Molecular analysis of opaque2 gene governing accumulation of lysine and tryptophan in maize endosperm

The recessive opaque2 (o2) gene enhances lysine and tryptophan by ~ twofold in maize kernels. Despite > 6 decades of research, no functional marker for o2 has been reported yet. Here, we sequenced 2848 bp of entire o2 gene in 10 diverse inbreds, and identified 7 exonic SNPs and 3 exonic InDels that distinguished the normal and quality protein maize (QPM) inbreds. Of these, three potential SNPs (SNP601_T/C, SNP2359_C/T and SNP2360_G/T) and three InDels (InDel_1, InDel_8 and InDel_9) were responsible for modification of amino acids. Clustering analysis grouped 24 QPM and 24 normal inbreds into two clusters, and led to the identification of 24 haplotypes of o2. Insertion of alanine was found to regulate the binding of o2 protein with promoter of zein genes. QPM inbreds possessed significantly higher lysine (0.271–0.406%) and tryptophan (0.069–0.101%) compared to normal (lysine: 0.124–0.213%; tryptophan: 0.029–0.047%). A functional marker (MGU-InDel10-o2) was developed, and it clearly differentiated the QPM and normal inbreds. MGU-InDel10-o2 successfully identified desirable plants in BC₁F₁, BC₂F₁ and BC₂F₂ populations. BC₂F₃ progenies possessed higher lysine (0.379%) and tryptophan (0.084%) over original inbreds (lysine: 0.189% and tryptophan: 0.039%). Among haplotypes, Hap-3 showed the highest average lysine (0.380%) and tryptophan (0.094%), while Hap-14 and Hap-16 had the lowest lysine (0.124%) and tryptophan (0.029%), respectively. This is the first report on understanding allelic variation, haplotypes and functional marker development for o2 gene in maize.