AFP is a negative regulator of ABA signaling that promotes ABI5 protein degradation and weakens regulation of ABA signaling by targeting upstream genes of ABI5, and TaABI5 gene was seed-specific, and accumulated during wheat grain maturation and dormancy acquisition, which played an important role in seed dormancy; TaAFP has a conserved domain with AFP, so TaAFP may also play an important role in seed dormancy in wheat. Two allelic variants of TaAFP were identified on chromosome 2BS in common wheat, and designated as TaAFP-B1a and TaAFP-B1b. Sequence analysis showed a 4-bp deletion in the 5’UTR region of TaAFP-B1b compared with TaAFP-B1a. Based on the 4-bp deletion, a co-dominant functional marker of TaAFP-B was developed and designated as AFPB. The genotype generating a 203-bp fragment (TaAFP-B1b) was more resistant to pre-harvest sprouting than the genotype producing a 207-bp fragment (TaAFP-B1a) in a test of 91 white-grained Chinese wheat cultivars and advanced lines. The average germination index(GI) values of TaAFP-B1a and that of TaAFP-B1b were 45.18 and 30.72%, respectively, indicating a significant difference (P < 0.001). Moreover, the 4-bp deletion located in the 5’UTR not only affected the transcription level of TaAFP-B but also affected the mRNA decay, reduced the translation level of GUS and tdTomatoER and GUS activity in wheat leaves of transient expression. The transcript expression and the mRNA half-life value of TaAFP-B1a in developing seeds and mature seeds were much higher than those of TaAFP-B1b. We identified a 4-bp InDel in the 5’UTR of TaAFP-B, which affected the mRNA transcription level, mRNA decay, translation levels of GUS and tdTomatoER, GUS activity, and was significantly associated with seed dormancy in common wheat. A functional marker was developed and validated based on this InDel.

Background: The low molecular weight glutenin subunits (LMW-GS) of wheat have great effects on food processing quality, but the resolution of LMW-GS and the scoring of their alleles by direct analysis of proteins are difficult due to the larger number of expressed subunits and high similarity of DNA sequences. It is important to identify and classify the LMW-GS genes in order to recognize the LMW-GS alleles clearly and develop the functional markers. Methods: The LMW-GS genes registered in GenBank were searched at NCBI, and 593 Glu-3 genes with complete coding sequences were obtained, including 146 Glu-A3, 136 Glu-B3, and 311 Glu-D3. Sequence analysis and characterization of DNA and deduced amino acids were performed using the software DNAman. Results: The alignment and classification showed that there were at least 9 genes with 69 allelic variants at the Glu-A3 locus, 11 genes with 64 allelic variants at the Glu-B3 locus, and 10 genes with 96 variants at the Glu-D3 locus, respectively. Furthermore, the specificity of some Glu-3 genes and their variations was analyzed. Conclusions: The results were beneficial to understanding the LMW-GS genes fully and to developing the functional markers and will provide a theoretical reference for the quality improvement of wheat variety.

Background: Grain length is a key determinant of yield and quality in barley (Hordeum vulgare L.) and is typically governed by complex quantitative traits. Methods: In this study, a diverse natural population comprising 198 barley accessions was evaluated across two years to investigate the genetic basis of grain length. Results: Phenotypic analysis revealed continuous variation with near-normal distribution, indicating polygenic control. Genome-wide association study (GWAS) identified 84 stable single nucleotide polymorphism (SNP) loci significantly associated with grain length, predominantly enriched on chromosome 7. RNA sequencing (RNA-seq) was conducted using two contrasting genotypes at four developmental stages. Differentially expressed genes (DEGs) were mainly enriched in structural constituent of chromatin, protein heterodimerization activity, and the starch and sucrose metabolism. Integration of GWAS and RNA-seq identified 7 key candidate genes seven key candidate genes, including LOC123412467, LOC123408579, LOC123407599, LOC123410619, LOC123410954, LOC123411868, and LOC123426274. Sequence variation analysis further revealed functional polymorphisms, including non-synonymous mutations. The sequencing results show that LOC123412467 and LOC123410619 exhibited consistent allelic variation between long-grain and short-grain accessions, while LOC123426274 displayed stable differential expression across developmental stages, indicating their potential roles as key genes controlling grain length. Conclusions: Collectively, these findings suggest that chromosome 7 contains major regulatory loci controlling barley grain length and demonstrate that integrative multi-omics analysis is an effective strategy for identifying high-confidence candidate genes associated with complex agronomic traits. This study provides valuable insights into the genetic basis of grain length and offers key candidate genes for barley molecular breeding.

