Co-dominant SSR (Simple Sequence Repeat) marker closely linked with nornicotine gene qnNIC and application of co-dominant SSR marker

Abstract

The invention provides a codominant SSR (simple sequence repeat) marker closely linked with nornicotine gene qnNIC and an application method of the codominant SSR marker. The serial numbers of the codominant SSR marker closely linked with the nornicotine gene qnNIC are TM55416 and TMy85136, and the nucleotide sequences of PCR (Polymerase Chain Reaction) amplification products of the codominant SSR marker closely linked with the nornicotine gene qnNIC are respectively shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. The application is the application of the codominant SSR marker closely linked with the nornicotine gene qnNIC in detecting whether the nornicotine gene qnNIC exists in tobacco genome DNA (Deoxyribose Nucleic Acid) or not. Compared with a method in the prior art, the codominant SSR marker has the characteristics of accuracy, high efficiency, stability, convenience, low cost and no detection opportunity limitation, so that the molecular marker can be applied to nornicotine gene qnNIC molecular marker-assisted selection in tobacco breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a co-dominant SSR marker tightly linked to a nicotine-reducing gene qnNIC and an application thereof. Background Art

[0002] Tobacco is a leaf-growing cash crop. Tobacco leaves contain a variety of chemical elements. Currently, more than 3,000 chemical components have been identified. However, with the application of modern separation and analysis instruments such as gas chromatography (GC), liquid chromatography (LC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), infrared spectroscopy (IR), nuclear magnetic resonance (NMR) and high-resolution mass spectrometry in tobacco research, the trace components that were once hidden will be revealed, and the number of chemical components in tobacco leaves will increase dramatically. As the raw material of tobacco leaves, the basis of the cigarette industry, its quality directly affects the quality of finished cigarettes. The quality of tobacco leaves is the result of the combined effects of various chemical components in its leaves. Therefore, the chemical composition of tobacco leaves is an important indicator for the identification of tobacco leaf quality, and it is also the manifestation of appearance quality and internal quality in appearance characteristics and smoke characteristics.

[0003] So far, the research on tobacco leaf chemical components has mainly focused on the identification of tobacco leaf chemical components and the close relationship between the main chemical components and smoking quality, appearance quality, physical properties and safety, while there are few studies on genetic analysis and gene / QTL positioning of tobacco leaf chemical components. Julio et al. (E.Julio, B.Denoyes-Rothan, J.-L.Verrier, F.Dorlhac de Borne, Detection of QTLs linked to leaf and smoke properties in Nicotiana tabacum based on a study of 114recombinant inbred lines.MolBreeding, 2006, DOI 10.1007 / s11032-006-9019-0) were the first to use AFLP, ISSR, SSAP and SCAR markers to conduct gene / QTL positioning research on tobacco leaf chemical components at the molecular level. They conducted preliminary gene / QTL analysis on six tobacco leaf chemical components, including total alkaloids, nicotine, nornicotine, anabasine, citric acid and malic acid. However, the results of the gene / QTL localization analysis are incomplete and inaccurate. The main reasons are: first, the tobacco genetic linkage map used for gene / QTL localization analysis by Julio et al. (2006) was of low quality, with only 18 linkage groups constructed from 184 markers, which did not meet the complete requirement of 24 chromosomes (linkage groups) of tobacco; second, they only obtained unilateral markers linked to the above-mentioned 6 tobacco leaf chemical components, and failed to obtain bilateral markers linked to the target gene / QTL (6 tobacco leaf chemical components) for effective localization; finally, the genetic distance between the obtained unilateral linkage marker and the target gene / QTL was large, resulting in low accuracy of the gene / QTL localization results. Subsequently, Xiao Bingguang et al. (Xiao Bingguang, Lu Xiuping, Jiao Fangchan, Li Yongping, Sun Yuhe, Guo Zhaokui. Preliminary analysis of QTLs for several chemical components in flue-cured tobacco. Acta Agronomica Sinica, 2008, 34(10): 1762-1769) used 11 ISSR markers and 158 RAPD markers to construct a low-quality tobacco genetic linkage map containing 27 linkage groups, and conducted gene / QTL analysis on three tobacco leaf chemical components, including total sugar, nicotine and potassium oxide.Subsequently, Cai Changchun et al. (Cai Changchun, Chai Liguang, Wang Yi, Xu Fangsen, Zhang Junjie, Lin Guoping, Construction of molecular marker genetic map of burley tobacco and genetic analysis of some traits. Acta Agronomica Sinica, 2009, 35(9): 1646-1654), Li Lihua et al. (Li Lihua, Chen Meixia, Zhou Dongxin, Chen Shunhui, Tao Aifen, Li Yankun, Ma Hongbo, Qi Jianmin, Guo Yuchun, QTL positioning of six important traits of tobacco. Acta Agronomica Sinica, 2011, 37(9): 1577-1584) and Liu Yingchao et al. (Liu Yingchao, Fang Dun Huang, Xu Haiming, Tong Zhijun, Xiao Bingguang, QTL positioning of tobacco alkaloid traits. Acta Agronomica Sinica, 2024, 50(1): 42-54) A low-quality tobacco genetic linkage map with a small number of markers was constructed using AFLP and SRAP markers, ISSR and SRAP markers, and SNP markers, respectively. Gene / QTL analysis was performed on nine tobacco leaf chemical components, including nicotine, total nitrogen, total sugar, total chlorine, total potassium, total alkaloids, nornicotine, anabasine, and anatabine. The accuracy of the positioning analysis results was still low.

