CN114032325B - SSR marker closely linked with V-type tobacco sucrose ester gene qBMVSE-499 - Google Patents

SSR marker closely linked with V-type tobacco sucrose ester gene qBMVSE-499 Download PDF

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CN114032325B
CN114032325B CN202111394234.5A CN202111394234A CN114032325B CN 114032325 B CN114032325 B CN 114032325B CN 202111394234 A CN202111394234 A CN 202111394234A CN 114032325 B CN114032325 B CN 114032325B
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陈学军
肖炳光
焦芳婵
童治军
方敦煌
李勇
袁诚
冯智宇
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Yunnan Academy of Tobacco Agricultural Sciences
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Abstract

The invention distinguishes the genotypes of the V-type sucrose esters in three different types of tobacco sucrose esters for the first time, and provides a co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE 499. The genotype status of the V-type tobacco sucrose esters in the plants to be detected and the V-type tobacco sucrose esters in the plants to be detected can be qualitatively or quantitatively detected by using the markers. Compared with the prior art, the method has the advantages of higher detection accuracy, stable and reliable detection method, simple and convenient operation, low cost and no limit on detection time.

Description

SSR marker closely linked with V-type tobacco sucrose ester gene qBMVSE-499
Technical field:
The invention belongs to the technical field of biology, and particularly relates to a co-dominant SSR marker closely linked with a V-type tobacco sucrose ester gene qBMVSE-499 and application thereof.
The background technology is as follows:
The sucrose ester of the tobacco is one of the main chemical components on the surface of the leaf and is also an important potential aroma precursor substance in the tobacco, wherein the cultivated tobacco has higher content of sucrose tetraester, and the degradation of the sucrose tetraester can generate micromolecular volatile fatty acid and can greatly improve the aroma quality of the tobacco.
A large number of researches show that the sucrose tetraesters in common tobacco are further divided into 6 types (groups) according to the difference of molecular weight, namely, type I tobacco sucrose esters-type VI tobacco sucrose esters, wherein the type III tobacco sucrose esters-type VI tobacco sucrose esters are called BMVSE(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.). because of containing 3-methyl pentanoyl groups, the structural types and the content of the sucrose tetraesters in different cultivated tobacco types have large differences, and flue-cured tobacco, burley tobacco and maryland tobacco contain only a small amount of type I tobacco sucrose esters and type II tobacco sucrose esters and do not contain or contain trace BMVSE; while aromatic and cigars contain significant amounts of BMVSEs.
It has been found that BMVSE, after further analysis by silanized gas chromatography mass spectrometry (GC-MS), can be further classified into BMVSE471 (type III tobacco sucrose esters), BMVSE485 (type IV tobacco sucrose esters) and BMVSE499 (type V tobacco sucrose esters) according to the partial characteristic fragment ion size of glucose ring, and these 3 types of tobacco sucrose esters are also core concerns for the cultivation of new flue-cured tobacco varieties with high aroma quality containing high sucrose esters.
However, in the prior art, three different independent genes originally belonging to BMVSE471 (type III), BMVSE485 (type IV) and BMVSE499 (type V) respectively are regarded as one gene (and are collectively named as BMVSE) in error for genetic localization study. As a result, the positioning results obtained are inaccurate. Resulting in far from reaching the molecular Marker Assisted Selection (MAS) accuracy requirements.
For example, the BMVSE trait gene is dominant, is located in about 5.2cM region between SSR markers PT30354 and PT52061, PT61362, and is coseparated (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.). from SSR markers PT30209 and PT20315 by using the above 5 SSR markers to carry out BMVSE screening verification on tobacco materials of different genetic backgrounds and 192 recombinant inbred line populations, and the result shows that only 2 markers (PT 20135 and PT 30354) can be used, but the uniformity rate of the genotypes of the markers and BMVSE phenotype is about 92.165% (Chen Biao, chen Ming, li Yangyang, qu Yafang, cheng Lirui, yang Aiguo, hu Risheng, liu Dan, luo Chenggang, SSR molecular marker screening for toxygenpeng, feng Quanfu, chang Aixia, tobacco sugar esters and assisted breeding application in chinese tobacco science, 2019,40 (3): 8-15.). The main reason is that: the authors of the literature incorrectly considered three different independent genes originally belonging to BMVSE471 (type III), BMVSE485 (type IV) and BMVSE499 (type V), respectively, as one gene.
