Combining QTL mapping and RNA-Seq reveals candidate genes controlling flag leaf width in foxtail millet

The flag leaf, a crucial component of plant architecture, significantly influences final grain yield in crops, including foxtail millet (Setaria italica L.). Optimizing flag leaf size is considered an effective strategy for enhancing grain yield potential under higher planting densities. However, the genetic mechanism underlying flag leaf size, particularly flag leaf width (FLW), remains largely unknown under varying planting densities in foxtail millet.

An FLW phenotype variation analysis was conducted across multiple planting densities using a recombinant inbred line (RIL) population derived from Heizhigu (narrow leaf) and Changnong 35 (wide leaf). Based on a high-density genetic map with 3795 Bin markers, 11 flag leaf width (FLW) QTLs were identified on chromosomes 3, 5, and 6, explaining 2.35%-36.06%. Among these, qFLW5-2 was a major QTL, detected consistently across 3 environments and explaining a large proportion of FLW variation. The QTL was further validated with 9 InDel markers with its candidate region across different planting densities. Moreover, RNA-seq revealed 2,293 and 2,338 differentially expressed genes (DEGs) between biparents at heading stage and grain filling stage, respectively. There were 11 and 9 DEGs within the location range of qFLW5-2 among 2 comparison groups (HZG-H_vs_CN35-H and HZG-G_vs_CN35-G). Combining QTL mapping and RNA-seq, we speculated that Seita.5g134600 (encoding an auxin responsive protein Aux/IAA) and Seita.5G123900 (encoding a cytochrome P450 family protein) as key candidate genes for qFLW5-2. Furthermore, variation analysis confirmed that the lines or germplasm with Seita.5G134600^5UTR277+ allele, both within the RIL population and natural populations, exhibited significantly wider leaves than those with Seita.5G134600^5UTR277- allele. These findings advance our understanding of the genetic and molecular regulatory mechanisms governing flag leaf growth.

This study elucidates genetic and molecular mechanism regulating flag leaf growth and development in foxtail millet. The results provide a theoretical foundation for improving plant architecture and facilitating molecular marker-assisted breeding in this crop.