[0004] Although Julio et al. (2006) and Liu Yingchao et al. (2024) have reported on QTL positioning analysis of the nicotine-reducing gene qnNIC, they are only preliminary positioning analyses and the results are of low accuracy. Therefore, the genetic distance between the molecular marker and the nicotine-reducing gene qnNIC is large, which seriously affects the accuracy of molecular marker-assisted selection for nicotine-reducing content in tobacco breeding; in addition, the above-mentioned AFLP and SNP markers linked to qnNIC cannot be effectively used in tobacco breeding due to complicated operations, time-consuming and labor-intensive, and high costs. In short, the above-mentioned deficiencies in the genetic and positioning analysis of nicotine-reducing traits have seriously restricted the breeding process of new high-content nicotine-reducing tobacco varieties using markers closely linked to the nicotine-reducing gene qnNIC.

[0005] In view of this, the present invention firstly uses the high-quality flue-cured tobacco variety Y3 (with high content of nornicotine in the flue-cured tobacco leaves, containing the gene qnNIC) and the main flue-cured tobacco variety K326 (with no nornicotine in the flue-cured tobacco leaves, containing the gene qnnic) as parents, and constructs a tobacco recombinant inbred line (RILs_F) containing 269 strains through hybridization and continuous bagging and self-pollination. 8:9 ) as the mapping population; secondly, the large-scale SSR molecular markers developed by our laboratory based on the genome information of the high-quality flue-cured tobacco variety Y3 were used to map 269 RILs_F 8:9The genotypes of the strains were analyzed and a high-quality tobacco SSR molecular genetic linkage map was constructed. The map contained 2059 SSR markers and was evenly distributed on 24 linkage groups of tobacco, covering a genome length of 1759.74 cM. Secondly, after the tobacco matured and was harvested, the gas chromatography-mass spectrometry (GC-MS) method was used to detect the content of anabasine in the middle leaves of C3F grade tobacco after baking. Finally, the quantitative trait linkage analysis (QTL) method was combined with RILs_F 8:9 The genotypic data (genetic linkage map) and phenotypic data (nicotine-reducing content value) of tobacco were used to screen for co-dominant SSR markers tightly linked to the nicotine-reducing gene qnNIC in the whole tobacco genome, so as to make up for the shortcomings of the reported literature and accelerate the precise and efficient use of molecular marker-assisted selection (MAS) in the breeding of tobacco varieties with high anabasine content. Summary of the invention

[0006] The present invention is to solve the above-mentioned problems and defects, and provides a co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC and its application.

