Genetic Diversity And Protection Strategy Of Buxus.sinica Var.parvifolia

The revisory method CTAB was used to extract DNA from Buxus.sinica var. parvifolia plant-leaves which were gathered from Qingliangfeng in Huangshan city of Anhui province, Sanqingshan in Shangrao city of Jiangxi province, Tianzhushan and Yaoluoping in Anqing city of Anhui province, Wanfoshan in Liuan city of Anhui province. The recycling parameters were optimized and optimal reaction system suitable for RAPD-PCR amplification was established. 21 primers that could produce polymorphic, clear and bright bands, sound repeatability had been selected for RAPD amplification and analysis on 162 samples in 5 B.sinica var. parvifolia populations. RAPD assessment of genetic variation and genetic diversity were carried out.The influencing factors of RAPD of B.sinica var. parvifolia were studied. The optimal RAPD conditions were: 20μL reaction volume,containing 30ng template DNA, 2.5mmo1/L MgCl₂, 0.5μmol/L mol/L primer, 200μmol/LdNTP, 1.5U Taq DNApolymerase. Amplification program was: 5min at 94℃; 40s at 94℃, 30s at 36℃, 2min at 72℃, 39 cycles; 5min at 72℃, then stored at 4℃.There were 167 polymorphic loci among 209 RAPD loci of 21 primes selected from 400 random primers. As analyzed by POPGENE 1.31,the percentage of polymorphic loci (PPB) was 79.9%, the Nei's gene diversity(h) and Shannon diversity index(I) were 0.2946 and 0.4343 in the species respectively. These data indicated that genetic diversity of populations was high. The population of Sanqingshan(SQ) had the highest genetic diversity of the 5 examinated populations (PPB=66.03%, h =0.2390, I=0.3547), at the same time, the population of Tianzhushan(TZ) had higher diversity level(PPB=63.16%,h = 0.2227,I = 0.3311). Effective number of alleles (Ae) was 1.5184 and observed number of alleles (A) was 1.799 in the species level. The genetic diversity within species of B.sinica var. parvifolia was 0.2978, but 0.2194 for that within population. The coefficient of genetic differentiation (Gst) had reached up to 0.2623, and gene flow(Nm) had arrived at 1.4065. It was indicated that 26.23% of total variation for genetic variation among populations , though most of variation in B.sinica var. parvifolia exists within the populations,the cause of which was being short of effective gene flow due to influence by minor population effect and scattered distribution for B.sinica var. parvifolia,There were higher genetic similarities among 5 populations of B.sinica var. parvifolia, all of which were above 0.83, but the largest one has reached up to 0.9374 in 2 populations from Tianzhushan (TZ) and Yaoluoping (YL). And the smallest one was 0.8355 in populations from Yaoluoping (YL) and Qingliangfeng (QL). The dendrograms of genetic relationships among populations were constructed based on Nei's genetic distance showed that the 5 natural B. sinica var. parvifolia populations could be divided into 2 big groups, the first group from Qingliangfeng (QL) and Sanqingshan(SQ); the second group from Tianzhushan(TZ) , Yaoluoping(YL) and Wanfoshan(WF). Genetic Identity and Genetic Distance between populations were estimated, and the results indicated that genetic distance between populations was not significantly correlative to geographic distance.In the view of the genetic information available for B.sinica var. parvifolia, it was suggested that the Sanqingshan (SQ) and Tianzhushan (TZ) populations should be incorporated into nature reserve to long-timely and effectively preserve the living space of B.sinica var. parvifolia. Simutaneously, adult plants or seedings mutually should cross-transplant among populations to enchance the gene flow. By the means, the genetic diversity resources of species could be preserved to the greatest extent. Most populations should be sampled to retain as much of the genetic diversity of the species in exsitu preservation as possible.