| There is ancient Platycladus orientalis forest with the maximum cover area and best preserved in the Mausoleum of the Yellow Emperor.The study of genetic diversity is of great significance for the in-depth study of the genetic mechanism and epigenetic characteristics of ancient tree aging.In this study,we focused on the ancient Platycladus orientalis populations in the Mausoleum of the Yellow Emperor,and analyzed their genetic diversity,relationships and population structure using SSR,ISSR and SRAP markers.We also further analyzed the genetic relationship of the 19 most ancient Platycladus orientalis in the yard of XuanYuan Temple.The main findings are as follows:1.Successfully developed 24 pairs of SSR primers from the early transcriptome sequencing results,and 7 ISSR polymorphic primers and 33 pairs of SRAP polymorphic primers were screened out.The average numer of polymorphic loci for SSRs was 2.333.For the 7 ISSR markers,we detected a total of 85 bands and 69 of them were polymorphic,accounting for 81.18% of the total bands.For SRAP markers,we detected a number of 649 bands and 545 of them were polymorphic,representing 83.98% of the total bands.These results showed that these selected polymorphic SSR,ISSR and SRAP markers are effective to apply in the analysis of the genetic diversity of ancient Platycladus orientalis.2.Based on 24 pairs of SSR markers,the genetic differences of Platycladus orientalis populations of different tree ages in huangdi mausoleum were studied.The result showed that ancient Platycladus orientalis population in the Mausoleum of the Yellow Emperor had a moderate genetic diversity(mean He = 0.324).In addition,ancient individuals had a higher genetic diversity than that of modern young individuals.The AMOVA analysis revealed that the majority of genetic variance was occurred within individuals,accounting for 93% of the total variance.This result indicated that there is large genetic variance among individuals in Mausoleum of the Yellow Emperor.Principal component analysis revealed that the 19 ancient individuals in the yard of XuanYuan Temple and ancient individuals aged around 2000 years had a higher geneic variance,which covered all the scope of the variance of individuals,aged around 1000 years and modern young individuals.Population structure revealed that most ofthe 19 ancient individuals in the yard of XuanYuan Temple had a much more diverse genetic pool,compared with other individuals.3.Correlations of co-characterization matrix and similarity coefficient matrix for SSR,ISSR and SRAP were 0.798,0.777 and 0.828,respectively,indicating that SRAP markers had the best performance in revealing the genetic relationship for the 19 ancient P.orientalis.Mantel tests of the co-representation matrix and similarity coefficient matrix of combining three marker types revealed that the correlation factor(0.820)was similar to that of SRAP(0.828),,but higher than that of SSR and ISSR.This result indicated that intergrating three markers had a better performance,compared with SSRs and ISSRs.Moderate genetic diversity was observed by using SRAP markers.The mean value of the expected heterogeneity(He)and Shannon’s information index(I)was 0.285 and 0.429,respectively.Population structure revealed by SRAP markers showed that these 19 ancient individuals could be mainly subdividied into three groups.Among them,No.1 called ‘Cypress planted by the Yellow Emperor’ and No.2 called ‘Han Emperor WuDi’s Cypress for Hanging Armor’represented the two main subgroups.Dendrogram analysis based on unweighted pair-group method with arithmetic means(UPGMA)revealed a similar pattern,compared with principal component analysis(PCA)and population structure,and them were basically the same as that of the three markers,all showing that ‘Cypress planted by the Yellow Emperor’ and ‘Han Emperor WuDi’s Cypress for Hanging Armor’ had a distinct genetic background and different genetic information. |
