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.

The influence mechanism of varying nitrogen (N) rates on the carbon (C) and N content of soil aggregate, as well as wheat yield were investigated in this study, so as to provide a scientific basis for the rational application of N fertilizer.

The 11-year experiment was conducted in Zhangpan Town, Xuchang City, Henan Province, with four different N levels, including 0 (N0), 180 kg·hm^-2 (N1), 240 kg·hm^-2 (N2), and 300 kg·hm^-2 (N3). The study systematically analyzed changes in soil carbon and nitrogen content, cluster distribution and their carbon and nitrogen content in different soil layers as a result of long-term N application, and investigated the regulatory pathways of long-term N application on wheat yield and its composition.

There was a transformation in the composition of soil aggregates in every soil layer, specifically from larger macroaggregates (>0.25 mm) to microaggregates (0.25-0.053 mm) and silt and clay particles (<0.053 mm), as well as an increase in N rate. Additionally, the application of N resulted in a significant decrease in the mean weight diameter (MWD). As N application rates increase, the C and N content of the soil increased in the 0-20 cm layer, the C and N content of the soil in the 20-40 cm soil layer showed the trend to increase at first and then decrease. Compared with the N0 treatment, N application increased soil organic carbon (SOC) and soil total nitrogen (STN) content by 13.1%-37.2% and 19.4%-29.4% in the 0-20 cm layer and by 15.3%-32.2% and 6.1%-29.3% in the 20-40 cm layer, respectively. The N treatment significantly increased the SOC content of each particle size aggregates compared with N0 treatment, with the SOC content of macroaggregates increasing by 31.6%-62.0%, the SOC content of microaggregates increasing by 8.7%-61.2% and the SOC content of silt and clay increasing by 14.0%-81.7%. As N application rates increased, the STN content of the soil increased in the 0-20 cm layer. With the STN content of macroaggregates increasing by 32.6%-51.0%, the STN content of microaggregates increased by 25.7%-35.9% and the STN content of silt and clay increased by 3.2%-9.7%, the N3 treatment had the highest STN content of all particle size aggregates. In the 20-40 cm soil layer, the STN content of all particle size aggregates tended to increase at first and then decrease. With the STN content of macroaggregates increasing by 17.6%-35.2%, the STN content of microaggregates increased by 11.7%-24.0% and the STN content of silt and clay increased by 1.1%-12.9%, and the N1 treatment had the highest STN content of all particle size aggregates. The study results indicated that long-term nitrogen application had a significant impact on the spike number and grain number per spike in wheat, resulting in increased yield. Compared with the N0 treatment, the application of N1, N2, and N3 treatments resulted in a significant increase in wheat yield, with improvements of 188.1%, 177.3%, and 173.2%, respectively. The correlation and structural equation modelling analyses revealed a significant and positive correlation between wheat yield and soil carbon and nitrogen content, as well as carbon and nitrogen content in aggregates. Additionally, the long-term application of nitrogen was found to influence wheat yield formation by affecting carbon and nitrogen content in microaggregates.

In summary, the application of nitrogen over a long period of time raised the content of carbon and nitrogen in both soil and aggregates, enhanced soil fertility, ultimately promoting wheat yield. The optimal nitrogen application rate was 180 kg·hm^-2 under the condition of this experiment.