Fingerprints Construction And Genetic Analysis Of Nicotiana Tabacum Based On SSR Markers

Tobacco(Nicotiana tabacum L.)as one of the most important economic crops and common model crop,which was used for the research of plant-virus and molecular biology.However,in the process of using tobacco,there are many problems such as small seeds,similar plant architecture and easy mixing seeds.Therefore,accurate and effective identification methods are very important for tobacco’s application.And it is of great significance to study the genetic background and the relationship of tobacco germplasm resources in order to elevate the genetic diversity of tobacco and cultivate elite new germplasm.In this research,SSR markers are used to construct fingerprints and analyze genetic diversity of 160 different types of tobacco germplasm,which aims at providing reference for the identification and breeding of tobacco varieties.Based on the current tobacco genome data,10 pairs of SSR markers with clear band and stable polymorphism are selected.160 varieties(including 38 main varieties)were scanned and their fingerprints were structured by using the10 pairs of SSR markers.Meanwhile,the genetic diversity and genetic relationship of 160germplasm were analyzed,and the specific results are as follows:(1)Using SSR markers to scan 11 tobacco genome sequences and Arabidopsis genome sequences,which can be found that among the 12 genomes,the number of six nucleotide repeat SSR accounted for the majority,accounting for 57.58%of all SSR,followed by seven nucleotide repeat SSR,accounting for 17.42%;and the number of five nucleotide repeats SSR was the least,accounting for 2.26%.(2)By using e-PCR and other methods,1245 pairs of common markers were found and 120 pairs of markers with high PIC value were selected.There are 2 to 4 representative varieties(only 1 Nicotianal rustica varietie)selected from each tobacco type to verify the SSR markers above,finally 10 pairs of specific markers with high PIC value,good polymorphism and distinct band are screened.(3)160 different types of germplasm materials were scanned with the selected 10 pairs of specific markers to construct their fingerprints,and the fingerprints of 38 main varieties were transformed into fingerprint QRcode for their spreading and application.(4)According to the results of scanning 160 tobacco germplasms(including 38 main varieties) with 10 pairs of specific markers,the average effective number of allelic variation(N_a)of 160 tobacco germplasms was 7.1;the average effective number of allelic variation(N_e)was 4.01;the average observed heterozygosity(H_o)was 0.278;the average expected heterozygosity(H_e)was 0.722;the average Nei’s diversity index(H)was 0.72;the average Shannon’s information index(I)was 1.502 and the average polymorphism information content(PIC)was 0.675.It proved that the selected markers had good polymorphism and the selected tobacco germplasm materials were rich in genetic diversity.(5)The results of UPGMA cluster analysis showed that 160 different tobacco varieties (including 38 main varieties)could be divided into two groups,which N.quadrrivalvis(N.bigelovii)tobacco forms one group and the other groups can be divided into two subgroups called Subgroup A and Subgroup B.Among them,Subgroup A had 62 varieties and Subgroup B had 97 varieties,which most of sun-cured tobacco were in subgroup A while most flue-cured tobacco were in group B.The 38 main varieties were mainly distributed in Subgroup B,while the remaining different types of germplasm materials were evenly distributed in different subgroups.It can be concluded that 122 tobacco germplasm materials have rich genetic background compared with the main varieties,which can be used to broaden and improve the genetic background of the mainvarieties.(6)Through principal component analysis and population structure analysis,160 tobacco germplasms could be divided into two groups,and the distribution of germplasms in the groups were basically similar to UPGMA clustering results,which further proved that compared with the main varieties,the genetic diversity of different tobacco germplasm types was higher,and the elite genetic resources could be selected from them for tobacco improving and breeding.