| Sophora japonica Linn.is an important medicinal plant resource in China.Almost all organizations,such as roots,branches,barks,leaves,flowers and fruits,which contain flavonoids,are almost used as medicine.Among them,the buds of S.japonica contain rutin(about 13%~28%),quercetin,kaempferol and glycosides,etc.In addition to the constituents above,the fruts of S.japonica contain isoflavones.The functions and components of the organizations of S.japonica which can be used as medicine have the pharmacological effects of clearing heat and purging fire,hemostasis,lowering blood pressure,antioxidant,delaying senility,anti-inflammatory,analgesia,anti-osteoporosis,anti-cancer,eye-opening and qi-invigorating.Aslo,S.japonica is important ornamental and honey-source tree species,which have great application prospects in the collection and excavation of medicinal germplasm,the development of modern forestry and landscape ecology.However,its germplasm resources lack research technology,accurate identification and systematic evaluation.The purpose of this paper is to establish molecular identification technology and molecular identity card database of the clones of S.japonica,by developing g SSR,EST-SSR and cp SSR markers and analyse the old and cultivated populations of S.japonica.using STRUCTURE population analysis,molecular variance analysis,cluster analysis and genetic relationship analysis of The research results showed that the level of the genetic diversity of old and modern cultivars of S.japonica was relatively high.186 clones were screened,based on the strategy of maximizing the number of allele loci,which could be used as part of construction of the core germplasm of S.japonica.in the future.The strategies of collecting and classifying the germplasm resources of S.japonica were propounded,which provided the basis and support for the protection,evaluation and innovative utilization of S.japonica germplasm resources.The major results of the study are the following::(1)Development,validation and screening of SSR primers.Based on magnetic beads enrichment and sequencing,30 sequences were obtained from nuclear genome.41 pairs of g SSR primers were designed,28 pairs were selected by polyacrylamide gel electrophoresis,and 9 pairs were selected.De novo sequencing of the clones of S.japonica clones was carried out through high-throughput sequencing platform,and 100 pairs of primers were screened from 7520 EST-SSR loci.59 pairs of primers were selected,30 pairs were screened and 12 pairs were finally selected.The de novo sequencing of chloroplast genome was carried out by high-throughput sequencing platform.51 primers were selected from 171 cp SSR loci of the whole genome of S.japonica var.violacea Carr.,10 pairs were selected by polyacrylamide gel electrophoresis,6 pairs were selected,and 3 pairs were finally selected.Different primers from different sources and different primers from the same source have some differences in genetic parameters evaluation,genetic relationship analysis and STRUCTURE subpopulation division.It is appropriate to select parental genetic markers and single-parent genetic markers for the study of S.japonica.The average commonality of 3 pairs of cp SSR,9 pairs of g SSR and 12 pairs of EST-SSR primers was over 66% among Sophpra and Maackia,and the polymorphism level of these primers was higher in 100 individuals of five S.japonica cultivation populations,but the variation was small,mainly from among individuals(74%),followed by within populations(22%),with the lowest among populations(4%);the genetic relationship was relatively close,among which the cultivation population of S.japonica in Beijing has the closest genetic relationship with the cultivation population of S.japonica in Liaoning Province.The genetic relationship of cultivation population of S.japonica in Shandong Province,cultivation population of S.japonica in Henan Province and cultivation population of S.japonica in Shanxi Province is distant from above populations in turn.NJ clustering method divides 100 individuals into two groups.Group I contains 17 individuals and Group II contains 83 individuals.STRUCTURE analysis also suggests that they should be divided into two sub-groups.There are fewer immigrant individuals in the old cultivation populations of S.japonica.(2)Clonal identification of S.japonica based on specific alleles and alleles.64 clones of S.japonica were identified by specific allele fragments.24 pairs of SSR primers amplified allele fragment length of small value,large value and size of binary information increased in turn,and 2 clones among 535 of S.japonica may be synonymous.The molecular identity information database of 534 clones of S japonica was constructed by using 14 pairs of SSR primers,and the molecular identity information and file information of each clone were transformed into a visual two-dimensional code graphics database.(3)Evaluating genetic diversity of S.japonica and formulating strategies for conservation and utilization.The old tree of S.japonica has obvious population structure and rich genetic diversity.The genetic diversity in longitude direction is higher than that in latitude direction.The longitude direction gradually decreased from west to east,then increased slightly,and the genetic differentiation was at a low level;latitude direction decreased from middle to south and north,and the level of genetic differentiation was generally low.Its variation originated from inter-individual(68%-69%),intra-populations(28%-30%)and inter-populations(1%-2%);the relationship between the class ? old tree cultivation population and the class?old tree cultivation population of S.japonica was the closest.The genetic relationship of class ? old tree cultivation population,modern clones and cultivars of S.japonica was gradually distant,and the above-mentioned genetic relationship remained unchanged after adding two exogenous taxa of S.davidii(Franch.)Skeels and S.secundiflora(Ort.)Lag ex.DC.and the germplasm resources of S.japonica from the United States and South Korea,respectively.Its genetic distance is small,genetic consistency is large,and its genetic relationship is relatively close.It may come from two STRUCTURE subpopulations.The selection of superior trees is the main way to utilize its variation.The conservation and utilization of S.japonica germplasm resources should be carried out by combining in-situ conservation with in-situ conservation.(4)Based on the strategy of maximizing the number of allele loci,stepwise clustering and random sampling,the core collection of S.japonica germplasm resources was constructed.Based on the strategy of maximizing the number of allele loci and stepwise clustering,the core germplasm of S.japonica could be obtained better.Among them,the core germplasm(186 clones)screened based on the strategy of maximizing the number of allele loci had the highest number of alleles(378 clones)with a retention rate of 97.4%,followed by the core germplasm(155 clones)obtained by the step-by-step clustering method,with an allele retention rate of 95.8%.The 186 clones screened were evenly distributed in the principal coordinate and three-dimensional distribution maps of the original germplasm of S.japonica.Although the t-test results did not reach a significant level,the related data supported that the selected germplasm with the strategy of maximizing the number of allele loci was an important part of the core germplasm construction of S.japonica in the future. |
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