In view of this, the invention distinguishes three types of tobacco sucrose esters, including BMVSE471 (III), BMVSE485 (IV) and BMVSE499 (V), contained in BMVSE, and uses high-quality multi-resistant cigar variety Beinhart-1 (the quality of high aroma in leaves is controlled by BMVSE, while BMVSE is controlled by quantitative trait genes qBMVSE (III), qBMVSE485 (IV) and qBMVSE499 (V)) and flue-cured tobacco variety safflower macrogold (comprehensive characters are excellent but BMVSE is not contained) as parents, a tobacco recombinant inbred line (RILs_F 7:8) containing 341 strain is constructed as a mapping population through hybridization and continuous bagging selfing, and dominant SSR markers closely linked with V-type tobacco sucrose ester genes qBMVSE MASs spectrum (GC-MS) are obtained by screening in the whole genome range of tobacco, so as to make up the defects of the prior art and reported literature, accelerate molecular markers (auxiliary selection) and high-efficiency utilization of high quality tobacco in high-quality MAS.
Therefore, three types of tobacco sucrose esters, namely BMVSE471 (III type), BMVSE485 (IV type) and BMVSE499 (V type), contained in the BMVSE are distinguished, and the co-dominant SSR marker closely linked with a certain independent gene of the tobacco sucrose ester is accurately obtained, so that the method is an important way for cultivating a new variety of flue-cured tobacco with high aroma quality and high sucrose ester content. Can accurately and efficiently accelerate the utilization of molecular Marker Assisted Selection (MAS) in the breeding of high-aroma varieties of tobacco.
Disclosure of Invention
The first object of the invention is to provide a co-dominant SSR marker closely linked to the V-type tobacco sucrose ester gene qBMVSE499,499; a second object is to apply the above co-dominant SSR markers to detect the presence or absence of the V-type tobacco sucrose ester gene qBMVSE499,499 in tobacco genomic DNA.
The innovation point of the invention is that: the present invention has found a significant misunderstanding in the prior art.
The inventors found that the literature of blending type III-V tobacco sucrose esters into one gene (BMVSE) for localization was inaccurate, even erroneous, and that one type should be specifically selected from among three types of BMVSE for accurate localization. The error or deficiency of the prior art severely restricts the development of the breeding process of the new variety of the high-aroma tobacco by utilizing the close linkage markers of the III-V BMVSE genes.
Studies by the inventors have shown that BMVSEs of type III-V are typical quantitative traits and are controlled by QTLs on different chromosomes or at different locations on the same chromosome within the Beinhart1000-1 genome, these genes/QTLs being temporarily designated qBMVSE471, qBMVSE485 and qBMVSE499.
The present invention further compares the 5 SSR markers closely linked to BMVSE reported in the literature with Beinhart1000-1 genomes, and shows that even though there is a physical distance of about 15Mb between PT30329 and PT20315 markers co-separated from BMVSE, i.e., the result of the literature targeting type III-V tobacco sucrose esters as one gene (BMVSE) is inaccurate, the targeting result is most likely only one of three types of BMVSE. Therefore, only 2 closely linked markers are available in BMVSE screening verification by a certain domestic tobacco breeding worker, and the condition that the marker genotype is consistent with the BMVSE phenotype cannot be accurately realized.
1. The invention distinguishes three types of tobacco sucrose esters, namely BMVSE471 (III type), BMVSE485 (IV type) and BMVSE499 (V type), contained in BMVSE, and uses a high-quality multi-resistant cigar variety Beinhart-1 as a parent and a flue-cured tobacco variety safflower big gold element to construct a tobacco recombination inbred line (RILs_F 7:8) containing 341 parts of lines as a mapping population through hybridization and continuous bagging selfing.
The high aroma quality in the leaf of the cigar resistant variety Beinhart1000-1 is controlled by BMVSE, which is controlled by quantitative trait genes qBMVSE471 (type iii), qBMVSE485 (type IV) and qBMVSE499 (type V). The flue-cured tobacco variety safflower Dajinyuan has excellent comprehensive properties, but does not contain BMVSE.
