Abstract:
Three non-chemical conditions of preservation, storage at room temperature (that is benchtop), in a freezer and at simulated herbarium were examined to determine which is most suitable for the preservation of cowpea leaves for molecular studies. Under each storage condition, the effect of duration of storage and the difference in leaf age (that is flush or mature) on DNA yield and purity were assessed. Periodically, DNA was extracted from both flush and mature leaf samples from 6 cowpea (Vigna unguiculata L. Walp.) varieties, Amantin, Asontem, Ayiyi, Bengpla, Ejura Red and Soronko beginning from the day of harvest (day 1), through day 7, 21, 35 and 49 after storage. At room temperature and simulated herbarium conditions, the yield and purity of the DNA extracted from both flush and mature leaves decreased during the period of the study. Time was found to correlate inversely with the purity and yield of DNA in all cases. Under these conditions, the yield of DNA extracted from day 1 samples (fresh samples) of both leaf types was significantly different from the DNA yield from day 7 to 49 samples. Also, no significant difference was found in DNA yield from day 7 to 49 samples. However, in most cases, more DNA was obtained from flush leaves than from their corresponding mature leaves. Of the three conditions, leaves stored at room temperature yielded the least amount of DNA. Samples kept under simulated herbarium conditions yielded more DNA than kept in the freezer but the difference was not significant. Also, a fairly constant yield and purity was obtained for DNA extracted from frozen samples. Frozen samples yielded relatively purer DNA than their corresponding samples stored either at room temperature or at simulated herbarium though the observed differences were not significant. Therefore, storage in a freezer provided the best non-chemical preservation condition among the three assessed. Only 2 (GA and AR) out of 15 random primers assayed using the technique of Random amplified polymorphic DNA-polymerase Chain Reaction, (RAPD-PCR) amplified segments of the genomic DNA extracted from the 6 varieties. Further analysis (Restriction Fragment Length Polymorphism studies) was done on the GAprimed 1200 bp PCR product using eight restriction endonucleases, Alul, Dral, ￡coRI, EcoRV, Haelll, Hinfl, Kpnl and Pvul. Four enzymes, Alul, Haelll. Hinfl and Kpnl digested the product. However, for each enzyme, identical banding patterns were observed on both agarose and polyacrylamide gels in all the six varieties Single stranded conformation polymorphism analysis (SSCP) was conducted on the two ARprimed products, 369 bp and 545 bp. Only the 545 bp was denatured but the profiles of the bands in all the six varieties were also identical. Therefore, these molecular techniques could not be used to identify the individual varieties
