| Sinowilsonia henryi Hemsl.,an endangered tree in family Hamamelidaceae,is a rare plant endemic to China.Resulting of deforestation and habitat changes,the number of the species has decreased dramatically.It has been classified as the protected plants in classⅡin China and the key conservation plant in Shaanxi Province.It is also listed as a vulnerable species by International Union for Conservation of Nature.As a relic species of the Tertiary relic species and endemic species in China,S.henryi is of important value in ecology,economy and scientific research.In this study,the microsatellite molecular markers of S.henryi have been developed based on the second generation sequencing technology.In order to provide theoretical reference for species conservation,the microsatellite molecular markers has been applied to population genetic analysis of S.henryi.The Illumina second generation sequencing technology was used to establish the genome library and the Microsatellite Library of S.henryi.The composition of the microsatellite was analyzed,and the primers of S.henryi were designed.The polymorphism was analyzed by the amplification and detection of 5 S.henryi populations.As the results,38942660 sequences longer than 125bp were returned by the second generation sequencing;among them,200386 splice sequences were obtained by mass pruning and splicing,the number of length is greater than 335bp is 7614.And 694 microsatellite loci were detected in the 7614 sequences,in which the single nucleotide repeat was the most and the number of A/T repeated in the single nucleotide repeat was the most;the dinucleotide length variation was the most abundance.The number of AG/CT repeats was the largest,and the variation in repeat length was positively correlated with microsatellite abundance.The primers were designed for high repetition of 36 S.henryi microsatellite sequences.By PCR amplification and polyacrylamide gel electrophoresis,20 pairs of primers were rich in polymorphism and with clear bands.The average number of alleles（NA）ranged from 3 to 6,with the average of4;the polymorphism information content（PIC）ranged from 0.5355 to 0.7540,with an average of 0.6155.The population genetic analysis of 5 S.henryi populations showed that the genetic diversity of the species was high（h=0.6975,I=1.4368,HE=0.7022）,and the population genetic differentiation was significant(Fst=0.374).The result of population genetic analysis for 5 populations of S.henryi showed that the primers which were developed had good usability.In this study,the primers of microsatellite markers of S.henryi were established,which laid the foundation for the molecular genetics of S.henryi.In this study,20 pairs of microsatellite primer were selected to detect 201 samples which collected from 19 wild populations in the main distribution area of S.henryi.Experimental data was counted,and genetic diversity and genetic differentiation were analyzed.In order to analyze the genetic relationships among the populations,genetic parameters were calculated,such as Nei’s distances between populations,the expected heterozygosity.The index of genetic differentiation and genetic structure among populations were analyzed and calculated.The results show that:（1）20 pairs of primers of S.henryi performed high polymorphism.The allele number of primers（NA）ranged from 7 to 18,and 239 mutation alleles were detected,with the averaging 11.95 mutation alleles per pair of primers.The expected heterozygosity（HE）ranged from 0.6790 to 0.8604,and the average of 0.7993.The index of Shannon’s information（I）ranged from 1.4279 to 2.2222,with an average of 1.8937.The index of Nei’s genetic diversity（h）ranged from 0.6773 to 0.8583,with an average of 0.7973,which suggested that the genetic diversity of S.henryi was high at the species level.（2）The results of AMOVA analysis showed that the genetic variation of S.henryi was mainly in the populations,and the genetic differentiation was significant between populations(Fst=0.3439).（3）The result of UPGMA showed that the populations with similar genetic distances had relatively close geographical distribution.The results of Mantel Test（R=0.288,P=0.010）,indicated there was a significant correlation between genetic distance and geographical distance of S.henryi.The result of STRUCTURE showed that 19 populations of S.henryi were clustered into 4 groups.Geographical isolation,limited gene flow,fragmentation in population history and bottleneck effect may be the main reasons leading to significant genetic differentiation.Based on the results of this study,an in situ protection strategy was put forward for the species. |
