ISSR Analysis For Genetic Diversity Of Platanthera Minor In Fujian Province

Platanthera minor is an annual herb belonging to the Platanthera(Orchidaceae),being native to Fujian,Guangdong,Guangxi,Zhejiang and Taiwan provinces,and growing on grassy hillside,forest edge or palisades in the humid areas.However,its resource distribution and genetic diversity at molecular level have not been reported in Fujian.Hence,a comprehensive survey of wild resources for P.minor in Fujian Province is conducted firstly.On this basis,three gene fragments,including ITS,mat K and rbc L,are applied to auxiliary identification for the samples of P.minor.And then,ISSR molecular marker technology is used to carry out the genetic diversity analysis of P.minor with 78 samples in 12 populations,involving the genetic structure,genetic similarity coefficient and genetic distance UPGMA cluster analysis among populations.This study is aimed at exploring adaptability and evolutionary potential of P.minor to environmental changes,and providing theoretical basis for the conservation,utilization and genetic improvement of wild germplasm resources of P.minor.And the main findings can be concluded as follows:(1)The results presents that the wild resources of P.minor are distributed in Fuzhou,Nan ping and other cities in Fujian Province,ranging from 250 meters to 1930 meters,but concentrating in the middle and high elevation area(700 m meters to 1400 meters).From the perspective of habitat,the coenotype of the P.minor are diverse and the species of associated plants rich,containing more than ten species of other orchids,such as Tainia dunnii,Herminium lanceum,Goodyera schlechtendaliana,Pogonia minor,Spathoglottis pubescens.(2)Three gene fragments,namely ITS,mat K and rbc L,are employed to auxiliary identification for the P.minor collected in the field.The results demonstrate that in the NJ and ML topological trees constructed based on ITS sequences,the collected samples are clustered into a branch with P.minor on NCBI.The phylogenetic tree constructed based on the other two gene fragments fails to achieve resolving effect and the reason might be that the evolutionary rate of these two gene sequences is not so fast as that of nuclear genes,which are more appropriate to the phylogenetic studies researching genus and taxon at higher rank.In conclusion,ITS gene is more suitable for molecular identification of P.minor on the basis of morphological identification.(3)A total of 12 primers with rich polymorphism screened from 100 ISSR primers are used in the ISSR-PCR amplification experiment of P.minor.As shown in the experiment,a total of 104 bands are amplified by 12 primers,of which 98 are polymorphic,and the ratio of polymorphism is 94.23%.The results analyzed by POPGENE 1.31 are presented as follows: PPF=96.15%,Na=1.9615,Ne=1.6321,H=0.3609,I=0.5319.Additionally,there is high genetic differentiation among populations of P.minor(Gst =0.2335),and the genetic variation of intra-population(76.65%)is more abundant than that of inter-population(23.35%).And on account of Nm(1.6409)being at a high leveland the frequent gene exchange among populations,the genetic differentiation could be effectively prevented.It can be found that the P.minor has rich genetic diversity,strong adaptability to the environment as well as great evolutionary potentiality.(4)On the basis of genetic similarity coefficient of the populations,UPGMA cluster analyses in the populations of P.minor are conducted with NTSYS-pc software.Moreover,The range of genetic distance and genetic similarity coefficient among the populations are0.0503-0.4582 and 0.6324-0.9510 respectively.Besides,the minimum genetic distance exists between XCZ(1)and XCZ populations,which is 0.0503;The maximum exists between HXX and BYX populations,namely 0.4582.And then UPGMA cluster analyses illustrates that the 12 populations could be divided into two branches when the threshold value is 0.76,one of which is HXX and GDZ,and the rest of which is another branch.When the threshold value is 0.848,the 10 populations of the second largest branch could be grouped into four subbranches: BYX,HXZ and JTZ,PYZ and LXX,YYZ,HKZ,XCZ(1),XCZ and WYSZ.The clustering results assumes that most of the geographically similar populations are clustered together,indicating that genetic distance is closely connected to geographical distance.To sum up,from the view of ISSR molecules,P.minor possesses rich genetic diversity and strong adaptability to the environment.Furthermore,the genetic variation within populations is greater than that among populations and the level of gene flow is high.In addition,the P.minor has great evolutionary potentiality,whose genetic distance is closely associated to the geographical distance.The genetic characteristics mentioned above are consistent with the widespread distribution of this kind of species in Fujian Province,and the appropriate countermeasure is to protect those fields where P.minor grows.In future,the systematic study of genetic diversity on P.minor could be carried out on a national scale,and the artificial reproduction and application of P.minor could be further explored.