CN117604144A - Co-dominant SSR marker closely linked with type II tobacco sucrose ester gene qSE457 and application - Google Patents

Co-dominant SSR marker closely linked with type II tobacco sucrose ester gene qSE457 and application Download PDF

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CN117604144A
CN117604144A CN202311528494.6A CN202311528494A CN117604144A CN 117604144 A CN117604144 A CN 117604144A CN 202311528494 A CN202311528494 A CN 202311528494A CN 117604144 A CN117604144 A CN 117604144A
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tobacco
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童治军
肖炳光
方敦煌
李勇
黄昌军
曾建敏
隋学艺
袁诚
张谊寒
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Yunnan Academy of Tobacco Agricultural Sciences
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Abstract

The invention relates to a co-dominant SSR marker closely linked with a type II tobacco sucrose ester gene qSE457 and application thereof, wherein the numbers of the co-dominant SSR markers closely linked with the type II tobacco sucrose ester gene qSE457 are TM39680 and TMc58652, and the nucleotide sequences of PCR amplified products are respectively shown as SEQ ID No.1 and SEQ ID No.2, and SEQ ID No.3 and SEQ ID No. 4. The application is the application of the co-dominant SSR marker closely linked with the II-type tobacco sucrose ester gene qSE457 in detecting whether the II-type tobacco sucrose ester gene qSE457 exists in the tobacco genome DNA. Compared with the prior art, the co-dominant SSR marker has the characteristics of accuracy, high efficiency, stability, convenience, low cost and no detection time limit, so that the molecular marker can be used as an auxiliary selection application of a type II sucrose ester gene qSE457 molecular marker without 3-methyl valeryl in high-aroma quality breeding of tobacco.

Description

Co-dominant SSR marker closely linked with type II tobacco sucrose ester gene qSE457 and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a co-dominant SSR marker closely linked with two sides of a type II tobacco sucrose ester gene qSE457 and application thereof.
Background
The tobacco sucrose ester is one of the main chemical components on the surface of tobacco leaves, is also an important potential aroma precursor substance in tobacco, can generate micromolecular volatile fatty acid after being degraded, and can greatly improve the aroma quality of the tobacco leaves.
Researches show that sucrose esters in cultivated tobacco are mainly sucrose tetraesters, and can be classified into 6 types according to the molecular weight, namely, I-VI type tobacco sucrose esters; while 6 types of sucrose tetraesters can be further classified into 2 general classes depending on whether or not the sucrose tetraesters contain an esterified 3-methylpentanoic acid group (3-methylpentanoyl): I. sucrose esters without 3-methylpentanoyl (SE) type II and sucrose esters with 3-methylpentanoyl (BMVSE) type III-VI (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.). The structure types and the content of sucrose tetraesters in different cultivated tobacco types are greatly different, and flue-cured tobacco, burley tobacco and maryland tobacco contain I-type and II-type tobacco Sucrose Esters (SE) and do not contain or contain trace BMVSE; whereas aromatic and cigar cigarettes are the exact opposite, contain BMVSE, and contain no or trace amounts of SE.
After sucrose esters in cultivated tobacco are analyzed by silanization gas chromatography mass spectrometry (GC-MS), 2 Sucrose Esters (SE) without 3-methyl pentanoyl can be divided into SE443 (type I tobacco sucrose esters) and SE457 (type II tobacco sucrose esters) according to partial characteristic fragment ion sizes of glucose rings, and the 2 types of tobacco sucrose esters are core attention points for cultivating new varieties of flue-cured tobacco with high aroma quality and high sucrose esters.
However, the prior art often incorrectly considers 6 different independent genes originally belonging to SE443 (type I), SE457 (type II), BMVSE471 (type III), BMVSE485 (type IV), BMVSE499 (type V) and BMVSE513 (type VI) as one gene (and collectively named BMVSE) for genetic mapping studies (Vontimita 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 tracks. Journal of Agricultural and Food Chemistry,2010, 58:294-300.). As a result, the positioning result obtained is erroneous. Resulting in far less than 100% precision requirements for molecular Marker Assisted Selection (MAS).
