CN118326078A - Codominant SSR marker closely linked with carbon monoxide release amount gene qCO in tobacco mainstream smoke and application - Google Patents
Codominant SSR marker closely linked with carbon monoxide release amount gene qCO in tobacco mainstream smoke and application Download PDFInfo
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Abstract
The invention provides a co-dominant SSR marker closely linked with a carbon monoxide release amount gene qCO in tobacco mainstream smoke and an application method thereof. The co-dominant SSR markers closely linked with the carbon monoxide release amount gene qCO in the main stream smoke of tobacco are numbered IDYK 0477 and TMy80405, and the nucleotide sequences of PCR amplified products are respectively shown as SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4. The application is the application of the co-dominant SSR marker closely linked with the carbon monoxide release gene qCO in detecting whether the carbon monoxide release gene qCO 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 application of auxiliary selection of the carbon monoxide release gene qCO molecular marker in low-harm tobacco breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a co-dominant SSR marker closely linked with a carbon monoxide release amount gene qCO in tobacco mainstream smoke and application thereof.
Background
Tobacco is a leaf-type commercial crop that is widely planted because it contains nicotine that is highly desirable and pleasant to the smoker when consumed. However, smoking is unhealthy and makes tobacco a controversial commercial crop. Therefore, the cultivation of low-harm tobacco varieties becomes an important direction of the breeding of new varieties of tobacco (Wang Yuanying, zhou Jian. Zhongmei main tobacco variety parent analysis and tobacco breeding. Chinese tobacco science report, 1995,3 (2): 11-22). The release amount of harmful components in the main stream smoke of the tobacco is a core basis for judging the success or failure of the low-harm tobacco variety breeding (Xie Jianping, liu Huimin, zhu Maoxiang, and the like. Research on the smoke hazard index of the cigarette. Tobacco science and technology, 2009, 2:5-15). Studies have shown that tobacco, especially finished cigarette smoke, is an extremely complex mixture produced by combustion, cracking and distillation of tobacco during smoking of the cigarette (Wang Keqing, qin Yanhua, wu Yang, etc. research on ammonia release characteristics in heated cigarette smoke, china tobacco science, 2021, 42 (6): 74-78; zhao Yunchuan, liao Xiaoxiang, chen Ran, etc. influence of microwave expanded cut stems on release amounts and hazard indexes of 7 smoke harmful components of the cigarette, tobacco science, 2015, 48 (11): 53-58; ning, liu Jiagong, dumei, etc. analysis of main harmful components of tobacco mainstream smoke in different producing areas of China, china tobacco science, 2017, 38 (1): 85-90; summer Smart, ma Lina, huang Gongyi, etc. comparative hazard indexes of different brands of cigarette samples at home and abroad [ J ]. Tobacco science, 2012, 3:37-40). Xie Jian et al (Xie Jianping, liu Huimin, zhu Maoxiang, et al. Cigarette smoke hazard index study. Tobacco science, 2009, 2:5-15) determined that the 7 harmful ingredient indices that most affect tobacco mainstream smoke hazard were ammonia (NH 3), carbon monoxide (CO), hydrocyanic acid (HCN), 4-methylnitrosamine-1-3-pyridinyl-1-butanone (NNK), benzopyrene (BAP), phenol (PHE), crotonaldehyde (CRO). In addition, in view of the great harm of nicotine (CIC) and TAR (TAR) in the main stream smoke of the tobacco to human health, the tobacco industry at home and abroad comprehensively considers the 9 harmful components, and a set of scientific method for evaluating the harm in the main stream smoke of the tobacco is further constructed.
