Fine mapping qKRN5.04 provides a functional gene negatively regulating maize kernel row number.

KEY MESSAGE: Zm00001d016075 was identified by fine mapping qKRN5.04. The function of Zm00001d016075, negatively modulating maize (Zea Mays L.) kernel row number (KRN), was verified by CRISPR-Cas9. InDel308 located in the promoter of Zm00001d016075 has potential for use as a molecular marker to identify KRN in maize breeding. Kernel row number (KRN), controlled by multiple quantitative trait loci (QTLs), is one of the most important traits that relate to maize production and domestication. Here, fine mapping was conducted to study a major QTL, qKRN5.04, to a 65-kb genomic region using a progeny test strategy in an advanced backcross population, in which Nong531 (N531) and the inbred line of Silunuo (SLN) were employed as the recurrent and the donor parent, respectively. Within this region, there was only one gene (Zm00001d016075) based on the B73 reference genome. Furthermore, we performed regional association mapping using a panel of 236 diverse inbred lines and observed that all significant SNPs were located within Zm00001d016075. The expression of Zm00001d016075 was significantly higher in N531 and qKRN5.04N531 than qKRN5.04SLN, resulting from the different promoter activity of Zm00001d016075. Sequence analysis revealed that InDel308, located in the promoter of Zm00001d016075, was related to the KRN variation in different maize inbred lines. Using the CRISPR-Cas9 strategy, we determined Zm00001d016075 played a role in negatively regulating KRN and had a moderate effect on 10-kernel width, 100-kernel weight, kernels per ear, and grain yield per ear. These results provide critical insights on the genetic basis and quantitative variation for KRN.