CN113957169B - Codominant SSR molecular marker closely linked with cis-cold shirt gene qAbl and application thereof - Google Patents

Abstract

The invention discloses a co-dominant SSR molecular marker closely linked with a cis-cold shirt alcohol gene qAbl and application thereof; the co-dominant SSR molecular markers closely linked with the cis-cold shirt gene qAbl are numbered TMc43307 and TMc43341, and the nucleotide sequences of PCR amplified products of the co-dominant SSR molecular markers are respectively shown by SEQ ID No.1 and SEQ ID No.2, and SEQ ID No.3 and SEQ ID No. 4; the application of a co-dominant SSR molecular marker closely linked with a cis-cold shirt alcohol gene qAbl in detecting whether the cis-cold shirt alcohol gene qAbl exists in tobacco genome DNA. The co-dominant SSR molecular marker closely linked with the cis-cold shirt gene qAbl has the characteristics of accuracy, high efficiency, stability, convenience and low cost, so that the molecular marker can be used as the application of the cis-cold shirt gene qAbl molecular marker for auxiliary selection in high-aroma quality breeding of tobacco.

Description

Codominant SSR molecular marker closely linked with cis-cold shirt gene qAbl and application thereof

Technical Field

The invention relates to the technical field of molecular genetic breeding, in particular to a co-dominant SSR molecular marker closely linked with a cis-cold shirt alcohol gene qAbl and application thereof.

Background

Cold shizanol (Abienol) is a terpene compound with fragrance secreted by plant surfaces, especially leaf surfaces. To date, the cold-shirt alcohols present in nature are Cis-cold-shirt alcohols (Cis-Abienol), and there are no reports on trans-cold-shirt alcohols. Cis-cryptazinol in tobacco is mainly lysibadanol, which is not only the main component of glandular hair secretion on the surface of tobacco leaves, but also an important tobacco aroma precursor. Early genetic studies on cis-cold-shirt alcohol in tobacco showed that cis-cold-shirt alcohol belongs to a quality trait and is under the control of a dominant single gene (Abl) (TomitaH, satoM, kawashima n.inhereitance of labdanoid producing ability in Nicotiana tabacum.agric Biol Chem,1980,44:2517-2518;KuboT,SatoM,TomitaH.Identificationofthechromosomecarryingthegenefor cis-abienol production byte useofmonosomicin nicotianabata ml.tobnit., 1982,184 (23): 57-59.). Based on this, sucrose ester gene (BMVSE) and cis-cold shirt gene (Abl) controlling aroma quality in cigar variety Beinhart1000 were located on linkage group 15 using tobacco Doubled Haploid (DH) population, vontimitta et al, and the genetic distance between genes controlling the above two traits was about 9cM, for cis-cold shirt gene (Abl) was located between SSR markers PT61373 and PT55091 about 2.6cM, and marker PT10324 was located on cis-cold shirt gene (Abl), i.e., co-segregating with Abl (Vontimitta V, danehower D A, steede T, moon H S, lewis R S, analysis of a Nicotiana tabacum L. Genomic region controlling two leaf surface chemistry traits.journal of Agricultural and Food Chemistry,2010,58:294-300. With continued intensive research into the cis-cold shirt synthesis pathway, sallaud et al succeeded in cloning 2 key genes (NtABS and NtCPS 2) controlling the tobacco cis-cold shirt contract pathway from the aromatic tobacco genome in 2012, and further confirmed by complementation experiments that the NtABS gene and the NtCPS2 gene jointly code for the synthesis of cis-cold shirt in tobacco, but the authors also elucidated that there were still other genes in addition to the 2 genes that were responsible for synthesizing cis-cold shirt in tobacco (Sallaud C, giacalone C, topfer R, goepfert S, bakaher N, rosti S and Tissier A. Characterization of two genes for the biosynthesis of thelabdane diterpene Z-abienol in tobacco (Nicotiana tabacum) gland derivative Journal,2012, 72:1-17.). As can be seen, the cis-frigories trait in tobacco is controlled by dominant multiple genes, and not by single gene control reported in earlier studies (TomitaH, satom, kawashima N.Inhereitance of labdanoid producing ability in Nicotiana tabacum. Agric Biol Chem,1980,44:2517-2518;KuboT,SatoM,TomitaH.Identificationofthechromosomecarryingthegenefor cis-abienol production byte of monosomicin Nicotianatabacul mL. TobInt.,1982,184 (23): 57-59;Vontimitta V,Danehower D A,Steede T,Moon H S,Lewis R S,Analysis of a Nicotiana tabacum L.genomic region controlling two leaf surface chemistry traits.Journal of Agricultural and Food Chemistry,2010,58:294-300.). On the other hand, detection of cis-cold shirt by simple use of the NtABS gene or/and the NtCPS2 gene in the cis-cold shirt synthesis pathway is not strict, because of the great risk of missed detection (Sallaud C, giacalone C, topfer R, goepfert S, bakaher N, rosti S and Tissier A. Characial of two genes for the biosynthesis of thelabdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glabrous trichler. The Plant Journal,2012,72:1-17; wang Guoping. Analysis of tobacco cold shirt alcohol synthesis gene function and development of functional markers. National academy of agricultural sciences, 2016, pp 4-5.). In addition, in order to realize the cultivation of a new line of tobacco having high aroma quality with cis-cold shirt-containing by using molecular Marker Assisted Selection (MAS), chang Aixia et al developed SNP markers for detecting the presence or absence of cis-cold shirt in tobacco based on the sequence of the key gene NtCPS2 in the cis-cold shirt synthesis pathway and issued patents (Chang Aixia, wang Yuanying, wang Guoping, liu Dan, yang Aiguo, sun Huiqing, cao Jianmin, li Yijiang, luo Chenggang, feng Quanfu, liu Guanshan. Specific primers and detection methods for detecting single nucleotide mutation of the key gene NtCPS2 for tobacco cold shirt synthesis. Authorized bulletin No. cn105177162b. It is obvious that the SNP marker provided by the patent can only detect whether the tobacco contains the NtCPS2 gene, but cannot be used for detecting whether cis-choledochol exists in tobacco plants to be detected, and because the gene for controlling cis-choledochol synthesis in tobacco is not only one, extremely high detection false negatives exist (namely, even if the tobacco plants to be detected do not contain NtCPS2, cis-choledochol is still contained in a great probability). The defects or errors seriously restrict the development of the breeding process of the new variety of the high-aroma tobacco by utilizing the markers closely linked with the cis-cold shirt alcohol gene qAbl.

