Analysis The Genetic Diversity Of Commom Bean By Botanical Character And ISSR

In this research,29kidney bean (Phaseolus vulgaris) cultivars and1small soybean (Vigna angularis) cultivar were investigated according to their morphological traits, including growth type,the first flowering node, flower color, pod color, pod length, pod width, pod thick, and hundred-grain weight. The DNA was extracted by CTAB method with a little modification. The reaction condition of ISSR was also optimized. At last, cluster analysis of these30materials was performed using both morphological traits and ISSR markers. The results are as follows:1. The DNA isolated by the modified CTAB method was suitable for ISSR-PCR, which show that all DNAs were integrity by agarose gel electrophoresis and DNA OD260/OD280of all materials were1.95-2.14.2.22valid ISSR primers were screened from100ISSR primers. In the30materials,262bands were obtained using these22primers (averagely every primer generated11.9bands). Of these262bands, there were243polymorphic bands, the Polymorphism rate was92.7%, averagely every primer generated11.0polymorphic bands.3. The results of the annealing temperature screened show that,the best annealing temperature of most primers is54℃. There are many clear electrophoretic bands with good polymorphism at this temperature.4. The PCR condition was optimized according to Tantasawat et al., and the total reaction system was determined to20ul, including60ng DNA,10μl2×MasterMix,0.5μmol/L primer. Cycling conditions consisted of1min initial denaturation at94℃, followed by1min denaturing at94℃,1min annealing at54℃and1min extension at72℃repeated for36cycles and10min extension at72℃, then Stored at4℃.5. The ISSR data was analyzed by NTSYS2.10software. Cluster analysis was based on UPGMA method. At the similarity coefficient of0.700, these30materials could be divided into five groups.The first group:including14materials. They are No.1、3、4、5、2、6、7、10、11、9、13、14、12、15. At Similarity coefficient of0.75, this group can be divided into three subgroups. The first subgroup include No.1-Sijidou-1(black seeds), No.3-Weiyuansijidou, No.4-YJ005309, No.5-YJ009765, No.2-Jiagong Ⅱ common bean, No.6-Sijidou-1, No.7-Sijidou-3, the pod color, pod shape of these cultivars are similar with each other. The second subgroup include No.10-YJ009763and No.11-YJ005307, these two cultivars have similar hundred-grain weight, and the pod color are green. The third subgroup include No.9-YJ005308, No.13-YJ005310, No.14-Xuedou-3, No.12-Hanyuanhuadou, No.15-YJ004616(pinto seeds), which had a similar pod size and hundred-grain weight.The second group:including No.18-YJ005312(pinto seeds), No.19-YJ005312-2, No.23-Xuedou-2, No.20-YJ004616, which have a similar pod shape and seed shape.The third group included No.16-Sijidou-2, No.17-YJ009764, No.21-YJ009766, No.24-Zhujiedou, No.25-Henanroudou, No.22-YJ005311,No.26-Chuanhongjiadou, No.27-Honghuabaijia, No.28-Wangzhongwangjiadou. The cultivars in this group have relatively long pods.The fourth group included No.29-Jinhuangsejia and No.30-Taiguodou. They have the same first flowering node and have similar flower color.The fifth group:including No.8-YJ004614,which was small soybean, has the furthest genetic distance to other materials. The seed size is the smallest and it was used as a control in this experiment.6. Based on the results of morphological cluster, at the genetic distance of9.261, these30materials could be divided into five groups.The first group:including12materials. They are No.14,19,23,15,20,12,6,7,25,30,24,28. At the genetic distance of8.0, this group can be divided into two subgroups. The first subgroup include No.14-Xuedou-3, No.19-YJ005312, No.23-Xuedou-2, No.15-YJ004616(pinto seeds), No.20-YJ004616, No.12-Hanyuanhuadou, No.6-Sijidou-1, No.7-Sijidou-3. These eight materials have similar color, their main color are all white. No.25-Henanroudou, No.30-Taiguodou, No.24-Zhujiedou, No.28-Wangzhongwangjiadou belonged to the second subgroup. The common feature of this subgroup is mainly manifested in the size of the pod and hundred-grain weight.The second group:including No.27-Honghuabaijia, No.1-Sijidou-1(black seeds), No.16-Sijidou-2, No.26-Chuanhongjiadou. They are all sprawling type, have similar pods in size and their colour of flower is all purple.The third group included No.16-Sijidou-2, No.17-YJ009764, No.21-YJ009766, No.24-Zhujiedou, No.25-Henanroudou, No.22-YJ005311, No.26-Chuanhongjiadou, No.27-Honghuabaijia, No.28-Wangzhongwangjiadou. The cultivars in this group have relatively long pods.The fourth group included No.29-Jinhuangsejia and No.30-Taiguodou. They have the same first flowering node and have similar flower color.The fifth group:including No.8-YJ004614,which was a small soybean and has the furthest genetic distance to other materials. The seed size is the smallest and it was used as a control in this experiment.7. There are some differences between the clustered results of morphological marker and ISSR molecular marker. The ISSR molecular marker which uses DNA as an object to study at the molecular level, is less affected by the environment and with good polymorphism. In order to ensure the correctness and repeatability of the experimental results beterly,we need to use multiple methods together.