[0007] The first purpose of the present invention is to provide a co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC; the second purpose is to apply the above co-dominant SSR marker to detect whether the nicotine-reducing gene qnNIC exists in tobacco genomic DNA and the genotype status of nicotine-reducing in the test plant.

[0008] The present invention is implemented by the following technical solution.

[0009] A co-dominant SSR marker tightly linked to a nicotine-reducing gene qnNIC. The co-dominant SSR marker tightly linked to a nicotine-reducing gene qnNIC of the present invention is numbered TM55416 and TMy85136, and the nucleotide sequences of the PCR amplification products thereof are shown in SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, respectively.

[0010] SEQ ID No.1:

[0011] CCATACACAGGTCATGAGACGAATAATTAATAATTAGATAAATTCATAGTTATTTAATGGGTATTATCACTTTTAGCCTGTGTAAAAATTTATTTATATTTGGTAGCCAAAAAAGTGTATATAATTTTTATATAACATACAAAATATATATATATATATATATATAAATACATTTTCGATTATTTTAAGAGAGACTACAATGTTATTTTCCTTATTATATAATACTGACACGTCTCATGATCGTATTGACTGCACATACGC。

[0012] SEQ ID No.2:

[0013] CCATACACAGGTCATGAGACGAATAATTAATAATTAGATAAATTCATAGTTATTTAATGGGTATTATCACTTTTAGCCTGTGTAAAAATTTATTTATATTTGGTAGCCAAAAAAGTGTATATAATTTTTATATAACATACAAAATATATATATATATATATATATATATATATATATAAATACATTTTCGATTATTTTAAGAGAGACTACAATGTTATTTTCCTTATTATATAATACTGACACGTCTCATGATCGTATTGACTGCACATACGC。

[0014] SEQ ID No.3:

[0015] GCAAGGACAGGATTGAGACCATCTTTTAACGACGGGCAAATGTGCCCAATTTCGTATAGTTCACCGGCAGGAATGAGCCTTTTCCGTCATTATTATTATTATTATTACTATTATTATTAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATA ATAATAATAATAATAATAATAATAATAACCGTTTTGATATATTAGAGGTAAATTTCAATATTTTGTTTGTCTATACGACCGTTATAGCAAATGCCAGAGTCAAGCTAGTCAGTTGTTACTCGAAGGGTATATTAAGATGAAAGATAACGATGGGGATTTTCATGA.

[0016] SEQ ID No.4:

[0017] GCAAGGACAGGATTGAGACCATCTTTTAACGACGGGCAAATGTGCCCAATTTCGTATAGTTCACCGGCAGGAATGAGCCTTTTCCGTCATTATTATTATTATTATTACTATTATTATTATTAATAATAATAATAATAATAATAATAATAATAATAATAATAATA ATAATAATAATAATAATAATAATAACCGTTTTGATATATTAGAGGTAAATTTCAATATTTTGTTTGTCTATACGACCGTTATAGCAAATGCCAGAGTCAAGCTAGTCAGTTGTTACTCGAAGGGTATATTAAGATGAAAGATAACGATGGGGATTTTCATGA.

[0018] The co-dominant markers TM55416 and TMy85136 of the present invention are located on both sides of the target gene qnNIC.

[0019] The primer sequences of the two sites corresponding to the molecular markers described in the present invention are:

[0020] The primer sequences for amplifying TM55416 are:

[0021] TM55416F: 5'-GCGTATGTGCAGTCAATACGAT-3' (SEQ ID NO.5),

[0022] TM55416R: 5'-CCATACACAGGTCATGAGACG-3' (SEQ ID NO. 6);

[0023] The primer sequences for amplifying TMy85136 are:

[0024] TMy85136F: 5'-GCAAGGACAGGATTGAGACC-3' (SEQ ID NO.7),

[0025] TMy85136R: 5'-TCATGAAATCCCCCATCGTT-3' (SEQ ID NO. 8).

