CN110499388B

CN110499388B - Codominant marker primer group for identifying RTSW allele type of tobacco anti-spotted wilt locus, identification method and application thereof

Info

Publication number: CN110499388B
Application number: CN201910933715.5A
Authority: CN
Inventors: 黄昌军; 刘勇; 童治军
Original assignee: Yunnan Academy of Tobacco Agricultural Sciences
Current assignee: Yunnan Academy of Tobacco Agricultural Sciences
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2023-03-24
Anticipated expiration: 2039-09-29
Also published as: CN110499388A

Abstract

The codominant marker primer group for identifying the type of RTSW allele of the tobacco anti-spotted wilt locus, the identification method and the application thereof, wherein the primer group consists of four single-stranded DNAs of a primer 1, a primer 2, a primer 3 and a primer 4. The co-dominant marker primer can specifically mark and identify the tobacco genome DNA, detect the allele type of RTSW in the tobacco material genome DNA, not only can identify whether the anti-spotted wilt locus RTSW contains the allele type of the anti-spotted wilt locus RTSW, but also can identify the allele type of the anti-spotted wilt locus RTSW of the tobacco, is reliable and convenient to use, has the advantages of close linkage with a target gene RTSW, high accuracy, low cost, high detection efficiency and the like compared with the traditional anti-spotted wilt marker, can greatly improve the screening efficiency of the anti-spotted wilt gene RTSW variety of the tobacco, greatly shortens the breeding cycle of the anti-spotted wilt tobacco variety, and improves the breeding efficiency.

Description

Codominant marker primer group for identifying RTSW allele type of tobacco anti-spotted wilt locus, identification method and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker primer capable of identifying a tobacco anti-spotted wilt gene locus RTSW and application thereof in positioning, cloning or breeding tobacco anti-spotted wilt varieties of the tobacco RTSW disease-resistant gene.

Background

Tobacco spotted wilt disease is a tobacco disease caused by infection with the virus of the genus Orthospovirus (Orthospovirus). The Virus belongs to a plant Virus with the widest host range and the most serious occurrence, and the American type representative species Tomato Spotted Wilt Virus (TSWV) of the Virus poses a great threat to tobacco leaf production in Yunnan tobacco regions of China. In recent years, from the results of sample collection and detection in counties (states and cities) in Yunnan, TSWV on tobacco in Yunnan province has been distributed and tends to expand and aggravate. The main cultivated flue-cured tobacco varieties in Yunnan province do not resist TSWV, and field investigation finds that the existing main cultivated flue-cured tobacco varieties K326, honghua Dajinyuan, yunyan 87 and the like can be infected by TSWV and become potential factors of TSWV epidemic and outbreak. The commonly used prevention and treatment means for the tobacco spotted wilt mainly depends on the prevention and treatment of the virus-transmission mediator thrips, but the thrips have the characteristics of short development period, small and easily concealed individuals, easy generation of drug resistance to pesticides and the like, so the existing prevention and treatment measures are difficult to obtain ideal control effect, and therefore, the breeding of the tobacco variety for resisting the spotted wilt is the most economic and effective means.

The wild tobacco resource contains rich resistance genes. Studies have shown that floral tobacco (Nicotiana alata) has very good resistance to TSWV. Inoculated TSWV showed symptoms of allergic necrosis only in inoculated leaves, and no virus was detected in systemic leaves. Through a series of conventional hybridization and backcross transformation, researchers have transformed the resistance gene from floral tobacco resistance to flue-cured tobacco varieties into a disease-resistant breeding intermediate material Polalta. Resistance to TSWV by Nicotiana alata and Polalta is controlled by a dominant single gene (designated RTSW) site.

Unfortunately, the disease resistance gene RTSW has not been cloned. Under the condition that the disease-resistant gene can not be cloned in a short time, developing a molecular marker tightly linked with the disease-resistant gene becomes an important means for the disease-resistant breeding of the tobacco spotted wilt. Until now, only H.Moon and J.S.Nicholson (2007) develop corresponding molecular markers internationally, but the AFLP markers exist and are far away from the resistance gene inheritance, the linkage is not tight, and the developed SCAR markers have the problem of generating false positive easily in practical application. Moreover, neither of these markers can distinguish between homozygous/heterozygous genotypes at the resistance locus, limiting the large-scale application of these markers. In the breeding process of using the RTSW to resist the spotted wilt of the tobacco, different genotype single plants of the gene can be separated from the filial generation or the backcross generation, so that the genotype of the single plant is required to be detected from the filial generation plant or the backcross generation plant, and the single plant homozygous or heterozygous for the RTSW is selected for further backcross or selfing. Therefore, a co-dominant marker for identifying the type of RTSW allele of the tobacco anti-spotted wilt locus is developed, a simple, convenient, rapid, accurate and sensitive method for identifying the tobacco anti-spotted wilt gene can be established, and the defects of the prior art are overcome.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a codominant marker primer group for identifying the RTSW allelic gene type of a tobacco anti-spotted wilt locus, a method for identifying the RTSW allelic gene type of the tobacco anti-spotted wilt locus by adopting the primer group and specific application of the primer group in positioning, cloning or breeding tobacco anti-spotted wilt disease resistance genes.