Background: Barley prolamin is a protein affecting end-use quality, malt production, and feeding value. Barley B-hordeins account for 70-80% of total prolamins and have significant effects on barley quality. It is of great importance to study genes encoding B-hordeins. Methods: Eighteen different barley varieties were used to identify new B-hordein genes. Genomic DNA was extracted from barley seedlings using a modified CTAB procedure, and PCRs were performed to amplify B-hordein genes using newly designed primers based on the previously published B-hordein gene sequences (Accessions X03103 and X87232). The B-hordein genes registered in GenBank were also obtained from NCBI. Gene sequences were analyzed using the software DNAman. Phylogenetic analysis was conducted by the method of Maximum Likelihood. Results: A total of 20 complete B-hordein gene sequences were obtained, including 14 putative active genes and 6 pseudogenes. Sequence variations were identified in all 20 genes compared with 72 B-hordein genes from GenBank (including 27 genes we previously identified). The deduced amino-acid sequences of 13 genes had a typical set of 8 conserved cysteine residues, and the other one had 7 cysteines and this gene may have positive effects on barley quality. Sequence analysis and phylogenetic analysis indicated obvious differences between the three kinds of B-hordeins ended with an amino acid V, H or Y. Conclusion: 20 new B-hordein gene sequences were identified, including one with 7 cysteine residues. 92 B-hordein genes were classified into three types (V-type, H-type and Y-type), of which the V-type should be the dominant, original B-hordein genes, while the H-type and the Y-type might be the variants of the V-type. The results provided a reference for further characterizing the B-hordein genes and for breeding quality barley varieties.

Leaf length is an important component of barley plant architecture, but its genetic basis remains incompletely understood. Here, we integrated genome-wide association study (GWAS), RNA sequencing (RNA-seq), and sequence variation analyses to investigate the genetic and transcriptional basis of barley leaf-length variation. A panel of 198 barley accessions was evaluated across two growing seasons, and contrasting long- and short-leaf accessions were subjected to RNA-seq. GWAS identified a major association signal on chromosome 4H, with a core interval of approximately 941.73 kb defined by local linkage disequilibrium analysis. RNA-seq identified 3507 and 3878 differentially expressed genes (DEGs) in flag leaves and second leaves below the flag leaf, respectively, including 2648 shared DEGs. Functional enrichment and gene set enrichment analyses indicated differences in developmental, signaling, metabolic, and temperature-responsive processes. Integration of GWAS and RNA-seq data prioritized three candidate genes, LOC123447426, LOC123449930, and LOC123450878, with different levels of supporting evidence. LOC123447426 and LOC123450878 were located within the core association interval, whereas LOC123449930 was located in the extended candidate-screening region, approximately 2.2 Mb from the lead SNP. LOC123447426 showed the strongest integrated support, with evidence from GWAS and differential expression in both leaf positions. Sequence variation, and qRT-PCR analyses provided further support for these candidates. Overall, this study prioritizes a major chromosome 4H region and three candidate genes associated with barley leaf-length variation, providing a basis for further investigation of their potential roles in leaf development. However, the current evidence does not establish causal relationships between these genes and leaf-length variation, and their biological functions remain to be experimentally verified.