For example, the BMVSE gene was located within about 5.2cM of the region between SSR markers PT30354 and PT52061, PT61362 and was co-separated from SSR markers PT30209 and PT20315 (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 tracks. Journal of Agricultural and Food Chemistry,2010, 58:294-300.). However, the use of the 5 SSR markers for BMVSE screening and verification of tobacco materials of different genetic backgrounds and 192 recombinant inbred line populations shows that only 2 markers (PT 20135 and PT 30354) can be used, and the genotype of the 2 available markers and the phenotype of BMVSE have a consistency rate of only about 92.165% (Chen Biao, chen Ming, li Yangyang, qu Yafang, cheng Lirui, yang Aiguo, hu Risheng, liu Dan, luo Chenggang, shipeng, feng Quanfu, chang Aixia, SSR molecular marker screening of tobacco sugar esters and assisted breeding applications. Chinese tobacco science, 2019,40 (3): 8-15.) far does not reach the 100% consistency rate required for assisted selection of molecular markers. The main reason is that: the authors of the literature have mistakenly regarded the genes which originally belong to six different independent genes (types I-VI) as one gene. In addition, researches on sucrose esters (BMVSE) containing 3-methyl pentanoyl have been reported, and corresponding patents such as three types of tobacco glycolipid genes of type III tobacco sucrose esters BMVSE471 (ZL 2021 1346741.1), type IV tobacco sucrose esters BMVSE485 (ZL 2021 1363676.3) and type V tobacco sucrose esters BMVSE499 (application number 202111394234.5) have been respectively genetically mapped and obtained with two side closely linked markers which can be used for guiding high-aroma quality breeding of tobacco. However, so far, no studies have been reported on genetic localization of 2 sucrose esters of tobacco (SE; sucrose esters type I SE443 and type II SE 457) without 3-methylpentanoyl.
In view of this, the present invention first distinguishes whether sucrose tetraesters in cultivated tobacco contain 3-methylpentanoyl, i.e., type I to type II tobacco Sucrose Esters (SE) that do not contain 3-methylpentanoyl from type III to type VI tobacco sucrose esters (BMVSE) that contain 3-methylpentanoyl; secondly, taking a high-quality multi-resistant cigar variety Beinhart1000-1 (containing BMVSE and containing no SE) and a flue-cured tobacco variety safflower Dajinyuan (containing SE and containing no BMVSE) as parents, and constructing a tobacco recombinant inbred line (RILs_F) containing 341 parts of strains through hybridization and continuous bagging selfing 8:9 ) Is a mapping population; and thirdly, screening and obtaining co-dominant SSR markers closely linked with the II-type tobacco sucrose ester gene qSE457 in the whole genome range of tobacco by using a quantitative trait linkage analysis (QTL) method and a silanization gas chromatography MASs spectrometry (GC-MS) method so as to make up for the defects of the prior art and reported documents and accurately and efficiently utilize an acceleration molecular Marker Assisted Selection (MAS) in the breeding of tobacco high-aroma varieties.
Therefore, the method carries out differentiation and genetic localization research on SE443 (I type) and SE457 (II type) 2 types of tobacco sucrose esters contained in SE without 3-methyl valeryl for the first time, not only effectively fills the blank of genetic localization analysis on SE443 (I type) and SE457 (II type) at home and abroad, but also can accurately obtain co-dominant SSR markers closely linked with two sides of a certain independent gene (qSE 457 or qSE 457), and is an important way for accurately and efficiently utilizing molecular Marker Assisted Selection (MAS) to cultivate new varieties of flue-cured tobacco with high aroma quality and containing high sucrose esters.
Disclosure of Invention
The invention aims to solve the defects, and provides a co-dominant SSR marker closely linked with a type II tobacco sucrose ester gene qSE457 and application thereof.
The first object of the invention is to provide a co-dominant SSR marker closely linked with a type II tobacco sucrose ester gene qSE457; the second aim is to apply the co-dominant SSR marker to detect whether the II type tobacco sucrose ester gene qSE457 exists in the tobacco genome DNA and the genotype state of the II type tobacco sucrose ester in the plant to be detected.
The innovation point of the invention is that: the invention carries out genetic and positioning analysis for the type II tobacco sucrose ester gene qSE457 which does not contain 3-methyl pentanoyl for the first time; a significant misunderstanding of the prior art has also been found.
The inventors found that the incorporation of type I-VI tobacco sucrose esters into one gene (BMVSE) for targeting in the literature was inaccurate, and even erroneous, and that one type should be specifically selected from the 6 types of tobacco sucrose esters to be precisely targeted. The error or deficiency of the prior art, lack of genetic localization analysis on the tobacco Sucrose Ester (SE) without 3-methyl pentanoyl, severely restricts the development of the breeding process of new varieties of high-aroma tobacco by utilizing the markers closely linked with both sides of I, II type tobacco sucrose ester genes (qSE 457 and qSE 457).