In view of the above, it is necessary to study a co-dominant SSR molecular marker closely linked to the cis-cold shirt gene qAbl and its application to solve the above technical problems.

Disclosure of Invention

The first object of the invention is to provide a co-dominant SSR molecular marker closely linked to a cis-cold shirt gene qAbl, and the second object of the invention is to provide application of the co-dominant SSR molecular marker closely linked to the cis-cold shirt gene qAbl. The invention aims to accelerate the process of breeding new varieties of high-aroma quality tobacco by utilizing a marker closely linked with a cis-cold shirt alcohol gene qAbl.

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

the first object of the present invention is achieved by a co-dominant SSR molecular marker closely linked to a cis-cold shirt gene qAbl, which is numbered TMc43307 and TMc43341, and the nucleotide sequences of PCR amplification products thereof are shown by SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, respectively.

Preferably, the primer sequences corresponding to TMc43307 and TMc43341 are respectively:

the TMc43307 primer sequences are:

TMc43307F：5’-ATGGCGTACAACCCTATCCA-3’，

TMc43307R：5’-CTGCACGTCACATCGAGTTT-3’；

the TMc43341 primer sequences are:

TMc43341F：5’-ATGGAAAACCTCGTGCACTT-3’，

TMc43341R：5’-GACGGATCTTCCGTTGACAT-3’。

the second object of the invention is achieved by the use of a co-dominant SSR molecular marker in close linkage with the cis-cold shirt alcohol gene qAbl in the detection of the presence or absence of the cis-cold shirt alcohol gene qAbl in tobacco genomic DNA.