2. Screening in the whole genome range of tobacco by using quantitative trait linkage analysis (QTL) method and silanization gas chromatography mass spectrometry (GC-MS) method to obtain a co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE499,499.
The co-dominant SSR marker linked with the V-type tobacco sucrose ester gene qBMVSE499 gene obtained by screening can be used for auxiliary selection of the V-type tobacco sucrose ester gene qBMVSE499 so as to improve the efficiency of molecular marker auxiliary selection, and improve the efficiency of breeding of simply, precisely and efficiently selecting the V-type tobacco sucrose ester tobacco variety with high aroma quality potential.
The co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE is numbered TMc42886 sequence and TMc43016 sequence, and is characterized in that PCR amplified products of the co-dominant SSR marker are selected from one or more of the following nucleotide sequences: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
The primer sequences of the 2 sites corresponding to the TMc42886 sequence and the TMc43016 sequence are respectively as follows:
primer of TMc42886 sequence: SEQ ID NO. 5 and SEQ ID NO. 6;
primer of TMc43016 sequence: SEQ ID NO. 7 and SEQ ID NO. 8.
The co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE-499 can be applied to detection of tobacco genes, and particularly can be used for detecting whether the V-type tobacco sucrose ester gene qBMVSE-499 exists in the tobacco genome DNA.
The detection method comprises the steps of respectively amplifying the tobacco genome DNA to be detected by using the TMc42886 sequence primer and the TMc43016 sequence primer, detecting PCR amplification products, and judging according to the following mode:
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 V-type tobacco sucrose esters with high aroma quality, and the genotype is recorded as SE499SE499;
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 or contains trace homozygous alleles of the V-type tobacco sucrose esters, and the genotype is recorded as se499se499;
If the PCR amplification 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 tobacco plant to be tested contains heterozygous alleles of V-type tobacco sucrose ester, and the genotype is SE499SE499.
In order to simply and efficiently select the V-type tobacco sucrose ester tobacco variety with high aroma quality potential, the invention specifically and purposefully selects the offspring material containing the V-type tobacco sucrose ester gene qBMVSE499, and can be used for auxiliary selection of the V-type tobacco sucrose ester gene qBMVSE499 so as to improve the efficiency of molecular marker auxiliary selection and the efficiency of breeding the high-aroma tobacco variety. The co-dominant SSR markers closely linked with the V-type tobacco sucrose ester gene qBMVSE-499 can be used for qualitatively detecting whether the V-type tobacco sucrose ester gene qBMVSE-499 exists in tobacco genome DNA in any growth period, can accurately, efficiently, conveniently and low-cost quantitatively detect the content of the V-type tobacco sucrose ester in tobacco, can clearly identify the genotype state of the V-type tobacco sucrose ester in plants to be detected (namely, homozygous genotype SE499SE499 with the highest content value and stable inheritance, heterozygosity genotype SE499SE499 with medium content value and unstable inheritance, and homozygous genotype SE499SE499 without or with trace content value and stable inheritance), and further improves the scientificity and predictability of breeding new flue-cured tobacco varieties with high aroma quality and accelerates the breeding process.
Compared with the prior art, the invention has the beneficial effects that:
1. Correcting errors of the prior art
The invention effectively corrects and makes up the error and the deficiency of regarding three different types of BMVSE as one gene in the literature, and can accurately identify the genotype of the V-type tobacco sucrose ester;
2. High accuracy for detecting V-type tobacco sucrose ester
The molecular marker for detecting the V-type tobacco sucrose ester gene qBMVSE-499 is provided, whether the tobacco to be detected contains the V-type tobacco sucrose ester can be accurately determined, and compared with the method for detecting the content of the tobacco sucrose ester in the prior art, the method has higher detection accuracy.
3. The detection method is efficient, stable and reliable, and has simple operation and low cost
Compared with the existing GC-MS method for detecting the sucrose ester content of the tobacco by using low flux, high cost, time and labor consumption, the co-dominant SSR marker has the characteristics of high efficiency, stability, reliability, simplicity and low cost.
4. Unlimited detection timing
The method can detect the tobacco at any period of growth, greatly shortens the experimental period, and further accelerates the process of breeding new flue-cured tobacco varieties with high aroma quality.
