| Fragaria pentaphylla is a perennial herbaceous plant and belongs to Rosaceae,This species widely distributed in the southwest of China.There are two types of this species in the field.In this paper,the genetic diversity and genetic structure of wild Fragaria pentaphylla resources in 15 different populations in the southwest of China were deeply studied and analyzed by using EST-SSR molecular markers technology.It provides an important theoretical basis for the effective utilization and protection of the germplasm resources of Chinese wild Fragaria pentaphylla excellent traits.The main results obtained in this paper are follows:1.Successfully developed Fragaria pentaphylla EST-SSR primers.68,356,166 clean reads has been obtained from the Fragaria pentaphylla transcriptome sequencing,which were assembled to 53748 UniGenes.SSR locus screening was conducted using MISA(MicroSAtellite)software.A total of 9494 SSR loci were detected including 1-6 base repeat units using bioinformatics methods,which were distributed in 7892 UniGene.The frequency of SSR was 14.7%,indicating that the frequency of SSR loci in the transcriptome data was higher.Among them,the mononucleotide repeat unit was the most abundant type,accounting for 39% of all SSR loci,followed by dinucleotide,35% and trinucleotide 25%.The total number of tetranucleotide,pentanucleotide and hexanucleotide repeat units is less than 1%.According to the transcriptome UniGene sequence,172 pairs of primers were designed and synthesized using primer synthesis software Primer 5,and were detected by polyacrylamide gel electrophoresis.Seventeen pairs of primers with high polymorphism,good stability,and good discrimination were screened out.2.The genetic diversity of 15 wild populations of Fragaria pentaphylla was detected and analyzed using 17 pairs of EST-SSR markers.The results show that the total of 94 allele loci were detected using 17 pairs of EST-SSR primer sequences,and the average number of observed alleles per locus was 5.5.The number of effective alleles(Ne)was 41.089 and the average was 2.417.The average values of heterozygosity(He)and observed heterozygosity(Ho)were expected to be 0.536 and 0.423,respectively.The average polymorphism information content(PIC)was 0.494.The Nei's gene diversity index ranged from 0.178 to 0.802,with an average of 0.534.The above results show that the Fragaria pentaphylla has rich genetic diversity.At the same time,According to the Hardy-weinberg equilibrium test,13 of the 17 EST-SSR loci were significantly out of balance due to genetic drift,migration,selection,or interference from other evolutionary factors.3.Genetic structure analysis of different populations of Fragaria pentaphylla.UPGMA cluster analysis,NJ contiguous tree,PCA analysis and structure group assignment analysis all showed that the genetic differentiation of the Fragaria pentaphylla was more obvious.The population of Fragaria pentaphylla white fruit,such as MaYaZi,KuanChuan,ZhengJiaDian,and Gaolou mountain,is clearly clustered into one category.The Gaolou mountain red fruit is also close to the cluster due to its geographical position.Taibai mountain,Jiuzhaigou,Miyaluo road,Maiji mountain,QingXi,Tiantai mountain,the several red fruit populations are clearly grouped together.It shows that Fragaria pentaphylla population is in a moderate genetic differentiation.The results of AMOVA analysis based on the fixation index(Fst)of Fragaria pentaphylla showed the genetic variation of 16.64% of the Fragaria pentaphylla from the population,and 83.36% of the genetic variation from within the population.Although the Fragaria pentaphylla has strong clonality,the self-incompatible breeding system and long-distance pollinators make the gene flow between populations smooth and prevent serious differentiation among populations. |
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