Genetic Diversity Analysis Of Gentiana Crassicaulis (Gentianaceae) Using AFLP Markers

Objective:To study the genetic diversity of Gentiana crassicaulis Duthie ex Burk.,an alpine medicinal plant,and explore the phylogenetic relationships between them,to provide molecular biological evidence for the conservation of germplasm resources and the identification of the crude drug and the evaluation of its quality.Method:Using the AFLP(amplified fragment length polymorphism)technique,the genetic diversity of G.crassicaulis from 20 populations of different habitats was analyzed with Gentiana straminea and Halenia elliptica as outgroups.DNA fingerprints were obtained by enzymatic digestion,ligation,pre-amplification and selective amplification of genomic DNA.The products of selective amplification were separated by electrophoresis on 10%denatured polyacrylamide gel and were colored by silver staining.Read "0,1" are obtained,using POPGENE 1.32 software calculation polymorphism bands for each pair of primers,polymorphism rate and the effective number of alleles,Shannon information index,and NT SYS was used for UPGMA cluster analysis,classifying G.crassicaulis germplasm resources.PCA and AMOVA,Mantel test and spatial autocorrelation analysis were performed by GenAlEx software.Pearson correlation analysis is performed by SPSS 22.The correlation between genetic diversity and altitude,latitude and longitude were evaluated,and the correlation analysis revealed the relationship between genetic diversity and geographical factors.Results:From 64 pairs of AFLP primers,12 pairs were selected for amplification,and a total of 315 bands were amplified,among which 254 were polymorphic bands,accounting for 80.63%.High genetic differentiation was detected between populations(87%),and low within populations(13%).On the number of alleles between populations(Na)is 1.7270,the effective number of alleles(Ne)is 1.3150,Nei’s(1973)genetic diversity(H)is 0.1899,Shannon information index(Im)is 0.2927,gene flow(Nm)is 0.0719,the group of total variation coefficient(Ht)is 0.1853,the group of between coefficient of genetic differentiation(Gst)was 0.8743.UPGMA tree was topologically consistent with the treatments of traditional taxonomy at the species level,and the populations of G.crassicaulis were divided into two branches:one from Yunnan and Guizhou,the other from Tibet,Qinghai,Sichuan,and Gansu.PCA and Mantel test showed that there was a positive correlation between genetic distance and geographical distance.Also,combined with SSR and SNP markers within cpDNA,the genetic differentiation within Sichuan population S1 was validated.Pearson’s correlation analysis showed that the genetic diversity of G.crassicaulis populations significantly decreased with latitude and elevation increasing.Conclusion:The species of G.crassicaulis has certain genetic diversity and the genetic diversity between populations is more than the genetic diversity within populations.There was a positive correlation between genetic distance and geographical distance.The genetic diversity of G.crassicaulis populations has some correlation with geographical factors.