Sequence information:
SEQ ID NO. 1: the nucleotide sequence containing V-type tobacco sucrose ester is obtained by utilizing an SSR marker sequence with the number TMc42886 when PCR amplification is carried out in the genome DNA of tobacco to be detected.
SEQ ID NO. 2: the nucleotide sequence of trace amount of V-type tobacco sucrose ester is obtained when PCR amplification is carried out in the genome DNA of tobacco to be detected by utilizing an SSR marker sequence with the number of TMc 42886.
SEQ ID NO. 3: the nucleotide sequence containing V-type tobacco sucrose ester is obtained by utilizing an SSR marker sequence with the number TMc43016 when PCR amplification is carried out in the genome DNA of tobacco to be detected.
SEQ ID NO. 4: the nucleotide sequence of trace amount of V-type tobacco sucrose ester is obtained when PCR amplification is carried out in the genome DNA of tobacco to be detected by utilizing an SSR marker sequence with the number of TMc 43016.
SEQ ID NO. 5: is the nucleotide sequence of an SSR marker upstream primer with the serial number of TMc42886
SEQ ID NO. 6: is the nucleotide sequence of an SSR marker downstream primer numbered TMc42886
SEQ ID NO. 7: is the nucleotide sequence of an SSR marker upstream primer with the serial number of TMc43016
SEQ ID NO. 8: is the nucleotide sequence of an SSR marker downstream primer numbered TMc43016
Drawings
FIG. 1 is a graph of a QTL analysis of tobacco sucrose esters type III on linkage group 24 based on a tobacco recombinant inbred population (RILs_F 7:8; honghuadajinyuan X Beinhart 1000-1).
Wherein, the utilization software is: QTL ICIMAPPING V4.2.2; parameter setting: the positioning method is ICIM-ADD: inclusive Composite INTERVAL MAPPING of ADDitive (and dominant) QTL, number of iterations of 1000 (Permutation times =1000), significance of 0.01 (SIGNIFICANCE =0.01), step size of 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 (qBMVSE 499-24).
Detailed Description
The invention is further described below with reference to examples and figures. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the relevant product specifications. The reagents or apparatus used were conventional products available commercially without the manufacturer's attention.
The co-dominant SSR markers closely linked with the V-type tobacco sucrose ester gene qBMVSE are TMc42886 and TMc43016, and the nucleotide sequences of PCR amplified products are shown as SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 respectively.
The primer sequences of the 2 sites corresponding to the molecular markers are respectively as follows:
TMc42886 sequence TMc42886F:5'-GCATCTATTGTGGTCGGAAGA-3' the process of the preparation of the pharmaceutical composition,
TMc42886R：5’-TCGAACTTCTGGATCTCCCTTA-3’；
TMc43016 sequence TMc43016F:5'-GGCGATCCCTTGATGCTAT-3' the process of the preparation of the pharmaceutical composition,
TMc43016R：5’-CATTGGGAAAAGTCGACACA-3’。
The application of the co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE-499 is the application of the co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE-499 in detecting whether the V-type tobacco sucrose ester gene qBMVSE-499 exists in the genome DNA of tobacco.
The application of the co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE is that a primer of TMc42886 sequence and a primer of TMc43016 sequence are respectively used for amplifying the tobacco genome DNA to be detected, and PCR amplified products are detected, if the PCR amplified products contain sequences shown as SEQ ID NO. 1 and SEQ ID NO. 3 at the same time, the sequences are homozygous alleles SE499SE499 containing the V-type tobacco sucrose ester of the tobacco plant; if the PCR amplification product contains sequences shown as SEQ ID NO. 2 and SEQ ID NO. 4, the sequence is homozygous allele se499se499 of the V-type tobacco sucrose ester which is not contained or contains trace amount of the tobacco plant to be detected; 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 hybrid gene SE499SE499 containing V-type tobacco sucrose ester in the tobacco plant to be detected is obtained.