So far, researches on 9 representative harmful components in main stream smoke of tobacco mainly focus on the aspects of detection methods of the harmful components, evaluation of cigarette product quality and the like, and genetic analysis researches on the representative harmful components in the main stream smoke of tobacco are only reported in 1 case at home and abroad. Namely Tong Zhijun et al (children direct troops, tang Danyun, xu Yongming, fangdunhuang, chen Xuejun, feng Yingjie, yang Zongcan, liu Wenzhao, zhang Tingting, yang Jinchu, shangguang. Genetic analysis of harmful components of mainstream smoke of cigarettes. Chinese tobacco science, 2023, 44 (3): 16-22) adopts a main gene+polygenic SEA-DH combined analysis method and combines tobacco recombination inbred line population (RILs) to carry out genetic analysis on 9 representative harmful components in mainstream smoke of tobacco, and the result shows that the genetic variation of the characteristics of the 9 harmful components (NH 3, CO, HCN, NNK, BAP, PHE, CRO, CIC and TAR) of the mainstream smoke of tobacco is mainly determined by genetic factors, and the influence of environmental factors on the genetic variation is small; the existence of the main gene/QTL with extremely high genetic rate can help to further develop molecular marker assisted selection and gene/QTL positioning research of tobacco low-hazard component traits. Specifically, for the analysis and research of carbon monoxide release amount genes/QTL localization in the harmful components of 9 tobacco main stream smoke, no report has been reported at home and abroad. Therefore, the blank of the positioning analysis of the carbon monoxide release amount gene/QTL in the main stream smoke of the tobacco severely restricts the molecular marker auxiliary breeding of low-harm tobacco varieties, and especially severely restricts the development of the breeding process of new low-harm varieties of the non-carbon monoxide release amount tobacco by utilizing the close linkage markers with the carbon monoxide release amount gene qCO.
In view of the above, the invention firstly takes flue-cured tobacco resource Y3 (carbon monoxide with high release amount in main stream smoke and containing gene qCO) and main-cultivated low-hazard flue-cured tobacco variety K326 (no carbon monoxide release amount in main stream smoke and containing gene qCO) as parents, and constructs a tobacco recombinant inbred line (RILs_F 8:9) containing 269 lines as a mapping population through hybridization and continuous bagging inbreeding; secondly, carrying out genotype analysis on 269 RILs_F 8:9 strains by utilizing a large-scale SSR molecular marker developed by the laboratory based on flue-cured tobacco germplasm resource Y3 genome information, constructing and obtaining 1 Gao Zhiliang tobacco SSR molecular genetic linkage maps which contain 2059 SSR markers and are more uniformly distributed on 24 tobacco linkage groups, wherein the length of a whole genome is 1759.74cM; thirdly, shredding and manually rolling the tobacco into a single-material cigarette after the tobacco is ripe and roasted, putting the rolled cigarette on an SM450 type linear smoking machine for smoking according to the specification of GB/T19609-2004, and detecting the release amount of carbon monoxide (CO) in main stream smoke by adopting a gas chromatography-mass spectrometry (GC-MS) method, a High Performance Liquid Chromatography (HPLC) method and a near infrared spectrometry (GB/T23356-2009); finally, the co-dominant SSR markers closely linked with the carbon monoxide release genes qCO are obtained by screening in the whole genome range of the tobacco by utilizing a quantitative trait linkage analysis (QTL) method and combining genotype data (genetic linkage map) and phenotype data (carbon monoxide release) of RILs_F 8:9, so that the blank of locating and analyzing the carbon monoxide release traits QTL in main stream smoke of the tobacco at home and abroad is effectively filled, the co-dominant SSR markers closely linked with the carbon monoxide release genes qCO are also obtained, and the method is an important way for cultivating new low-hazard flue-cured tobacco varieties without carbon monoxide release by accurately and efficiently utilizing molecular Marker Assisted Selection (MAS).
Disclosure of Invention
The invention provides a co-dominant SSR marker closely linked with a carbon monoxide release amount gene qCO in tobacco mainstream smoke and application thereof, and aims to solve the blank of carbon monoxide release amount trait genes/QTL positioning analysis in tobacco mainstream smoke.