Preferably, the primer TMc43307F, TMc43307R of the TMc43307 sequence and the primer TMc43341F, TMc43341R of the TMc43341 sequence are used for amplifying the tobacco genome DNA to be detected respectively, and PCR amplification products are detected;

if the PCR amplification product contains sequences shown as SEQ ID No.1 and SEQ ID No.3, the tobacco plant to be detected contains homozygous alleles of cis-cryptate with high aroma quality, and the genotype is marked as AA;

if the PCR amplification product contains sequences shown as SEQ ID No.2 and SEQ ID No.4, the sequence is a homozygous allele of the tobacco plant to be detected without cis-cryptate, and the genotype is aa;

if the PCR amplified product contains sequences shown as SEQ ID No.1 and SEQ ID No.2, or contains sequences shown as SEQ ID No.3 and SEQ ID No.4, or contains sequences shown as SEQ ID No.1, SEQ ID No.3 and SEQ ID No.4, or contains sequences shown as SEQ ID No.3, SEQ ID No.1 and SEQ ID No.2, the heterozygous allele containing cis-colestolide is obtained, and the genotype is Aa.

In summary, compared with the prior art, the invention has the following beneficial effects:

1. in order to simply and efficiently select a cis-cold shirt-alcohol-containing tobacco variety with high aroma quality potential, a offspring material containing a cis-cold shirt-alcohol gene qAbl is selected in a targeted and specific manner, the invention provides molecular markers TMc43307 and TMc43341 for detecting the cis-cold shirt-alcohol gene qAbl, the molecular markers adopt a quantitative trait linkage analysis (QTL) method and an ultra-high liquid chromatography-ultraviolet (UPLC-UV) method, co-dominant SSR markers interlocked with the cis-cold shirt-alcohol gene qAbl are obtained through screening within the whole genome range of tobacco, and the co-dominant SSR markers can be used for auxiliary selection of the cis-cold shirt-alcohol gene qAbl so as to improve the efficiency of auxiliary selection of the molecular markers and the breeding efficiency of the tobacco variety with high aroma quality.

2. The invention takes cigar variety Beinhart1000-1 with high cis-cold shirt content and flue-cured tobacco variety safflower Dajinyuan (with good comprehensive properties) without cis-cold shirt content as parents, and constructs tobacco recombinant inbred line (RILs_F) through hybridization and continuous bagging inbreeding _7:8 ) For mapping population, quantitative trait linkage analysis (QTL) method and ultra-high liquid chromatography-ultraviolet (UPLC-UV) method are adopted to screen and obtain co-dominant SSR markers closely linked with cis-cold shirt alcohol gene qAbl in the whole genome range of tobacco so as to accelerate the accurate and efficient utilization of molecular Marker Assisted Selection (MAS) in tobacco high-aroma variety breeding.

3. Compared with the detection of the tobacco cis-cold shirt alcohol content by the markers reported in the literature, the molecular markers provided by the invention can accurately identify whether the tobacco to be detected contains cis-cold shirt alcohol or not, and compared with the markers reported in the literature, the molecular markers have higher detection accuracy, and effectively correct and make up errors and defects of the detection of the tobacco cis-cold shirt alcohol by using the gene NtCPS2 in the literature; compared with the existing method for detecting the content of the tobacco cis-cold shirt by adopting a UPLC-UV method with low flux, high cost and time and labor consumption, the co-dominant SSR marker has the characteristics of high efficiency, stability, reliability, simplicity and low cost, can be used for detecting at any period of tobacco growth, greatly shortens the experimental period, and further accelerates the process of breeding new varieties of high-aroma quality tobacco.

4. The co-dominant SSR marker is adopted, so that the molecular marker has the characteristics of accuracy, high efficiency, stability, convenience and low cost, and can be used as an application of qAbl gene molecular marker auxiliary selection in tobacco high-aroma quality breeding.

Drawings

FIG. 1 is a tobacco-based recombinant inbred population (RILs_F) of the invention _7:8 The method comprises the steps of carrying out a first treatment on the surface of the Figure of cis-cold shirt QTL analysis on linkage group 15 of safflower dajinyuan x bennhart 1000-1).

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

The invention provides a co-dominant SSR molecular marker closely linked with a cis-cold shirt gene qAbl, which is numbered TMc43307 and TMc43341, and the nucleotide sequences of PCR amplified products of the co-dominant SSR molecular marker are respectively shown by SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

The primer sequences corresponding to TMc43307 and TMc43341 are respectively as follows:

the TMc43307 primer sequences are:

TMc43307F：5’-ATGGCGTACAACCCTATCCA-3’(SEQ ID No.5)，

TMc43307R：5’-CTGCACGTCACATCGAGTTT-3’(SEQ ID No.6)；

the TMc43341 primer sequences are:

TMc43341F：5’-ATGGAAAACCTCGTGCACTT-3’(SEQ ID No.7)，

TMc43341R：5’-GACGGATCTTCCGTTGACAT-3’(SEQ ID No.8)。

the invention also provides application of the co-dominant SSR molecular marker closely linked with the cis-cold shirt alcohol gene qAbl in detecting whether the cis-cold shirt alcohol gene qAbl exists in the tobacco genome DNA.

