Genome-wide association and co-expression uncovered ZmMYB71 controls kernel starch content in maize

Starch plays a crucial role as a storage component, greatly affecting the grain yield and quality of maize (Zea mays L.). To meet future demands, it is essential to understand the genetic basis of the natural variation in kernel starch content (SC) for maize breeding. Through a genome-wide association study (GWAS), we identified 84 and 96 loci associated with kernel SC within two years that overlapped with 185 candidate genes. The candidate gene ZmMYB71, which encodes a myeloblastosis (MYB)-related transcription factor, exhibited the highest co-expression frequency with starch synthesis genes. We demonstrated that ZmMYB71 functions as a nuclear located transcription repressor, and the kernel SC of mutants increased by over 2.32%, with a minor effect on amylose content or 100-grain weight. Sh1, Sh2, and GBSSI showed up-regulation in mutants by 1.56-, 1.45- and 1.32-fold, respectively, consistently with the RNA sequencing result; their promoter activities may be directly repressed by ZmMYB71 through the GATATC and TTAGGG motifs. Furthermore, the ZmMYB71 elite haplotype Hap1 was present in over 55% of the high-starch maize sub-populations BSSS and PB but only in 7.14% of the low-starch sub-population PA. Comparing the Hap1 haplotype frequencies in different breeding stages found that its frequency in the inbred group released after 2010 is significantly higher at 40.28%, in contrast to 28.57% and 27.94% in the years 1980 and 1990, and 2000, respectively. The finding provides valuable information on the natural variation in SC of the maize kernel and suggests that ZmMYB71 serves as a negative regulator with the potential to be used to improve SC in the kernels.