对广西红锥种群的DNA进行提取和ISSRPCR扩增体系进行优化。分析了退火温度、模板DNA浓度、Mg2+浓度、dNTPs浓度、TaqDNA聚合酶用量对反应结果的影响。红锥ISSRPCR分析较适宜的扩增体系是:25μL PCR反应体积中,buffer(10 mM TrisHCl,pH 9.0,50 mM KCl,0.1%Triton X100),1.25 U Taq DNA聚合酶,4种dNTPs各0.2 mM,0.4μM引物,2.0mM MgCl2,50 ng模板DNA。
[Objective] This study aimed to investigate the genetic variation of wild and cultivated populations of Castanopsis hystrix. [Method] Genetic variation of five wild populations and three cultivated populations of Castanopsis hystrix, was investigated with ISSR-PCR amplification. Totally, 151 individuals were selected and analyzed by amplification using nine pairs of ISSR primers screened. [Result] Each primer pair produced 7-20 bands and 122 polymorphic bands were obtained. At population level, ISSR diversity in the wild populations (P=59.84%, HPOP=0.182 7, I=0.285 6) was higher than which in cultivated ones (P=54.87%, HPOP=0.136 6, and I=0.219 8). The genetic differentiation coefficient among wild populations (GST) was 0.99. The similar population structure was found in three cultivated populations (GST=0.127 5). According to the UPGMA cluster analysis, the genetic distance among wild populations became larger with the increase of geographical distance. [Conclusion] Compared with other seed plants, with either a similar life history or various breeding system attributes, relatively low level of genetic diversity was observed in these five wild populations, which was caused by population size reduction and habitat fragmentation related to human activities. The formation of population structure may be explained by the species’ breeding system.
[Objective] This research aimed to develop molecular identification method for Castanopsis hystrix,Castanopsis carlesii and Quercus griffithii.[Method] DNA fingerprints of C.hystrix,C.carlesii and Q.griffithii were established by using ISSR-PCR method.Cluster Analysis was carried out by using UPGMA method based on Nei＇s genetic distances among each individual.[Result] Six polymorphic primers were selected from 50 ISSR primers for ISSR-PCR amplification,and totally 86 discernible DNA bands were amplified with 53 polymorphic bands,accounting for 61.2% of the total.The average number of DNA bands amplified by each primer was 10.75.Specifically,totally 5 primers had amplified differential bands and specific bands,which were able to accurately identify C.hystrix,C.carlesii and Q.griffithii.As calculated by DPS v3.01 software,the genetic distances among test materials were ranged from 0.166 67 to 0.809 52,with an average of 0.563 57.[Conclusion] ISSR-PCR method can be used to identify C.hystrix,C.carlesii and Q.griffithii effectively.