In the context of the present invention, the term "anti-spotted wilt gene site RTSW" refers to a site comprising a chromosomal segment of wild tobacco leaf blight resistance gene (RTSW gene).

In order to achieve the purpose, the invention adopts the following technical scheme:

the codominant marker primer group is used for identifying the type of RTSW allele of the tobacco anti-spotted wilt locus and consists of four single-stranded DNAs (deoxyribonucleic acids) of a primer 1, a primer 2, a primer 3 and a primer 4; the sequence of the primer 1 is Seq ID No.1:

RTSW_Marker2_F 5’-GTTATAAATTGTGTGGAGGGCATG-3’；

the sequence of the primer 2 is Seq ID No.2:

RTSW_Marker2_R 5’-TCAAATTCCCGACCAGCAGTA-3’；

the sequence of the primer 3 is Seq ID No.3:

rtsw_Marker1_F 5’-CCGGAATAAGGGTAAGGTCTG-3’；

the sequence of the primer 4 is Seq ID No.4:

rtsw_Marker1_R 5’-CAACAACAACAATCGGGTGA-3’。

the method for identifying the RTSW allele type of the tobacco anti-spotted wilt locus by adopting the primer group comprises the following steps:

(1) Respectively taking the genomic DNA of tobacco to be identified, tobacco spotted wilt resistant Polalta and spotted wilt susceptible variety K326 as templates, carrying out PCR amplification on the genomic DNA by using a PCR primer group consisting of four single-stranded DNAs of a primer 1, a primer 2, a primer 3 and a primer 4, and obtaining a fragment with the size of 736bp by using the amplification of the primer 1 and the primer 2; the 736bp fragment is peculiar to a plant containing the RTSW gene, and the sequence of the fragment is SEQ ID No.5; the fragment obtained by amplification of the primer 3 and the primer 4 has the size of 214bp, and the 214bp fragment is specific to a plant without the RTSW gene and has a sequence of SEQ ID No.6;

(2) And (3) carrying out electrophoresis detection or sequencing on the amplified PCR product, and identifying and determining the type of the RTSW allele of the tobacco anti-spotted wilt locus to be identified according to the following method:

1) If the band of the PCR amplification product of the tobacco to be identified after electrophoresis or sequencing is the same as the band type of Polalta and the size is 736bp, the tobacco to be identified is the anti-spotted wilt tobacco or the candidate is the anti-spotted wilt tobacco, and the genotype is the homozygous resistance RTSW gene locus, namely RTSW/RTSW;

2) If the band of the PCR amplification product of the tobacco to be identified after electrophoresis or sequencing is the same as the band type of K326 and the size is 214bp, the tobacco to be identified is the tobacco with the spotted wilt or the candidate is the tobacco with the spotted wilt, and the genotype is homozygous RTSW gene locus, namely RTSW/RTSW;

3) If the bands of the PCR amplification product of the tobacco to be identified after electrophoresis or sequencing contain bands with the same types as Polalta and K326 bands, and the sizes of the bands are 736bp and 214bp, the tobacco to be identified is the anti-spotted wilt tobacco or candidate anti-spotted wilt tobacco, and the genotype is the heterozygosis of the anti-disease RTSW gene locus, namely RTSW/RTSW.

The invention provides application of the codominant marker primer group for identifying the type of RTSW allele of the tobacco anti-spotted wilt locus in positioning, cloning or breeding tobacco anti-spotted wilt disease-resistant genes.

Furthermore, when the invention extracts the tobacco DNA to be identified, the tissue of any one part or a plurality of parts of the seeds, leaves, roots and flower organs of the tobacco can be adopted.

Further, the invention uses the PCR primer composed of four single-stranded DNAs of primer 1, primer 2, primer 3 and primer 4 to perform PCR amplification on the genome DNA, and the reaction system of PCR is as follows: respectively taking the genomic DNA of tobacco to be identified, tobacco spotted wilt resistance Polalta and spotted wilt susceptible variety K326 as templates, and performing PCR amplification system: 2 XPremix Ex Taqmix PCR Buffer 12.5. Mu.L, 10. Mu. Mol/L primer 1, primer 2, primer 3 and primer 4 each 0.5. Mu.L, 50 ng/. Mu.L template DNA 1. Mu.L, adding sterile double distilled water to make the total volume 25. Mu.L; the PCR reaction program is: pre-denaturation at 94 ℃ for 5min; then 35 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; after circulation is finished, extension is carried out for 10min at 72 ℃; storing at 4 deg.C.

