Thinopyrum intermedium (JJJ^sJ^sStSt, 2n = 6x = 42), a wild relative of common wheat, possesses many desirable agronomic genes for wheat improvement. The production of wheat–Thinopyrum intermedium introgression lines is a key step for transferring these beneficial genes into wheat. In this study, we characterized three wheat–Thinopyrum intermedium introgression lines TA3681, TA5566, and TA5567 using non-denaturing fluorescence in situ hybridization, genomic in situ hybridization, PCR-based landmark unique gene, and intron targeting markers. Our results showed that TA3681 is a wheat–Thinopyrum intermedium 1St disomic addition line, TA5566 is a wheat–Thinopyrum intermedium non-Robertsonian translocation line carrying two pairs of 3A-7J^s translocation chromosomes, and that TA5567 is a wheat–Thinopyrum intermedium non-Robertsonian translocation line carrying a pair of 3A-7J^s translocation chromosomes. We developed 13, 36, and 15 Thinopyrum intermedium chromosome-specific markers for detecting the introgressed Thinopyrum chromosomes in TA3681, TA5566, and TA5567, respectively. Stem rust assessment revealed that TA3681 exhibited a high level of seedling resistance to Chinese-prevalent Puccinia graminis f. sp. tritici pathotypes, and both TA5566 and TA5567 were highly resistant to Australian P. graminis f. sp. tritici pathotypes, indicating that Thinopyrum intermedium chromosomes 1St and 7J^s might carry new stem rust resistance genes. Therefore, the new identified introgression lines may be useful for improving wheat stem rust resistance.

Amino acid permeases (AAPs) are proteins of the integral membrane that play important roles in plant growth, development, and responses to various stresses. The molecular functions of several AAPs were characterized in Arabidopsis and rice, but there is still limited information on wheat. Here, we identified 51 AAP genes (TaAAPs) in the wheat genome, classified into six groups based on phylogenetic and protein structures. The chromosome location and gene duplication analysis showed that gene duplication events played a crucial role in the expansion of the TaAAPs gene family. Collinearity relationship analysis revealed several orthologous AAPs between wheat and other species. Moreover, cis-element analysis of promoter regions and transcriptome data suggested that the TaAAPs can respond to salt stress. A TaAAP1 gene was selected and transformed in wheat. Overexpressing TaAAP1 enhanced salt tolerance by increasing the expression of ethylene synthesis genes (TaACS6/TaACS7/TaACS8) and accumulating more ethylene. The present study provides an overview of the AAP family in the wheat genome as well as information on systematics, phylogenetics, and gene duplication, and shows that overexpressing TaAAP1 enhances salt tolerance by regulating ethylene production. These results serve as a theoretical foundation for further functional studies on TaAAPs in the future.

Freezing stress is a major factor limiting production and geographical distribution of temperate crops. Elongator is a six subunit complex with histone acetyl-transferase activity and is involved in plant development and defense responses in Arabidopsis thaliana. However, it is unknown whether and how an elongator responds to freezing stress in plants. In this study, we found that wheat elongator subunit 4 (TaELP4) negatively regulates freezing tolerance through ethylene signaling. TaELP4 promoter contained cold response elements and was up-regulated in freezing stress. Subcellular localization showed that TaELP4 and AtELP4 localized in the cytoplasm and nucleus. Silencing of TaELP4 in wheat with BSMV-mediated VIGS approach significantly elevated tiller survival rate compared to control under freezing stress, but ectopic expression of TaELP4 in Arabidopsis increased leaf damage and survival rate compared with Col-0. Further results showed that TaELP4 positively regulated ACS2 and ACS6 transcripts, two main limiting enzymes in ethylene biosynthesis. The determination of ethylene content showed that TaELP4 overexpression resulted in more ethylene accumulated than Col-0 under freezing stress. Epigenetic research showed that histone H3K9/14ac levels significantly increased in coding/promoter regions of AtACS2 and AtACS6 in Arabidopsis. RT-qPCR assays showed that the EIN2/EIN3/EIL1-CBFs-COR pathway was regulated by TaELP4 under freezing stress. Taken together, our results suggest that TaELP4 negatively regulated plant responses to freezing stress via heightening histone acetylation levels of ACS2 and ACS6 and increasing their transcription and ethylene accumulation.