[0026] The application of the above-mentioned co-dominant SSR markers closely linked to the nicotine-reducing gene qnNIC is to detect whether the nicotine-reducing gene qnNIC exists in tobacco genomic DNA and the nicotine-reducing genotype status in the tested plants.

[0027] The above application of the present invention comprises the following steps: using primers of TM55416 and TMy85136 sequences to amplify the tobacco genome DNA to be detected, and detecting the PCR amplification products.

[0028] If the PCR amplification product of the present invention contains the sequences shown in SEQ ID No. 1 and SEQ ID No. 3 at the same time, it indicates that the tobacco plant to be tested has a homozygous allele with a high content of nornicotine, and the genotype is nNICnNIC.

[0029] The PCR amplification product of the present invention contains the sequences shown in SEQ ID No. 2 and SEQ ID No. 4 at the same time, which indicates that the tobacco plant to be tested has a homozygous allele with no nicotine reduction, and the genotype is nnicnnic.

[0030] If the PCR amplification product of the present invention contains the sequences shown in SEQ ID No.1 and SEQ ID No.4, or contains the sequences shown in SEQ ID No.2 and SEQ ID No.3, it means that the tobacco plant to be tested has a heterozygous allele with a medium content of nornicotine, and the genotype is nNICnnic.

[0031] In order to select tobacco varieties with high nicotine reduction potential simply and efficiently, the present invention selectively and specifically selects offspring materials containing the nicotine reduction gene qnNIC, which can be used for auxiliary selection of the nicotine reduction gene qnNIC, so as to improve the efficiency of molecular marker-assisted selection and the efficiency of breeding high-content nicotine reduction tobacco varieties. The two co-dominant SSR markers closely linked to the nicotine reduction gene qnNIC provided by the present invention can not only be used to qualitatively detect whether the nicotine reduction gene qnNIC exists in tobacco genomic DNA at any growth period, but also can clearly and accurately identify the nicotine reduction genotype state in the tested plant (i.e., homozygous genotype nNICnNIC with the highest content value and stable inheritance, heterozygous genotype nNICnnic with a medium content value and unstable inheritance, and homozygous genotype nnicnnic with no content value and stable inheritance), thereby improving the scientificity and predictability of breeding new flue-cured tobacco varieties with high nicotine reduction content, and accelerating the breeding process.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. High accuracy in detecting nornicotine

[0034] The invention provides a molecular marker for detecting the nicotine-reducing gene qnNIC, which can accurately determine whether the tobacco to be tested contains nicotine. Compared with the detection of nicotine content using molecular markers published in reported literature, the detection accuracy of the method is higher and the genotype status of nicotine in the tobacco plant to be tested can be clearly and accurately identified.

[0035] 2. The detection method is efficient, stable, reliable, simple to operate and low-cost

[0036] Compared with the existing low-throughput, high-cost, time-consuming and labor-intensive GC-MS method for detecting the nornicotine content after ripening and baking, the co-dominant SSR marker described in the present invention is characterized by high efficiency, stability, reliability, simplicity and low cost.

[0037] 3. No limit on the timing of testing

[0038] This method can be used for testing at any stage of tobacco growth, especially at the tobacco seedling stage, which can greatly shorten the experimental cycle and thus accelerate the process of breeding new flue-cured tobacco varieties with high nicotine content.

[0039] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The tobacco recombinant inbred line population (RILs_F 8:9 ; Y3×K326) in linkage group 5 for nicotine reduction QTL analysis.

[0041] The software used was QTL IciMapping v4.2; parameter settings: the positioning method was ICIM-ADD: Inclusive Composite Interval Mapping of ADDitive (and dominant) QTL, the number of iterations was 1000 (Permutation times = 1000), the significance was 0.01 (Significance = 0.01), and the step size was 0.5 cM (Walk speed = 0.5 cM). The horizontal axis is the genetic distance (unit: centimorgan cM); the vertical axis is the LOD value. The horizontal dotted line in the figure is the LOD value = 3.5744 at the significance threshold of 0.01; the highest point of the LOD curve is the dominant gene (qnNIC). DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the examples and drawings. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in accordance with the instructions of the relevant products are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased.

