Genome-wide association study (GWAS) and transcriptome analysis reveal new candidate genes for leaf size in alfalfa

Leaves are the most important organs of plants and serve as the primary harvest parts in alfalfa production. The size of the leaf area is a crucial factor influencing forage yield and quality. Therefore, elucidating the genetic factors governing leaf development is critical for breeding new alfalfa varieties with improved yield and quality. In this study, through genome-wide association study (GWAS), we examined SNPs significantly associated with the area (LA), length (LL), width (LW), and aspect ratio (L/W) of mature alfalfa leaves. Two SNPs on chromosome 2 and chromosome 8 were found to be associated with multiple leaf size traits, and 246 genes were located within 20 kb flanking regions. Transcriptome sequencing was utilized to identify 1119 differentially expressed genes in Xinjiang Daye and Nei 1 Nei 2 leaves at various developmental stages. By collectively analyzing the transcriptome and conducting GWAS, nine candidate genes were identified as being related to leaf size regulation, including GRF-interacting factor and some transcription factors. This study provides new candidate genes for leaf size regulation and lays the foundation for breeding efforts to improve forage quality and yield.