| Atractylodes lancea is a traditional Chinese herbal medicine,which is widely used in anti-aging,anti-cancer,anti-inflammatory,liver-protective,anti-ulcer and so on.Because A.lancea has low seed setting rate and seed survival rate is not high,so that the long-term use of tubers as a provenance for vegetative propagation has resulted in serious degradation of germplasm and frequent occurrence of diseases on a large scale in the artificial planting,which become the urgent practical problems of large-scale planting of A.lancea to be solved.In this study,the genetic diversity of 81 A.lancea materials and 5 Atractylodes macrocephala materials were analyzed using SSR molecular markers developed by ourselves,and a DNA fingerprinting map was constructed as well.At the same time,the phenotypic data and medicinal quality of 29 materials were investigated and the related chemical fingerprints were constructed.The results of the study are as follows:1.A total of 83 pairs of SSR markers with clear and polymorphic bands were identified from the 239 pairs of SSR primers used for genetic polymorphism analysis of the tested materials.The SSR primers of 83 pairs amplified a total of 420 loci.The number of polymorphic sites was approximately 408,accounting for 97.14%.The average values of polymorphism information content PIC,effective allele number Ne,Shannon's information index I,observation heterozygosity Ho,and expected heterozygosity He were 0.43,2.27,0.92,0.36,and 0.47,respectively.The results of the primers with high polymorphic showed that the genetic variation and heterozygosity of the tested materials were high,which were more favorable for cultivation and breeding for screening the materials to reduce the impact of varietal degradation on yield and quality of currently cultivated species.Among them,the highest polymorphic primer is PLD136 with 18 polymorphic loci;The Ho of primer PLD150 is 0,which showed that the sites amplified by this primer are conserved in all materials.2.The coefficient of genetic distance is 0.63 based on the cluster analysis of SSR markers,which could distinguish between A.lancea and Atractylodes macrocephala.81 A.lancea materials could be divided into two large groups when genetic distance was 0.59.In the first large group,there were 71 materials,including most of the tested materials;the second group contained 2 kinds of Bokang I materials and 8 kinds of Yingshan materials.The first large group minght be divided into 4 subgroups when the genetic distance was 0.49.Subgroup i included all the materials of Jingshan,Baokang II and 6 kinds of Baokang I;subgroup ii only had 1 kind of the material of Baokang I;subgroup iii had 13 kinds Yingshan materials;subgroup iv had only one kinds of Baokang? material.Meanwhile,the second group could be divided into two subgroups at a genetic distance of 0.56.Subgroup v had only one of the material of Baokang I;subgroup vi contained one kind of ByIa2 of Baokang I materials and 8 kinds of Yingshan materials.The results of cluster analysis showed that the genetic distance of Jingshan materials was the closest,and it was clustered in subgroup i.The cluster analysis result of BaoKang I was the most complex,what showed that the genetic information of Baokang I was the most abundant.Combined with the results of molecular marker and PCoA,it was found that the cluster analysis result was the same as that of the PCoA.The results of population structure analysis showed that the genetic background of Atractylodes macrocephala was the simplest in this study,but the genetic background of Baokang I was complex.3.Genetic analysis of population structure revealed that the genetic variation of different populations came from within the population,not between populations.Nm=1.597,it showed that genetic exchange could be performed normally among the A.lancea populations of Jingshan,Baokang,and Yingshan.Geographic isolation could not hinders gene exchange.The Nei's genetic distance(GD)among the populations of A.lancea was less than 0.1771,and the genetic identity(GI)was greater than 0.8377,indicating that the genetic similarity between the A.lancea populations was high and the variation came from within the population.The results of cluster analysis among populations showed that there was a large difference in the genetic basis between Atractylodes macrocephala and A.lancea.However,the differences between the populations of A.lancea were small,and it was also verified that the genetic variation came from within the population rather than from among the populations.4.Correlation analysis and cluster analysis of phenotypic data and medicinal quality data of the tested materials revealed that the significantly positive correlation(P<0.01)between the main stem diameter and plant height and the drying rate of rhizomes,and the correlation between the plant height and upper branches was found;the significant negative correlation displayed between the oil area,the total root area ratio Q and atractylodine content of rhizomes;and the significantly positive correlation between the ?-eucalyptus content and the ether extract content.5.The HPLC chemical fingerprints were established based on the test materials and the similarity of the chemical fingerprints was compared,and the clustering results after quantification of the common peaks' areas were analyzed.The result indicated that the similarity of the fingerprints of the test materials was between 0.80-1.00,which showed that the chemical compositions of the different populations of A.lancea were quite different.The chemical compositions of Atractylodes macrocephala was highly similar to that of A.lancea and its similarity reacheed 0.89.Atractylodes macrocephala were clustered into one group alone,and all A.lancea materials were clustered into one group,which was consistent with the clustering results of SSR markers. |
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