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Introgression of Opaque-2 Gene Into the Genetic Background of Popcorn Using Marker Assisted Selection P

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Introgression of Opaque-2 Gene Into the Genetic Background of Popcorn Using Marker Assisted Selection P International Research Journal of Biotechnology Vol. 5(1) pp 10-18, January, 2019 DOI: http:/dx.doi.org/https://www.interesjournals.org/biotechnology.html Available online http://www.interesjournals.org/IRJOB Copyright ©2018 International Research Journals Research Article Introgression of Opaque-2 Gene into the Genetic Background of Popcorn Using Marker Assisted Selection P. M. Adunola1*, B. O. Akinyele1, A. C. Odiyi1, L. S. Fayeun1 and M. G. Akinwale2 1Department of Crop, Soil and Pest Management, Federal University of Technology, P.M.B 704, Akure, Ondo State, Nigeria 2Chitedze Research Station, International Institute of Tropical Agriculture, Malawi ABSTRACT Biofortification of a natural snack like popcorn is important for improved nutrition and health of children and adults. This study was aimed at introgressing opaque-2 gene, a high lysine/tryptophan gene into the genetic background of popcorn. Field experiments were carried out at the Teaching and Research Farm (TRF) of the Federal University of Technology, Akure (FUTA) during the dry and wet seasons of 2016 while biochemical and molecular analyses were carried out in the Biochemistry Laboratory, FUTA and Bioscience Laboratory, International Institute of Tropical Agriculture (IITA) respectively. Nine parental genotypes were used which comprised three popcorn maize varieties and six Quality Protein Maize (QPM) varieties. F1 hybrids were developed by crossing the parental genotypes using North Carolina mating design I. The hybrids were then advanced to F2 by self-pollination. Segregated F2 kernels from each genotype were screened on the light box. Kernel modifications 2 (25% opaque) and 3 (50% opaque) were selected. Selected kernels were analysed biochemically for protein and tryptophan contents. Data obtained were subjected to Analysis of Variance (ANOVA) using Minitab software version 17. Significant means were separated using Tukeys HSD (P ≤ 0.05). The allelic state of opaque-2 gene in the selected F2s was determined using SSR marker phi 112. Protein and tryptophan contents and quality index for unpopped and popped F2 of crosses of QPM and popcorn were significantly (P ≤ 0.05) higher than popcorn checks. Unpopped protein content, unpopped tryptophan content and popped tryptophan content were highly correlated with one another. Phi 112 SSR marker showed dominant pattern of polymorphism, amplifying DNA fragments between 154bp and 160bp for the F2. Keywords: Introgression, Opaque-2, QPM, Popcorn, Biochemical properties. INTRODUCTION hard corneous endosperm with only a small portion of Maize (Zea mays L.) is one of the most important food soft starch and is characterized by its popping ability; security crops in developing countries (Avinashe et al., the kernel pops upon heating as a result of the unique 2012). More than 300 million Africans depend on quality of the endosperm (Acquaah, 2006). Popcorn is maize as their main food source (M’mboyi et al., 2010). a soft and tasty aliment that is appreciated in many In Sub–Sahara Africa (SSA), maize is used directly for countries (Rodovalho et al., 2008). The cultivation, human food with more than 100kg consumption per consumption and utilization of popcorn are becoming capita (Adeyemo et al., 2011). Maize can be classified popular especially in developing countries like Nigeria into specialty types based on kernel morphology, (Adunola et al., 2017). The advent of cinema, playing texture, usage, functionality and other characteristics, ground, arcade, modern schools and corn popping which include waxy maize, high protein maize, high oil technology (microwave) have been a major driver for maize, flour maize, sweet-corn and popcorn (Johnson, the increased consumption of popcorn in Nigeria 2000). Popcorn (Zea mays var. everta) is an extreme (Adunola et al., 2018). form of flint corn (Z. mays var. indurata) that has a very Adunola et al 11 Malnutrition is a serious health concern that has been endosperm) maize. Maize homozygous for the o2 linked to deficiency of diet in protein quantity, protein mutant has a quality value equivalent to 90% that of quality and some other essential nutrients such as milk (Vivek et al., 2008). vitamins, minerals etc. needed for body growth, The use of Marker-assisted selection (MAS) helps in development and maintenance (Adunola et al., 2018). phenotype screening and selection with high reliability. Maize, as a source of plant protein, is not just low in Breeding for QPM genotypes becomes straightforward protein quantity, but also low in some essential amino with the help of o2 locus-specific simple sequence acids such as lysine, tryptophan and niacin. The repeats (SSRs) (Gupta et al., 2013). phi57, phi112 and dependence of people on maize as a major food source umc1066 are the three SSRs predisposes them to the risk of malnutrition because (www.agron.missouri.edu) located within the o2 locus, these amino acids cannot be synthesized in human