The invention is further illustrated by the following examples:
Example 1 screening of Co-dominant SSR markers linked to the V-type tobacco sucrose ester Gene qBMVSE499
Screening co-dominant SSR markers linked with V-type tobacco sucrose ester gene qBMVSE499 in the whole genome range of tobacco by adopting quantitative trait linkage analysis (QTL) method and combining silanization gas chromatography mass spectrometry (GC-MS) method
1. Experimental materials
The flue-cured tobacco variety safflower Dajinyuan with excellent comprehensive properties and no V-type tobacco sucrose esters is taken as a female parent, cigar variety Beinhart1000-1 containing III-type, IV-type and V-type tobacco sucrose esters (the III-V-type tobacco sucrose esters are respectively controlled by qBMVSE, qBMVSE485 and qBMVSE499 genes) is taken as a male parent, and a recombination inbred line (RILs_F 7:8) of 341 strains is obtained through hybridization and continuous selfing as a genetic mapping population.
2. Acquisition of sucrose ester content data from V-type tobacco of parent and RILs_F 7:8 population
Transplanting the test material into a field after seedling formation, randomly selecting 10 strains of each strain when tobacco leaves in the field are mature, and selecting 3 middle leaves of each strain of tobacco; and stacking 30 middle blades of each plant, and randomly punching 2 holes in the middle position of each blade by using a puncher with the diameter of 1cm to obtain 60 circular blades with the diameter of 1 cm.
The obtained 60 round leaves were subjected to tobacco sucrose ester content detection according to the methods reported in literature (Cai Lili, xie Fu, liu Kejian, zhang Ying, xie Jianping, gas chromatography/mass spectrometry analysis of sucrose esters in aromatic tobacco, tobacco science, 2009,3:40-44, wang Ruiling, wang Yingying, mao Duo, gu Chunxiao, GC-MS analysis of sucrose tetraesters in tobacco, chemical research and application, 2011,23 (8): 1030-1035).
The content of the obtained 341 parts of RILs_F 7:8 group V-type tobacco sucrose esters is detected by a GC-MS method to be used as the phenotype value of the RILs_F 7:8 group for the subsequent QTL linkage analysis.
3. SSR 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 is a conventional system and can be referred to published literature, wherein the annealing temperature of the markers provided by the invention is 60 ℃; the PCR amplification program information can be referred to the relevant literature; electrophoresis detection is also carried out by a conventional method, and reference can be made to published relevant documents.
About 50000 SSR markers developed by the laboratory based on genome information of flue-cured safflower Dajinyuan and cigar Beinhart-1 respectively are utilized to carry out polymorphism screening on parents (safflower Dajinyuan and Beinhart 1000-1) and sub-generations (F 1) of RILs_F 7:8 population, and finally 2001 polymorphic SSR markers are obtained through screening.
And obtaining 2001 polymorphic SSR markers by screening, and carrying out genotype analysis on 341 RILs_F 7:8 samples.
Secondly, carrying out linkage analysis on genotype data of 341 RILs_F 7:8 samples by using genetic linkage mapping software JoinMap 4.0.0, drawing a high-quality cigar genetic linkage map which contains 24 linkage groups and is uniformly distributed with 1974 SSR markers, covering the length of tobacco genome as 3213.138cM, and taking the high-quality cigar genetic linkage map as genotype value of RILs_F 7:8 groups for subsequent QTL linkage analysis.
4. Whole genome QTL localization analysis of V-type tobacco sucrose esters (qBMVSE 499)
The full genome QTL scan was performed on the V-type tobacco sucrose ester gene qBMVSE499 using QTL localization analysis software QTL ICIMAPPING V4.2.2 on genotype data (constructed to obtain cigar genetic linkage map) and phenotype data (341 parts of V-type tobacco sucrose ester content of rils_f 7:8 population) of the rils_f 7:8 population.
Wherein, the relevant parameters are set as follows: the positioning method selects ICIM-ADD: inclusive Composite INTERVAL MAPPING of ADDitive (and dominant) QTL, number of iterations of 1000 (Permutation times =1000), significance of 0.01 (SIGNIFICANCE =0.01), step size of 0.5cM (WALK SPEED =0.5 cM).
Finally, under the condition of lod= 3.3733 over the whole genome, 1 major QTL (temporarily designated qBMVSE499—24) that is located at 61.00cM of linkage group No. 24, is mapped to obtain the V-type tobacco sucrose ester trait. The major QTL may explain a phenotypic variation of about 19.35% and a LOD value of about 7.70 at this point, as detailed in fig. 1 and table 1.
