Abstract
The advent and application of molecular markers technology enabled dissection of genomic regions underling important agronomic traits in crop plants. Isolation of sufficient quantity and good quality DNA from a large number of plant samples is a major challenge in most genetic and genomic analyses. Wheat is the world’s third most important food security crop on which most molecular breeding and genetic engineering researches have been undertaken. So far, several wheat DNA isolation protocols and commercial kits are available. However, in some, the cost is so prohibitive for small laboratories, and hence finding cost effective alternative makes small biotechnology laboratories of developing countries beneficial to the technology. The present study was targeted to provide alternative economical, reliable and safer genomic DNA isolation method in wheat. Accordingly, genomic DNA was isolated from 27 bread wheat recombinant inbred lines (RIL) raised in greenhouse with and without liquid nitrogen. From 100 mg, 20-day old leaf samples, averagely 1890.7 ng/μl sufficiently pure genomic DNA (A260/A280 ratio of 1.8) was extracted. In the extraction process, ammonium acetate and sodium acetate solutions were also avoided. Polymerase chain reaction (PCR) analysis of the extracted DNA using two simple sequence repeats (SSR) markers produced polymorphic bands confirming the suitability of the extracted DNA for PCR. Therefore, it is possible to deduce that we have adopted a simple, cost effective, reliable and safer DNA isolation method that can help to extract good quality and sufficiently pure genomic DNA for further molecular studies. Key words: Wheat, genomic DNA isolation, liquid nitrogen, polymerase chain reaction (PCR) analysis, simple sequence repeats (SSR) markers.
Highlights
Nowadays, molecular markers are widely used to promote the speed and efficiency of crop improvement program
To determine the reliability and efficiency of the current extraction method, genomic DNA was isolated from 27 bread wheat recombinant inbred lines (RIL) obtained from CIMMYT both with- and without liquid nitrogen (Table 1)
It was confirmed that the DNA extracted in the present method without liquid nitrogen is sufficiently pure for further molecular downstream studies
Summary
Molecular markers are widely used to promote the speed and efficiency of crop improvement program. Loci (QTLs) underling economically important agronomic traits, germplasm characterization, genetic diagnostics, characterization of transformants, study of genome organization, phylogenetic analysis, marker-assisted selection (Gupta et al, 1999). Several DNA marker systems have been developed and are routinely being applied in plant breeding program. Marker assisted crop improvement involving scanning of large sets might be hundreds to thousands of germplams, and isolation of sufficient quantity and high quality genomic DNA from such huge plant samples is the major bottleneck in most genetic and genomic studies (Xin and Chen, 2012). Wheat is the world’s third most important food security crop (Green et al, 2012) on which most molecular breeding and genetic engineering researches have been undertaken
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