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Maize protein lacks essential amino acids such as methionine, lysine, and tryptophan. Limited information exists on the composition and variation of kernel methionine. This study assessed methionine variability and its relationship with lysine and tryptophan in 48 maize inbreds at three locations. Analysis of variance revealed significant variations for kernel methionine (0.031%–0.305%), lysine (0.118%–0.389%), and tryptophan (0.014%–0.090%). CML591 (0.305%), CML596 (0.286%), and CML421 (0.284%) showed the highest methionine content. The average methionine contents were comparable in quality protein maize (0.156%) and normal (0.150%) lines. Methionine showed no correlation with lysine (r= 0.001) and tryptophan (r= 0.032), while lysine and tryptophan showed a strong positive correlation (r= 0.740). CML165 (methionine: 0.272%, lysine: 0.320%, and tryptophan: 0.090%), CML186 (methionine: 0.254%, lysine: 0.389%, and tryptophan: 0.083%), and HKI163 (methionine: 0.252%, lysine: 0.331%, and tryptophan: 0.084%) were promising genotypes considering all three amino acids. Multilocation experiment revealed genetic variation in methionine, lysine, and tryptophan among the 48 inbreds. No negative correlation between the amino acids indicates the possibility of developing cultivars high in methionine, lysine, and tryptophan. Identified nutrient‐rich inbreds will serve as valuable germplasm in maize biofortification programs. This is the first report on methionine variability and its relationship with lysine and tryptophan in subtropical maize.

Maize kernel protein is deficient in sulfur-containing essential amino acid such as methionine. The dzs18gene encodes methionine-rich 18-kDa δ-zein in maize kernels. In this study, we sequenced full-length of dzs18gene (820 bp) among 10 maize inbreds, revealing 43 SNPs and 22 InDels(average length-7.58 bp). Three InDels (4 bp at 113th, 15 bp at 463rd and 3 bp at 615th position) distinguished the wild-type (functional) from the mutant (non-functional) allele of dzs18. The 4 bp (TTAT) insertion caused a frameshift mutation, resulting in truncated DZS18 protein. The 15 bp insertion (ATG–TCT–TCG–ATG–ATA) added methionine–serine–serine–methionine–isoleucine, while the 3 bp deletion (CAA) led to loss of a glutamine residue in the mutant allele. Three gene-based PCR markers were developed for diversity analysis of dzs18gene among 48 inbreds, which had an average methionine content of 0.136 %. (range: 0.031–0.340 %). Eight haplotypes were identified with methionine content varying from 0.066 % (Hap7) to 0.262 % (Hap3). Haplotypes with 4 bp deletion accumulated more methionine (0.174 %) than haplotypes with 4 bp insertion (0.082 %). The average methionine in 15 bp deletion and insertion haplotypes was 0.106 % and 0.150 %, respectively. The 3 bp insertion had 0.140 % methionine, while the deletion possessed 0.117 % methionine. Protein–protein association analysis predicted that DZS18 protein interacts with 19-kDa α-zein, 27- and 16-kDa γ-zeins, WAXY and O2 protein. A paralogue of dzs18gene with 74 % sequence identity was identified. The functional markers reported here could facilitate the development of high methionine maize cultivars, which holds great significance to combat malnutrition, especially in developing countries.