| In present study, ISSR molecular markers were used to analysis the genetic diversity of Cymbidium ensifolium (Linn.) Sw in Jiangxi, The aim was to know more about the genetic diversity, genetic structure and genetic differentiation of C. ensifolium (Linn.) Sw populations. It was to provide theoretical proof for protecting and utilizing C. ensifolium (Linn.) Sw. The main results are as follows.The optimal ISSR-PCR reaction system for C. ensifolium (Linn.) Sw was established. It is25μl reaction volume containing1*PCR buffer,2.5mmol/L MgCl2,200ng templates DNA,160μmol/L dNTPs,1.25U Taq DNA polymerase and0.4μmol/L primer. The optimal amplification procedures were per-denaturing for5min at94℃, followed by40cycles of denaturing for45s at94℃, annealing for45s, extension for60s at72℃. Then extension step of7min at72℃.was performed.In this study,16ISSR primers were screened for the PCR amplification of C. ensifolium (Linn.) Sw,137bands were resulted, of which131were polymorphic bands, and the percentage of polymorphic bands was96.19%. POPGENE analysis indicated the genetic diversity of C. ensifolium (Linn.) Sw at the species level (PPF=76.21%, Na=1.7621, Ne=1.4763, Hpop=0.2730,I=0.4052) and at the population level (PPF=100.00%, Na=2.0000, Ne=1.6696, Hpop=0.3787,I=0.5564). Among the genetic diversity of all the populations, The one of Chongyi population (CY) was the highest (Hpop=0.3280) and the one of Xunwu population (XW) was the lowest (Hpop=0.2082).WINAMOVA analysis indicated the genetic differentiation of C. ensifolium (Linn.) Sw, It ((φST) was0.1557,which showed that the genetic differentiation within populations was greater than the genetic differentiation among populations. The genetic differentiation coefficients (φST) among the populations respectively located in different mountains were as follows from large to small:φST=0.1752among three populations located in Dayuling and Jiulian Mountains,(φST=0.1650among two populations located in Jiuling Mountains,φST=0.1616among eight populations located in Wuyi Mountains,φST=0.1088among four populations located in Luoxiao Mountains. It showed that the genetic differentiation within population separately located those Mountains were higher than the one among populations.Nei’s genetic distance (D) and genetic identity (I) of the seventeen C. ensifolium populations were accounted by POPGENE. It indicated that the genetic distance (D) each other in seventeen populations was in the range of0.0749to0.2819and the genetic identity (I) was the range of0.7543to0.9278in which the genetic distance (D=0.0749) between Jinggangshanshi (JGSS) population and Longnan (LN) population was the smallest and the one (D=0.2819) between Lichuan (LC) population and Yifeng (YF) population was the biggest. UPGMA cluster analysis of Nei’s genetic distance by NTSYS-pc and principal component analysis (PCA) of C. ensifolium (Linn.) Sw populations province by MVSP. That showed Jinggangshanshi (JGSS) population, Longnan (LN) population, Chongyi (CY) population, Ningdu (ND) population, Nanfeng (NF) population was a group, Wanzai (WZ) population, Yifeng (YF) population, Anfu (AF) population, Shaowu (SW) population, Wuyishanshi (WYSS) population, Zixi (ZX) population was a group, Xunwu (XW) population was a group, Guixi (GX) population and Shicheng (SC) population was a group, Anyuan (AY) population and Tonggu (TG) population was a group, and then those groups and Lichuan (LC) population clustered.Mantel statistics and analysis of genetic distance and geographical distance of seventeen C. ensifolium (Linn.) Sw populations was carried out by TFPGA and significance test was also carried out. The result was that negative correlation was presented between genetic distance and geographical distance (r=0.0907, p=0.8240>0.05), which showed that geographical distribution was not related to distance.Some conservation strategies were suggested according to research results and field trips. For example, Moved to protect, In situ conservation, Large-scale artificial cultivation of tissue culture for the Orchid market demand. |
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