Co-dominant SSR (simple sequence repeat) marker closely linked with beta carotene gene qbCar of tobacco and application of co-dominant SSR marker

Abstract

The invention provides a codominant SSR (Simple Sequence Repeat) marker closely linked with a tobacco beta carotene gene qbCar and an application method. The serial numbers of the codominant SSR marker closely linked with the tobacco beta carotene gene qbCar are TMy80084 and TMy81298, and the nucleotide sequences of PCR (Polymerase Chain Reaction) amplification products of the codominant SSR marker closely linked with the tobacco beta carotene gene qbCar 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 tobacco beta carotene gene qbCar in detecting whether the tobacco beta carotene gene qbCar exists in tobacco genome DNA (Deoxyribonucleic 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 auxiliary selection of the beta carotene gene qbCar molecular marker in tobacco breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a co-dominant SSR marker tightly linked to a tobacco beta-carotene gene qBCar 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, but the accuracy of the positioning analysis results was still low.

[0004] However, there has been no report on the QTL location analysis of tobacco β-carotene so far. Therefore, the lack of genetic research and location analysis of tobacco β-carotene traits has seriously restricted the breeding process of new tobacco varieties with high β-carotene content using markers closely linked to the tobacco β-carotene gene qbCar.

[0005] In view of this, the present invention firstly uses the high-quality flue-cured tobacco variety Y3 (with high content of tobacco β-carotene in flue-cured tobacco leaves, containing gene qbCar) and the main flue-cured tobacco variety K326 (without tobacco β-carotene in flue-cured tobacco leaves) 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 8:9 The 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 β-carotene content of the C3F middle leaves after baking was detected by high performance liquid chromatography (HPLC). Finally, quantitative trait linkage analysis (QTL) method was used in combination with RILs_F 8:9The genotype data (genetic linkage map) and phenotypic data (tobacco β-carotene content) were used to screen and obtain co-dominant SSR markers closely linked to the β-carotene gene qbCar in the whole tobacco genome. This not only effectively filled the gap in the QTL positioning analysis of tobacco β-carotene traits at home and abroad, but also obtained co-dominant SSR markers closely linked to the tobacco β-carotene gene qbCar. This is an important way to accurately and efficiently use molecular marker-assisted selection (MAS) to breed new flue-cured tobacco varieties with high β-carotene content. Summary of the invention

[0006] The present invention is to solve the blank of QTL location analysis of tobacco beta-carotene trait, and provides a co-dominant SSR marker closely linked to tobacco beta-carotene gene qbCar and its application.

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

[0008] Innovation of the present invention: The present invention is the first to conduct genetic and location analysis on the tobacco beta-carotene gene qbCar. The inventor's research shows that tobacco beta-carotene is a typical quantitative trait and is controlled by the QTL of the second chromosome in the genome of the flue-cured tobacco variety Y3. The gene / QTL is temporarily named qbCar.

[0009] The present invention is implemented by the following technical solution.

[0010] A co-dominant SSR marker tightly linked to a tobacco beta-carotene gene qbCar. The co-dominant SSR marker tightly linked to a tobacco beta-carotene gene qbCar of the present invention is numbered TMy80084 and TMy81298, 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.

[0011] SEQ ID No.1:

[0012] AATGAAGGGCAATGCAAGACATAGTTGAATGTATATATATATATATATATATATATATATATATATATATATATATATAGTAAATTTAACATTTTCCCCTTCTTTAATTAGCAGTCGTGTTAGTTAGTTGAGCCACTCACGTAGTCAATTGAAAGACTAGTTGATTGATACTTAGTCATTTATATTTACATATGGAAGATGAAAATTTGTTCCTTCAAGATTGGAGGTCAGGCATTTGTCTGCCCTAAACTCCC。

[0013] SEQ ID No.2:

[0014] AATGAAGGGCAATGCAAGACATAGTTGAATGTATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAGTAAATTTAACATTTTCCCCTTCTTTAATTAGCAGT CGTGTTAGTTAGTTGAGCCACTCACGTAGTCAATTGAAAGACTAGTTGATTGATACTTAGTCATTTATATTTACATATGGAAGATGAAAATTTTGTTCCTTCAAGATTGGAGGTCAGGCATTTGTCTGCCCTAAACTCCC。

[0015] SEQ ID No.3:

[0016] GAAGAGCCCAAATGTCGAAGAAACCGAACCATACCGATGCAAGCCGAAGTGTGCCCACTAGCATCAACCACCATCCTAACAGCTCCATCGCTC ...