And respectively amplifying the tobacco genome DNA to be detected by using a primer TMc43307F, TMc43307R of the TMc43307 sequence and a primer TMc43341F, TMc43341R of the TMc43341 sequence, and detecting PCR amplification products.

If the PCR amplification product contains sequences shown as SEQ ID No.1 and SEQ ID No.3, the tobacco plant to be detected contains homozygous alleles of cis-cryptate with high aroma quality, and the genotype is marked as AA.

If the PCR amplification product contains sequences shown as SEQ ID No.2 and SEQ ID No.4, the tobacco plant to be detected does not contain homozygous allele of cis-colestosterol, and the genotype is aa.

If the PCR amplified product contains sequences shown as SEQ ID No.1 and SEQ ID No.2, or contains sequences shown as SEQ ID No.3 and SEQ ID No.4, or contains sequences shown as SEQ ID No.1, SEQ ID No.3 and SEQ ID No.4, or contains sequences shown as SEQ ID No.3, SEQ ID No.1 and SEQ ID No.2, the heterozygous allele containing cis-colestolide is obtained, and the genotype is Aa.

The invention is further illustrated by the following examples.

Example 1:

this example 1 employs quantitative trait linkage analysis (QTL) in combination with ultra-high liquid chromatography-ultraviolet (UPLC-UV) to screen co-dominant SSR markers linked to the cis-cold shirt gene qAbl across the whole genome of tobacco.

1.1 Experimental materials

The tobacco variety Honghuadajinyuan with excellent comprehensive characters and no cis-cold shirt alcohol is taken as a female parent, the cigar variety Beinhart1000-1 with cis-cold shirt alcohol is taken as a male parent, and the recombinant inbred line (RILs_F) of 341 parts of strains is obtained through hybridization and continuous selfing _7:8 ) As a population of genetic mapping.

1.2 parent and RILs_F _7:8 Population cis-cryptazinol content data acquisition

After the test materials are grown into seedlings, the seedlings are transplanted to a field, after the tobacco leaves in the field are ripe, picked and baked, the tobacco cis-cold shirt alcohol content is detected according to a UPLC-UV method reported in the literature (No. Qiu Juan, dumei, wang Aihua, liu Yanhua, han Xiao and Zhang Xiaojing. Ultra-high liquid chromatography is used for measuring cis-cold shirt alcohol in tobacco, tobacco science and technology, 2020,53 (11): 64-68). 341 parts of RILs_F obtained by detection by UPLC-UV method _7:8 Cis-cold shirt alcohol content as rils_f _7:8 The phenotype values of the population are used for the QTL linkage analysis of the next step.

1.3SSR marker analysis

Extraction of tobacco genome DNA: the conventional CTAB method or the plant tissue DNA extraction kit can be adopted, and the method can be referred to the existing literature or the instruction in the kit.

PCR amplification and electrophoresis detection: the PCR amplification system was 20. Mu.L, which contained 2.0. Mu.L of 10 XBuffer (10 mmol/L Tris-Cl, pH=8.4, 50mmol/L KCl,1.5mmol/L MgCl) ₂ ) 200. Mu. Mol/L dNTPs (Takara Biotechnology Co. Ltd., dalia, china), 0.5. Mu. Mol/L upstream and downstream primer (Takara), 0.75U rTaq polymerase (Takara), 30-50 ng template DNA, and finally ddH ₂ O was made up to 20. Mu.L. The PCR reaction procedure was: pre-denatured at 95℃for 5min,30 cycles (denaturation at 95℃for 30s, renaturation at 60℃for 30s, extension at 72℃for 30 s), extension at 72℃for 5min, and storage at 4 ℃. PCR amplification product detection: the PCR amplification product was subjected to 1/6 volume of 6×loading Buffer, and 2.5. Mu.L of the mixture was subjected to 6% non-denaturing polyacrylamide gel (non-denaturing PAGE, 550V)2.5 h) separating by electrophoresis on DYY-6C electrophoresis apparatus (Beijing Liuyi factory), fixing, silver staining, rinsing, developing, rinsing, and performing photographing and film data processing.