which are useful for the QPM breeders to discard non- body. Hence, boosting the protein quality of maize, QPM genotypes at seedling stage and to select particularly in terms of lysine and tryptophan contents heterozygous and homozygous genotypes for o2 during will avail people living in the developing countries of marker-assisted breeding (Babu et al., 2005, Gupta et quality diet on regular basis. This will also help to al., 2009; Kostadinovic et al., 2015). Significant reduce nutrition related diseases, deaths and successes have been recorded with the use of MAS in significantly improve nutritional status of individuals QPM breeding in terms of selecting for o2 allele who depend primarily on maize for sustenance (Ado, genotypes alongside their modifier genes as well as full 1999). recovery of recurrent parent. Babu et al. (2005) used The search for means to boost protein quality of maize MAS for development of QPM parental lines of Vivek-9 met with an important discovery in 1963 when three hybrid and could develop QPM hybrid in less than half scientists (Edwin T. Mertz, Oliver E. Nelson, Jr., and the time required through conventional breeding. Lynn S. Bates) from Purdue University, Illinois, USA Danson et al. (2006) explored various markers to found a maize natural mutant with increased levels of introgress o2 gene into herbicide tolerant elite maize lysine and tryptophan, twice as much as the normal inbred lines. They found that using marker for QPM and maize. A single gene mutation called opaque-2 gene endosperm modification can greatly enhance the was found to be responsible for this superior protein selection efficiency for isolating fully modified kernels quality. However, other natural maize mutants in QPM background (Sofi et al., 2009). In view of the conferring higher lysine and tryptophan were identified foregoing, the objective of this study is the in the 1960s and 1970s, viz., floury-2 (fl2), opaque-7 introgression of opaque-2 gene into the genetic (o7), opaque-6 (o6), and floury-3 (fl3) (Vivek et al., background of popcorn using marker assisted 2008). Among these natural mutants, major selection. breakthrough has been the isolation of the opaque-2 mutant (o2), with superior nutritional properties, albeit MATERIALS AND METHODS undesirable agronomic traits (Mertz et al., 1964). Identification of the nutritional benefit of recessive Experimental site opaque-2 mutant present on chromosome 7, followed The research experiment was carried out on the by accumulation of endosperm-modifiers, led to Teaching and Research Farm (TRF) of the Federal development of Quality Protein Maize (QPM) at the University of Technology, Akure (FUTA) during the 2016 International Maize and Wheat Improvement Center dry and wet seasons. FUTA’s TRF is located at an (CIMMYT), Mexico (Vasal et al., 1980). Afterwards, altitude of 332 m above sea level, 7016’N, 5012’E in efficacy testing in humans and animals was rainforest south-western region of Nigeria. Biochemical enthusiastically undertaken (Bressani, 1992; Knabe et analysis and molecular analysis were carried out in the al., 1992). Subsequent conventional breeding efforts Biochemistry Laboratory, FUTA and Bioscience generated numerous cultivars with improved Laboratory, International Institute of Tropical agronomic characteristics, and these were referred to Agriculture (IITA), Ibadan respectively. as QPM (Krivanek et al., 2007; Vivek et al., 2008). Maize homozygous for the o2 (recessive) mutation was Description of parent genotypes shown to have substantially higher lysine and tryptophan content than maize that was either Nine parental genotypes were employed in this study heterozygous (O2o2) or homozygous dominant (O2O2) which comprised different varieties of popcorn maize for the opaque-2 locus (Crow and Kermicle, 2002). and QPM. The three popcorn maize varieties used as Bressani (1992) showed that increased concentrations female parents were PopAkr, PopLag and PopIb while of these two amino acids in the grain endosperm can the remaining six QPMs as male parents were double the biological value of maize protein. However, 2009TZEORSTRQPM, Pop66-SR/ACR-91-SUWAN-1- the amount of protein in such maize remains at about SR-C1, ART/98/SW1-OBQPM, ART/98/SW6, ILE-1-OB 10%, the same as that of common (or normal 12 Int.Res.J.Biotechnol. and ART/98/SW1-Y. The variety, source, type and opaque; 2 indicating 25% opaque; and 1 indicating description of the genotypes are presented in Table 1. 100% normal (vitreous). Endosperm modification scores were derived based on analysis of 100 Table 1: Description of parental genotypes. randomly chosen kernels from the ears of F2. Modification Type 2 and 3 segregating for endosperm Typ modifiers were selected. Laboratory analysis was done S/N Variety Source Parent e for the parents and selected F2 generation for their 1 2009TZEORSTRQPM IITA Male OPV protein and tryptophan content. The derived value was Pop66-SR/ACR-91-SUWAN-1- used to determine the lysine and protein quality index. 2 SR-C1 IITA Male OPV Biochemical analysis 3 ART/98/SW1-OBQPM IAR&T Male OPV Tryptophan content was determined using the 4 ART/98/SW6 IAR&T Male OPV colorimetric method of Eric et al.
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