The first aim of the invention is to provide a co-dominant SSR marker closely linked with a carbon monoxide release amount gene qCO in tobacco mainstream smoke; the second aim is to apply the co-dominant SSR marker to detect whether the genome DNA of the tobacco has the genotype state of the carbon monoxide release amount gene qCO and the carbon monoxide release amount character in the plant to be detected.
The innovation point of the invention is that: the invention carries out genetic positioning analysis for the carbon monoxide release amount gene qCO in tobacco mainstream smoke for the first time. The research of the inventor shows that the carbon monoxide release amount in tobacco mainstream smoke belongs to typical quantitative character and is controlled by the QTL of chromosome 5 in flue-cured tobacco germplasm Y3 genome, and the gene/QTL is temporarily named qCO.
The invention is realized by adopting the following technical scheme.
The co-dominant SSR markers closely linked with the carbon monoxide release gene qCO in the tobacco mainstream smoke are numbered IDYK 0477 and TMy80405, and the nucleotide sequences of PCR amplified products are respectively shown as SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
SEQ ID No.1：
AATATGCTTAGCTTGCGAGGATACATATTATATATATATATATATATATATATATATATATATAGTAGCTAATCCTTTCAAATTAAAAAATTAAAATGAAGCTATTACAGGCAAAGATATTAAAAAATATTTTTGAACAAAAATAATTATACAAATAAATATTTACAATCTCTTGGTGAATTTTTAATTACAAAAACACTTTCAGCATCATCTAGTTCTAAGACAGCA.
SEQ ID No.2：
AATATGCTTAGCTTGCGAGGATACATATTATATATATATATATATATATAGTAGCTAATCCTTTCAAATTAAAAAATTAAAATGAAGCTATTACAGGCAAAGATATTAAAAAATATTTTTGAACAAAAATAATTATACAAATAAATATTTACAATCTCTTGGTGAATTTTTAATTACAAAAACACTTTCAGCATCATCTAGTTCTAAGACAGCA.
SEQ ID No.3：
AAATGTAGCCACCCATCCAATTAGTTAAATTAGAAATGATGGATAAGTATGTAATATATATTTAATCTATATACAATAATAACTGCATATAATTAGTAGTATAATATATACATATATATATATATATATATATATATATATATATATATATATATATATATATATTAATTTTAAAAATAAATAATAAATTCGACATTTGTGTGGAATTGTGAACGGACAGGGCCCGAAACTTGGCCCGCTCTTAATCACAAAAATAGAAGAATATGCTTTCTTTGTAGCTTGTTTCGAAAGTGCAAAATAAAAAGCCCTAGCTCTCTCTCTCACGCAGCAGCTGTAG.
SEQ ID No.4：
AAATGTAGCCACCCATCCAATTAGTTAAATTAGAAATGATGGATAAGTATGTAATATATATTTAATCTATATACAATAATAACTGCATATAATTAGTAGTATAATATATACATATATATATATATATATATATATATATATATATATATATNTATATTAATTAAATAATATAAATTCGACATTTGTGTGGAATTGTGAACGGACAGGGCCCGAAACTTGGCCCGCTCTTAATCACAAAAATAGAAGAATATGCTTTCTTTGTAGCTTGTTTCGAAAGTGCAAAATAAAAAGCCCTAGCTCTCTCTCTCACGCAGCAGCTGTAG.
The co-dominant markers IDYK, 0477 and TMy, 80405 of the invention flank the gene qCO of interest.
The primer sequences of the 2 loci corresponding to the molecular markers are respectively as follows:
the primer sequences for amplification IDYK 0477 were:
IDYK04177F：5’-AATATGCTTAGCTTGCGAGGA-3’(SEQ ID NO.5)，
IDYK04177R：5’-TGCTGTCTTAGAACTAGATGATGC-3’(SEQ ID NO.6)；
the primer sequences for amplifying TMy and 80405 are as follows:
TMy80405F：5’-AAATGTAGCCACCCATCCAA-3’(SEQ ID NO.7)，
TMy80405R：5’-CTACAGCTGCTGCGTGAGAG-3’(SEQ ID NO.8)。
The application of the co-dominant SSR marker closely linked with the carbon monoxide release gene qCO in the main stream smoke of the tobacco is to detect whether the carbon monoxide release gene qCO exists in the genome DNA of the tobacco and the genotype state of the carbon monoxide release in the plant to be detected.