Studies by the present inventors have shown that type I and type II tobacco Sucrose Esters (SE) are typical quantitative traits and are controlled by QTLs of different chromosomes within the major golden-element genome of the flue-cured tobacco variety safflower, these genes/QTLs being designated temporarily as qSE457 and qSE457.
The invention is realized by adopting the following technical scheme.
The co-dominant SSR markers closely linked with the II-type tobacco sucrose ester gene qSE457 are numbered TM39680 and TMc58652, and the nucleotide sequences of PCR amplified products of the co-dominant SSR markers closely linked with the II-type tobacco sucrose ester gene qSE457 are respectively shown as SEQ ID No.1 and SEQ ID No.2 and SEQ ID No.3 and SEQ ID No. 4.
SEQ ID No.1:
TTCGACCCACTTTACAATACCCTTATTAGAGTTACACAAATAGCCATATTCATTTAAAGACATAGTACTTTATAACTTGGCCATATCTTACAAGTTTTATAAACACAAATACTATATATCATACTTCAAAACTCTTCAATCATATATATATATATATATATATATATATATATATAACATTATCATCAAGGCTTTTATATACCTATATACGTATAGACAACTAATCCTTTAAACCTAGTTTAAGTCCTATGGAGCTACCTCAGGTAAATTTTATGGGGTGCTACCACC。
SEQ ID No.2:
TTCGACCCACTTTACAATACCCTTATTAGAGTTACACAAATAGCCATATTCATTTAAAGACATAGTACTTTATAACTTGGCCATATCTTACAAGTTTTATAAACACAAATACTATATATCATACTTCAAAACTCTTCAATCATATATATATATATATATATATATATAACATTATCATCAAGGCTTTTATATACCTATATACGTATAGACAACTAATCCTTTAAACCTAGTTTAAGTCCTATGGAGCTACCTCAGGTAAATTTTATGGGGTGCTACCACC。
SEQ ID No.3:
CGAGGCATTTTCCTCCTCTAGTAATTCAGGATCCTCACTCGATTTCTTTTTGTAAGCCTCCTCGACACGAGGCTTGATTTTAGCAATCGCCGTCGGCTCAGAAGGTTTGGTAACCTTGGCGGCTTTCCTAGGTCGGACCGTCAGTGCCGAGGCATTTTCCTCCTCCTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCGTCTCTCAGTTTTTGGACCGAGTCC。
SEQ ID No.4:
GGACTCGGTCCAAAAACTGAGAGACGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAGGAGGAGGAAAATGCCTCGGCACTGACGGTCCGACCTAGGAAAGCCGCCAAGGTTACCAAACCTTCTGAGCCGACGGCGATTGCTAAAATCAAGCCTCGTGTCGAGGAGGCTTACAAAAAGAAATCGAGTGAGGATCCTGAATTACTAGAGGAGGAAAATGCCTCG。
The primer sequences of the 2 loci corresponding to the molecular markers are respectively as follows:
the primer sequences for amplifying TM39680 were:
TM39680F：5’-TTCGACCCACTTTACAATACCC-3’(SEQ ID NO.5)，
TM39680R：5’-GGTGGTAGCACCCCATAAAA-3’(SEQ ID NO.6)；
the primer sequences for amplifying TMc58652 are:
TMc58652F：5’-GGACTCGGTCCAAAAACTGA-3’(SEQ ID NO.7)，
TMc58652R：5’-CGAGGCATTTTCCTCCTCTA-3’(SEQ ID NO.8)。
the application of the co-dominant SSR marker closely linked with the II-type tobacco sucrose ester gene qSE457 is used for detecting whether the II-type tobacco sucrose ester gene qSE457 exists in the tobacco genome DNA and detecting the genotype state of the II-type tobacco sucrose ester in a plant to be detected.
The application of the invention comprises the steps of amplifying the tobacco genome DNA to be detected by using primers of TM39680 and TMc58652 sequences respectively, and detecting PCR amplification products.
The PCR amplification product of the invention contains sequences shown as SEQ ID No.1 and SEQ ID No.3, which shows that the tobacco plant to be detected contains homozygous alleles of type II tobacco sucrose esters with high aroma quality, and the genotype is SE457SE 457.
The PCR amplification product of the invention contains sequences shown as SEQ ID No.2 and SEQ ID No.4, and the sequence is the homozygous allele of the tobacco plant to be detected, which does not contain the II-type tobacco sucrose ester, and the genotype is se457se 457.