The PCR primer group consisting of four single-stranded DNAs of a primer 1, a primer 2, a primer 3 and a primer 4 comprises a primer group of basically the same DNA fragments obtained by amplifying 1-30 bases at the 5 'end or the 3' end of Seq ID No.1 or Seq ID No.2 or Seq ID No.3 or Seq ID No.4 respectively.

Compared with the prior art, the invention has the beneficial effects that:

the primer group is a marker closely linked with the RTSW gene locus, the molecular marker method established by the invention is a co-dominant marker generated based on a PCR technology, can be used for map-based cloning and molecular marker-assisted selection of a disease-resistant gene, predicts the resistance of tobacco to the spotted wilt by detecting the anti-spotted wilt gene locus, identifies the type of RTSW allele of the tobacco anti-spotted wilt locus, can carry out elimination selection at the seedling stage of the tobacco, not only saves the production cost, but also greatly improves the selection efficiency, and further accelerates the tobacco anti-spotted wilt breeding process.

The concrete points are as follows:

1. closely interlocked: experiments prove that the result of the auxiliary identification of the tobacco breeding material by using the method is completely consistent with the result of resistance identification, and the partial result and resistance identification are inconsistent by using the conventional anti-spotted wilt AFLP and SCAR markers, which shows that the method can be used for molecular marker auxiliary selection of the anti-spotted wilt tobacco breeding.

2. The method has high accuracy, and the result of the auxiliary identification of the tobacco breeding material by the method has clear bands and obvious band type difference, while the prior anti-spotted wilt SCAR marker amplified fragment is between 100 and 200bp and is very easy to be confused with the band position of a primer dimer. Compared with the prior anti-spotted wilt marker, the detection method overcomes the problems of high false positive, poor stability and the like, and the accuracy rate reaches 100%.

3. The cost is low: the research utilizes a common PCR system, and the PCR product can be detected through electrophoresis, so that the high-flux detection cost is effectively reduced.

4. The detection efficiency is high: compared with the prior anti-spotted wilt AFLP marker, the research only uses one-time common electrophoresis analysis, overcomes the defect that the prior detection needs polypropylene gel electrophoresis, and greatly improves the detection efficiency.

5. The operation is simple: the marking and detecting of the tobacco spotted wilt resistance gene RTSW of the breeding material by the invention has simple operation, not only saves the production cost, but also greatly improves the screening efficiency of the tobacco spotted wilt resistance gene RTSW variety, greatly shortens the breeding period of the disease resistance tobacco variety and improves the breeding efficiency.

Drawings

FIG. 1 is the electrophoresis detection result of the amplification product of the invention with molecular marker primer 1, primer 2, primer 3 and primer 4 as primer groups, lane M is marker which is 100bp DNA Ladder, lane 1 is disease-resistant parent Polalta, lane 2 is hybridization F1 single plant of tobacco spotted wilt resistant Polalta (male) and main susceptible variety K326 (female); lane 3 is the susceptible parent K326;

FIG. 2 is an electrophoresis detection result of 210 single-plant amplification products of a hybrid BC6F3 segregation population of a tobacco verticillium wilt resistant Polalta (male) and a main planting susceptible variety K326 (female) by taking a primer 1, a primer 2, a primer 3 and a primer 4 as primer sets.

Detailed Description

The present invention will be described in further detail with reference to examples.

The codominant molecular marker primer group for identifying the type of RTSW allele of the tobacco anti-spotted wilt locus is a sequence shown in Seq ID No.5 or Seq ID No.6, and the molecular marker can be obtained by amplifying a primer consisting of a primer 1, a primer 2, a primer 3 and a primer 4. The primer group consists of four single-stranded DNAs of a primer 1, a primer 2, a primer 3 and a primer 4.

The sequence of the primer 1 is Seq ID No.1:

RTSW_Marker2_F 5’-GTTATAAATTGTGTGGAGGGCATG-3’；

the sequence of the primer 2 is Seq ID No.2:

RTSW_Marker2_R 5’-TCAAATTCCCGACCAGCAGTA-3’；

the sequence of the primer 3 is Seq ID No.3:

rtsw_Marker1_F 5’-CCGGAATAAGGGTAAGGTCTG-3’；

the sequence of the primer 4 is Seq ID No.4:

rtsw_Marker1_R 5’-CAACAACAACAATCGGGTGA-3’。

sequence of Seq ID No. 5:

GTTATAAATTGTGTGGAGGGCATGCCCTTCCAATTTTCACAAACCTCTCATTTTAAAAAGAAATACGT ACTATTTTGTATTTTCCAGTTTAACATTTCTTTTTCTTTTGCTTTATTGCTATAGCGAATATTTAAAA AAATCAAAACACAAAACCCGAAGTTAATATTGAAAACTATAATGAAGCTTAAGTAGAGAAGATTGAAC CAAAAGACCTCTTCATGTCTTAAATTAACGGGAAAATAGCTATTTTAGTCCTTGTATTGTTGCTTAAT TTTAATTTTGATCCTTGTATTATTCAGCTAAACAAGTTTAACCTCTAATTAGTTTAAAAAGTGACTTA GATCCGTGCAACTAACAGAAGTTAGATATTACTAAGAACTATCATTATTTCAAGAATATATAAAATAG AAAAATAGAAGATGAAGAATTAGTTCTAAAGGCTTAAAGTTTCGCAGTAACTTGTTCATTTCCGTAGT TGTCCTTCCAAGTGCAAAATCAAATTCAAATGGGTCTAAGCGTCTAAAAAAGGAATTAAGCGAGGTGA GAACGTAGTTTTTAGAACTTAACACACACACACATTGCCTCTCTTCTCTCTACGAAAGTTTCTTTATT TGACTTTGAAATTTCGATTTGAGGTGAAAAATTGAAATTTAATGCTTCTCTTACTGAAATCGAGTAAG AAGACAATTGTTTGAGCTTAAACATTGAGAATATTTACTGCTGGTCGGGAATTTGA

sequence of Seq ID No. 6:

CCGGAATAAGGGTAAGGTCTGGATACATTTTACCTGCCCCAGACCCCACTTGTGGGAACTAGGGATGG CAAACGGTGCAGTGCGGTGCGATTGCGGGGCAAACCCGCACAAACCCATGAGGATTTTAATCCGCCCC GCCCCGCATAGCCTTAAATTCGCCCCGCCCCGCCCCATTTTCCATCCCTAGTGGGAACTCACCCGATT GTTGTTGTTG

the method for identifying the RTSW allelic gene type of the tobacco anti-spotted wilt locus by adopting the primer group comprises the following specific steps:

(1) Respectively taking the genomic DNA of tobacco to be identified, tobacco spotted wilt resistant Polalta and spotted wilt susceptible variety K326 as templates, carrying out PCR amplification on the genomic DNA by using a PCR primer consisting of four single-stranded DNAs of a primer 1, a primer 2, a primer 3 and a primer 4, and obtaining a fragment with the size of 736bp by using the amplification of the primer 1 and the primer 2; the 736bp fragment is peculiar to a plant containing the RTSW gene, and the sequence of the fragment is SEQ ID No.5; the fragment obtained by amplification of the primer 3 and the primer 4 has a size of 214bp, and the 214bp fragment is specific to a plant without the RTSW gene, and has a sequence of SEQ ID No.6. When the DNA of the tobacco to be identified is extracted, the tissue of any one part or a plurality of parts of seeds, leaves, roots and flower organs of the tobacco can be adopted. The extraction method of the tobacco DNA is a conventional extraction method in the field, and can be a CTAB method, an SDS extraction method, a ROSE one-tube method, a TPS extraction method and the like, and the DNA can also be extracted by directly adopting a commercial kit. It will be appreciated by those skilled in the art that the molecular markers of the present invention can also be obtained by chemical DNA synthesis.

When the genomic DNA is subjected to PCR amplification by using a PCR primer consisting of four single-stranded DNAs of the primer 1, the primer 2, the primer 3 and the primer 4, the reaction system of PCR is as follows: respectively taking the genomic DNA of tobacco to be identified, tobacco spotted wilt resistance Polalta and spotted wilt susceptible variety K326 as templates, and performing PCR amplification system: 2 XPremix Ex Taqmix PCR Buffer 12.5. Mu.L, 10. Mu. Mol/L primer 1, primer 2, primer 3 and primer 4 each 0.5. Mu.L, 50 ng/. Mu.L template DNA 1. Mu.L, adding sterile double distilled water to make the total volume 25. Mu.L; the PCR reaction program is: pre-denaturation at 94 ℃ for 5min; then 35 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; extending for 10min at 72 ℃ after circulation is finished; storing at 4 ℃.

1) If the band of the PCR amplification product of the tobacco to be identified after electrophoresis is the same as the band type of Polalta and the size is 736bp, the tobacco to be identified is the anti-spotted wilt tobacco or the candidate is the anti-spotted wilt tobacco, and the genotype is the homozygous resistance RTSW gene locus (RTSW/RTSW);

2) If the band of the PCR amplification product of the tobacco to be identified after electrophoresis is the same as the band type of K326 and the size is 214bp, the tobacco to be identified is the tobacco suffering from the spotted wilt or the candidate is the tobacco suffering from the spotted wilt, and the genotype is the homozygous (RTSW/RTSW) of the susceptible RTSW gene locus;

3) If the bands of the PCR amplification product of the tobacco to be identified after electrophoresis contain bands with the same types as those of Polalta and K326 and the sizes are 736bp and 214bp, the tobacco to be identified is the anti-spotted wilt tobacco or candidate anti-spotted wilt tobacco, and the genotype is the anti-disease RTSW gene locus heterozygosis (RTSW/RTSW).