The application of the invention comprises the steps of amplifying tobacco genome DNA to be detected by using IDYK 0477 and TMy80405 sequence primers 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 has homozygote allele with high carbon monoxide release amount, and the genotype is COCO.
The PCR amplification product of the invention contains sequences shown as SEQ ID No.2 and SEQ ID No.4, and the sequences are homozygous alleles of the tobacco plants to be detected without carbon monoxide release amount, and the genotype is coco.
The PCR amplified product of the invention contains sequences shown as SEQ ID No.1 and SEQ ID No.4 at the same time or contains sequences shown as SEQ ID No.2 and SEQ ID No.3 at the same time, so that the heterozygous allele with medium carbon monoxide release amount of the tobacco plant to be detected is shown, and the genotype is COco.
In order to quickly and efficiently select the low-harm tobacco variety without carbon monoxide release, the invention pertinently selects the tobacco offspring material without the carbon monoxide release gene qco, and can be used for auxiliary selection of the gene qco without carbon monoxide release so as to improve the efficiency of molecular marker auxiliary selection and the breeding efficiency of the low-harm tobacco variety without carbon monoxide release. The 2 co-dominant SSR markers closely linked with the carbon monoxide release gene qCO in the tobacco mainstream smoke provided by the invention can be used for qualitatively detecting whether the carbon monoxide release gene qCO exists in the tobacco genome DNA in any growth period, can clearly and accurately identify the genotype state of the carbon monoxide release in the plant to be detected (namely, the homozygous genotype COCO with the highest carbon monoxide release value and stable inheritance, the heterozygous genotype COco with the medium carbon monoxide release value and unstable inheritance and the homozygous genotype COCO without the carbon monoxide release amount and stable inheritance), thereby improving the scientificity and predictability of the breeding of the new variety of the low-harm flue-cured tobacco without the carbon monoxide release amount and accelerating the breeding process.
Compared with the prior art, the invention has the beneficial effects that:
1. fills the blank of research on genetic localization of the carbon monoxide release gene qCO in tobacco mainstream smoke at home and abroad.
2. Genotype state for clearly and accurately detecting carbon monoxide release amount in main stream smoke of tobacco
The molecular marker for detecting the carbon monoxide release amount gene qCO in the main stream smoke of the tobacco is provided, so that whether the carbon monoxide release amount exists in the tobacco to be detected can be accurately determined, and the genotype state of the carbon monoxide release amount in the tobacco plant to be detected can be clearly identified.
3. The detection method is efficient, stable and reliable, and has simple operation and low cost
Compared with the existing method for detecting the release amount of 9 representative harmful components (NH 3, CO, HCN, NNK, BAP, PHE, CRO, CIC and TAR) in tobacco main stream smoke by adopting a low-flux, high-cost, time-consuming and labor-consuming GC-MS method, an HPLC method and a near infrared spectrum analysis method, 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 at any period of tobacco growth, especially at the tobacco seedling stage, thoroughly and perfectly avoids the processes of long tobacco growth period, harvesting, baking, leaf shredding, cigarette rolling, smoking machine sucking, time-consuming and labor-consuming detection and the like, remarkably shortens the detection period, and further accelerates the breeding process of new low-harm flue-cured tobacco varieties without carbon monoxide release.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a graph of QTL analysis of carbon monoxide emissions in tobacco mainstream smoke based on linkage group 5 of a tobacco recombinant inbred population (RILs_F 8:9; Y3XK326).
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= 3.3066 at the 0.01 significance threshold; the highest point of the LOD curve is the major gene (qCO).
