Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Marker-assisted pyramiding of opaque2 and novel opaque16 genes for further enrichment of lysine and tryptophan in sub-tropical maize Konsam Sarika a,1 , Firoz Hossain a, ⁎ , Vignesh Muthusamy a , Rajkumar U. Zunjare a , Aanchal Baveja a , Rajat Goswami a , Jayant S. Bhat b , Supradip Saha c , Hari S. Gupta a a Maize Genetics Unit, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India b Regional Research Centre, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India c Division of Agricultural Chemicals, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India ARTICLE INFO Keywords: QPM opaque2 opaque16 Lysine Tryptophan MABB ABSTRACT 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 tryp- tophan 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. 1. Introduction Malnutrition caused by inadequate consumption of balanced diet is pervasive in both under-developed and developing world afflicting an estimated two billion people [1]. An estimated 45% of deaths of chil- dren under age five are linked to malnutrition [2]. Malnutrition con- tributes to global burden of disease, and loss in annual gross domestic product (GDP) in Asia and Africa to an extent of 11%. Maize is a pre- ferred staple cereal food crop, and together with rice and wheat, it provides 30% of food-calorie intake of more than 4.5 billion people in as many as 94 developing countries [3]. Apart from being a vital source of calories and proteins to humans, a major portion of maize is used as animal feed, besides serving as source to many industrial products [4,5]. The demand for cereals will continue to increase and the demand for maize will be doubled in developing world by the end of 2050 [6]. India too would require a double in production of maize to meet the domestic demand in next decade [7]. However, maize endosperm protein is poor in nutritional value due to reduced availability of essential amino acids such as lysine and tryptophan. These amino acids are therefore supplemented in diet for proper growth and development in humans, and monogastric animals such as pigs and poultry birds [8]. The best sources of quality protein are meat, poultry, fish and dairy products, which are neither affordable nor can it be made available to poorer section of the population on a regular basis. This warrants genetic amelioration to enhance the con- tent of these essential amino acids. Development of biofortified crop varieties with enhanced nutritional value through conventional breeding approaches provides a sustainable and cost-effective solution https://doi.org/10.1016/j.plantsci.2018.04.014 Received 30 January 2018; Received in revised form 7 April 2018; Accepted 15 April 2018 ⁎ Corresponding author. 1 Present address: ICAR Research Complex for Northeast Hill Region, Umiam, Meghalaya, 793103, India. E-mail address: fh_gpb@yahoo.com (F. Hossain). Abbreviations: QPM, quality protein maize; o2, opaque2; o16, opaque16; MABB, marker-assisted backcross breeding; MAS, marker-assisted selection; RPG, recurrent parent genome Plant Science 272 (2018) 142–152 Available online 18 April 2018 0168-9452/ © 2018 Elsevier B.V. All rights reserved. T