Sep 22, 2011 - lysine ketogluterate reductase dehydrogenase as a consequence of reduced levels of ... geneâbased SSR markers in lysine, tryptophan pathway and opaque2 ...... Laurent, D., D. Dervins, A. M. Jacob, J. Joets, J. P. Martinant,.
Plant Breeding 131, 20—27 (2012) 2011 Blackwell Verlag GmbH
doi:10.1111/j.1439-0523.2011.01919.x
Identification of candidate gene–based SSR markers for lysine and tryptophan metabolic pathways in maize (Zea mays) B A N I S E T T I K . B A B U 1 , P A W A N K . A G R A W A L 1 , H A R I S . G U P T A 3 , A N I L K U M A R 2 and J a g a d e e s h C . B H A T T 1 1 Vivekanand Institute of Hill Agriculture (VPKAS), ICAR, Almora 263 601, Uttarakhand, India, E-mail: kalyan_biotek@ yahoo.co.in; 2G. B. Pant University of Agriculture and Technology, Department of Molecular Biology and Genetic Engineering, Pantnagar, U. S. Nagar 263149, India; 3Present address: Indian Agricultural research Institute, Pusa, New Delhi 110012, India
With 3 figures and 5 tables Received February 2, 2011/Accepted September 22, 2011 Communicated by T. Lu¨bberstedt
Abstract Identification of candidate gene–based SSR markers is extremely useful in genetic enhancement of crop plants. A total of twenty-four SSR loci were developed for the candidate genes of lysine and tryptophan metabolic pathways and for opaque2 (o2) modifiers. Of the 24 SSR loci, 23 yielded amplicons of expected size; however, fifteen were found polymorphic among the 22 maize genotypes including normal and quality protein maize inbreds from India and exotic inbreds from CIMMYT, Mexico. The polymorphism information content of primers was in the range of 0.16–0.72, with a mean of 0.49. The gene diversity ranged from 0.17 to 0.75, and the inbreeding coefficient was in the range of 0.48–1.0. The candidate genes of lysine metabolism showed higher polymorphism levels and genetic diversity than other candidate genes. The SSR loci developed from the candidate genes of aspartate kinase 2 and homoserine dehydrogenase were found to have high potential. Thus, the SSR loci identified in this study added useful markers for fine mapping and high-density mapping of opaque2 modifiers.
Key words: candidate gene–based SSR markers — opaque2 modifiers — gene diversity — lysine and tryptophan metabolism — quality protein maize Cereal endosperm proteins are deficient in essential amino acids such as lysine and tryptophan and generally contain 1.5– 2% lysine and 0.25–0.5% tryptophan. However, 5% lysine and 1.1% tryptophan are required for optimal human nutrition (Young et al. 1998). The opaque2 (o2) in maize leads to decrease in the synthesis of prolamine (zein) protein, thereby resulting in increase in lysine and tryptophan content (Munck 1992). The loci that alter the soft and starchy texture, named as Ôo2 modifiersÕ (Opm), proved to be highly complex in genetic nature but effective in ameliorating the negative features of maize traits (Vasal et al. 1980) and lead to development of quality protein maize (QPM) at International Center for Wheat and Maize Improvement (CIMMYT), Mexico (Vasal et al. 1980) and Pietermaritzburg, South Africa (Geevers and Lake 1992). The o2 mutation in maize is associated with an increased level of free amino acids in the mature endosperm. Results from several studies have shown that key enzymes involved in amino acid metabolism are altered in o2 mutants. The activity of aspartate kinase (AK) is up-regulated by o2 (Brennecke et al. 1996) and is an important enzyme in the synthesis of several amino acids, including lysine (Bryan 1990). The o2 mutant leads to the down-regulation of the activity of lysine ketogluterate reductase dehydrogenase as a consequence
