Open Access Maydica 58-2013 Short Comunication RECEIVED 04/29/2013 Prospects of endosperm DNA in maize seed characterization Alhassan D Halilu 1 *, Shehu G Ado 1 , Inuwa S Usman 1 , David Appiah-Kubi 2 1 Department of Plant Science, Institute for Agricultural Research, Ahmadu Bello University, Samaru, Nigeria 2 Council for Scientific and Industrial Research, Crop Research Institute, Ghana *Corresponding author: E-mail: adhalilu@gmail.com Keywords: genomic DNA, maize endosperm, genotyping, SSR-PCR DNA based characterisation of maize germplasm has become the easiest and fastest approach to identify genetic diversity as compared to phenotyping. The conventional DNA source for genotyping is the leaf which required at least 2 weeks waiting period from seed planting to leaves sampling. This work exploits the use of endosperm DNA (EDNA) for the genotyping of maize germplasm. Maize endosperm was excised from maize seeds using pli- ers, ground and used for Genomic DNA extraction (gDNA). Leaves DNA (LDNA) was also extracted concurrently. The extracted LDNA and EDNA were quantified and subjected to SSR-PCR. The mean concentrations of DNA extracted were 1575 ng/ul for the leaves and 526 ng/ul for endosperm. Though the difference in quantity of EDNA and LDNA were highly significant, the quality (A260/A280) for both EDNA and LDNA fall within 1.6-1.8 range of pure DNA index. SSR-PCR products using phi032 were similar for both EDNA and LDNA, indicating the usability of EDNA in genotyping. This seed based method of gDNA extraction takes less than 24 hours from sampling to quantification and genotyping. It also allows germination of sampled seeds, selection before planting, avoids the delay of planting and waiting in leaf sampling and saves field space. Abstract Introduction DNA markers have the advantage of detecting un- limited number of polymorphisms randomly distribut- ed in the genome without environmental effects and without influence of plant physiological development. Thus DNA based methodologies for seed purity as- sessment, genetic diversity study and selections are becoming more popular (Salgado et al, 2006; Menkir et al, 2005; Senior et al, 1998). Simple sequence repeat (SSR) markers have been extensively used for most of these studies in maize (Smith et al, 1997; Reif et al, 2003; Pinto et al, 2003; Menkir et al, 2004). SSR motifs are 2, 3, or 4 nucleotides that are found in abundance in the genomes of eukaryotic plant species and these units are tandemly repeated many times in the DNA sequence (Morgante and Olivieri, 1993; Enoki et al, 2002; Menkir et al, 2004). However, extracting DNA samples from leaves, the conventional method, which include collecting, processing leaf tissue and tracking back to source plants is the most significant rate limiting factor in ge- notyping and marker assisted selection. It is also time consuming and costly. According to Crouch (2007) ‘use of leaf tissue means that lab analysis of mark- ers has been “after the fact” in essence, scientists need to wait for plants to develop to obtain samples’. Meanwhile, only a few plants may contain the desired genes from the large number that must be grown. Seed DNA-based genotyping is now considered an important alternative (Salgado et al, 2006; Gao et al, 2008). It involves a non-destructive sampling method in extracting DNA that allows germination of sampled seeds and permits selection to be carried out in ad- vance of planting. It also saves field space and cre- ates the possibility of working with larger effective populations for complex agronomic traits. This study was carried out to extract DNA from maize endosperm using simpler, non-destructive and cheaper sampling technique. In Salgado et al (2006), whole seeds were ground in liquid nitrogen while Gao et al (2008) soaked the seeds for few hours to soften without stimulating germination for sampling. Materials and Methods Genetic materials Five open pollinating maize varieties derived from the maize breeding unit of Institute for Agricultural Research, Samaru Nigeria, were used for this study. Endosperm Sampling Seed endosperm sampling was done using plier to excise small pieces of the endosperm from the other side of the embryo. The seeds were not soaked for this sampling. 50 mg of dry endosperm pieces ex- cised from 5 seeds per varieties were ground into fine powder using genogrinder at 1,000 strokes min -1 for 2 minutes. Leaves Sampling 20 seeds of each variety were planted separately in a screen house at room temperature using normal garden soil. Leaves from 2 weeks old seedlings were freeze dried and ground using genogrinder at 1,200 strokes minute -1 for 2 minutes.