Rice spotted-leaf mutants are ideal materials to study the molecular mechanism underlying programmed cell death and disease resistance in plants. LOC_Os07g04820 has previously been identified as the candidate gene responsible for the spotted-leaf phenotype in rice Spotted-leaf 26 (Spl26) mutant. Here, we cloned and validated that LOC_Os07g04820 is the locus controlling the spotted-leaf phenotype of Spl26 by reverse functional complementation and CRISPR/Cas9-mediated knockout of the mutant allele. The recessive wild-type spl26 allele (Oryza sativa spotted-leaf 26, Osspl26) is highly conservative in grass species and encodes a putative G-type lectin S-receptor-like serine/threonine protein kinase with 444 amino acid residuals. OsSPL26 localizes to the plasma membrane and can be detected constitutively in roots, stems, leaves, sheaths and panicles. The single base substitution from T to A at position 293 leads to phenylalanine/tyrosine replacement at position 98 in the encoded protein in the mutant and induces excessive accumulation of H₂O₂, leading to oxidative damage to cells, and finally, formation of the spotted-leaf phenotype in Spl26. The formation of lesions not only affects the growth and development of the plants but also activates the defense response and enhances the resistance to the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae. Our results indicate that the gain-of-function by the mutant allele OsSpl26 positively regulates cell death and immunity in rice.

Rice spotted-leaf mutants serve as valuable resources for studying plant programmed cell death (PCD) and disease resistance mechanisms, making them crucial for research on disease resistance in rice. Map-based cloning was used to identify and clone the spotted-leaf gene OsSPL42. Then, functional complementation and CRISPR/Cas9 techniques were also employed to further validate the function of this gene. By applying leaf clippings for bacterial blight (BB) inoculation, the BB resistance of different rice lines was assessed. The results in this study were as follows: The OsSPL42 behaved as a recessive nuclear gene and was narrowed down to a 111 kb region on chromosome 8. All T₀ transgenic rice plants in the complementation experiments exhibited a wild-type phenotype, without any lesion spots on the rice leaves. This suggests that the LOC_Os08g06100 encoding O-methyltransferase is the candidate gene for the mutant spl42. The OsSpl42 is widely expressed and the OsSPL42-GFP protein is mainly localized in the cytoplasm. OsSPL42 overexpression lines are more susceptible to BBs, which indicates that OsSPL42 may act as a negative regulator of rice resistance to BB. In summary, we speculate that OsSPL42 plays an important role in the regulation of pathogen response, providing new insights into plant defense mechanisms.

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.

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.