The codominant marker primer group for identifying the type of the RTSW allele of the tobacco anti-spotted wilt locus can be applied to positioning of the tobacco anti-spotted wilt disease genes, cloning or breeding of tobacco anti-spotted wilt varieties. By using the primer group of the invention and taking the to-be-detected tobacco genome DNA as a template to carry out PCR, whether the tobacco contains the tobacco anti-spotted wilt genetic locus RTSW can be detected, and the allele type of the tobacco anti-spotted wilt genetic locus RTSW can be identified. The detection may be a method of PCR detection, and specifically, the genomic DNA may be subjected to PCR amplification using PCR primers consisting of four single-stranded DNAs of primer 1, primer 2, primer 3 and primer 4. The detection can also detect whether the primer group sequence and the amplification product of the invention are contained by a high-throughput sequencing method.

As is well known to those skilled in the art, in the sequence of Seq ID No.1, seq ID No.2, seq ID No.3 or Seq ID No.4, 1 to 30 bases may be added at the 5 'end or 3' end thereof, respectively, and the type of the added bases may be determined according to the base type of the region on the tobacco genomic DNA which matches with Seq ID No.1, seq ID No.2, seq ID No.3 or Seq ID No.4 and according to the base pairing rules, whereby the resulting primer set is substantially the same as the amplification product of Seq ID No.1, seq ID No.2, seq ID No.3 or Seq ID No.4 (the DNA sequence between the upstream and downstream primers is the same). Therefore, the primer sets that can amplify substantially the same DNA fragments by adding 1 to 30 bases to the 5 'end or the 3' end of Seq ID No.1, seq ID No.2, seq ID No.3, or Seq ID No.4, respectively, are included in the primer sets of the present invention.

The invention utilizes the TSWV-resistant tobacco material containing RTSW anti-spotted wilt gene loci: polalta, a TSWV material K326 not containing RTSW anti-spotted wilt gene loci, and BC6F3 offspring obtained by hybridization, backcross and selfing of tobacco spotted wilt resistant Polalta (male) and a main planting infected variety K326 (female). The tobacco materials are common tobacco germplasm resources, and can be obtained by the public from tobacco germplasm resource storage units or tobacco agriculture science research institutes in Yunnan province. Restriction enzymes, kanamycin, spectinomycin, taq DNA polymerase 2 × Premix Ex Taqmix were purchased from Dalibao Bio. Other chemicals were all commercially available. A reference genomic sequence for N.tobacterium (K326) has been disclosed in (Edwards et al, 2017, A reference genome for a mammalian bacterium access map-based cloning of a mammalian gene localization in simulation efficiency. Bmc Genomics 18,448.) and publicly available from https:// solgenomics.

One specific example of the operation of the present invention is as follows:

1. and (3) constructing an anti-influenza pool by utilizing the tobacco resistance identification of the segregation population and sequencing the transcriptome.

And (3) taking 210 BC6F3 progeny obtained by hybridizing, backcrossing and selfing the tobacco verticillium wilt resistance Polalta and the main susceptible variety K326, and performing TSWV disease resistance identification by using a non-toxic gene infiltration method. And more specifically, the method for identifying the disease resistance is described in Chinese patent application 201710414755. X (a method for identifying the resistance of tobacco using the tomato spotted wilt virus NSm gene). When 4-5 leaves of the tobacco seedling are planted, the top leaf which is completely unfolded is selected, and three positions are selected on the same leaf to be respectively inoculated with pK2-35S-NSm, pK2-35S-NSs and pK2-35S-NSm + p 2300-35S-Sw-5 b. The control method is characterized in that pK2-35S-NSs is used as a negative control, pK2-35S-NSm + p2300-35S-S w-5b is used as a positive control, and the positive control can generate allergic necrosis on all tobaccos. Culturing the tobacco seedlings in a light culture room at 20-28 ℃ for 72h after inoculation, and investigating and observing the anaphylactic necrosis (HR reaction) caused by pK2-35S-NSm on the tobacco leaves to be detected. Among 210 strains, 161 strains developed an HR reaction and were resistant individuals, while 49 strains developed no HR reaction and were susceptible individuals. The results are shown in Table 2

40 plants of 161 resistant individuals and 49 susceptible individuals are respectively randomly selected to construct a resistant pool (R-pool) and a sensitive pool (S-pool), the construction method comprises the steps of respectively taking 0.1g of leaves from each of the selected 40 resistant individuals, mixing 4g of leaves to construct the resistant pool (R-pool), grinding the leaves into powder, and then delivering the powder to a sample for transcriptome sequencing. A sensitive pool (S-pool) was constructed and sent for sequencing using the same method.

The transcriptome sequencing is carried out by selecting a BGI500 sequencing platform of Huada gene, and each sample of the resistance/pool is sequenced to obtain 12Gb sequencing data.