About 50000 SSR markers developed by the laboratory based on flue-cured safflower Dajinyuan and cigar Beinhart1000-1 genome information are utilized for RILs_F _7:8 Parents (safflower Dajinyuan and Beinhart 1000-1) and sub-generations (F) ₁ ) And carrying out polymorphism screening, and finally, screening to obtain 2001 polymorphic SSR markers. Then 2001 polymorphic SSR markers are obtained by screening, and 341 parts of RILs_F are subjected to _7:8 The samples were genotyped. Next, the genetic linkage mapping software JoinMapv4.0 vs. 341 parts RILs_F was used _7:8 Carrying out linkage analysis on genotype data of a sample, drawing a high-quality cigar genetic linkage map which contains 24 linkage groups and is uniformly distributed with 1974 SSR markers and covers the length of a tobacco genome of 3213.138cM, and taking the cigar genetic linkage map as RILs_F _7:8 Genotype values of the population were used for the next QTL linkage analysis.

1.4 Whole genome QTL positioning analysis of cis-Cold-Cardigan (qAbl)

RILs_F pairs using QTL positioning analysis software QTL IcinMapping v4.2 _7:8 Genotype data (constructed to obtain cigar genetic linkage map) and phenotype data (341 RILs_F) _7:8 Cis-cryptazinol content of the population), the cis-cryptazinol gene qAbl was subjected to whole genome QTL scan. Wherein, the relevant parameters are set as follows: the positioning method selects ICIM-ADD: inclusive Composite Interval Mapping of ADDitive (and domitant) QTL, number of iterations 1000 (Permutation times =1000), significance 0.01 (signalicance=0.01), step size 0.5cM (Walk speed=0.5 cM). The abscissa is the genetic distance (unit: centimorgan cM); the ordinate is LOD value. The horizontal dashed line in the figure is LOD value= 3.3733 at the 0.01 significance threshold; the highest point of the LOD curve is the major gene (qabl_15). Finally, under genome-wide lod= 3.3733 conditions, cis-cold was obtained by localization at 83.00cM of linkage group 151 major QTL for the paclitaxel trait (designated temporarily qabl_15). The major QTL may explain a phenotypic variation of about 39.15% and a LOD value of about 9.48 at this time. Based on tobacco recombinant inbred populations (rils_f) _7:8 The method comprises the steps of carrying out a first treatment on the surface of the The graph of cis-cold shirt QTL analysis on linkage group 15 of safflower dajinyuanx bennhart 1000-1) is shown in fig. 1, and the statistics of cis-cold shirt gene qAbl (qabl_15) information are shown in table 1.

TABLE 1

QTL

Chromosome

Position/cM

LeftMarker

RightMarker

LOD

PVE(％)

Add

qAbl_15

15

83.00

TMc43307

TMc43341

9.4796

39.1478

0.3501

Note that: PVEs are the effector value of a QTL, i.e., the QTL can account for the percentage of phenotypic variation; add is an additive effect.

Example 2

This embodiment provides co-dominant linkage markers at RILs_F _8:9 Verification in population individuals.

Using the obtained co-dominant SSR markers TMc43307 and TMc43341 closely linked to both sides of the cis-cold shirt gene qAbl, RILs_F for seedling stage _8:9 Genotyping the individual plants of the population (Honghuadajinyuan X Bennhart 1000-1) to obtain RILs_F _8:9 Genotype data for each individual of the population; on the other hand, waiting for RILs_F _8:9 After the tobacco leaves of the colony grow mature and are picked and roasted, the UPLC-UV method is adopted to detect the cis-cold shirt alcohol content in the leaves of each strain, namely RILs_F is obtained _8:9 Phenotype values for each strain of the population. Finally, 341 parts of RILs_F are analyzed _8:9 Genotype data of the population and cis-cold shirt content phenotype values find that the genotype values of the two co-dominant SSR markers TMc43307 and TMc43341 disclosed by the invention completely coincide with the phenotype values, namely, the coincidence rate reaches 100%.

