A rapid and reproducible method for isolating genomic DNA from a few crop plants suitable for polymerase chain reaction-based genotyping
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Abstract
As most of the molecular markers in crop molecular breeding programmes are successful based on polymerase chain reaction (PCR), the isolated genomic DNA must be suitable for the same. Though PCR is a robust method and in most of the cases requires only a minute amount of genomic DNA as template, removal of potential PCR-inhibitory factors is quite important. The present work reports the optimization of a rapid genomic DNA isolation method, suitable for PCR-based genotyping of plants. As very minute amount of the genomic DNA isolated in this rapid method was found to be sufficient for PCR, a researcher is capable to go for several hundred independent PCR from single isolation. The method was validated in 4 different crops (wheat, tomato, brinjal and cauliflower) using different PCR-based molecular markers. In case of wheat, genomic DNA isolated in this method was found to be suitable PCR using the specific marker for the detection of the Lr34 gene. For tomato, genomic DNA isolated in this method was successfully used with the molecular markers for the detection of resistance alleles for yellow leaf curl disease and root knot disease. In case of brinjal, the isolated genomic DNA was found to be suitable for simple sequence repeat (SSR) marker assay. In a similar way, genomic DNA isolated in this method from cauliflower leaves was observed to be suitable for amplifying a gene of ~1.5 kb length. Thus, this method will be quite helpful to expedite marker assisted selection of plants in plant molecular breeding programmes.
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Genomic DNA, Molecular markers, polymerase Chain Reaction, Rapid method, SDS-potassium acetate method
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