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 carbon monoxide release amount gene qCO in tobacco mainstream smoke are numbered IDYK 0477 and TMy80405, and the nucleotide sequences of PCR amplified products are respectively shown as SEQ ID NO.1 and 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 amplification IDYK 0477 were:
IDYK04177F：5’-AATATGCTTAGCTTGCGAGGA-3’，
IDYK04177R：5’-TGCTGTCTTAGAACTAGATGATGC-3’；
the primer sequences for amplifying TMy and 80405 are as follows:
TMy80405F：5’-AAATGTAGCCACCCATCCAA-3’，
TMy80405R：5’-CTACAGCTGCTGCGTGAGAG-3’。
the application of the co-dominant SSR marker closely linked with the carbon monoxide release gene qCO in the tobacco mainstream smoke is the application of the co-dominant SSR marker closely linked with the carbon monoxide release gene qCO in detecting whether the carbon monoxide release gene qCO exists in the tobacco genome DNA.
The application of the co-dominant SSR marker closely linked with the carbon monoxide release amount gene qCO in the main stream smoke of the tobacco is that the primer of IDYK 0477 sequence and the primer of TMy80405 sequence are respectively used for amplifying the genome DNA of the tobacco to be detected, the PCR amplified product is detected, and if the PCR amplified product contains sequences shown as SEQ ID NO.1 and SEQ ID NO.3 at the same time, the sequence is the homozygous allele qCOqCO with high carbon monoxide release amount 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 qcoqco of the tobacco plant to be detected without carbon monoxide release amount; 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 qCOqco with medium carbon monoxide release amount of the tobacco plant to be detected is obtained.
Example 1 Co-dominant SSR marker screening linked to carbon monoxide Release Gene qCO in tobacco mainstream Smoke
Screening co-dominant SSR markers linked with carbon monoxide release amount gene qCO in tobacco mainstream smoke in the whole genome range of tobacco by adopting quantitative trait linkage analysis (QTL) method and combining gas chromatography-mass spectrometry (GC-MS) method, high Performance Liquid Chromatography (HPLC) method and near infrared spectrometry
1. Experimental materials
The low-harm flue-cured tobacco variety K326 with excellent comprehensive properties and no carbon monoxide release amount in main stream smoke is taken as a female parent, the flue-cured tobacco germplasm Y3 with high carbon monoxide release amount is taken as a male parent, and the recombinant inbred line (RILs_F 8:9) of 269 strains is obtained through hybridization and continuous selfing as a genetic mapping population.
2. Acquisition of carbon monoxide release amount data in main stream smoke of parent and RILs_F 8:9 groups
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, and selecting 3 middle leaves per plant of tobacco to carry out listing numbering and baking; after baking, 30C 3F grade middle leaves of each of the above strains were mixed together to prepare shreds.
Cutting 30C 3F-level middle leaves of each strain into tobacco shreds with the width of 1.0mm and the length of 15-20mm by using a tobacco cutter, and manually rolling into normal rolled cigarettes with the length of 84 mm. Each sample is manually rolled into 20 cigarettes, the length of each cigarette is strictly controlled to be 84mm plus or minus 0.02mm, and the weight of each cigarette is strictly controlled to be 0.95g plus or minus 0.01g.
According to GB/T19609-2004, placing rolled cigarettes in a constant temperature and humidity box with temperature of 22+ -2deg.C and relative humidity of 60+ -5%, balancing for 48 hr, and selecting weight and suction resistance; suction was applied to a SM450 type 20-channel linear type smoking machine for 2s at suction intervals of 58s with a suction capacity of 35mL, and the total particulate matters in the smoke were trapped with a Cambridge filter.
The total particulate matters of the trapped main stream smoke are dissolved in an extracting agent, and the carbon monoxide release amount in the main stream smoke is measured by adopting a gas chromatography-mass spectrometry (GC-MS) method, a High Performance Liquid Chromatography (HPLC) method and a near infrared spectrometry method, wherein each sample is measured for 2 times, and the average value of 2 times is calculated as the carbon monoxide release amount in the main stream smoke of the sample.
