| Glyptostrobus pensilis(Staunt.)Koch)is a first-class protected plant in China.Its molecular genetic research lags behind other conifer species.At present,there are no studies or reports on SSR molecular markers.In this study,EST-SSR markers were developed by sequencing the transcriptomes of Glyptostrobus pensilis.Using the polymorphic sites developed,genetic variation analysis was performed on representative populations of all existing distribution areas of Glyptostrobus pensilis(China,Vietnam,and Laos)to detect their genetic diversity levels and genetic structure,and to understand their evolutionary history and differentiation information.Relevant research work is of great significance in understanding the crucible crisis system of Glyptostrobus pensilis and carrying out population science protection.(1)A total of 106 901 Unigene were obtained after the assembly of the transcriptome sequences of Glyptostrobus pensilis,and the total length was 93.240 Mb,and the average length was 872.240 bp.12 279 SSR loci were screened out using the MISA software to search for EST-SSR loci,distributed on 9597 Unigenes with an average distribution distance of 1/7.590 kb.The transcriptome SSR of Glypostrobus pensilis was dominated by three and four nucleotide repeats.(2)SSR sequences with a motif greater than or equal to 8 were screened and a total of 170 pairs of primers were designed(8 of them were provided by the Royal Botanic Garden,Edinburgh,UK).Among them,130 pairs showed clear bands with an amplification efficiency of 76.47%.Sixteen samples of Glyptostrobus pensilis showed that 27 loci were polymorphic with a polymorphism ratio of 20.76%.The genetic parameters of the 23 loci selected indicated that the average observed heterozygosity(Ho)and expected heterozygosity(He)were 0.273 and 0.161,respectively,and the average polymorphism information content(PIC)was 0.322.The results of PCA analysis showed that the 23 sites developed had strong ability to distinguish different groups and individual plants and could be used for genetic diversity analysis of subsequent populations.(3)Analysis of genetic variation in 14 groups(343 samples)from China,Vietnam,and Laos.The results of genetic diversity showed that the overall genetic diversity of Glyptostrobus pensilis was low.The average allele(Na)was 2.335,the Shannon information index(I)was 0.459,the average observation heterozygosity(Ho)was 0.317,and the average expected heterozygosity(He)was 0.272,and the inbreeding index(Fis)was-0.175;the genetic diversity of the domestic population was lower than that of the foreign population,and the genetic diversity of the artificial population was higher than that of the near-natural population.The genetic differentiation of Glyptostrobus pensilis was high,45.10%of the genetic variation existed among populations.The standard genetic differentiation coefficient(Gst)was 0.429,and the total genetic variation(Ht)between populations was 0.481.The genetic variation(Hs)was 0.278.The results of molecular genetic variance(AMOVA)analysis showed that genetic variation within the population was higher than between groups and between groups.The average value of Nm was 0.304,and the degree of gene flow among the populations was not significant.Mantel's test results showed that there was a significant correlation between genetic differentiation and geographic distance(r=0.613).The results of the bottleneck tests under the "TPM" models all showed that all Glyptostrobus pensilis had experienced the recent bottleneck effect.The results of STRUCTURE analysis and CLUMPP analysis show that 13 groups are divided into two groups,Guangdong group,Laos group,and Vietnamese group;Fujian group,Jiangxi group,and Changsha group are another group.The phylogenetic tree analysis results based on NJ method is the same as those of STRUCTURE analysis.The dynamic analysis of the population history shows that when K=2,Scenario3 is optimal(PP=0.870),which indicates that 218 000 years ago,Glyptostrobus pensilis was differentiated from Fujian,Jiangxi,and Laos in Guangdong.When K=3,Scenario 1 is the optimal model,and the subsequent probability(PP)is 0.573,which indicates that in 756000 years ago,Glyptostrobus pensilis retreated to the Jiangxi region in Fujian,and the population in Fujian and Jiangxi Province was divided into 280 000 years ago.Out of Cantonese groups and groups in Vietnam and Laos.The results of the historical dynamic analysis suggest that the time for the differentiation of the water pine was approximately the same as that of the Quaternary glacier. |
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