2. And (4) screening resistance linked molecular markers.

And respectively comparing the anti-susceptible pool data obtained by the transcriptome with the K326 reference genome, and finding out 60 SNP loci with the Dealta value larger than 0.4 among the anti-susceptible pools by SNP typing, wherein the total number of the SNP loci is 47, and the SNP loci are mainly concentrated on the No. 7 chromosome of the K326, so that the anti-spotted wilt gene is positioned on the No. 7 chromosome. Reads were retrieved for the presence of SNPs and sequence alignment was performed based on the genome of K326 as a reference genome. The sequence derived from the sensitive pool (S-pool) SNP is consistent with K326, the sequence derived from the resistant pool (R-pool) SNP and K326 are rich in SNPs, and the assembled sequences are used for designing primers, so that a total of 12 pairs of primers are designed, wherein 3 pairs of sequences derived from the sensitive pool (S-pool) SNP are used for amplifying, and 9 pairs of sequences derived from the resistant pool (R-pool) SNP are used for amplifying. Extracting the genomic DNA of a tobacco anti-spotted wilt parent material Polalta, a susceptible spotted wilt parent material K326, a tobacco spotted wilt anti-source Polalta (female) and a main susceptible variety K326 (female) hybridized F1, designing a primer group according to the obtained 12 sequences, carrying out PCR amplification, carrying out electrophoresis, and screening the polymorphic markers between parents. Finally, the PCR primer group consisting of the four single-stranded DNAs of the primer 1, the primer 2, the primer 3 and the primer 4 is obtained to carry out PCR amplification, and obvious polymorphism exists among parents.

PCR amplification System: 2 XPremix Ex Taqmix PCR Buffer 12.5. Mu.L, 10. Mu. Mol/L upstream primer 1 and downstream primer 2 each 0.5. Mu.L, 25 ng/. Mu.L template DNA 2. Mu.L, adding sterile double distilled water to make the total volume 25. Mu.L; PCR reaction procedure: pre-denaturation at 94 ℃ for 5min; then 35 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 3 s; after circulation is finished, extension is carried out for 10min at 72 ℃; storing at 4 ℃.

The electrophoresis result shows that: the results shown in fig. 1 show that when the PCR labeled primer consisting of four single-stranded DNAs of primer 1, primer 2, primer 3 and primer 4 is used to perform PCR amplification on the above Polalta, K326 and F1 materials, all the materials are amplified, no false negative result appears, which indicates that the PCR reaction system verified by the research institute is completely normal and meets the detection requirements. Performing electrophoretic comparison analysis on the PCR product, wherein the lane 1 is a disease-resistant parent Polalta, and the PCR amplification product has an obvious specific band after electrophoresis, and the band is single and has the size of 736bp; the Lane 2 is a tobacco spotted wilt resistant Polalta (male parent) and main planting susceptible variety K326 (female parent) hybrid F1 single plant, and after electrophoresis, a PCR amplification product has two obvious specific bands with the sizes of 736bp and 214bp respectively; lane 3 is the susceptible parent K326, and the PCR amplification product has an obvious specific band after electrophoresis, the band is single, and the size is 214bp. Indicating the presence of a polymorphism between the parents using this marker.

3. And verifying the polymorphic markers by using 210 single plants of BC6F3 progeny segregation populations obtained by hybridizing, backcrossing and selfing tobacco verticillium wilt resistant Polalta (male parent) and main susceptible variety K326 (female parent).

The individual plants are numbered, DNA samples of 210 different individual plants are respectively extracted, and the PCR amplification is carried out on the genomic DNA by using the primers according to the conventional nucleic acid extraction method CTAB method and the purified genomic DNA as a template. PCR amplification System: 2 XPremix Ex Taq Mix PCR Buffer 12.5. Mu.L, 10. Mu. Mol/L primer 1, primer 2, primer 3 and primer 4 each 0.5. Mu.L, 25 ng/. Mu.L template DNA 2. Mu.L, adding sterile double distilled water to make the total volume 2. Mu.L; PCR reaction procedure: pre-denaturation at 94 ℃ for 5min; then 35 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; extending for 10min at 72 ℃ after circulation is finished; storing at 4 ℃. And taking the PCR reaction product for electrophoresis detection and verification.

The primer 1 is as follows:

RTSW_Marker2_F 5’-GTTATAAATTGTGTGGAGGGCATG-3’(Seq I D No.1)

the primer 2 is as follows:

RTSW_Marker2_R 5’-TCAAATTCCCGACCAGCAGTA-3’(Seq ID No. 2)

the primer 3 is as follows:

rtsw_Marker1_F 5’-CCGGAATAAGGGTAAGGTCTG-3’(Seq ID N o.3)

the primer 4 is as follows:

rtsw_Marker1_R 5’-CAACAACAACAATCGGGTGA-3’(Seq ID No. 4)

the electrophoresis result shows that: FIG. 2 is an electrophoretic analysis of molecular markers established by the applicant for rapid identification of the tobacco anti-spotted wilt genetic locus RTSW. From the analysis result, the strip brightness is high, the strip is clear and easy to identify, and the strip size difference is obvious. If the band of the PCR amplification product of the tobacco to be identified after electrophoresis is the same as the band of Polalta and the size is 736bp, the tobacco to be identified is the anti-spotted wilt tobacco or the candidate is the anti-spotted wilt tobacco, the genotype is homozygous of the resistant RT SW gene locus (RTSW/RTSW), if the band of the PCR amplification product of the tobacco to be identified after electrophoresis is the same as the band of K326 and the size is 214bp, the tobacco to be identified is the anti-spotted wilt tobacco or the candidate is the anti-spotted wilt tobacco and the genotype is homozygous of the infectious RTSW gene locus (RTSW/RTSW), if the band of the PCR amplification product of the tobacco to be identified after electrophoresis contains the bands the same as the Polalta and K326 band and the sizes are 736bp and 214bp, the tobacco to be identified is the anti-spotted wilt tobacco or the candidate is the anti-spotted wilt tobacco, and the genotype is the anti-heterozygous RTSW gene locus (RTSW/rtSW).

As a result of marker detection, it was found that 39 individuals (resistant homozygous, RTSW/RTSW) were found to be single plants containing only the 736bp band pattern, 50 individuals (susceptible homozygous, RTSW/RTSW) were found to be single plants containing only the 214bp band pattern, and 121 individuals (resistant locus heterozygous) were found to be heterozygous. The resistance detection purity identification result shows that 160 of 210 single plants are disease-resistant plants, 50 of the single plants are disease-susceptible plants, the resistance detection purity identification result is consistent with the marking detection result, and the accuracy rate reaches 100%. The molecular marker result and the resistance identification result of the rapid identification tobacco anti-spotted wilt genetic locus RTSW established by the invention are compared, and then the resistance identification results of 210 samples are completely consistent, which shows that the method has stable, reliable and extremely high accuracy, and no false positive result (see table 1).

TABLE 1 statistics of resistance identification and marker detection results of BC6F3 populations

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4. Verification of anti-spotted wilt locus RTSW allele type

Selecting resistance RTSW gene locus homozygosis (RTSW/RTSW) from 210 single plants of the BC6F3 group in the 3 rd point randomly, wherein the genotype is the susceptible RTSW gene locus homozygosis (rts w/RTSW), and the genotype is the disease resistance RTSW gene locus heterozygosis (RTSW/RTSW), each 5 plants are selfed and reserved, BC6F4 seeds are obtained, about 30 plants are sown in each plant, disease resistance detection is carried out on each single plant of BC6F4, and the allele type of the BC6F4 single plant is determined. As can be seen from Table 2, the anti-infection/infection segregation ratio of randomly selected BC6F3 single plant offspring completely accords with the mark detection result of BC6F3 single plant, and the method is stable, reliable and extremely high in accuracy, and has no false positive result.

Table 2 part of disease resistance detection of BC6F4 individuals after BC6F3 individuals are selfed and reserved

Therefore, the method for detecting the resistance of the tobacco to the spotted wilt is reliable, simple, convenient and practical, has important application prospect in the evaluation of tobacco germplasm resources and the auxiliary selection of breeding markers, and provides reference basis for the cultivation of tobacco varieties with high resistance to the spotted wilt.

The specific techniques or conditions not indicated in the examples are performed according to the techniques or conditions described in the literature in the field or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.

The above examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and all equivalent variations and modifications made in the claims of the present invention should be covered by the present invention.

Sequence listing

<110> research institute of tobacco agricultural science in Yunnan province

<120> codominant marker primer group for identifying RTSW allele type of tobacco anti-spotted wilt locus, identification method and application thereof

<141> 2019-09-29

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> RTSW_Marker2_F

<400> 1

gttataaatt gtgtggaggg catg 24

<210> 2

<211> 21

<212> DNA

<213> RTSW_Marker2_R

<400> 2

tcaaattccc gaccagcagt a 21

<210> 3

<211> 21

<212> DNA

<213> rtsw_Marker1_F

<400> 3

ccggaataag ggtaaggtct g 21

<210> 4

<211> 20

<212> DNA

<213> rtsw_Marker1_R

<400> 4

caacaacaac aatcgggtga 20

<210> 5

<211> 736

<212> DNA

<213> research institute of tobacco agricultural science in Yunnan province ()