[0043] The co-dominant SSR markers tightly linked to the nicotine-reducing gene qnNIC of the present invention are numbered TM55416 and TMy85136, and the nucleotide sequences of the PCR amplification products thereof are shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0044] The primer sequences of the two sites corresponding to the molecular markers are:

[0045] The primer sequences for amplifying TM55416 are:

[0046] TM55416F: 5'-GCGTATGTGCAGTCAATACGAT-3',

[0047] TM55416R: 5'-CCATACACAGGTCATGAGACG-3';

[0048] The primer sequences for amplifying TMy85136 are:

[0049] TMy85136F: 5'-GCAAGGACAGGATTGAGACC-3',

[0050] TMy85136R: 5'-TCATGAAATCCCCATCGTT-3'.

[0051] The application of the codominant SSR marker tightly linked to the nicotine-reducing gene qnNIC described in the present invention is the application of the codominant SSR marker tightly linked to the nicotine-reducing gene qnNIC in detecting whether the nicotine-reducing gene qnNIC exists in tobacco genomic DNA.

[0052] The application of the co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC is to amplify the genomic DNA of the tobacco to be tested with primers of the TM55416 sequence and the TMy85136 sequence, respectively, and detect the PCR amplification product. If the PCR amplification product contains the sequences shown in SEQ ID NO.1 and SEQ ID NO.3 at the same time, it means that the tobacco plant has a homozygous allele qnNICqnNIC with a high nicotine-reducing content; if the PCR amplification product contains the sequences shown in SEQ ID NO.2 and SEQ ID NO.4 at the same time, it means that the tobacco plant to be tested has a homozygous allele qnnicqnnic with no nicotine-reducing content; if the PCR amplification product contains the sequences shown in SEQ ID NO.1 and SEQ ID NO.4, or contains the sequences shown in SEQ ID NO.2 and SEQ ID NO.3, it means that the tobacco plant to be tested has a heterozygous gene qnNICqnnic with a medium nicotine-reducing content.

[0053] Example 1 Screening of co-dominant SSR markers linked to the nicotine-reducing gene qnNIC

[0054] The quantitative trait linkage analysis (QTL) method combined with gas chromatography-mass spectrometry (GC-MS) was used to screen for co-dominant SSR markers linked to the nicotine-reducing gene qnNIC in the whole tobacco genome.

[0055] 1. Experimental Materials

[0056] The flue-cured tobacco variety K326 with excellent comprehensive traits but no nicotine was used as the female parent, and the high-quality flue-cured tobacco variety Y3 with high nicotine content was used as the male parent. After hybridization and continuous self-pollination, 269 recombinant inbred lines (RILs_F 8:9 ) as a genetic mapping population.

[0057] 2. Parents and RILs_F 8:9 Obtaining data on nicotine content in the population

[0058] After the test materials have grown into seedlings, they are transplanted to the field. When the tobacco leaves in the field are mature, 10 plants are randomly selected from each strain, and 3 middle leaves from each tobacco plant are selected for labeling and numbering, and then baked. After baking, the 30 middle leaves of each strain are mixed together and ground into powder.

[0059] The C3F grade powdered tobacco samples obtained from the middle of each strain were used to detect the nornicotine content in the tobacco leaves after baking according to the tobacco industry standard YCT 383-2010 of the People's Republic of China (determination of nicotine, nornicotine, anatabine, myosamine and anabasine in tobacco and tobacco products by gas chromatography-mass spectrometry).

[0060] 269 ​​RILs_F obtained by GC-MS 8:9 Population nicotine content as RILs_F 8:9 The phenotypic values ​​of the population were used for the next QTL linkage analysis.

[0061] 3. SSR marker analysis

[0062] Extraction of tobacco genomic DNA: Either the conventional CTAB method or a plant tissue DNA extraction kit can be used. The method can be referred to in existing literature or the instructions in the kit.

