Akram Ghaffari; Mohammad Reza Jazayeri; Bita Oskouei
Abstract
Introduction: Reaching an appropriate DNA isolation protocol is a great challenge in chickpea seeds due to its richness in protein and carbohydrates. In this research, a chickpea seed ...
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Introduction: Reaching an appropriate DNA isolation protocol is a great challenge in chickpea seeds due to its richness in protein and carbohydrates. In this research, a chickpea seed collection from various seed classes of candidate varieties was tested for genetic purity. Some off-type seeds could not germinate properly and using seedlings are not available for DNA isolation. Thus, seven experiments were designed to find a proper protocol for chickpea seed DNA extraction.
Methods: SDS based seed extraction buffer along with protein kinase K, various levels of DTT, and PVP including, 2% PVP with 0.2% , 0.4%, and 0.8% DTT as well as applying organic reagents including phenol: chloroform (25:24, v/v) and chloroform: isoamyl alcohol (24:1, v/v) were compared with a DNA extraction kit. Machine learning techniques contain regression linear modeling and discriminant analysis (DA) employed to evaluate the protocols.
Results: The results indicated that seed extraction buffer with 0.8% DTT showed the highest efficiency in DNA concentration, but the lowest in purity. Thus, using DTT can increase DNA yield in chickpea seeds but not great impact on diminishing protein residuals. Both 0.4% DTT and protein kinase K presented similar purity results. DTT 0.4% can be the proper alternatives of protein kinase K to remove protein residuals from chickpea seeds in DNA isolation. However, the best DNA purity achieved by using 0.4% DTT and organic reagents that was similar to the kit. Both 260/280 and 260/230 ratios were above 2 and the DNA yield was higher than the kit. Furthermore, Discriminant analysis (DL) demonstrated that applying 0.4% DDT with organic reagents and kit categorized in the same group.
Conclusions: This method of DNA isolation is the reliable and efficient protocol can be employed for seeds that are rich in protein.