The PCR amplified product of the invention contains the sequences shown as SEQ ID No.1 and SEQ ID No.4 at the same time or contains the sequences shown as SEQ ID No.2 and SEQ ID No.3 at the same time, so that the PCR amplified product is a heterozygous allele of the type II tobacco sucrose ester with high aroma quality of tobacco plants to be detected, and the genotype is SE457SE 457.
In order to simply and efficiently select the type II tobacco sucrose ester tobacco variety with high aroma quality potential, the invention specifically and purposefully selects the offspring material containing the type II tobacco sucrose ester gene qSE457, and can be used for auxiliary selection of the type II tobacco sucrose ester gene qSE457 so as to improve the efficiency of molecular marker auxiliary selection and the efficiency of breeding the high aroma tobacco variety. The 2 co-dominant SSR markers closely linked with the two sides of the II-type tobacco sucrose ester gene qSE457 can be used for qualitatively detecting whether the II-type tobacco sucrose ester gene qSE457 exists in tobacco genome DNA of any growth period, can also scientifically and accurately identify the genotype state of the II-type tobacco sucrose ester in a plant to be detected (namely, the homozygous genotype SE457SE457 with the highest content value and stable inheritance, the heterozygous genotype SE457 with the medium content value and unstable inheritance, and the homozygous genotype SE457SE457 without containing or with trace values and stable inheritance), so that the scientificity and predictability of breeding new flue-cured tobacco varieties with high aroma quality are improved, and the breeding process is accelerated.
Compared with the prior art, the invention has the beneficial effects that:
1. fills the blank of the genetic and positioning analysis of the type II tobacco sucrose ester gene qSE457 which does not contain 3-methyl pentanoyl at home and abroad.
2. Correcting errors of the prior art
The invention effectively corrects and makes up the error and the deficiency of regarding 6 different types of tobacco sucrose ester genes as one gene (BMVSE) in the literature, and can accurately identify the genotype of the type II tobacco sucrose ester.
3. High accuracy for detecting II-type tobacco sucrose ester
The molecular marker for detecting the II-type tobacco sucrose ester gene qSE457 is provided, whether the tobacco to be detected contains II-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.
4. 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 tobacco in the maturity stage by adopting 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.
5. 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.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a tobacco-based recombinant inbred population (RILs_F) 8:9 The method comprises the steps of carrying out a first treatment on the surface of the Figure of the QTL analysis of sucrose esters of tobacco type II linked group No. 24 of Honghuada Jinyuan X Bennhart 1000-1).
Wherein, the utilization software is: QTL IciMapping v4.2; parameter setting: the positioning method is 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= 3.3806 at the 0.01 significance threshold; the highest point of the LOD curve is the major gene (qSE 457).
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 two sides of the II-type tobacco sucrose ester gene qSE457 are numbered TM39680 and TMc58652, and nucleotide sequences of PCR amplified products of the co-dominant SSR markers are respectively shown as 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 molecular markers are respectively as follows:
the primer sequences for amplifying TM39680 were:
TM39680F：5’-TTCGACCCACTTTACAATACCC-3’，
TM39680R：5’-GGTGGTAGCACCCCATAAAA-3’；
the primer sequences for amplifying TMc58652 are:
TMc58652F：5’-GGACTCGGTCCAAAAACTGA-3’，
TMc58652R：5’-CGAGGCATTTTCCTCCTCTA-3’。
the application of the co-dominant SSR marker closely linked with the II-type tobacco sucrose ester gene qSE457 is the application of the co-dominant SSR marker closely linked with the II-type tobacco sucrose ester gene qSE457 in detecting whether the II-type tobacco sucrose ester gene qSE457 exists in the genome DNA of tobacco.
The application of the co-dominant SSR marker closely linked with the II-type tobacco sucrose ester gene qSE457 is that primers of TM39680 sequences and primers of TMc58652 sequences are used for amplifying the genomic DNA of tobacco to be detected, PCR amplified products are detected, and 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 qSE457qSE457 containing II-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 a homozygous allele qse457qse457 of the tobacco plant to be detected which does not contain the type II tobacco sucrose ester; if the PCR amplified product contains sequences shown as SEQ ID NO.1 and SEQ ID NO.4 or contains sequences shown as SEQ ID NO.2 and SEQ ID NO.3, the heterozygous gene qSE457qSE457 containing the type II tobacco sucrose ester in the tobacco plant to be detected is obtained.
