Marker-assisted pyramiding of opaque2 and novel opaque16 genes for further enrichment of lysine and tryptophan in sub-tropical maize

•In the present manuscript, a novel opaque16 gene has been pyramided with opaque2 allele to further enhance the protein quality.•This is the first ever report on accelerated pyramiding of opaque2 and opaque16 in sub-tropical genetic background.•The introgressed inbreds and their hybrids possess 50–60% more lysine and tryptophan over their respective QPM parental inbreds and hybrids.•Since opaque16 does not enhance the opaqueness, combining opaque2 and opaque16 assumes great significance in QPM breeding programme.•The improved hybrids possess great potential to address protein energy malnutrition more effectively than opaque2 based QPM hybrids. The improvement of protein quality in maize so far has been based on recessive opaque2 (o2) mutant that along with endosperm-modifiers led to development of quality protein maize (QPM). Recent discovery of nutritional benefits of recessive opaque16 (o16) mutant was of immense significance for further improvement of protein quality. In the present study, o16 was introgressed into o2-based parental inbreds (HKI161, HKI193-1, HKI193-2 and HKI163) of four commercial QPM hybrids (HQPM-1, HQPM-4, HQPM-5 and HQPM-7) released in India, using marker-assisted backcross breeding. Background selection led to high recovery of recurrent parent genome (RPG) to maximum of 95%, and introgressed progenies showed considerable phenotypic resemblance for plant-, ear- and grain- characteristics to their respective recurrent parents. Selection of markers for o2 and o16 led to development of pyramided lines (o2o2/o16o16) that possessed as high as 76% and 91% more lysine and tryptophan over the recurrent parents, respectively. Reconstituted hybrids showed an average enhancement of 49% and 60% in lysine and tryptophan over the original hybrids, with highest enhancement amounting 64% and 86%, respectively. This is first report of enhancement of both lysine and tryptophan by o16 in maize genotypes adaptable to sub-tropics. Moderate variation in lysine and tryptophan was also observed in pyramided lines. Multi-location evaluation of reconstituted hybrids revealed similar grain yield and attributing traits to their original versions. This study signified the role of o16 as supplementary to o2 for nutritional quality enhancement in maize, and improved elite inbreds and hybrids developed here hold great significance in maize biofortification programme.