Development Of SSR Primers And Analysis Of Genetic Diversity Of Germplasm Resources

Juema is a Rosacrae,Potentilla,a variant of Potentilla anserina L.,Perennial herb,Tibetan Plateau is the unique plant resources,is the Qinghai-Tibet Plateau region unique resources of plants,Juema root was swollen only in the Qinghai-Tibet Plateau and the nearby areas.Juema middle or end of the root of the bulging,forming a spherical,spindle-shaped or line-like,in the autumn and winter season,Juema root bark brown or brown,fleshy white,slightly waxy,sweet.Research purpose and significance:Development of SSR primers,The genetic diversity of Potentilla germplasm resources was studied from physiological and biochemical and molecular level,Is the key to the underlying Juema germplasm in situ conservation and ex situ conservation,Screening for the Qinghai-Tibet Plateau Juema excellent germplasm resources directed breeding,To protect the Qinghai-Tibet Plateau Juema wild grown resources,The research on the platycladus orientalis system in the Qinghai-Tibet Plateau provides a scientific theoretical basis for further development and utilization.Research content:(1)using magnetic beads enrichment method developed Potentilla SSR primers orthogonal experiments were used to screen primers with high polymorphism,The annealing temperature of the SSR reaction system and the primer were optimized,Which laid the foundation for the follow-up study of SSR molecular markers.(2)using SSR molecular markers of 14 populations of 127 parts of the genetic diversity Juema material,Analysis of population genetic structure and genetic diversity within Juema Juema populations and among populations.(3)We used the technique of polyacrylamide gel electrophoresis analysis of the peroxidase isozyme of 75 potentilla anserina.Genetic diversity was analyzed from the level of the protein.The main conclusions are:(1)Development of Juema SSR Primers by Magnetic Beads Enrichment.According to the sequencing results,Primer5.0 software for primer design,the final design of 40 pairs of primers.According to the screening and PAGE of agarose gel electrophoresis,20 pairs of primers with high amplification effect and high polymorphism were selected.The length of the target fragment was 71 bp ~ 3160 bp.(2)In this paper,the concentration of template DNA,Mg2 +,dNTP,primers and Taq DNA polymerase were optimized by orthogonal design.L16(45)was used to optimize the five factors.Finally,the optimal reaction system for PCR was establishedTaq enzyme 0.7U,dNTP0.09 mmol × L-1,Mg2 + 1.8 mmol × L-1,primer 0.4 ?mol × L-1,the optimal concentration of template DNA is 125 ng,2?L10 × PCR Buffer.(3)20 pairs of SSR primers were found in 414 alleles were polymorphic loci,the percentage of polymorphic loci was 100%.The average number of PIC was 0.8275,the Na was 7.878,Nei's(1973)was 0.818,and the Shannon's information index was 2.218.20 pairs of primers can reflect the genotype diversity of Potamogeton germplasm resources,and can be used as molecular markers for the genetic diversity analysis of Potamogeton germplasm resources.(4)The similarity coefficient of 127 materials in 14 populations was 0.7971 ~ 0.9976,the average value was 0.8651,and the genetic relationship among the materials was close.Cluster analysis showed that the similarity coefficient 0.8500 when divided into two categories.(5)The Fst is 0.096,Fis is 0.031,The mean Nm of gene flow was 2.358 and the total gene diversity was 0.109.The population genetic diversity(Hs)was 0.086.That is,the genetic variation among populations is 9.6% of the total genetic variation,and 90.4% of the variants exist between individuals,which can show that the genetic and variation of Potentilla arundinaceus occurs mainly between individuals,and the variation among groups is low.The level of gene flow among the populations was high.The results of the population structure show that when K = 2,127 materials were divided into two categories,and the maximum likelihood of Q-matrix was selected.The genetic range of 14 populations was 0.9872 and the genetic distance was 0.0218.According to the genetic distance,the UPGMA method was used to obtain the clustering results of 14 populations,which were divided into two groups,(6)A total of 14 bands were obtained by POD electrophoresis staining of 75 species of Potentilla chinensis germplasm resources.There are 3 to 10 different bands of different ferns.There were 4 bands in the first region and 5 bands in the zone ?.The percentage of polymorphic loci in the zone 14(with 5 bands)was 100%.75% of the material had a similarity coefficient of 0.214 and 1.000,The average value of 0.681,in the similarity coefficient of 0.61 is divided into two categories.To sum up,the SSR molecular markers and peroxidase isoenzymes were used to analyze the genetic diversity of Potamogeton germplasm resources.The results of cluster analysis using isoenzyme and SSR molecular markers were the same.Juema high genetic diversity.The genetic diversity among the populations was lower than that of the populations.The allele variation of the total population of Potentilla anserina was large,and the genetic diversity of the population was very abundant.The Qinghai-Tibet Plateau and its surrounding areas,due to the special environmental conditions make the distribution of Potentilla anserina in the Qinghai-Tibet Plateau has been in the wild state,In order to survive and survive,Potentilla needs to adapt to a variety of microenvironment,and harsh environmental conditions are important causes of rich genetic diversity.