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Genetic Diversity Analysis And Varietal Identification Among 68 Chinese Asparagus Bean (Vigna. Unguiculata Ssp. Sesquipedialis) Cultivars Based On RAPD, ISSR And Morphological Markers

Posted on:2015-10-02Degree:MasterType:Thesis
Country:ChinaCandidate:H Q TanFull Text:PDF
GTID:2283330482475487Subject:Vegetables learn
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In this experiment, five classic plant DNA isolation methods, including three CTAB methods and two SDS methods, were compared and evaluated using cowpea cultivar"Chengjiang No 7" as material, while isolation using a commercial kit was also undertaken, for the sake of an ideal method to extract DNA from cowpea for downstream PCR analysis. Afterwards, we used RAPD and ISSR markers, separately and in combination, as well as morphological markers, to evaluate the level of genetic diversity within 68 Chinese asparagus bean cultivars. The main results were as following:(1) The cowpea DNA yield by the SDS methods (Aljanabi et al.,1997; Dellaporta et al., 1983) were significantly higher than those obtained by the CTAB methods (Saghai-Maroof et al.,1984; Rogers et al.,1985; Doyle et al.,1987) and E.Z.N.A.(?) plant DNA Kit (Tukey’s, P<0.05). The extraction method had a significant effect on the DNA yield (F= 29.20, df= 5, P<0.01) and OD260/280 ratio (F= 15.69, df= 5, P<0.01).When extracting DNA for restriction enzymes digestion or PCR based downstream molecular manipulations, the RNase treatment is not needed.(2) After evaluating the cost and time, yield, purity, integrity and functionality among the six methods, the SDS method described by Dellaporta et al., was considered an ideal protocol to isolate DNA from Vigna unguiculata. The cost and time required in this method was relatively low. Besides, the quantity and the quality of the DNA extracted by this method were high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning. In addition, it had the added advantage of not requiring any phenol or chloroform extraction.(3) We used Premix Taq in RAPD and ISSR analysis among asparagus bean cultivars and optimized the reaction conditions. Based on their performance on agarose gel, the optimized PCR reactions was determined, which contained 40 ng DNA,0.8 μmol/L primer,12.5 μl Premix Taq, and ddH2O up to 25 μl.(4) We screened the annealing temperature between 44-55℃ to obtain clear and distinct band patterns. Based on their band patterns on agarose gel, the optimized annealing temperature for each primer was chosen. The GC content of selected primers was between 44%-55%. Some primers generated clear band patterns at lower annealing temperature while some obtained better results at higher annealing temperature, which has no relationship with GC content.(5) 9 of 22 RAPD primers were applied to PCR analysis in 68 asparagus bean cultivars. A total of 56 bands ranging from 200 to 2000 bp were amplified, of which 21 were polymorphic (37.50%). While 14 of 18 ISSR primers were employed to PCR analysis. A total of 116 bands ranging from 220 to 2000 bp were scored, corresponding to an average of 8.28 bands per primer, of which 38 (32.76%) were polymorphic.(6) By the standard two-sample t test, no significant differences were detected between the average value of PIC (t=1.37, p=0.186), MI (t=-0.04, p-0.967) and RP (t=0.11, p = 0.913), indicating that the RAPD and ISSR technique have almost the same ability to detect polymorphism and to determine diversity. However, ISSR markers have a little advantage over RAPD markers, because of its better stability, higher band intensity and more no. of polymorphic bands per primer generated.(7) A high degree of congruence (r>0.67) between RAPD and ISSR and a slight and significant correlation between molecular and morphology (r<0.2366) were found by Mantel test. In particular, three morphological traits, including pod color, pod length and pod weight, were significantly correlated to all the molecular data.(8) Statistical analysis of morphological traits showed that there exists considerable variation in the phenotype of asparagus bean. Whereas the pair-wise Nei’s genetic distance ranged between 0.00-0.18 and 0.00-0.14 for RAPD and ISSR markers, respectively. This indicated that the genetic base was very low. Therefore, the combination of molecular markers and morphological markers is essential for a comprehensive understanding of the amount and pattern of genetic variation that exists within and between the available accessions.(9) In addition, an extremely low diversity was found among Chinese asparagus bean cultivars. Diverse landrace forms of asparagus bean are found throughout Asia, which generally have a wider genetic basis compared to improved cultivars. Hence, introgression of alien genes from landrace sources or ssp.unguiculata needs to be further conducted to enhance the germplasm diversity for elite cultivar development in asparagus bean.
Keywords/Search Tags:asparagus bean, DNA extraction, genetic diversity, molecular and morphological markers
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