| Due to the characteristics of rapid growth,strong adaptability and excellent wood properties,Larix olgensisis,the main tree species for afforestation in my country.In recent years,the genetic improvement of L.olgensis has developed rapidly by establishing clone seed orchards.However,the differences between clones are small and difficult to distinguish effectively,which affects variety selection and promotion.Molecular markers are not affected by factors such as the environment,and are widely used in the identification of forest tree species and the analysis of genetic relationship.In this study,the clones and individual plants of the seed orchards of primary clones of L.olgensis were used as the research materials,and SSR and IS SR markers were used to analyze the genetic diversity of seed orchards,and the SSR molecular marker technology was used to identify and fingerprint the clones of L.olgensis.The map construction provides a theoretical basis for the genetic improvement and variety identification of Larix.The results of the study are as follows:SSR primer universality analysis.Using 270 pairs of SSR primers to carry out PCR amplification on a single plant of L.olgensis,there were 108 pairs of primers that could amplify the bands,with a common rate of 40%,50 pairs of primers were polymorphic,and the rate of polymorphic primers was 46.3%.Among them,Pinus dabeshanensis,Pinus armandii and Pinus koraiensis in the Pinus which are far related to Larix,have an average primer universal rate of 24.67%and an average polymorphism rate of 5.92%.The universal rates of Larix kaempferi and Larix gmelinii were 67.16%and 28%,respectively,and the polymorphism rates were 62.5%and 14.28%,respectively.The universal rate of primers for Larix principisrupprechtii was 95%,and the polymorphism rate was 73.68%.The SSR primers of the related species of Larix are highly versatile.Evaluation of genetic diversity of individual plants in primary seed orchards.Using 30 pairs of highly polymorphic SSR primers to analyze the genetic diversity of 105 L.olgensis samples,258 pairs of alleles were detected,ranging from 100bp to 300bp,and the polymorphic site ratio was as high as 99.05%.The number of observed alleles(Na)in the population was 2.833,the number of effective alleles(Ne)was 1.814,the observed heterozygosity(Ho)was 0.404,and the expected heterozygosity(He)was 0.401;the average value of the inbreeding coefficient Fis within the population and the total population inbreeding coefficient Fit is negative,which indicates a slight excess of heterozygotes in the population,the Shannon’s diversity index(I)was 0.694,the Nei’s gene diversity index(H)is 0.4006,and the polymorphism information content PIC value range is 0.0866-0.6486;11 pairs of highly polymorphic ISSR primers were used to analyze the genetiic diversity of 347samples of L.olgensis,and 172 loci were detected,ranging from 100bp to 3000bp.Each primer amplified an average of 15.63 loci,there are 169 polymorphic loci,and the polymorphic loci ratio is as high as 98.26%,the population number of observed alleles(Na)was 1.9826,the number of effective alleles(Ne)was 1.4795,the Shannon’s index(I)was 0.4360,the Nei’s gene diversity index(H)is 0.2855,the genetic diversity parameters of 105 individual plants were calculated,the ratio of polymorphic loci was as high as 91.28%,the number of observed alleles(Na)per plant was 1.9128,the number of effective alleles(Ne)was 1.3400,and the Shannon index(I)was 0.3481,Nei’s gene diversity index(H)was 0.2178.Through comparative analysis of genetic diversity between SSR and ISSR showed that the polymorphism percentage of SSR markers(99.05%)was higher than that of ISSR markers(98.26%and 91.28%),and the rest of the genetic parameters were that SSR markers were higher than ISSR markers,indicating that the average information content of SSR markers is higher,the bivariate correlation analysis results of the two markers show that the two markers are significantly correlated,and the correlation coefficient is 0.979,so the results of the two markers can be verified with each other.It has high consistency and reliability,indicating that both markers can be used for the genetic diversity study of Larix,and the L.olgensis seed orchard has high genetic diversity.Analysis of clone diversity in primary seed orchards.Genetic diversity analysis of 16 clones,the average number of alleles(Na)was 2.6923,the average number of effective alleles(Ne)was 1.6474,and the polymorphism information content(PIC)of primers was between 0.110-0.624.The Nei’s gene diversity index(H)of the clone population of L.olgensis was 0.3349,with a range of 0.1172-0.6758,and the average Shannon’s information index(I)was 0.5909,with a range of 0.2338-1.2420.In order to further analyze the genetic relationship among them,the UPGMA cluster diagram of L.olgensis was constructed,the genetic similarity coefficient was between 0.5882 and 0.9444.Combined with the results of principal coordinate analysis and population structure analysis,the 16 clones were divided into 3 groups.The genetic parameters of the 16 clones were compared with the genetic parameters of a single plant,and it was found that the polymorphism information content of primers(0.297)was slightly lower than that of a single plant(0.354),and the rest of the genetic parameters were less than the genetic diversity of a single plant.However,the genetic diversity parameters obtained by the two markers showed the same trend on the whole,and the differences were not significant,indicating that the genetic diversity of seed orchards based on the individual plant and clone levels was high.Identification of clones in primary seed orchards and construction of fingerprints.Using 13 pairs of SSR primers with high polymorphism and good stability,132 individual plants were identified by DNA typing detected by capillary electrophoresis and bands by polyacrylamide gel electrophoresis.With reference to the clone configuration of the seed orchard,the participating samples were identified as 16 clones.Each pair of SSR primers can distinguish 25 clones of L.olgensis.Using the primer combination method for further identification,only 13 pairs of primers can distinguish 16 clones of L.olgensis.Based on this,the fingerprints of 16 L.olgensis clones based on 13 pairs of SSR primers were established,which provided an effective molecular marker technology for the selection of new varieties of L.olgensis clones and the identification of clones.In conclusion,the SSR primers of the related species of L.olgensis have high versatility,and both SSR and IS SR molecular marker techniques can be used to analyze the genetic diversity of L.olgensis,and seed orchards have high genetic diversity.The SSR primers can be used to molecularly identify the L.olgensis clones,and construct the fingerprint of L.olgensis.The above research results provide theoretical guidance for the selection of hybrid breeding parents and high-generation breeding,and lay a foundation for formulating long-term breeding strategies for L.olgensis. |
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