Genetic basis of maize kernel protein content revealed by high-density bin mapping using recombinant inbred lines

Maize with a high kernel protein content (PC) is desirable for human food and livestock fodder. However, improvements in its PC have been hampered by a lack of desirable molecular markers. To identify quantitative trait loci (QTL) and candidate genes for kernel PC, we employed a genotyping-by-sequencing strategy to construct a high-resolution linkage map with 6,433 bin markers for 275 recombinant inbred lines (RILs) derived from a high-PC female Ji846 and low-PC male Ye3189. The total genetic distance covered by the linkage map was 2180.93 cM, and the average distance between adjacent markers was 0.32 cM, with a physical distance of approximately 0.37 Mb. Using this linkage map, 11 QTLs affecting kernel PC were identified, including qPC7 and qPC2-2, which were identified in at least two environments. For the qPC2-2 locus, a marker named IndelPC2-2 was developed with closely linked polymorphisms in both parents, and when tested in 30 high and 30 low PC inbred lines, it showed significant differences (P = 1.9E-03). To identify the candidate genes for this locus, transcriptome sequencing data and PC best linear unbiased estimates (BLUE) for 348 inbred lines were combined, and the expression levels of the four genes were correlated with PC. Among the four genes, Zm00001d002625, which encodes an S-adenosyl-L-methionine-dependent methyltransferase superfamily protein, showed significantly different expression levels between two RIL parents in the endosperm and is speculated to be a potential candidate gene for qPC2-2. This study will contribute to further research on the mechanisms underlying the regulation of maize PC, while also providing a genetic basis for marker-assisted selection in the future.