Development Of EST-SSR Markers For Taxus Chinensis Var.mairei And Application In Paternal Analysis

Taxus chinensis var.mairei is an endangered and rare species in China.However,genetic analysis of this species lagged behind those of other conifers species due to lack of suitable molecular markers.Based on the transcriptome sequences of four Taxus species from NCBI database,we developed a series of EST-SSR markers for Marie yew,also tested their transferability across species and carrying out paternity analysis.The study have important significance for Taxus conservation and molecular breeding.The results were as belows:(1)The characterization of transcriptome sequences of four Taxus from NCBI(National Center of Biotechnology Information)database showed that 2 344,2 361,1 357,2 312 SSR loci were found in Taxus chinensis var.mairei,Taxus cuspidata,Taxus baccata,Taxus media,respectively.In 2-6bp SSRs,Hexa-and Tri-nucleotide were the most abundant SSR motifs in Taxus chinensis var.mairei and Taxus media,while in other two species it were Tri-and Hexa-nucleotide.Moreover,the AG/CT,AAG/CTT and AAGAGG/CCTCTT were predominant motifs for Di-,Tri-and Hexa-nucleotide,respectively.(2)112 polymorphic loci were got from 150 primers based on the number of repeat units?9.The polymorphic rate was 74.67%.59,17,13 and 23 polymorphic primers were developed form Taxus chinensis var.mairei,Taxus cuspidata,Taxus baccata and Taxus media,respectively.(3)A total of 462 putative alleles were generated from 103 polymorphic loci based on 16 Taxus chinensis var.mairei individuals,the number of alleles per locus ranged from 2 to 11 with a mean of 4.41 alleles.The observed heterozygosity(Ho)varied from 0 to 1 with an average of 0.459;The expected heterozygosity(He)ranged from 0.0625 to 0.891 with mean value is 0.513;The inbreeding coefficient(Fis)ranged from-0.654 to 1 with an average of 0.085.The polymorphism information content(PIC)ranged from 0.11 to 0.754 with a mean of 0.453.14 loci having null alleles(Oosterhout value)with the frequency ranged from 0.135 to 0.470.22 loci deviated from Hardy-Weinberg equilibrium(HWE).(4)The average transferability of 107 loci were 96.07%among 5 species.Only the loci Ma-25497-83A did not successfully amplified,the amplification rate of four loci(C-52641-193B,Ma-1402-721B,Ma-186-234D,Ma-6383-968B)were lower than 50%while 100 loci were 100%.(5)PCA analysis for 48 Taxus individuals were consistent with the classification of the species while the phylogenetic analysis of 6 Taxus were similar to the results of other markers.These results all proved the validity and accuracy of the polymorphic loci.(6)Conducting paternity analysis with 10 polymorphic loci by the CERVUS software.Under 95%confidence level(CL),it could assign the real paternity for its 12(accounting for 37.5%of the total offspring),21(accounting for 84.00%of the total offspring)and 7(accounting for 18.91%of the total offspring)progenies from Qiyang,Youxian and Badagongshan populations,respectively.The cumulative exclusion probability for three populations were 98.32%,93.80%,97.40%,respectively.The effective pollen disperal distance ranged from 10 to 25m.