[0063] PCR amplification and electrophoresis detection: The PCR amplification system is a conventional system and can refer to published literature. Among them, the label annealing temperature provided by the present invention is 60°C; the PCR amplification program information can refer to relevant literature; electrophoresis detection also adopts conventional methods and can refer to published relevant literature.

[0064] Using approximately 50,000 SSR markers developed by our laboratory based on the Y3 flue-cured tobacco genome information, we identified RILs_F 8:9 The parents (K326 and Y3) and offspring (F 1 ) were used for polymorphism screening, and finally, 2158 polymorphic SSR markers were obtained.

[0065] 2158 polymorphic SSR markers were obtained by screening and 269 RILs_F 8:9 The samples were subjected to genotyping analysis.

[0066] Finally, the genetic linkage mapping software JoinMap 4.0 was used to map the 269 RILs_F 8:9 The genotype data of the samples were subjected to linkage analysis, and a high-quality flue-cured tobacco SSR molecular genetic linkage map containing 24 linkage groups and 2059 evenly distributed SSR markers, covering a tobacco genome length of 1759.74 cM, was drawn as RILs_F 8:9 The genotype values ​​of the population were used for the next step of QTL linkage analysis.

[0067] 4. Genome-wide QTL analysis of nornicotine (qnNIC)

[0068] The QTL localization and analysis software QTL IciMapping v4.2 was used to analyze the RILs_F8:9 The genotype data (construction of the genetic linkage group map of flue-cured tobacco) and phenotype data (269 RILs_F 8:9 The nicotine-lowering gene qnNIC was scanned for genome-wide QTL.

[0069] Among them, the relevant parameters are set as follows: the positioning method is ICIM-ADD: Inclusive Composite Interval Mapping of ADDitive (and dominant) QTL, the number of iterations is 1000 (Permutation times = 1000), the significance is 0.01 (Significance = 0.01), and the step size is 0.5 cM (Walk speed = 0.5 cM).

[0070] Finally, under the condition of LOD=3.2983 in the whole genome, a major QTL (temporarily named qnNIC) for the nicotine-reducing trait was located at 77.50 cM on linkage group 5. This major QTL can explain about 11.219% of the phenotypic variation, and the LOD value at this time is about 7.6709, see Figure 1 and Table 1.

[0071] Table 1 Statistics of nicotine-reducing QTL (qnNIC) information

[0072] QTL Chromosome Position / cM Left Marker Right Marker LOD PVE(%) Add qnNIC 5 77.50 TM55416 TMy85136 7.6709 11.219 17.7959

[0073] Note: PVE is the effect value of QTL, that is, the percentage of phenotypic variation that can be explained by the QTL; Add is the additive effect.

[0074] Example 2 Co-dominant linkage markers in RILs_F 9:10 Verification in individual plants of the population

[0075] Using the co-dominant SSR markers TM55416 and TMy85136, which were closely linked to the nicotine-reducing gene qnNIC, we stained the RILs_F 9:10 The genotypes of individual plants in the population (K326×Y3) were analyzed to obtain RILs_F 9:10 Genotype data of each individual plant in the population.

[0076] On the other hand, when RILs_F 9:10 After the tobacco leaves of the group grow to maturity and are harvested and cured, the middle C3F grade cured leaves of each strain are ground into powder by GC-MS and tested for nicotine content. 9:10 Phenotypic values ​​of each strain in the population.

[0077] Finally, 269 RILs_F were analyzed 9:10 The genotype data of the population and the nicotine-reducing phenotype values ​​showed that the genotype values ​​and phenotype values ​​of the two co-dominant SSR markers TM55416 and TMy85136 published in the present invention were completely consistent with each other, that is, the consistency rate reached 100%.