Example 1
Screening of co-dominant SSR markers linked to tobacco sucrose ester gene type II qSE457
Screening co-dominant SSR markers linked with type II tobacco sucrose ester gene qSE457 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 tobacco variety Honghua Dajinyuan with excellent comprehensive characters and containing type II tobacco sucrose esters is taken as a female parent, the cigar variety Beinhart1000-1 without the type II tobacco sucrose esters 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 8:9 ) As a population of genetic mapping.
2. Parent and rils_f 8:9 Group I tobacco sucrose ester content data acquisition
Transplanting the test material into a field after seedling formation, randomly selecting 10 plants per plant line when tobacco leaves in the field are mature (before baking), and selecting 3 middle leaves per plant 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).
341 parts of RILs obtained by GC-MS detection_F 8:9 Group II tobacco sucrose ester content as RILs_F 8:9 The phenotype values of the population are used for the QTL linkage analysis of the next step.
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 flue-cured safflower Dajinyuan and cigar Beinhart1000-1 genome information are utilized for RILs_F 8:9 Parents (safflower Dajinyuan and Beinhart 1000-1) and sub-generations (F) 1 ) And (3) carrying out polymorphism screening, and finally, screening to obtain 2093 polymorphic SSR markers.
Then 2093 polymorphic SSR markers are obtained by screening, and 341 parts of RILs_F are subjected to 8:9 The samples were genotyped.
Next, 341 parts of RILs_F were mapped using the genetic linkage mapping software JoinMap 4.0 pair 8:9 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 2000 SSR markers and covers the length of a tobacco genome of 3097.319cM, and taking the cigar genetic linkage map as RILs_F 8:9 Genotype values of the population were used for the next QTL linkage analysis.
4. Whole genome QTL localization analysis of type II tobacco sucrose esters (qSE 457)
RILs_F pairs using QTL positioning analysis software QTL IcinMapping v4.2 8:9 Genotype data (constructed to obtain cigar genetic linkage map) and phenotype data (341 RILs_F) 8:9 Group II tobacco sucrose ester content), a full genome QTL scan was performed on the tobacco sucrose ester gene type II qSE457.
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).
Finally, under genome-wide lod= 3.3806 conditions, 1 major QTL (transiently designated as qSE 457) for the type II tobacco sucrose ester trait was located at 60.50cM of linkage group 24. The major QTL may explain the phenotype variability of about 9.5344% and the LOD value at this point is about 6.8168, see in detail fig. 1 and table 1.
TABLE 1 tobacco sucrose ester QTL (qSE 457) information statistics
QTL Chromosome Position/cM Left Marker Right Marker LOD PVE(％) Add
qSE457 24 60.50 TM39680 TMc58652 6.8168 9.5344 0.0169
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 Co-dominant linkage marker at RILs_F 9:10 Verification in population individuals
RILs_F for seedling stage are obtained by utilizing co-dominant SSR markers TM39680 and TMc58652 which are closely linked with two sides of type II tobacco sucrose ester gene qSE457 9:10 Genotyping the individual plants of the population (Honghuadajinyuan X Bennhart 1000-1) to obtain RILs_F 9:10 Genotype data for each individual of the population.
On the other hand, waiting for RILs_F 9:10 And (3) growing tobacco leaves of the population to a mature period (before baking), sampling mature middle leaves of each strain by adopting a GC-MS method, and detecting the content of the type II tobacco sucrose esters in the leaves. That is, RILs_F is obtained 9:10 Phenotype values for each strain of the population.
Finally, 341 parts of RILs_F are analyzed 9:10 Genotype data of the population and a type II tobacco sucrose ester phenotype value show that the genotype values of the two co-dominant SSR markers TM39680 and TMc58652 disclosed by the invention are completely matched with the phenotype values, namely, the coincidence rate is 100%.
