Development Of Specific Molecular Markers For Chromosome 9 Of N.plumbaginifolia Based On Transcriptome Sequencing

Tobacco(Nicotiana tabacum Lin.)(2n=4x=48)is Nicotinan(solanacene)plant,which is one of the important economy crops in the world and our country.However,It has been harmed by black shank disease and other diseases,often causing huge losses.N.plumbaginifolia(2n=2x=20)has strong resistance to tobacco black shank,and is cross compatible with common tobacco.It is an excellent source of resistance to tobacco black shank.Distant hybridization is a common method of modern crop breeding,which can introduce excellent characters from the materials of close relatives.In the process of crossing and backcrossing,a large number of non target genes were introduced at the same time of introducing target genes,which may have a negative impact on the good characters of crops.The main content of distant hybridization is to select materials with target genes but less non target genes.Molecular markers can be used for early identification of materials quickly,accurately and on a large scale,and have been widely used in crop breeding.Molecular marker assisted method can be used to screen target materials in distant hybridization.After years of research,TP-1 has been successfully created,which is an alien monomer addition line with chromosome 9 of N.plumbaginifolia.In the future,it will be used to cultivate a new high-quality flue-cured tobacco line resistant to black shank,and then to cultivate a new high-quality resistant variety.In order to speed up the screening progress and improve the screening accuracy,it is necessary to obtain the specific molecular markers of chromosome 9 in the background of tobacco genome.However,there are few studies on N.plumbaginifolia and few available markers.In order to speed up the progress of breeding for tobacco black shank disease by using N.plumbaginifolia,and accurately select new strains with excellent resistance,this study uses transcriptome sequencing,expression quantity analysis to select specific expression sequence,design primers by using specific expression sequence,amplify and verify the designed primers,so as to obtain the specific marker of N.plumbaginifolia chromosome 9 under the background of tobacco genomeThe main test results are as follows:1.Transcriptome data analysisThe young leaves of N.plumbaginifolia(chromosome donator),TP-1(monosomic alien addition line,the yunyan87 genome is attached to chromosome 9 of N.plumbaginifolia)and yunyan87(chromosome receptor)were selected.Total RNA was extracted by conventional methods,and transcriptome data were sequenced by Illumina Hiseq high-throughput sequencing platform after reverse transcription.9 samples obtained a total of 73.73 Gb Clean Data,and the Clean Data of each sample reached 7.89 Gb.A total of 100121 unigene were obtained by sequence assembly without reference genome.The Cout Numbers of different sequences were compared and counted,and the value of FPKM(Fragments per kilobase per million)was calculated.2.Specific expression sequence selection and primer designSelection in the yunyan 87(no additional exogenous chromosomes of host materials)expression in three repeat quantity(FPKM)values are 0,in N.plumbaginifolia(chromosome source species)and TP-1(monosomic alien addition line)expression in three repeat quantity(FPKM value)at least two repeated expression is the sequence of 0 is specifically expressed sequence.942 specific expression sequences were obtained.Primer5.0 software was used to design primers for the selected specific expression sequences,among which 718 specific expression sequences could obtain more suitable primers,and more than 90 primers were selected.Two or more primers were obtained for some specific expression sequences,and 740 primers were finally obtained.3.Verification of primersThe genomic DNA of N.plumbaginifolia,TP-1 and yunyan87 were extracted from the their young leaves by the CTAB method.Using N.plumbaginifolia,TP-1 and yunyan87 total genome DNA as templates.The primers were selected to select suitable amplification system for PCR amplification,and polypropylene gel electrophoresis was used to detect the products.The results showed that 51 pairs of primers could not amplify the products in 3 materials,472 pairs of primers could amplify the products in 3 materials,166 pairs of primers could amplify the special spots of blue Jasmine tobacco in TP-1,which accounted for 22.43% of the total primers.The gene number is c557171?c0 has two pairs of specific primers and the gene number is c63766?c0 have three pairs of specific primers.The 163 specific sequence of primer is the specific expression sequence of chromosome 9 and the genomic DNA sequence of the primer is the specific genomic DNA sequence of chromosome 9.4.Product recovery verificationTo further validate the screened markers,10 pairs of primers were randomly selected from 166 pairs of specific primers.After PCR amplification,agarose gel electrophoresis was used for detection,and the products in the specific bands were recovered and sequenced.The sequences of specific products amplified from the genomic DNA of N.plumbaginifolia and TP-1 genomic DNA were compared.The results showed that the sequence similarity of the same pair of primers amplified in both N.plumbaginifolia and TP-1 exceeded 95%.It is confirmed that the previously validated marker is indeed the specific marker of chromosome 9 in N.plumbaginifolia.5.Expression quantity and annotation of specific sequencesIn order to preliminarily understand the function of the sequence in which these 166 specific molecular markers are located,multiple databases were used to annotate the sequences.Only 27.71% of the sequences had annotation information.The results of expression analysis showed that among the 163 specific sequences,most of the sequences(143)had less than 10 expression amount(FPKM value)in N.plumbaginifolia,and 55 had less than 1 expression amount.In TP-1,there were 161 specific sequences with expression less than 10,and 109 sequences with expression less than 1.In general,the expression of specific sequence in TP-1 was lower than that in N.plumbaginifolia,and the expression of specific sequence in TP-1 was still lower than that in N.plumbaginifolia after the weighted comparison of chromosome number.It can be seen that the expression sequences of specific markers are mostly functionally unknown,and the expression of specific sequences is at a low level.Moreover,under the new genomic background,the expression of specific genes on chromosome 9 of N.plumbaginifolia may be inhibited.In summary,it is feasible to develop specific molecular markers for chromosome 9 of N.plumbaginifolia under the background of tobacco genome by using specific expression sequence.166 specific markers have been obtained in this study,which provides an important reference for the cultivation of high-quality new strains of tobacco resistant to black shank disease by using heteromonomer addition line TP-1.