The carbon monoxide release amount in 269 parts of tobacco main stream smoke of RILs_F 8:9 group is obtained as the phenotype value of RILs_F 8:9 group and used 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 flue-cured tobacco Y3 genome information are utilized to carry out polymorphism screening on parents (Y3 and K326) and sub-generations (F 1) of RILs_F 8:9 population, and finally 2158 polymorphic SSR markers are obtained through screening.
Then 2158 polymorphic SSR markers are obtained by screening, and genotype analysis is carried out on 269 RILs_F 8:9 samples.
Finally, carrying out linkage analysis on genotype data of 269 RILs_F 8:9 samples by using genetic linkage mapping software JoinMap 4.0.0, drawing a high-quality flue-cured tobacco SSR molecular genetic linkage map which contains 24 linkage groups and is uniformly distributed with 2059 SSR markers and covers the length of tobacco genome 1759.74cM, and taking the genetic linkage map as genotype value of the RILs_F 8:9 group for subsequent QTL linkage analysis.
4. Whole genome QTL positioning analysis of carbon monoxide release amount (qCO) in mainstream smoke
The QTL localization analysis software QTL ICIMAPPING V4.2.2 was used to perform a whole genome QTL scan of the carbon monoxide release gene qCO on genotype data (constructed to obtain a genetic linkage map of flue-cured tobacco) and phenotype data (269 parts of carbon monoxide release in mainstream smoke of the rils_f 8:9 population) of the rils_f 8:9 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= 3.3066 in the whole genome range, 1 major QTL (temporarily named qCO) of the carbon monoxide release profile in the mainstream smoke was located at 16.00cM of linkage group No. 5. The major QTL may explain the phenotype variability of about 10.2492% and the LOD value at this point is about 4.8932, see in detail fig. 1 and table 1.
TABLE 1 statistics of carbon monoxide Release QTL (qCO) information in tobacco mainstream smoke
| QTL | Chromosome | Position/cM | Left Marker | Right Marker | LOD | PVE(％) | Add |
| qCO | 5 | 16.00 | IDYK04177 | TMy80405 | 4.8932 | 10.2492 | 0.6766 |
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 9:10 population individuals
And (3) carrying out genotype analysis on the single plants of the RILs_F 9:10 group (Y3X K326) in the seedling stage by utilizing the obtained co-dominant SSR markers IDYK 0477 and TMy80405 which are closely linked with both sides of the carbon monoxide release amount gene qCO in the main stream smoke of the tobacco to obtain genotype data of each single plant of the RILs_F 9:10 group.
On the other hand, after tobacco leaves of RILs_F 9:10 group grow to maturity and are roasted, the middle C3F class roasted leaves of each strain are shredded and rolled into cigarettes by adopting a GC-MS method, an HPLC method and a near infrared spectrum analysis method, and carbon monoxide release amount in main stream smoke is detected. That is, the phenotype values of each strain of the RILs_F 9:10 population are obtained.
Finally, the genotype data of 269 RILs_F 9:10 population and the carbon monoxide release profile value are analyzed, and the genotype value and the phenotype value of the two co-dominant SSR markers IDYK 0477 and TMy80405 disclosed by the invention are completely matched, namely, the coincidence rate reaches 100%.