[0017] SEQ ID No.4:

[0018] GGGTCAATGGGCATGTTTATCGGCCCCTTACTATTTAGTGGTTGAAAAATGAAAACAGAGGGAGAAAACAGAAAAAGTAACTCTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGA GAGAGAGAGAGAGAGAGAGAGAGAGAGAGCGATGGAGCTGTTAGGATGGTGGTTGATGCTAGTGGGCACACTTCGGCTTGCATCGGTATGGTTCGGTTTCTTCGACATTTGGGCTCTTC.

[0019] The co-dominant markers TMy80084 and TMy81298 of the present invention are located on both sides of the target gene qBCar.

[0020] The primer sequences of the two sites corresponding to the molecular markers described in the present invention are:

[0021] The primer sequences for amplifying TMy80084 are:

[0022] TMy80084F: 5'-AATGAAGGGCAATGCAAGAC-3' (SEQ ID NO.5),

[0023] TMy80084R: 5'-GGGAGTTTAGGGCAGACAAA-3' (SEQ ID NO. 6);

[0024] The primer sequences for amplifying TMy81298 are:

[0025] TMy81298F: 5'-GAAGAGCCCAAATGTCGAAG-3' (SEQ ID NO.7),

[0026] TMy81298R: 5'-GGGTCAATGGGCATGTTTAT-3' (SEQ ID NO. 8).

[0027] The application of the above-mentioned co-dominant SSR marker tightly linked to the tobacco β-carotene gene qbCar is to detect whether the tobacco β-carotene gene qbCar exists in tobacco genomic DNA and the genotype status of tobacco β-carotene in the tested plants.

[0028] The above application of the present invention comprises the following steps: using primers of TMy80084 and TMy81298 sequences to amplify the tobacco genome DNA to be detected, and detecting the PCR amplification products.

[0029] 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 tobacco beta-carotene, and the genotype is bCarbCar.

[0030] If 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, the tobacco plant to be tested has no homozygous allele of tobacco beta-carotene content, and the genotype is bcarbcar.

[0031] 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, the tobacco plant to be tested has a heterozygous allele with a medium content of tobacco beta-carotene, and the genotype is bCarbcar.

[0032] In order to select tobacco varieties with high β-carotene content potential simply and efficiently, the present invention selectively selects offspring materials containing tobacco β-carotene gene qbCar, which can be used for auxiliary selection of tobacco β-carotene gene qbCar, so as to improve the efficiency of molecular marker-assisted selection and the efficiency of high-content β-carotene tobacco variety breeding. The two co-dominant SSR markers closely linked to tobacco β-carotene gene qbCar provided by the present invention can not only be used to qualitatively detect whether tobacco β-carotene gene qbCar exists in tobacco genomic DNA at any growth period, but also can clearly and accurately identify the genotype state of tobacco β-carotene in the tested plants (i.e., homozygous genotype bCarbCar with the highest content value and stable inheritance, heterozygous genotype bCarbcar with medium content value and unstable inheritance, homozygous genotype bcarbcar with no content value and stable inheritance), thereby improving the scientificity and predictability of the breeding of new flue-cured tobacco varieties with high β-carotene content, and accelerating the breeding process.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. It fills the gap in the research on the inheritance and positioning of tobacco β-carotene gene qbCar at home and abroad.

[0035] 2. Clear and accurate detection of tobacco beta-carotene genotype status

[0036] The invention provides a molecular marker for detecting the tobacco beta-carotene gene qbCar, which can not only accurately determine whether the tobacco to be tested contains tobacco beta-carotene, but also clearly identify the genotype status of beta-carotene in the tobacco plants to be tested.

[0037] 3. The detection method is efficient, stable, reliable, simple to operate and low-cost

[0038] Compared with the existing low-throughput, high-cost, time-consuming and labor-intensive HPLC method for detecting the β-carotene content of mature and cured tobacco, the co-dominant SSR marker described in the present invention is highly efficient, stable, reliable, simple and low-cost.

[0039] 4. No limit on the timing of testing

[0040] 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 β-carotene content.