of reduced levels of mRNA (Kemper et al. 1999). Reduction of lysine ketogluterate reductase dehydrogenase is an important factor responsible for high free lysine in the maize endosperm. Based on these results, it is understood that the o2 mutant leads to the regulation of lysine and tryptophan pathway enzymes, which results in higher lysine and tryptophan. Candidate genes are sequenced genes of known biological action involved in the development or physiology of a trait. Candidate genes have been effectively used in several molecular breeding programmes such as QTL mapping, association mapping and marker-assisted selection (MAS) and in transgenics for improvement in agronomically important traits in crop plants (Barret et al. 2004). In maize, candidate genes associated with lysine metabolism were identified by Wang et al. (2007), and they found lys-sensitive Asp kinase is a better candidate gene for the quantitative trait locus affecting free amino acid content than Thr-sensitive AK-HSDH2. Whereas, enzyme activities were measured and QTLs were observed for Aspartate kinase 2 and AK-homo-Serdehydrogenase (HSDH) 2 on the long arm of chromosome 2 (Wang and Larkins 2001). Laurent et al. (2005) reported linkage mapping of 1454 new maize candidate gene loci that corresponded to 954 cDNA probes that were previously unmapped. Identification of SSR markers in candidate genes has become a novel approach in the association mapping, QTL mapping and their effective utilization in further crop improvement programmes through MAS. Recently, Sharma and Chauhan (2008) identified candidate gene–based SSR and SNP markers in the zinc and iron transporter sequences of maize. They identified 34 SSRs of di-, tri- and tetranucleotide repeats in 28 candidate genes for zinc and iron transporters of maize. The exponential increase in publicly available databases made it possible to identify SSR that are desirable DNA markers, because they are abundant in plant genomes, highly polymorphic, rapid, less expensive and can be used to identify specific chromosomal regions across populations (Chin et al. 1996). This is the first report of identification of SSR markers in the candidate genes of lysine, tryptophan pathway and opaque2 modifiers and is further useful to find the opaque2 modifiers, which play a very important role in the development of quality protein maize. Thus, the present study aimed at (i) identifying candidate gene–based SSR markers in lysine, tryptophan pathway and opaque2 modifiers and (ii) testing their extent of polymorphism across a range of maize inbred lines (normal and QPM lines)
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Identification of candidate gene–based SSR markers
including Indian and exotic lines (CIMMYT), Mexico, with the ultimate goal of utilizing these markers in our QTL mapping studies for o2 modifiers.
Materials and Methods Plant materials: Twenty-three maize inbred lines consisting of eight normal inbred lines obtained from Vivekananda Institute of Hill Agriculture (VPKAS), ICAR, Almora, India; eight QPM inbreds developed by VPKAS; and seven exotic QPM inbred lines obtained from CIMMYT, Mexico, were used in the study. The details of the inbred lines are shown in Table 1. Identification of candidate genes and sequences: Candidate genes have been selected from the metabolic pathways of lysine and tryptophan and opaque2 modifiers in maize (Fig. 1) as previously reported by Wang and Larkins (2001) and Wang et al. 2007, and aspartate kinase 2 (Ask2) is a good candidate gene that regulates the reaction of aspartate to aspartate 4-semialdehyde, which in turn is the source for lysine production. Aspartate kinase homoserine dehydrogenase 2 (Akh2) is also a good candidate gene, mapped on the long arm of chromosome 2 (Bin 2.06), while Ask2 was mapped on bin location 2.05. The candidate gene HSDH is involved in regulation of synthesis of lysine. Chorismate synthase and tryptophan synthase are good candidate genes involved in the synthesis of anthranilate from chorismate and thus tryptophan from anthranilate, respectively. Opaque2 modifier genes for designing primers were selected from the earlier studies (Reina et al. 1990, Lopes et al. 1995, Woo et al. 2001, Wu et al. 2010). Sequences for these candidate genes were selected from the Arabidopsis thaliana clones available in the NCBI database (http:// www.ncbi.nlm.nih.gov). Sequences of these clones were BLASTed against the publicly available EST sequences database to find the similar sequences in maize and other closely related organisms using the database search tool BLASTn (http://blast.ncbi.nlm.nih.gov). We selected all the contig sequences from Zea mays that showed more than 85% similarity at