<400> 5

gttataaatt gtgtggaggg catgcccttc caattttcac aaacctctca ttttaaaaag 60

aaatacgtac tattttgtat tttccagttt aacatttctt tttcttttgc tttattgcta 120

tagcgaatat ttaaaaaaat caaaacacaa aacccgaagt taatattgaa aactataatg 180

aagcttaagt agagaagatt gaaccaaaag acctcttcat gtcttaaatt aacgggaaaa 240

tagctatttt agtccttgta ttgttgctta attttaattt tgatccttgt attattcagc 300

taaacaagtt taacctctaa ttagtttaaa aagtgactta gatccgtgca actaacagaa 360

gttagatatt actaagaact atcattattt caagaatata taaaatagaa aaatagaaga 420

tgaagaatta gttctaaagg cttaaagttt cgcagtaact tgttcatttc cgtagttgtc 480

cttccaagtg caaaatcaaa ttcaaatggg tctaagcgtc taaaaaagga attaagcgag 540

gtgagaacgt agtttttaga acttaacaca cacacacatt gcctctcttc tctctacgaa 600

agtttcttta tttgactttg aaatttcgat ttgaggtgaa aaattgaaat ttaatgcttc 660

tcttactgaa atcgagtaag aagacaattg tttgagctta aacattgaga atatttactg 720

ctggtcggga atttga 736

<210> 6

<211> 214

<212> DNA

<213> research institute of tobacco agricultural science in Yunnan province ()

<400> 6

ccggaataag ggtaaggtct ggatacattt tacctgcccc agaccccact tgtgggaact 60

agggatggca aacggtgcag tgcggtgcga ttgcggggca aacccgcaca aacccatgag 120

gattttaatc cgccccgccc cgcatagcct taaattcgcc ccgccccgcc ccattttcca 180

tccctagtgg gaactcaccc gattgttgtt gttg 214

Claims

1. The codominant marker primer group for identifying the RTSW allele type of the tobacco anti-spotted wilt locus is characterized by consisting of four single-stranded DNAs (deoxyribonucleic acids) of a primer 1, a primer 2, a primer 3 and a primer 4; the sequence of the primer 1 is Seq ID No.1:

RTSW_Marker2_F 5’-GTTATAAATTGTGTGGAGGGCATG-3’；

the sequence of the primer 2 is Seq ID No.2:

RTSW_Marker2_R 5’-TCAAATTCCCGACCAGCAGTA-3’；

the sequence of the primer 3 is Seq ID No.3:

rtsw_Marker1_F 5’-CCGGAATAAGGGTAAGGTCTG-3’；

the sequence of the primer 4 is Seq ID No.4:

rtsw_Marker1_R 5’-CAACAACAACAATCGGGTGA-3’。

2. the method for identifying the type of RTSW allele of tobacco anti-spotted wilt locus by using the primer set as claimed in claim 1, which comprises the following steps:

(1) Respectively taking the genomic DNA of tobacco to be identified, tobacco spotted wilt resistant Polalta and spotted wilt susceptible variety K326 as templates, carrying out PCR amplification on the genomic DNA by using a PCR primer group consisting of four single-stranded DNAs of a primer 1, a primer 2, a primer 3 and a primer 4, and obtaining a fragment with the size of 736bp by using the amplification of the primer 1 and the primer 2; the 736bp segment is peculiar to a plant containing RTSW gene, and the sequence thereof is SEQ ID No.5; the fragment obtained by amplification of the primer 3 and the primer 4 has the size of 214bp, and the 214bp fragment is specific to a plant without the RTSW gene and has a sequence of SEQ ID No.6;

1) If the band of the PCR amplification product of the tobacco to be identified after electrophoresis or sequencing is the same as the band type of Polalta and the size is 736bp, the tobacco to be identified is the anti-spotted wilt tobacco or the candidate anti-spotted wilt tobacco, and the genotype is homozygous resistance RTSW gene locus, namely RTSW/RTSW;

3. The use of the co-dominant marker primer set for identifying the type of the RTSW allele of the tobacco anti-spotted wilt locus as claimed in claim 1 in the positioning, cloning or breeding of the tobacco anti-spotted wilt disease resistance gene.

4. The method for identifying the type of RTSW allele of tobacco anti-spotted wilt locus using primer set as claimed in claim 2, wherein the DNA of tobacco to be identified is extracted using the tissue of any one or more parts of the seed, leaf, root and floral organ of tobacco.

5. The method for identifying the type of RTSW allele of tobacco anti-spotted wilt locus by using the primer set as claimed in claim 2, wherein the PCR amplification of the genome DNA is carried out by using the PCR primer consisting of four single-stranded DNAs of primer 1, primer 2, primer 3 and primer 4, and the reaction system of the PCR is as follows: respectively taking the genomic DNA of tobacco to be identified, tobacco spotted wilt resistance Polalta and spotted wilt susceptible variety K326 as templates, and performing PCR amplification system: 2 XPremix Ex Taqmix PCR Buffer 12.5. Mu.L, 10. Mu. Mol/L primer 1, primer 2, primer 3 and primer 4 each 0.5. Mu.L, 50 ng/. Mu.L template DNA 1. Mu.L, adding sterile double distilled water to make the total volume 25. Mu.L; the PCR reaction program is: pre-denaturation at 94 deg.C for 5min; then 35 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s; after circulation is finished, extension is carried out for 10min at 72 ℃; storing at 4 ℃.