[0041] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The tobacco recombinant inbred line population (RILs_F 8:9 ; Y3×K326) in linkage group 2 of tobacco β-carotene QTL analysis curve.

[0043] 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 major effect gene (qbCar). DETAILED DESCRIPTION

[0044] 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.

[0045] The co-dominant SSR markers tightly linked to the tobacco beta-carotene gene qbCar of the present invention are numbered TMy80084 and TMy81298, 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.

[0046] The primer sequences of the two sites corresponding to the molecular markers are:

[0047] The primer sequences for amplifying TMy80084 are:

[0048] TMy80084F: 5'-AATGAAGGGCAATGCAAGAC-3',

[0049] TMy80084R: 5'-GGGAGTTTAGGGCAGACAAA-3';

[0050] The primer sequences for amplifying TMy81298 are:

[0051] TMy81298F: 5'-GAAGAGCCCAAATGTCGAAG-3',

[0052] TMy81298R: 5'-GGGTCAATGGGCATGTTTAT-3'.

[0053] The application of the codominant SSR marker tightly linked to the tobacco beta carotene gene qbCar of the present invention is the application of the codominant SSR marker tightly linked to the tobacco beta carotene gene qbCar in detecting whether the tobacco beta carotene gene qbCar exists in tobacco genomic DNA.

[0054] The application of the codominant SSR marker tightly linked to the tobacco beta-carotene gene qbCar is to amplify the tobacco genomic DNA to be tested with primers of TMy80084 sequence and primers of TMy81298 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 qbCarqbCar with a high tobacco beta-carotene 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 no homozygous allele qbcarqbcar with a tobacco beta-carotene 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 qbCarqbcar with a medium content of tobacco beta-carotene.

[0055] Example 1 Screening of co-dominant SSR markers linked to tobacco β-carotene gene qbCar

[0056] The quantitative trait linkage analysis (QTL) method combined with high performance liquid chromatography (HPLC) was used to screen for co-dominant SSR markers linked to the tobacco β-carotene gene qbCar in the whole tobacco genome.

[0057] 1. Experimental Materials

[0058] The flue-cured tobacco variety K326, which has excellent comprehensive traits but does not contain tobacco β-carotene, was used as the female parent, and the high-quality flue-cured tobacco variety Y3, which has a high content of tobacco β-carotene, was used as the male parent. Through hybridization and continuous self-pollination, 269 recombinant inbred lines (RILs_F 8:9 ) as a genetic mapping population.

[0059] 2. Parents and RILs_F 8:9 Data acquisition of β-carotene content in tobacco population

[0060] 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.

[0061] The obtained C3F grade powdered tobacco samples from the middle of each strain were tested for β-carotene content in tobacco leaves after baking according to the tobacco industry standard YCT 382-2010 of the People's Republic of China (high performance liquid chromatography method for the determination of somatic pigments in tobacco and tobacco products).

[0062] 269 ​​RILs_F obtained by HPLC 8:9 β-carotene content of tobacco as RILs_F 8:9 The phenotypic values ​​of the population were used for the next QTL linkage analysis.

[0063] 3. SSR marker analysis

[0064] Tobacco genomic DNA extraction: 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.

[0065] 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.

[0066] 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 the first generation (F1) of the population were screened for polymorphism, and ultimately, 2158 polymorphic SSR markers were obtained.

[0067] 2158 polymorphic SSR markers were obtained by screening and 269 RILs_F 8:9 The samples were subjected to genotyping analysis.

[0068] 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.

[0069] 4. Genome-wide QTL analysis of tobacco β-carotene (qbCar)

[0070] The QTL localization and analysis software QTL IciMapping v4.2 was used to analyze the RILs_F 8:9 The genotype data (construction of the genetic linkage group map of flue-cured tobacco) and phenotype data (269 RILs_F 8:9 The β-carotene content of tobacco populations was analyzed and a genome-wide QTL scan of tobacco β-carotene gene qbCar was performed.

[0071] 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).

[0072] Finally, under the condition of LOD=3.5744 in the whole genome, a major QTL (temporarily named qbCar) for tobacco β-carotene trait was located at 73.50 cM on linkage group 2. This major QTL can explain about 16.5918% of the phenotypic variation rate, and the LOD value at this time is about 42.2094, see Figure 1 and Table 1.

