grain weight, and shallower roots at tillering stage, and have been positively selected in the wheat breeding process. TaTIFY11c-4A-SNP-A and TaSRL1-4A-SNP-C genotypes synergistically reduced plant height and enhanced thousand grain weight.

TaTIFY11c-4A encodes a nuclear-localized JAZ protein. It is expressed in various tissues of wheat and involved in responses to ABA, IAA, MeJA, as well as abiotic stresses such as drought, extreme temperature, and high salinity. The TaTIFY11c-4A-SNP is associated with plant height and thousand grain weight under multiple environments, and root depth. SNP-A allele has been positively selected in the wheat breeding process. The superior genotypes and combinations of TaTIFY11c-4A and TaSRL1-4A provide genetic resources for breeding high-yield and stress-resistant wheat cultivars.

Enolase is a key rate-limiting enzyme in the process of glycolysis, and plays a crucial role in plant growth and development, and response to abiotic stress. The function of common wheat enolase gene TaENO1-5B was revealed and molecular markers were developed to provide genetic resources for improving wheat through molecular breeding.

TaENO1-5B was cloned from wheat variety Hanxuan 10. The domains of its encoded protein were analyzed on the SMART website. The secondary and tertiary structures of the protein were predicted by Phyre2 software. Real-time quantitative fluorescent PCR was used to analyze the expression levels of the target gene in wheat tissues at different developmental stages and its expression patterns under phytohormone treatment and abiotic stress. Thirty wheat germplasm with rich genetic diversity were used as plant materials to analyze the gene sequence polymorphisms, and develop molecular markers. The association analysis between TaENO1-5B haplotypes and phenotypic traits was carried out in a natural population consisting of 323 wheat accessions. The trend of breeding selection of superior haplotype in different wheat production zones in China was analyzed by using a landrace population and a modern variety population.

TaENO1-5B gene consists of 17 exons and 16 introns encoding 446 amino acids and contains a conserved N-terminal domain and a C-terminal TIM (triose-phosphate isomerase) barrel domain. TaENO1-5B was expressed in all tissues of wheat at seedling, jointing, heading and flowering stages, and the expression level was higher in roots, root bases and spikes. The TaENO1-5B promoter region contains a variety of cis-acting elements, including elements responding to plant hormones, such as abscisic acid (ABA), auxin (IAA), and methyl jasmonate (MeJA), as well as elements responding to drought and low temperature. The expression of TaENO1-5B was significantly induced by phytohormones and abiotic stress in wheat. Four SNPs were detected in the promoter region and three SNPs in the gene region of the TaENO1-5B gene, which constituted three haplotypes, i.e., Hap-5B-1, Hap-5B-2, and Hap-5B-3. Among them, Hap-5B-2 was a favorable haplotype highly associated with shorter plant height, more spikelets per spike, and more grains per spike under various environments such as drought and high temperature, and had been positively selected in the breeding history of major wheat production zones in China. The KASP (kompetitive allele-specific PCR) marker developed based on the SNP (2 399 bp, G/A) of the TaENO1-5B promoter region was significantly correlated with the spikelet number per spike in multiple environments.

TaENO1-5B gene responds to phytohormones and abiotic stress, and is significantly correlated with plant height, spikelet number per spike and grain number per spike under various environments such as drought and high temperature. Hap-5B-2 is a favorable haplotype with shorter plant height and more number of spikelets and grains per spike. Molecular markers developed based on the variation sites of TaENO1-5B gene sequence can be used for genetic improvement of plant height and related yield traits in wheat.