Genetic Diversity Of Ammopiptanthus Mongolicus Based On ISSR Molecular Markers

Ammopiptanthus mongolicus belongs to the evergreen broad-leaved shrub of the genus Ammopiptanthus.It is the only super-algaline evergreen broad-leaved shrub in the Alashan desert region.It is as a national three-level rare and endangered plant.The population distribution showed a patchy distribution,and its population was significantly reduced due to its fragile ecosystem,extreme climate and human activities.Therefore,exploring the genetic structure and genetic diversity of population under climate change has important reference value not only for the rational utilization of genetic resources and breeding of Ammopiptanthus mongolicus but also for the relationship between the evolution direction and the environment of its growing area under different micro-habitats.The guiding significance laid the foundation for the subsequent research on Ammopiptanthus ongolicus The specific research results are as follows:1.The ISSR-PCR reaction system of Ammopiptanthus mongolicus was optimized by orthogonal test.The optimal reaction system(20 ?L)for the determination of Ammopiptanthus mongolicus was:10?L mix,1 ?L primer,5 ?L DNA template,4?L water.Primers were screened based on this system.A total of 15 primers suitable for Ammopiptanthus mongolicus amplification were screened from 100 universal primers published by Columbia University,namely UBC808,UBC826,UBC827,UBC856,UBC857,UBC888,UBC889 and UBC890.The optimum annealing temperature of the 15 primers was determined by a gradient PCR instrument.The optimal reaction system was finally determined to be:10 u L of mix;1 u L of primer;1 u L of template;8 u L of ddH20.The optimal PCR reaction procedure was:pre-denaturation at 95? for 5 min;denaturation at 95? for 1 min;annealing at 53? for 1 min;extension at 72? for 1 min,35 cycles;extension at 72? for 10 min;preservation at 4?.2.Polymorphic loci in the population of Ammopiptanthus mongolicus showed that the percentage of polymorphic loci(%P)in the population ranged from 9.86(EJNYG)to 29.58%(ALSY and WH).In the results of genetic diversity of 200 samples,the overall expected heterozygosity(HE)was lower than 0.12 and the Shannon index(?)was lower than 0.16,is two populations(WLTH and ALSY)for farthest distance.Although the genetic diversity of the entire species is slightly higher than the genetic diversity within the population,it is still low,especially for HE.3.The genetic variation within the population was small,but the difference between the groups was large.The principal component analysis and cluster analysis of 200 Mongolian sand holly samples from 10 different regions were performed by GenALEx 6.5 software.The results showed that they could be divided into three groups.The first group is the Dengkou of Bayannaoer and the Suhaitu of Alashan.The second group is Nuergong of Alashan,Wulatehouqi,Helanshan,Wulateqianqi,Alashany Youqi,Ejina Yagan,Alashany Zuoqi,Wuhai.The genetic similarity(GS)of Sabina in 10 different regions ranged from 0.747 to 0.911 with a mean of 0.822,and the genetic distance(GD)ranged from 0.094 to 0.291 with a mean of 0.199.The data showed that the genetic similarity of Ammopiptanthus mongolicus was higher in 10 regions,and the genetic distance was closer.From the perspective of genetic similarity,among the different regions,the highest similarity was Nuergong of Alashan and Ejina Yagan(0.9115),the lowest similarity is the Wulateqianqi and Helanshan(0.747).From the genetic distance,the farthest genetic distance between the different regions is the Wulatehouqi and Ejina Yagan(0.291).The closest genetic distance is Nuergong of Alashan and Ejina Yagan(0.094).4.The Mantel test was performed by the vegan data analysis module in the R language to test the correlation between the genetic and geographic distances of environmental difference control and the correlation between them.The results showed that the genetic structure and climatic factors were middle-age precipitation(bio 12)and the wettest.Monthly precipitation(bio 13),wettest season precipitation(bio 16),hottest season precipitation(bio18)(bio12:r=0.563,p=0.005;bio13:r=0.554,p=0.003;bio 16:r=0.567,p=0.004;bio 18:r=0.553,p=0.007)is significantly correlated,and these climatic factors are bioclimatic factors related to precipitation,so the key factor affecting the genetic structure of Ammopiptanthus mongolicus is the difference in precipitation between populations.Geographic isolation is not a condition for genetic diversity among populations.There are genetic exchanges among populations in different geographical regions,and precipitation in the environment is the dominant factor leading to the genetic diversity of its population.