Wheat pathogens, especially those causing powdery mildew and stripe rust, seriously threaten yield worldwide. Utilizing newly identified disease resistance genes from wheat relatives is an effective strategy to minimize disease damage. In this study, chromosome-specific molecular markers for the 3S^b and 7S^b chromosomes of Aegilops bicornis were developed using PCR-based landmark unique gene primers for screening wheat–A. bicornis progenies. Fluorescence in situ hybridization (FISH) was performed to further identify wheat–A. bicornis progenies using oligonucleotides probes Oligo-pSc119.2-1, Oligo-pTa535-1, and Oligo-(GAA)₈. After establishing A. bicornis 3S^b and 7S^b chromosome-specific FISH markers, Holdfast (common wheat)–A. bicornis 3S^b addition, 7S^b addition, 3S^b(3A) substitution, 3S^b(3B) substitution, 3S^b(3D) substitution, 7S^b(7A) substitution, and 7S^b(7B) substitution lines were identified by the molecular and cytological markers. Stripe rust and powdery mildew resistance, along with agronomic traits, were investigated to evaluate the breeding potential of these lines. Holdfast and Holdfast–A. bicornis progenies were all highly resistant to stripe rust, indicating that the stripe rust resistance might derive from Holdfast. However, Holdfast–A. bicornis 3S^b addition, 3S^b(3A) substitution, 3S^b(3B) substitution, and 3S^b(3D) substitution lines showed high resistance to powdery mildew while Holdfast was highly susceptible, indicating that chromosome 3S^b of A. bicornis carries previously unknown powdery mildew resistance gene(s). Additionally, the transfer of the 3S^b chromosome from A. bicornis to wheat significantly increased tiller number, but chromosome 7S^b has a negative effect on agronomic traits. Therefore, wheat germplasm containing A. bicornis chromosome 3S^b has potential to contribute to improving powdery mildew resistance and tiller number during wheat breeding.

Male‐sterile genes and mutants are critical for hybrid seed production in monocotyledonous crops. Lipids are essential structural components of male reproductive organs, such as the anther and pollen. Here, we show that the pollen‐preferential gene TaRIP2 is essential for wheat anther development and pollen formation. RT‐qPCR analysis revealed TaRIP2 is specifically expressed during the callose and tetrad stages. Using CRISPR/Cas9, we generated TaRIP2 mutants ( rip2 ), which displayed smaller, wilted anthers with defective cuticles and a low proportion of viable pollen grains (~5.7%). Microscopy revealed that the mutant Rip2 microspores had a smaller size, a smooth exine lacking sculptural elements and fewer organelles. RNA‐seq identified differentially expressed genes (DEGs) enriched in pathways related to pollen wall formation. KEGG analysis showed these DEGs are involved in cutin, suberine and wax biosynthesis and fatty acid degradation. Fatty acids C16:0, C18:0 and C18:2 were significantly elevated in rip2 anthers. These phenotypes coincided with the downregulation of genes involved in lipid metabolism and anther development. Dual‐luciferase and EMSA assays confirmed TaRIP2 is directly regulated by the transcription factor MYB80. Together, our results show TaRIP2 regulates pollen wall formation through the MYB80‐control lipid metabolic pathways.

Aegilops sharonensis, a wild relative of wheat, harbors diverse disease and insect resistance genes, making it a potentially excellent gene source for wheat improvement. In this study, we characterized and evaluated six wheat-A. sharonensis derivatives, which included three disomic additions, one disomic substitution + monotelosomic addition and two disomic substitution + disomic additions. A total of 51 PLUG markers were developed and used to allocate the A. sharonensis chromosomes in each of the six derivatives to Triticeae homoeologous groups. A set of cytogenetic markers specific for A. sharonensis chromosomes was established based on FISH using oligonucleotides as probes. Molecular cytogenetic marker analysis confirmed that these lines were a CS-A. sharonensis 2S^sh disomic addition, a 4S^sh disomic addition, a 4S^sh (4D) substitution + 5S^shL monotelosomic addition, a 6S^sh disomic addition, a 4S^sh (4D) substitution + 6S^sh disomic addition and a 4S^sh (4D) substitution + 7S^sh disomic addition line, respectively. Disease resistance investigations showed that chromosome 7S^sh of A. sharonensis might harbor a new powdery mildew resistance gene, and therefore it has potential for use as resistance source for wheat breeding.