| Pinus massoniana,whose plantation area ranks the first in China,is one of the most important timber resources in the south of China for its high quality.At present,a variety of molecular marker technology has been used for the genetic diversity of Pinus massoniana,but none of the co-dominant genetic marker based on the sequence of Pinus massoniana had yet been developed so far,which did not meet the needs of molecular marker-assisted breeding and genetic diversity establishment of P.Massoniana.In order to elucidate its genetic information comprehensively and optimize its breeding,transcriptome based simple sequence repeats(SSR)markers and genetic diversity of P.massoniana germplasm were developed and established in this work.Based on the sequencing results of the transcription group of Pinus massoniana,the distribution types and characteristics of the transcriptome sequence were analyzed.The SSR primers were designed and identified.Totally,143 germplasm,which were collected from three P.massoniana Germplasm Conservation Centers were used here in,systematic analysis i.e.genetic diversity,genetic relationship,the degree of inter-and intra-germplasm similarity,as well as the genetic structure of group population,were conducted,which might added new pieces to the genetic puzzle of P.massoniana and provided new insights for its germplasm collection and conservation.The main results are as follows:1)Original SSR primers of P.massoniana were developed independently.Totally,3,329 SSR loci were detected in 7,086 gene clusters(Unigene),distributed on 3,074 Unigenes,with a frequency of 4.69% and an average distance of 20.94 kb.The dominant repeat motifs in the transcriptome were mononucleotides,trinucleotides and dinucleotides,accounting for 40.16%,32.83% and 20.94% of the total SSR loci,respectively.A/T,AT/AT and AAG/CTT were the dominant repeat elements of mononucleotides,dinucleotide and trinucleotide,respectively,which accounted for 97.68%,71.45% and 21.70% of the total SSR repeats.According to the sequence of transcripts,200 pairs of primers were designed and synthesized,and the amplification rate of primers was 78.5%.The product of 24 pairs of high polymorphic primers was between 100 bp and 500 bp.(2)Genetic divisity of P.massoniana SSR sites were detected.137 alleles had been detected from 24 SSR sites.The polymorphism information content(PIC)was 0.703.The mean value of the primer index(Marker index,MI)was 4.530.The average value of Resolving power(Rp)was 2.35.According to the PIC value,the validity of the 24 pairs of primers was sorted as: Pms164?Pms142(Pms184)?Pms114?Pms122?Pms134?Pms109 ? Pms165 ? Pms120 ? Pms190 ? Pms130 ? Pms22 ? Pms107 ? Pms117 ?Pms126 ? Pms54 ? Pms65 ? Pms13 ? Pms55(Pms77)? Pms49 ? Pms88 ? Pms97 ?Pms33.Polymorphism index showed that the SSR loci were highly polymorphic.(3)A germplasm DNA fingerprint was constructed.The optimal primer combinations(Pms164 and Pms184)were used to distinguish 27 clones completely,the relevant fingerprints and manual comparisons identification diagram(MCID)were constructed.Fingerprints and polymorphism indications SSR primers based on the sequence design of the transcripts could obtain high polymorphism potential and rich genetic information by fewer primers,which indicated that SSR markers based on transcriptome information were feasible.(4)Totally,141 polymorphic bands were amplified from 143 germplasm,the percentage of polymorphism(PPL)was 100%,Shannon's information diversity index(I)ranged from 0.5992 to 1.4554,Nei's genetic diversity(H)ranged from 0.4090 to 0.7344,and the heterozygosity(Ho)ranged from 0.2630 to 0.5895,the average expected heterozygosity(He)in the range of 0.4105 to 0.7370,meanwhile,the genetic diversity of pine germplasm resources was abundant.The UPGMA cluster diagram of 143 germplasm showed that the genetic similarity coefficient ranged from 0.45 to 1.00.When the similarity coefficient was 0.64,three categories could be obtained for the tested materials.The first cluster contained all fast-growing clones of Duyun Forest Farm and all the fast-growing and red-heartwood of Wuyi Forest Farm in Fujian.The second categories were 43 fast-growing clones from Nanning,Guangxi.The third categories included high-yield masson pine from Nanning and Zhangping.With 0.73 as the threshold,all the tested germplasm could be divided into 10 sub-categories,which mainly clustered according to the fast-growing germplasm that from Duyun and Zhangping.From the whole cluster diagram,the first division could be divided into three categories: Guizhou Duyun,Fujian Zhangping and Nanning,Guangxi.In addition,the basic clustering was divided based on the materials.(5)The results of genetic diversity among populations showed that Nei's genetic diversity(H)ranged from 0.48 to 0.56 and Shannon diversity index(I)were 0.79 to 0.94.The genetic diversity of Guizhou Duyun(FG-DY)was the highest,and the high yield(HYR-NN)was the lowest.The mean values of observed heterozygosity(Ho)and expected heterozygosity(He)were 0.4517 and 0.5483,respectively.Among which,the heterozygosity(Ho)of the HYR-NN was the smallest,except for the FG-ZP,the average expected heterozygosity(He)of the other five groups was higher than the observed heterozygosity(Ho).The genetic similarity coefficient of the six populations was in the range of 0.15 to 0.43,and the threshold was 0.22,which could be divided into three groups: FG-DY for the first class,HY-ZP and FG-NN constituted the second group,RW-ZP,FG-ZP and HYR-NN formed a third category.Fast-growing resources collected by the seed park were gathered in the ?,??,??? class.The genetic distance between the six populations was in the range of 0.0344 to 0.4274,and the genetic consistency ranged from 0.6522 to 0.9662.RW-ZP and FG-ZP had a close genetic distance,while FG-ZP and HYR-NN showed a farthest distance.The population variation of AMOVA showed that the genetic variation of the tested materials was the most from the group,reaching 93% and 7% of the genetic variation came from all groups.The degree of genetic differentiation between populations was 0.084,indicating that the degree of differentiation among populations was moderate.Gene flow(Nm)was 2.727,demonstrated a certain gene exchange between the populations and had a role of homogenization.Group inbred coefficient(Fis)was-0.2013 in the six populations,and the heterozygote was excessive in the populations.The allele richness(AR)was 2.6545.(6)Structure analysis showed that when K=3,the optimal genetic structure of the tested germplasm was divided into three groups.The first group was mainly composed of FG-DY,RW-ZP and FG-ZP,the second group was mainly composed of FG-NN,and FG-DY,HYR-ZP and HYR-NN constituted the third group.All of the three groups were basically divided according to the provenances,and the degree of mixing was relatively low.However,the mixing degree between FG-DY and RW-ZP was relatively high,and the exchange of genetic material among the groups was relatively large.Only 8 of the tested materials had a Q value less than 0.6,which accounted for 5.6% of the total materials,indicating that most of the germplasm relationships were relatively simple.(7)The results of single germplasm cluster analysis,PCA principal component analysis,intergroup AMOVA analysis and Structure population genetic structure showed that different geographical environment was critical in germplasm evolution,while the genetic relationship and materiality of different germplasm significant correlation is relatively low. |
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