[0073] Table 1 Statistics of tobacco β-carotene QTL (qBCar) information

[0074] QTL Chromosome Position / cM Left Marker Right Marker LOD PVE(%) Add qbCar 2 73.50 TMy80084 TMy81298 42.2094 16.5918 -4.1056

[0075] 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.

[0076] Example 2 Co-dominant linkage markers in RILs_F 9:10 Verification in individual plants of the population

[0077] Using the codominant SSR markers TMy80084 and TMy81298, which were closely linked to the tobacco β-carotene gene qbCar, 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.

[0078] On the other hand, when RILs_F 9:10 After the tobacco leaves of the group grew to maturity and were harvested and cured, the middle C3F grade cured leaves of each strain were ground into powder using HPLC and the β-carotene content was tested. 9:10 Phenotypic values ​​of each strain in the population.

[0079] Finally, 269 RILs_F 9:10 The genotype data of the population and the tobacco β-carotene phenotype values ​​showed that the genotype values ​​and phenotype values ​​of the two co-dominant SSR markers TMy80084 and TMy81298 published in the present invention were completely consistent with each other, that is, the consistency rate reached 100%.

[0080] The specific analysis method is: when the beta-carotene content of each tobacco strain obtained by HPLC is higher than or equal to that of the parent Y3, the genotype of the strain also presents the sequences shown in SEQ ID NO.1 (258 bp) and SEQ ID NO.3 (273 bp) at the same time, which is the homozygous genotype bCarbCar;

[0081] When the beta-carotene content of each tobacco strain obtained by the test is equal to that of the parent K326 (i.e., the beta-carotene content is 0), the genotype of the strain also presents the sequences shown in ID NO.2 (278 bp) and SEQ ID NO:4 (247 bp) at the same time, which is the homozygous genotype bcarbcar;

[0082] When the beta-carotene content of each tobacco strain obtained by detection is between that of the parents Y3 and K326, that is, close to the content of the first generation (F1), the genotype of the strain also simultaneously presents 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, which is a heterozygous genotype bCarbcar.

[0083] Experimental conclusion:

[0084] The above results showed that the codominant markers TMy80084 and TMy81298 were tightly linked to the tobacco β-carotene gene qbCar, and the two markers were located on both sides of the target gene (qbCar).

[0085] By using the above two co-dominant tightly linked SSR markers, not only can the tobacco β-carotene content at any growth stage of tobacco be detected accurately, efficiently, conveniently and at low cost, but also the genotype status of tobacco β-carotene in the tested plants can be clearly identified (i.e., the homozygous genotype bCarbCar with the highest content value and stable inheritance, the heterozygous genotype bCarbcar with a medium content value and unstable inheritance, and the homozygous genotype bcarbcar with no content value and stable inheritance), thereby improving the scientificity and predictability of the breeding of new varieties with high tobacco β-carotene content and accelerating the breeding process.

[0086] 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 tobacco β-carotene gene qbCar, characterized in that: The co-dominant SSR markers tightly linked to the tobacco beta-carotene gene qbCar are numbered TMy80084 and TMy81298, and the nucleotide sequences of their PCR amplification products 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 tobacco β-carotene gene qbCar according to claim 1, characterized in that: The co-dominant markers TMy80084 and TMy81298 are located on both sides of the target gene qbCar.

3. The co-dominant SSR marker tightly linked to the tobacco β-carotene gene qbCar according to claim 1, characterized in that: The primer sequences of the two sites corresponding to the molecular markers are: The primer sequences for amplifying TMy80084 are: TMy80084F: 5'-AATGAAGGGCAATGCAAGAC-3', TMy80084R: 5'-GGGAGTTTAGGGCAGACAAA-3'; The primer sequences for amplifying TMy81298 are: TMy81298F: 5'-GAAGAGCCCAAATGTCGAAG-3', TMy81298R: 5'-GGGTCAATGGGCATGTTTAT-3'.

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

5. The use according to claim 4, characterized in that: The application method is to amplify the tobacco genome DNA to be detected using primers of TMy80084 and TMy81298 sequences respectively, and detect the PCR amplification products.

6. The use according to claim 5, 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 tobacco beta-carotene, and the genotype is bCarbCar.

7. The use according to claim 5, 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, the tobacco plant to be tested has no homozygous allele for tobacco beta-carotene content, and the genotype is bcarbcar.

8. The use according to claim 5, 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 tobacco beta-carotene, and the genotype is bCarbcar.

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