TABLE 1 statistics of tobacco sucrose ester QTL (qBMVSE _24) information for type V
QTL Chromosome Position/cM Left Marker Right Marker LOD PVE(％) Add
qBMVSE499_24 24 61.00 TMc42886 TMc43016 7.6973 19.3518 0.2618
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 verification of Co-dominant linkage markers in RILs_F 8:9 population individuals
And (3) carrying out genotype analysis on single plants of RILs_F 8:9 group (Honghua Dajinyuan X Beinhart 1000-1) in seedling stage by utilizing the obtained co-dominant SSR markers TMc42886 and TMc43016 which are closely linked with two sides of the V-type tobacco sucrose ester gene qBMVSE to obtain genotype data of each single plant of the RILs_F 8:9 group.
On the other hand, before tobacco leaves of RILs_F 8:9 group grow to maturity, picking and baking, sampling mature middle leaves of each strain by adopting a GC-MS method, and detecting the content of type III tobacco sucrose esters in the leaves. That is, the phenotype values of each strain of the RILs_F 8:9 population are obtained.
Finally, the genotype data of 341 RILs_F 8:9 groups and the phenotype value of the V-type tobacco sucrose esters are analyzed, and the genotype values of the two co-dominant SSR markers TMc42886 and TMc43016 disclosed by the invention are completely matched with the phenotype values, namely, the coincidence rate reaches 100%.
The specific analysis method comprises the following steps: when the sucrose ester content of the V-type tobacco of each strain obtained by GC-MS detection is higher than or equal to the content of the parent Beinhart-1, the genotype of the strain also shows sequences shown as SEQ ID NO.1 (266 bp) and SEQ ID NO.3 (313 bp) to be homozygous genotype SE499SE499;
When the sucrose ester content of the V-type tobacco of each strain is equal to or lower than the major golden element content of the parent safflower, the genotype of the strain simultaneously shows sequences shown as ID No.2 (310 bp) and SEQ ID No. 4 (339 bp), namely homozygous genotype se471se471;
When the sucrose ester content of each strain V-type tobacco obtained by detection is between the parent safflower Dajinyuan and Beinhart1000-1, namely the content of the strain V-type tobacco is similar to that of a sub-generation (F 1), the genotype of the strain also simultaneously shows 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, namely the heterozygous genotype SE499SE499.
Conclusion of experiment:
The above results indicate that co-dominant markers TMc42886 and TMc43016 are closely linked to the tobacco sucrose ester V gene qBMVSE499, respectively, and that the two markers flank the gene of interest (qBMVSE 499-24).
By utilizing the two co-dominant closely linked SSR markers, the detection of the sucrose ester content of V-type tobacco in any growth period of tobacco can be accurately, efficiently, conveniently and inexpensively realized, the genotype state of the sucrose ester of V-type tobacco in a plant to be detected (namely, the homozygous genotype SE499SE499 with the highest content value and stable inheritance, the heterozygous genotype SE499SE499 with the medium content value and unstable inheritance, and the homozygous genotype SE499SE499 without or with trace content value and stable inheritance) can be clearly identified, so that the scientificity and predictability of the breeding of new flue-cured tobacco varieties with high aroma quality are improved, and the breeding process is accelerated.
