Genetic Diversity Of An Endangered Plant Chimonanthus Grammatus Endemic To Jiangxi Province

Chimonanthus grammatus is an extremely endangered plant endemic to Jiangxi province, China. The plant may reproduce by sexual (seeds) and asexual (rhizomes) means. The methods of CTAB, modified CTAB, SDS, High salt low pH, SDS-CTAB and SDS-Proteinase K were used to be compared in order to isolate high-quality genomic DNA from Ch.grammatus plant-leaves which were gathered from 7 populations in Jiangxi province. The recycling parameters were optimized and optimal reaction system suitable for ISSR amplification was established in the paper. ISSR assessment of 213 species was carried out.The results showed that the modified CTAB was the best one, in which the extract DNA had an ideal OD₂₆₀/OD₂₈₀ ratio and the highest concentration. The optimal reaction system of ISSR for Ch.grammatus was determined as follows: In total 20μL reaction volume, 1×PCR buffer, template DNA 50 ng, 1.5 mmol/L MgCl₂, primer 0.6μmol/L, dNTPs 0.15μmol/L and Taq DNA polymerase 2.0 U. The optical amplification program was also obtained: 94°C 5 min, 1 cycle; 94°C 30 s, 50.8⁵8.3°C anneal 45 s, 72°C 2 min, 40 cycles; 72°C 5 min, 1 cycle.100 ISSR primers were used to screen suitable primers with 7 samples from every population for assessing genetic diversity of Ch.grammatus, of which 10 ISSR primers with high resolution and multiple polymorphic bands were screened, and were employed to assess genetic diversity of 100 samples from 7 populations. The total 74 ISSR bands were amplified, 39 of which were polymorphic bands. The percentage of polymorphic bands is 52.7%.0, 1 matrix constructed according to amplified maps was inputted into computer. The result of analysis indicated low genetic variation and high clonal diversity at species level ( P =52.70%, H = 0.1572, I = 0.2408, NC = 1.1390, PD = 0.8780, D = 0.9980, E = 0.9330) and at population level (P = 21.62%～41.89%, H = 0.0958～0.1538, I = 0.0946～0.2220, Nc = 1.0000～1.2310, PD = 0.8620～1.0000, D = 0.9840～1.0000, E = 0.0000～0.7840). It exhibited a high degree of population differentiation among seven populations with Gst value of 0.2489. Analysis of molecular variance (AMOVA) revealed that 26.37% of ISSR variation resided among populations, while 73.63% resided within populations. There was significant association between genetic and geographical distances (r=0.712,p<0.001) suggesting that gene flow was restricted geographically. The program of genetic relationships among populations was constructed based on Nei's gene distance. Four populations of An Yuan clustered in a clade, three populations of Hui Chang clustered in a clade.The clonal diversity was very high in populations, revealing that clonal propagation was not the major regeneration mechanism. A number of factors including mating system, gene flow, genetic drift and evolutionary history might have affected genetic variation and clonal diversity of Ch.grammatus populations. Based on the data, efficient and practical conservation strategies were proposed to preserve this endangered species.