The specific analysis method comprises the following steps: when the content of cis-cold shirt alcohol of each strain obtained by UPLC-UV detection is higher than or equal to that of the parent Beinhart1000-1, the genotype of the strain simultaneously shows sequences shown as SEQ ID NO.1 (216 bp) and SEQ ID NO.3 (239 bp), namely homozygous genotype AA; when the cis-cold shirt alcohol content of each strain is equal to or lower than the parent safflower big gold element content, the genotype of the strain simultaneously shows sequences shown as ID NO.2 (212 bp) and SEQ ID NO.4 (305 bp), namely homozygous genotype aa; when the cis-cold shirt alcohol content of each strain obtained by detection is between the parent safflower Dajinyuan and Beinhart1000-1, namely the cis-cold shirt alcohol content is equal to that of the sub-generation (F) ₁ ) When the content is similar, the genotype of the strain is also represented by sequences shown as SEQ ID NO.1 and SEQ ID NO.2, or sequences shown as SEQ ID NO.3 and SEQ ID NO.4, or sequences shown as SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4, or sequences shown as SEQ ID NO.3, SEQ ID NO.1 and SEQ ID NO.2 are contained, so that the heterozygous genotype Aa is obtained.

The above results indicate that co-dominant markers TMc43307 and TMc43341 are closely linked to the cis-cold shirt gene qAbl, respectively, and that the two markers flank the gene of interest (qabl—15). By utilizing the two co-dominant closely linked SSR markers, the detection of the cis-cold shirt alcohol content of tobacco in any growth period can be accurately, efficiently, conveniently and at low cost, the genotype state of cis-cold shirt alcohol in a plant to be detected (namely, the homozygous genotype AA with the highest content value and stable inheritance, the heterozygous genotype AA with the medium content value and unstable inheritance and the homozygous genotype AA without the content or the trace value and stable inheritance) can be clearly identified, and the scientificity and predictability of breeding new varieties of tobacco with high aroma quality are improved, and the breeding process is accelerated.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims (3)

1. Cis-cryptate geneqAblThe closely linked co-dominant SSR molecular marker is characterized in that: the and cis-cold shirt alcohol geneqAblThe numbers of the co-dominant SSR molecular markers which are tightly linked are TMc43307 and TMc43341, and the nucleotide sequences of PCR amplified products of the co-dominant SSR molecular markers are respectively shown by SEQ ID No.1 and SEQ ID No.2, and SEQ ID No.3 and SEQ ID No. 4.

2. The gene of cis-cold shirt according to claim 1qAblClosely linked co-dominanceThe SSR molecular marker is characterized in that primer sequences corresponding to TMc43307 and TMc43341 are respectively as follows:

the TMc43307 primer sequences are:

TMc43307F：5’- ATGGCGTACAACCCTATCCA -3’，

TMc43307R：5’- CTGCACGTCACATCGAGTTT -3’；

the TMc43341 primer sequences are:

TMc43341F：5’- ATGGAAAACCTCGTGCACTT -3’，

TMc43341R：5’- GACGGATCTTCCGTTGACAT -3’。

3. a gene of cis-cold shirt of claim 2qAblThe application of the closely linked co-dominant SSR molecular marker is characterized in that the gene and cis-cold shirt alcohol geneqAblClosely linked co-dominant SSR markers for detecting whether cis-chrysanthemumol genes exist in tobacco genome DNAqAblIs applied to the application of the system;

respectively amplifying tobacco genome DNA to be detected by using a primer TMc43307F, TMc43307R of the TMc43307 sequence and a primer TMc43341F, TMc43341R of the TMc43341 sequence, and detecting PCR amplification products;

if the PCR amplification product contains sequences shown as SEQ ID No.1 and SEQ ID No.3, the tobacco plant to be detected contains homozygous alleles of cis-cryptate with high aroma quality, and the genotype is marked as AA;

if the PCR amplification product contains sequences shown as SEQ ID No.2 and SEQ ID No.4, the sequence is a homozygous allele of the tobacco plant to be detected without cis-cryptate, and the genotype is aa;

if the PCR amplified product contains sequences shown as SEQ ID No.1 and SEQ ID No.2, or contains sequences shown as SEQ ID No.3 and SEQ ID No.4, or contains sequences shown as SEQ ID No.1, SEQ ID No.3 and SEQ ID No.4, or contains sequences shown as SEQ ID No.3, SEQ ID No.1 and SEQ ID No.2, the heterozygous allele containing cis-colestolide is obtained, and the genotype is Aa.