Genomic DNA Extraction from Sesame (Sesamum indicum L.)

Abstract:

Sesame (Sesamum indicum L.) is an ancient oilseed crop valued for its edible oil and nutritional properties. Genetic diversity among genotypes is vital for breeding and SSR markers are widely used due to their abundance and efficiency in the sesame genome. Successful application of these markers depends on extracting high-quality genomic DNA. This study aimed to standardize an efficient DNA extraction protocol by evaluating two different methods and determining the optimal seedling stage for sesame. Three sesame genotypes (YLM-146, YLM-66, and TPTS-55) were germinated and harvested at three different intervals: seven, 10, and 12 days. Two extraction protocols were compared: a standard CTAB 2.5× Method (Method-1) and a Modified CTAB Method (Method-2). Method-1 involved overnight incubation and additional purification steps, requiring approximately two days to complete. In contrast, Method-2 utilized a higher salt concentration (5 M NaCl) and immediate RNase treatment, allowing for completion within a single day. The results demonstrated that while both methods produced DNA of sufficient quality, Method-2 was significantly faster and more convenient for routine laboratory use. Spectrophotometric analysis using a Nanodrop revealed that DNA yield and purity were consistent across all seedling stages tested. Seven-day-old seedlings were identified as the most efficient tissue source because they reduced the overall experimental timeframe without compromising DNA integrity. DNA purity, as measured by A₂₆₀/A₂₈₀ ratios, remained close to the ideal value of 1.8, indicating minimal contamination from polysaccharides or phenolic compounds. The high concentrations obtained (ranging from 1014.87 ng/µL to 2241.59 ng/µL) and excellent purity levels confirm that the modified CTAB protocol is highly suitable for downstream applications such as PCR amplification and SSR marker analysis in sesame.