The specific analysis method comprises the following steps: when the sucrose ester content of the type II tobacco of each strain obtained by GC-MS detection is higher than or equal to the major golden element content of the flue-cured tobacco type parent safflower, the genotype of the strain also shows sequences shown as SEQ ID NO.1 (288 bp) and SEQ ID NO.3 (256 bp) to be homozygous genotype SE457SE 457;
when the sucrose ester content of the tobacco of each strain II obtained by detection is equal to or lower than the Beinhart1000-1 content of a cigar type parent, the genotype of the strain also shows sequences shown as ID No.2 (280 bp) and SEQ ID No.4 (250 bp) to be homozygous genotype se457se 457;
and when the sucrose ester content of each strain II tobacco obtained by detection is between that of the tobacco in bakingThe tobacco type parent safflower Dajinyuan is between the cigar type parent Beinhart1000-1, i.e. with the sub-generation (F) 1 ) When the content is similar, the genotype of the strain also presents the sequences shown as SEQ ID NO.1 and SEQ ID NO.4 at the same time, or contains the sequences shown as SEQ ID NO.2 and SEQ ID NO.3, namely the heterozygous genotype SE457SE 457.
Conclusion of experiment:
the above results indicate that co-dominant markers TM39680 and TMc58652 are closely linked to the tobacco sucrose ester gene type II qSE457, respectively, and that the two markers flank the gene of interest (qSE 457).
By utilizing the 2 co-dominant closely linked SSR markers, the detection of the sucrose ester content of the type II tobacco in any growth period of the tobacco can be accurately, efficiently, conveniently and inexpensively realized, the genotype state of the type II tobacco sucrose ester in the plant to be detected (namely, the homozygous genotype SE457 with the highest content value and stable inheritance, the heterozygous genotype SE457SE457 with the medium content value and unstable inheritance and the homozygous genotype SE457SE457 without the content or the content value and stable inheritance) can be clearly identified, and the scientificity and predictability of the breeding of new variety of flue-cured tobacco with high aroma quality are improved, and the breeding process is accelerated.
What has been described above is only a part of the specific embodiments of the present invention, and the specific contents or common knowledge known in the art are not described herein too much (including but not limited to shorthand, abbreviations, units commonly used in the art). It should be noted that the above embodiments do not limit the present invention in any way, and it is within the scope of the present invention for those skilled in the art to obtain the technical solution by equivalent substitution or equivalent transformation. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims (8)

1. The co-dominant SSR marker closely linked with the tobacco sucrose ester gene II qSE457 is characterized in that the numbers of the co-dominant SSR markers closely linked with the two sides of the tobacco sucrose ester gene II qSE457 are TM39680 and TMc58652, 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.
2. A co-dominant SSR marker closely linked to both sides of the tobacco sucrose ester gene type II qSE457 according to claim 1, wherein the co-dominant markers TM39680 and TMc58652 flank the gene of interest qSE457.
3. The co-dominant SSR marker closely linked to both sides of the sucrose ester gene qSE457 of type II tobacco according to claim 1, wherein the primer sequences of the 2 sites corresponding to the molecular marker are respectively:
the primer sequences for amplifying TM39680 were:
TM39680F：5’-TTCGACCCACTTTACAATACCC-3’，
TM39680R：5’-GGTGGTAGCACCCCATAAAA-3’；
the primer sequences for amplifying TMc58652 are:
TMc58652F：5’-GGACTCGGTCCAAAAACTGA-3’，
TMc58652R：5’-CGAGGCATTTTCCTCCTCTA-3’。
4. use of a co-dominant SSR marker closely linked to both sides of the tobacco sucrose ester type II gene qSE457 according to claim 1 or 2 or 3 for detecting the presence or absence of the tobacco sucrose ester type II gene qSE457 in tobacco genomic DNA and the genotype status of tobacco sucrose ester type II in the plant to be tested.
5. The method according to claim 4, wherein the PCR amplification products are detected by amplifying the tobacco genomic DNA to be detected with primers of the sequences TM39680 and TMc58652, respectively.
6. The use according to claim 5, wherein the PCR amplification product contains both sequences shown as SEQ ID No.1 and SEQ ID No.3, which indicates that the tobacco plant to be tested contains homozygous alleles of type II tobacco sucrose esters with high aroma quality, and the genotype is SE447 SE457.
7. The use according to claim 5, wherein the PCR amplification product contains both sequences shown as SEQ ID No.2 and SEQ ID No.4, which are homozygous alleles of type II tobacco sucrose esters, the tobacco plants to be tested do not contain high aroma quality, and the genotype is se457se 457.
8. The use according to claim 5, wherein the PCR amplification product contains both sequences shown as SEQ ID No.1 and SEQ ID No.4 or both sequences shown as SEQ ID No.2 and SEQ ID No.3, and the genotype is SE457SE 457.
CN202311528494.6A 2023-11-16 2023-11-16 Co-dominant SSR marker closely linked with type II tobacco sucrose ester gene qSE457 and application Pending CN117604144A (en)

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