The maize (Zea mays ssp. mays) wild ancestor, teosinte, has three times the seed protein content of most modern inbreds and hybrids, but the mechanisms responsible for this trait are unknown. We created a contiguous haplotype DNA sequence of a teosinte, Zea mays ssp. Parviglumis, with Trio-Binning, and map-based cloned a major high-protein QTL, teosinte high protein 9 (Thp9) on chromosome 9. Thp9 encodes an asparagine synthetase 4 that is highly expressed in teosinte, but not in the B73 inbred, where a deletion in the 10 th intron of Thp9-B73 causes incorrect splicing of Thp9-B73 transcripts. Transgenic expression of Thp9-teosinte in B73 significantly increased seed protein content. Introgression of Thp9-teosinte into modern maize inbreds and hybrids greatly enhanced free amino acid accumulation, especially asparagine, throughout the plant, increasing seed protein content without affecting yield. Thp9-teosinte appears to increase nitrogen utilization efficiency, important for promoting a high yield under low nitrogen conditions.

The mechanism that creates vitreous endosperm in the mature maize kernel is poorly understood. We identified Vitreous endosperm 1 (Ven1) as a major QTL influencing this process. Ven1 encodes β-carotene hydroxylase 3, an enzyme that modulates carotenoid composition in the amyloplast envelope. The A619 inbred contains a nonfunctional Ven1 allele, leading to a decrease in polar and an increase in non-polar carotenoids in the amyloplast. Coincidently, the stability of amyloplast membranes is increased during kernel desiccation. The lipid composition in endosperm cells in A619 is altered, giving rise to a persistent amyloplast envelope. These changes impede the gathering of protein bodies and prevent them from interacting with starch grains, creating air spaces that cause an opaque kernel phenotype. Genetic modifiers were identified that alter the effect of Ven1A619, while maintaining a high β-carotene level. These studies provide insight for breeding vitreous kernel varieties and high vitamin A content in maize.