Sequence listing
<110> Tobacco agricultural science institute of Yunnan province
<120> SSR marker closely linked to the V-type tobacco sucrose ester gene qBMVSE499
<130> 20211123
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 266
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
gcatctattg tggtcggaag atattgctat gcacatatga attatgcggt acatcatgta 60
gttacatctt gggggtatga ttatgctttg atacagcttg ttgagattga tatagttata 120
tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 180
tatatatata cacacacaca cacacacaca cacacacaca cacacatata taagaatcat 240
atgttaaggg agatccagaa gttcga 266
<210> 2
<211> 310
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
gcatctattg tggtcggaag atattgctat gcacatatga attatgcggt acatcatgta 60
gttacatctt gggggtatgt ttgacaatgc ttcttgttga gcaagagaac tccatattat 120
gctttgatac agcttgttga gattgatata gttatatata tatatatata tatatatata 180
tatatatata tatatatata tatatatata tatatatata tatatatata tatacacaca 240
cacacacaca cacacacaca cacacacaca tatataagaa tcatatgtta agggagatcc 300
agaagttcga 310
<210> 3
<211> 313
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
ggcgatccct tgatgctata tttagggagc actttacacc attttactct atttcatgta 60
gttgcatttg atataaatat atatatatat atatatatat atatatatat atatatatat 120
atatatatat atatatatat atatatatat atagtatgtt ttatttacat tatatagtat 180
gttttatgta tgatgtttta ttttgttggc aatatccttg gtttctttgt accacagttc 240
ttttttcaaa gatgtcaatt tatttttgaa ccaggtattt gtgggtaaat gtttgtgtcg 300
acttttccca atg 313
<210> 4
<211> 339
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
ggcgatccct tgatgctata tttagggagc actttacacc attttactct atttcatgta 60
gttgcatttg atataaatat atatatatat atatatatat atatatatat atatatatat 120
atatatatat atatatatat atatatatat atatatatat atatatatat atatatatag 180
tatgttttat ttacattata tagtatgttt tatgtatgat gttttatttt gttggcaata 240
tccttggttt ctttgtacca cagttctttt ttcaaagatg tcaatttatt tttgaaccag 300
gtatttgtgg gtaaatgttt gtgtcgactt ttcccaatg 339
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
gcatctattg tggtcggaag a 21
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
tcgaacttct ggatctccct ta 22
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
ggcgatccct tgatgctat 19
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 8
cattgggaaa agtcgacaca 20

Claims (7)

1. The application of the nucleic acid fragment combination with the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 in detecting the tobacco V-type tobacco sucrose ester gene qBMVSE 499.
2. The nucleotide sequence of the primer combination for detecting the tobacco V-type tobacco sucrose ester gene qBMVSE499 is shown as SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.
3. The co-dominant SSR marker closely linked with the V-type tobacco sucrose ester gene qBMVSE-499 comprises a TMc42886 sequence and a TMc43016 sequence, and is characterized in that the nucleotide sequence of a PCR amplification product of the co-dominant SSR marker is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
4. A co-dominant SSR marker according to claim 3, wherein the primer sequences for the 2 sites corresponding to the TMc42886 sequence and TMc43016 sequence are:
primer of TMc42886 sequence: SEQ ID NO. 5 and SEQ ID NO. 6;
primer of TMc43016 sequence: SEQ ID NO. 7 and SEQ ID NO. 8.
5. Use of a co-dominant SSR marker according to claim 3 or 4 for detecting the tobacco sucrose V-ester gene qBMVSE499,499.
6. The use of claim 5, wherein the detection of tobacco type V tobacco sucrose ester gene qBMVSE is to detect the presence or absence of type V tobacco sucrose ester gene qBMVSE499, the content of type V tobacco sucrose ester and the genotype status of type V tobacco sucrose ester in the plant to be tested in the tobacco genomic DNA.
7. The use as claimed in claim 6, wherein the primers of TMc42886 and TMc43016 are used to amplify the genomic DNA of tobacco to be detected, respectively, and the PCR amplification product is detected and judged as follows:
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 V-type tobacco sucrose esters with high aroma quality, and the genotype is recorded as SE499SE499;
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 or contains trace homozygous alleles of the V-type tobacco sucrose esters, and the genotype is recorded as se499se499;
If the PCR amplification 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 tobacco plant to be tested contains heterozygous alleles of V-type tobacco sucrose ester, and the genotype is SE499SE499.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197101A (en) * 2018-11-20 2020-05-26 云南省烟草农业科学研究院 Codominant SSR marker closely linked with tobacco leafy gene mLN and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197101A (en) * 2018-11-20 2020-05-26 云南省烟草农业科学研究院 Codominant SSR marker closely linked with tobacco leafy gene mLN and application thereof

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Title
VIJAY VONTIMITTA等.Analysis of a Nicotiana tabacum L. Genomic Region Controlling Two Leaf Surface Chemistry Traits.J. Agric. Food Chem..2009,第58卷第294-300页. *
陈彪 等.烟草糖酯SSR分子标记筛选及辅助育种应用.中国烟草科学.2019,第40卷(第3期),第8-15页. *

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