Development And Correlation Analysis Of EST-SSR Primers Of Pinus Massoniana Lamb Fat-producing Traits

Pinus massoniana lamb belongs to the evergreen tree of Pinaceae,Pinus and subgenus of two vascular bundles.Being a coniferous tree species widely distributed in China,it is an important afforestation tree species and is also the dominant tree species in southern China.Both the resin and wood of Pinus massoniana can be used.It is the main fat-producing tree species in China,and the rosin accounts for more than 70% of the national resin production.In today's market,the demand for pine resin is large,however the high-yield strain of Pinus massoniana can be promoted are few,which cannot meet the market's demand.In this case,genetic improvement of the pine traits of Pinus massoniana is of great important.The traditional improvement method is through clones hybridization,but its period is too long.By using methods such as genome association analysis,molecular markers that are significantly related to the fat-producing traits of Pinus massoniana are developed,and molecular marker-assisted selection can shorten the breeding cycle,improve breeding efficiency,and speed up the process of breeding high-yield refined varieties.In this study,three groups of Pinus massoniana clones will be transcriptome sequenced to analyze the type,frequency and distribution characteristics of SSR repeat units in the transcriptome sequence.The study will screen,design and synthesize EST-SSR primers based on the difference in lipid production sequence,and verify the 259 pairs of primers developed.EST-SSR primers with polymorphism were selected for genotyping detection of clones and correlation analysis of fat-producing phenotype data.The aim of this study was to search for the molecular markers significantly related to the lipid production characters of Pinus massoniana,to provide tools for molecular markers selection of Pinus massoniana,to shorten the genetic cycle of lipid production characters of Pinus massoniana,and to accelerate the breeding process of superior varieties.The results of this research are as follows:1.EST-SSR primers for Pinus massoniana based on transcriptome sequencing was developed.Transcriptome sequencing was performed on 6 tissues of needles,terminal buds and phloem of high lipid clones and common lipid clones of Pinus massoniana.The analysis of 124,345 Unigene sequences obtained from transcriptome sequencing revealed that 14,461 SSR sites were distributed in 12,077 Unigene,indicating that the occurrence frequency of SSR was 9.71%.The maximum number of repeat sequences was 10~24 bp in SSR sequence length.The repeat primitive types of SSR are mononucleotide repeats,followed by dinucleotide and trinucleotide repeats.Mononucleotide repeats accounted for 58.18% of the total SSR sequences,and the percentages of dinucleotide and trinucleotide repeats were 20.32% and 19.7%,respectively.The dominant repeating primitive elements were A/T,AT/AT and AGC/CTG.A total of 3873 differentially expressed genes with SSR sequence,including 281 differentially expressed genes in needles,terminal buds and phloem,were obtained from the analysis of the expression difference sequences of high fat and common fat producing Pinus massoniana.A total of 259 pairs of primers were designed and synthesized based on the screening of differential genes.2.Polymorphism detection of candidate gene EST-SSR primers was conducted?PCR was conducted to detect the effectiveness and polymorphism of the designed and synthesized 259 pairs of EST-SSR primers.It was found that 147 pairs of primers could amplify clear bands,and the amplification efficiency of primers was 56.76%.It was also found that 65 pairs of primers had polymorphism,and the polymorphism rate was 25.10%.It is proved that the EST-SSR primer is effective and feasible based on the sequence design of the difference in fat production of Pine masson.3.For the correlation analysis of candidate genes for lipid production traits of Pinus massoniana,the admixture software was first used to analyze the structure of Pinus massoniana population,and the optimal group number was obtained by cross validation based on the clustering results.The result was K=1.Explain that the group structure is simple.Then,four analysis models of EMMAX,GAPIT,TASSEL-GLM and TASSEL-MLM were used to analyze the correlation of lipid production(CZL),daily average lipid production(QRC)and expected daily average lipid production(RC)of Pinus massoniana respectively.Eight significant correlation markers(P<0.05)were detected,and the phenotypic contribution rate ranged from 0.01% to 1.61%.EMMAX and GAPIT models were used to carry out correlation analysis to obtain 1,4,3 and 2,respectively,significantly correlated with lipid production,daily average lipid production and expected daily lipid production of Pinus massoniana lamb.In the GLM model correlation analysis,6 markers were significantly correlated with the lipid production of Pinus massoniana,7 markers were significantly correlated with the daily average lipid production,and 7 markers were significantly correlated with the expected daily lipid production.The MLM model was used for correlation analysis and three markers were significantly correlated with the lipid production of Pinus massoniana,three markers were significantly correlated with the daily average lipid production,and four markers were significantly correlated with the expected daily lipid production.In both models,there were 3 significant markers associated with lipid production,3 significant markers associated with average daily production and 4 significant markers associated with expected daily production.