Genetic Diversity Of Cymbidium Kanran Makino From Jiangxi Province Revealed By Inter Simple Sequence Repeats

In order to know more about the genetic diversity, genetic structure and genetic differentiation of Cymbidium kanran Makino populations and provide basis for the resource protection and development of C. kanran, ISSR molecular markers were used to analysis the genetic diversity of C. kanran, mainly from Jiangxi Province. C. kanran could be divided into three types, spring, summer, and autumn, by their flowering seasons, and two types, the large leaves and small leaves by their leaves size. The main results are as follows.The ISSR-PCR reaction system and amplified procedures for C. kanran were established. It is 25μl reaction volume containing 1* PCR buffer,2.0 mmol/L MgCl2,100 ng templates DNA,200μmol/L dNTP,1.4 U Taq DNA polymerase and 0.4μmol/L primer. The optimal amplification process is as follows. After a pre-denaturation of 5 min at 94℃,40 cycles were performed with denaturing for 45 s at 94℃, annealing for 45 s due to the denaturing temperature selected for each primer,72℃extension for 80 s at 72℃, and then a final extention step at 72℃for 7 min.In this study,12 ISSR primers were screened for the amplification of a totle of 194 individuals of C. kanran from different regions of Jiangxi province and Fujian province,119 individuals from Jiangxi and 75 individuals from Fujian. And 123 bands were resulted, of which 97 were polymorphic bands, and the percentage of polymorphic bands was 78.9%.The ISSR analysis revealed a higher genetic diversity at the species level (PPF= 79.67%, Na= 1.7967, Ne= 1.4461,Hpop= 0.2669,I= 0.3995), and a lower genetic diversity at the population level (PPF= 43.97%, Na= 1.4397,Ne= 1.2478,Hpop= 0.1462,I= 0.2204). It could be found that the genetic diversity of the population from Wuyishan, the small leaves (WYXH), was the highest (PPF=52.85%,Hpop =0.1732,I= 0.2622). From the results of genetic structure analysis, it could be found that the high level of genetic differentiation was found among C. kanran populations from Jiangxi Province, and the genetic differentiation coefficient (GST) was 0.4719, which showed that the genetic differentiation within population were higher than the genetic differentiation among populations. UPGMA cluster analysis and principal component analysis also showed that C. kanran populations in Jiangxi were divided into four branches, branch I contained three populations located in Yifeng and Jing'an of Jiuling Moutains (YH, AH) and Jinggangshan of Luoxiao Mountains (JGS); branch II contained three populations located in Wuyi and Zixi of Wuyi Mountains (ZX, WY, WYXH); branche III contained three populations located in Shaowu of Wuyi Mountains(SW, SWXH); branch IV contained three populations located in Anyuan of Dayuling and Jiulian Mountains (GZ-A), Chongyi of Luoxiao Mountains (GZ-C) and Shicheng of Wuyi Mountains (SC). And then branch I and branch II clustered,branch III and branch IV clustered. This also showed that the genetic diversity structures were different in different mountains, and Mantel test also showed that geographical distribution was not related to distance(r= 0.3791).For C. kanran populations of different flowering seasons from Yifeng, GST among populations was 0.5231, which showed that the genetic differentiation among populations were higher than which of within populations. UPGMA cluster analysis and principal component analysis showed that there was significant difference in the different flowering seasons of C. kanran on the genetic diversity.For C. kanran populations of the different breadth leaves, GST among populations was 0.0363, the level of gene flow Nm was 13.2655, gather than 1, which indicated that gene flow between the two types were frequently. The percentage of the genetic difference of the two types was 7.64%. UPGMA cluster analysis and principal component analysis revealed that genetic differences among C. kanran populations with the large leaves and small leaves were not obvious, from which it could be presumed that they were at the same evolutionary level, and they belonged to the same species, but only the leaves'breadth were different.For the populations from different regions of Wuyi Mountains, GST among populations was 0.3781, which showed the genetic differentiation within population were higher than that among populations. UPGMA cluster analysis and principal component analysis revealed that the structures of the populations of the same mountain range section were different, and Mental test also showed that geographical distribution in Wuyi Mountains was not related to distance (r= 0.5854).In addition, some protection strategies for C. kanran were suggested according to the its genetic diversity in the paper, such as protecting C. kanran in its native environment, indraughting new genus et al..