Development And Preliminary Application Of SNP Markers Based On Tobacco Transcriptome

Tobacco(Nicotiana tabacum L.)is one of the most important economic crops in China.However,tobacco breeding is faced with many problems,such as lack of parents,narrow genetic background,blindness and repeatability.SNP markers are widely used in the identification of genetic relationship,the construction of genetic map,and molecular marker assisted selection of crop germplasm resources.High throughput transcriptome sequencing provides a lot of information for the development of SNP markers.It has been successfully applied to the genetic and Breeding Research of rice,wheat,radish and other crops,but its application in tobacco has not been reported.Therefore,in this study,based on a large number of unigenes obtained from tobacco transcriptome sequencing,a large number of SNPs were identified by bioinformatics methods,and then AS-PCR and kasp markers were developed according to SNP site information,which were applied to the genetic diversity analysis of tobacco germplasm and the fingerprint construction of core germplasm.The specific research results are as follows:1.High throughput Illumina hiseqtm was used to sequence the transcriptome of 10 tobacco materials.A total of 108.04 G clean bases were obtained,with an average of10.084 g per sample.The total number of SNP loci was 121416.Among them,there are 78236 transformed SNPs,accounting for 64.4%;42842 transformed SNPs,accounting for 35.3%;the ratio of the two is 1.83:1,close to 2:1.In the transformation type,C-> t is the majority,while G-> T is the majority.2.SNP was detected by AS-PCR,ARMS-PCR and KASP.The results showed that both AS-PCR and kasp can detect SNP effectively,with good repeatability and stability.The results showed that the optimal PCR system for SNP detection was as follows: 5 ul 2×Taq mastermix?,FA/FB primer 0.25 ul,DNA 30 ng,ddH2 O complement to 10 ul.The optimal PCR amplification procedures were: pre denaturation at 95 ? for 3 min,denaturation at 95 ? for 30 s,annealing at the optimal annealing temperature for 30 s,extension at 72 ? for 1 min,35 cycles,and extension at 72 ? for 5 min.3.265 groups of AS-PCR primers and 30 groups of kasp were designed.The optimized AS-PCR reaction conditions and kasp technology were used to detect 30 SNPs at the same time.Results: 42 groups of AS-PCR primers could detect 8polymorphic SNPs,and 11 groups of kasp primers could detect 11 polymorphic SNPs,indicating that the detection efficiency of kasp was higher.However,compared with AS-PCR,kasp has the advantages of high cost,complicated instruments and long time consumption.4.The ORF finder in NCBI database was used to predict the open reading frames of 11 SNP sites.The results showed that: among the 11 SNPs,S368 was located in the 5'UTR region,s382 was in the 3'UTR region,and the other nine SNPs were in thecoding region.Six SNPs could cause missense mutations.According to the GO annotation and KEGG enrichment analysis of transcriptome data,it was found that the functions of 11 polymorphic SNP genes were related to cyanamide acid metabolism,starch and sucrose metabolism,secondary metabolite biosynthesis,phenylpropane biosynthesis,plant pathogen interaction and other biological processes.5.A total of 22 different allelic loci were detected in 94 tobacco materials with 16 SNP primers.The pic value of polymorphism information content ranged from 0.022 to 0.375,and the average pic value was 0.287.The genetic similarity coefficient of 94 tobacco materials ranged from 0.22 to 1.00,and the genetic diversity was rich.When the similarity coefficient is 0.73,94 tobacco materials can be divided into four categories;when the similarity coefficient is 1.00,94 tobacco materials can be divided into 34 categories.Eleven polymorphic SNP markers could distinguish 24 tobacco materials.The fingerprints of 34 tobacco germplasms were constructed using 11 polymorphic SNP markers.