Development Of EST-derived SSR Markers And Analysis Of Genetic Diversity In Tobaccos(Nicotiana Tabacum L.)

Tobacco(Nicotiana tabacum L.)is not only an important economic crop in the world,but also a very classical model plant used in molecular biology and genetic engineering.China is the largest country in the world for tobacco production and consumption,and the quality of tobacco has a direct impact on the national economy.However researches in tobacco genetics and molecular breeding,because of its narrow genetic bauckground and shortage of genetically diverse parents,particularly less harmful or harmless breeding,are lagging behind.Molecular markers-aided breeding technology is widely studied and applied owing to its great capacity for increasing breeding efficiency.SSR(Simple Sequence Repeat)marker technology initially developed in 1990s and based on PCR technology is one of many marker technologies.With the advantages of large number and uniform distribution in the whole genome,high polymorphisms,repeatability and co-dominant character,SSR is widely used in various fields of life sciences.Therefore,the large-scale development of SSR markers in tobacco will have great significance on genetic diversity study,construction of genetic linkage map,tagging main agronomic traits loci and molecular marker-assisted breeding of Nicotiana tabacum L.In this study,based on EST sequences and genome sequence of N.sylvestris and N.tomentosiformis,EST-SSR and genomic SSR were identified and the distribution frequency and other characteristics of EST-SSR and genomic SSR markers were analyzed.And further EST-SSR markers were successfully developed experientally.Genetic diversity among 20 germplasm resource was assessed by using these EST-SSR markers.The main results are as follows:1.The frequency and characteristics of EST-SSR.A total of 429,869 ESTs from tobacco were downloaded from NCBI(http://www.ncbi.nih.gov/).Totally 38,165 SSRs were identified from 379,967 Uni-ESTs,accounting for 10.04%and including 124 SSR motifs.The frequency of SSR was 1/5.52kb.Analysis of SSR repeat types revealed that the mononucleotide and tricleotides were the dominant ones,accounting for 40.53%and 34.51%,respectively.Among all of the motif,A/T was the most common motif and accounted for 36.61%,followed by AGC/GCT and AG/CT,accounting for 14.83%and 12.16%,respectively.With the genomic SSRs scanned from genomic sequences of N.sylvestris and N.tomentosiformis,its comparison with EST-SSRs was conducted in terms of a number of characteristics.2.Development of the EST-SSR markers in tobacco.By blasting with genomic sequences of N.sylvestris and N.tomentosiformis,the unique match of the EST-SSR primers were obtained.By aligning these unique EST-SSR primers and target genes to the genomic sequences,the primers which near the target genes were selected.Finally 86 primer pairs including 38 primers near the genes were designed for detecting the polymorphism among 2 pairs of parents of two recombinant populations.Among these primers,47 pairs PCR amplified with at least one fragment(70-300 bp),and the others failed to produce PCR amplification bands or produced ambiguous fragments.Fifteen pairs of primers(17.44%)showed polymorphisms in 2 pairs of parent materials.3.Genetic diversity in tobacco germplasm.15 polymorphic primers were used to assess genetic diversity of 20 tobacco accessions.In total,58 alleles were detected in the 20 accessions,each locus producing two to six bands with an average of 3.86.And the polymorphism information content(PIC)for individual primers ranged from 0.16 to 0.71 with a mean of 0.54,indicating a moderate or high level of polymorphisms.From the UPGMA analysis,the genetic similarity coefficients of the 20 accessions range from 0.490-0.93,indicating obvious diversity among these accessions.In a point with a similarity coefficient of 0.49,these accessions are divided into two main groups,I and II.Group I contains two accessions of N.rustica,whereas group II comprised all accessions of N.tabacum,indicating great genetic differentiation between N.rustica and N.tabacum.Group ? was further divided into two subgroups,IIa and IIb,and oriental tobacco(IIa)separated from other N.tabacum accessions(IIb).Despite this,the genetic relationships between the accessions of N.tabacum were very close.In addition,in the PCA scatter plot,the accessions of N.rustica were obviously distinct from the accessions of N.tabacum,strongly supporting the results of the UPGMA analysis in this study.In this study,based on EST sequences and genome sequence of tobacco,15 polymorphic EST-SSR primers were developed.And these SSR markers will provide useful information for the follow-up studies on tagging main agronomic traits loci,construction of genetic linkage map,and molecular marker-assisted breeding of N.tabacum.