[0078] The specific analysis method is: when the nornicotine content of each strain obtained by GC-MS detection is higher than or equal to the parent Y3 content, the genotype of the strain also presents the sequences shown in SEQ ID NO.1 (266 bp) and SEQ ID NO.3 (339 bp) at the same time, which is the homozygous genotype nNICnNIC;

[0079] When the nicotine content of each strain obtained by the test is equal to that of the parent K326 (i.e., the nicotine content is 0), the genotype of the strain also presents the sequences shown in ID NO.2 (278 bp) and SEQ ID NO:4 (333 bp) at the same time, which is the homozygous genotype nnicnnic;

[0080] When the nicotine content of each strain was between that of the parents Y3 and K326, that is, the same as that of the first generation (F 1 ) content is similar, the genotype of the strain also presents the sequences shown in SEQ ID NO.1 and SEQ ID NO.4 at the same time, or contains the sequences shown in SEQ ID NO.2 and SEQ ID NO.3, which is a heterozygous genotype nNICnnic.

[0081] Experimental conclusion:

[0082] The above results showed that the co-dominant markers TM55416 and TMy85136 were closely linked to the nicotine-reducing gene qnNIC, and the two markers were located on both sides of the target gene (qnNIC).

[0083] Utilizing the above two co-dominant tightly linked SSR markers, not only can the detection of nicotine-reducing content in tobacco at any growth stage be realized accurately, efficiently, conveniently and at low cost, but also the genotype status of nicotine-reducing in the tested plants can be clearly identified (i.e., the homozygous genotype nNICnNIC with the highest content value and stable inheritance, the heterozygous genotype nNICnnic with a medium content value and unstable inheritance, and the homozygous genotype nnicnnic with no content value and stable inheritance), thereby improving the scientificity and predictability of the breeding of new varieties with high nicotine-reducing content and accelerating the breeding process.

[0084] The above are only some specific embodiments of the present invention. The known specific contents or common sense in the scheme are not described in detail here (including but not limited to abbreviations, abbreviations, and units commonly used in the art). It should be pointed out that the above embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC, It is characterized in that The co-dominant SSR markers closely linked to the nicotine-reducing gene qnNIC are numbered TM55416 and TMy85136, and the nucleotide sequences of the PCR amplification products thereof are shown in SEQ ID No. 1 and SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

2. The co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC according to claim 1, It is characterized in that The co-dominant markers TM55416 and TMy85136 are located on both sides of the target gene qnNIC.

3. The co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC according to claim 1, It is characterized in that The primer sequences of the two sites corresponding to the molecular markers are: The primer sequences for amplifying TM55416 are: TM55416F: 5'-GCGTATGTGCAGTCAATACGAT-3', TM55416R: 5'-CCATACACAGGTCATGAGACG-3'; The primer sequences for amplifying TMy85136 are: TMy85136F: 5'-GCAAGGACAGGATTGAGACC-3', TMy85136R: 5'-TCATGAAATCCCCATCGTT-3'.

4. The application of the co-dominant SSR marker tightly linked to the nicotine-reducing gene qnNIC as claimed in claim 1, 2 or 3 is to detect whether the nicotine-reducing gene qnNIC exists in tobacco genomic DNA and the genotype status of the nicotine-reducing gene in the test plant.

5. The use according to claim 4, It is characterized in that The application method is to amplify the tobacco genome DNA to be detected using primers of TM55416 and TMy85136 sequences respectively, and detect the PCR amplification products.

6. The use according to claim 5, It is characterized in that If the PCR amplification product contains the sequences shown in SEQ ID No. 1 and SEQ ID No. 3 at the same time, it indicates that the tobacco plant to be tested has a homozygous allele with a high content of nornicotine, and the genotype is nNICnNIC.

7. The use according to claim 5, It is characterized in that If the PCR amplification product contains the sequences shown in SEQ ID No. 2 and SEQ ID No. 4 at the same time, it is a homozygous allele of the tobacco plant to be tested that has no nicotine reduction content, and the genotype is nnicnnic.

8. The use according to claim 5, It is characterized in that If the PCR amplification product contains the sequences shown in SEQ ID No.1 and SEQ ID No.4, or contains the sequences shown in SEQ ID No.2 and SEQ ID No.3, it means that the tobacco plant to be tested has a heterozygous allele with a medium content of nornicotine, and the genotype is nNICnnic.

Images