| Dendrocalamopsis oldhami Munro is a cluster of bamboos in the Gramineae Bambusoideae Dendrocalamopsis,It is the most widely distributed and most cultivated species of bamboo in Dendrocalamopsis.Dendrocalamopsis oldhami Munro is one of the excellent fast-growing sympodial bamboo species for shoot and timber in southern China,which has high ornamental value and economic value.In recent years,Fujian Province introduced Dendrocalamopsis oldhami Munro in coastal sandy lands,and constructed coastal shelter forest along with Casuarina equisetifolia Forst,and played the ecological benefits of bank protection berms and windbreaks and sand fixation.According to the distribution status of Dendrocalamus oldhami Munro resources,20 representative populations in 4 provinces were selected in this study.The genetic diversity and phylogenetic relationship were studied by using IS SR molecular marker technique,and DNA fingerprinting was established to identify the germplasm resources of Dendrocalamopsis oldhami Munro effectively.The main study results are as follows:1.Using the BioSpin plant Genomic DNA extraction Kit(CAT#BSC13S1)method by Hangzhou Bori Company,and the method was adjusted slightly to establish a method of extraction of high purity and integrity of DNA.2.The concentration of dNTPs,Mg2+,Taq DNA polymerase,primer,template DNA,annealing temperature and cycle times were screened by combining orthogonal design with single factor design.The optimized system was used to screen 100 IS SR primers.Ultimately determine the best reaction system as follows:In 20μL reaction system containing 0.2mmol/L dNTPs,2.0 mmol/L Mg2+,1U Taq DNA polymerase,0.4μmol/L primer,50 ng DNA template,2.0μL10×Taq Buffer,and ddH2O completed.The order of the effect by the factors was in Mg2+>dNTPs>DNA template>Taq DNA polymerase>primers。The optimal amplified procedure was as follows:after a pre-denaturing of 5min at 94℃,38 cycles were performed with denaturing of 45 s at 94℃,annealing of 30s due to denaturing temperature of different primer,extension of 90 s at 72℃,a final extension step of 10 min at 72℃ and hold at 4℃.According to this PCR system,12 primers of 100 primers were chosen for their clarity,high polymorphism and repetition.3.The DNA samples from 20 different populations of Dendrocalamus oldhami Munro were analyzed by using the selected primers of ISSR,a total of 158 loci were amplified,of which the polymorphic sites were 125,the percentage of polymorphic loci(PPL)was 79.11%.In 20 different populations of Dendrocalamus oldhami Munro,the percentage of polymorphic loci in Zhongshan population was the lowest in 31.65%,and the highest in Fuqing population was about 53.16%,the average percentage of polymorphic loci was 44.46%.The result showed that number of observed allele(Na)was 1.8734,number of effective alleles(Ne)was 1.3043,the average value of the Nei’s genetic diversity(h)was 0.1958 and the Shannon’s information index(I)was 0.3186.The differentiation index(Dst)was 0.7648,gene flow coefficient(Nm)was 0.1537.The results showed that 76.48%of the total variation existed among populations,and only 23.52%of the genetic variation within populations.4.The genetic distance of different populations ranged from 0.0117 to 0.7931,the genetic coefficients ranged from 0.4525 to 0.9884.By using the UPMGA cluster analysis,20 populations of Dendrocalamus oldhami Munro can be divided into 3 main groups and 5 small classes.Mantel test for the genetic distance and geographic distance among populations of Dendrocalamus oldhami Munro showed that r=0.4220,p=0.0350<0.05,and shows a significant correlation between genetic distance and geographic distance.5.The fingerprints of 20 germplasm resources of Dendrocalamus oldhami Munro were constructed using ISSR markers.Using the combination of primers UBC857,UBC895 and UBC899,20 populations could be effectively identify with the least number of primers.6.The genetic variation of Dendrocalamus oldhami Munro was small and the genetic relationship was close.This shows that its ability to adapt to environmental changes is not very strong.Therefore,it is necessary to strengthen the protection of the population with high genetic diversity,and to pay attention to the selection and protection of fine tree species in the population,and the preservation of high-quality resources. |
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