The specific analysis method comprises the following steps: when the carbon monoxide release amount of each strain obtained by detection of a GC-MS method, an HPLC method and a near infrared spectrum analysis method is higher than or equal to the content of parent Y3, the genotype of the strain also shows sequences shown as SEQ ID NO.1 (228 bp) and SEQ ID NO.3 (337 bp) to be homozygous genotype COCO;
When the carbon monoxide release amount of each strain obtained by detection is equal to the content of the parent K326 (namely, the carbon monoxide release amount is 0), the sequences shown as SEQ ID NO.2 (214 bp) and SEQ ID NO. 4 (323 bp) are simultaneously presented in the genotype of the strain, namely, the homozygous genotype coco;
When the carbon monoxide release amount of each strain obtained by detection is between the parent Y3 and K326, namely the content of the strain is similar to that of a sub-generation (F 1) plant, the genotype of the strain also simultaneously presents sequences shown as SEQ ID NO.1 and SEQ ID NO.4, or the genotype COco contains sequences shown as SEQ ID NO.2 and SEQ ID NO. 3.
Conclusion of experiment:
The above results indicate that co-dominant markers IDYK 0477 and TMy80405 are closely linked to the carbon monoxide release gene qCO in tobacco mainstream smoke, respectively, and that the two markers flank the gene of interest (qCO).
By utilizing the two co-dominant closely linked SSR markers, the detection of the carbon monoxide release amount in the mainstream smoke of any growth period of tobacco can be accurately, efficiently, quickly and at low cost, the genotype state of the carbon monoxide release amount in the plant to be detected (namely, the homozygous genotype COCO with the highest carbon monoxide release amount value and stable inheritance, the heterozygous genotype COco with medium carbon monoxide release amount value and unable to be inherited stably and the homozygous genotype COCO without carbon monoxide release amount and stable inheritance) can be clearly and accurately identified, and the scientificity and predictability of the breeding of the new variety of the low-harm tobacco without carbon monoxide release amount are improved, and the breeding process is accelerated.
What has been described above is only a part of the specific embodiments of the present application, 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 application in any way, and it is within the scope of the present application for those skilled in the art to obtain the technical solution by equivalent substitution or equivalent transformation. The protection scope of the present application is 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 carbon monoxide release amount gene qCO in the tobacco mainstream smoke is characterized in that the numbers of the co-dominant SSR markers closely linked with the carbon monoxide release amount gene qCO in the tobacco mainstream smoke are IDYK 0477 and TMy80405, and the nucleotide sequences of PCR amplified products are respectively shown as SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
2. A co-dominant SSR marker closely linked to a carbon monoxide release amount gene qCO in tobacco mainstream smoke according to claim 1 wherein the co-dominant markers IDYK 0477 and TMy80405 flank the gene qCO of interest.
3. The co-dominant SSR marker closely linked to a carbon monoxide release amount gene qCO in tobacco mainstream smoke according to claim 1, wherein primer sequences of 2 sites corresponding to the molecular marker are respectively:
the primer sequences for amplification IDYK 0477 were:
IDYK04177F：5’-AATATGCTTAGCTTGCGAGGA-3’，
IDYK04177R：5’-TGCTGTCTTAGAACTAGATGATGC-3’；
the primer sequences for amplifying TMy and 80405 are as follows:
TMy80405F：5’-AAATGTAGCCACCCATCCAA-3’，
TMy80405R：5’-CTACAGCTGCTGCGTGAGAG-3’。
4. The use of co-dominant SSR markers closely linked to the carbon monoxide release gene qCO in tobacco mainstream smoke according to claim 1 or 2 or 3 for detecting the presence or absence of the carbon monoxide release gene qCO in tobacco genomic DNA and the genotype status of carbon monoxide release in the plant to be tested.
5. The use according to claim 4, wherein the PCR amplification products are detected by amplifying the tobacco genomic DNA to be detected with the primers IDYK 0477 and TMy80405 sequences, 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 has homozygous allele with high carbon monoxide release, and the genotype is COCO.
7. The method according to claim 5, wherein the PCR amplification product contains homozygous alleles having the sequences shown in SEQ ID No.2 and SEQ ID No.4, which are the non-carbon monoxide release levels of the tobacco plants to be tested, and the genotype is coco.
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, which is a heterozygous allele with moderate carbon monoxide release in the tobacco plant to be tested, and the genotype is COco.
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