CN114480703A - Co-dominant InDel marker for identifying burley tobacco types and application thereof - Google Patents
Co-dominant InDel marker for identifying burley tobacco types and application thereof Download PDFInfo
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- CN114480703A CN114480703A CN202210013453.2A CN202210013453A CN114480703A CN 114480703 A CN114480703 A CN 114480703A CN 202210013453 A CN202210013453 A CN 202210013453A CN 114480703 A CN114480703 A CN 114480703A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention discloses a codominant InDel marker for identifying burley tobacco types and application thereof, wherein the nucleotide sequence of the codominant InDel marker is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; the co-dominant InDel marker disclosed by the invention can realize accurate identification of the burley tobacco type, has the characteristics of stability, reliability, simplicity, convenience, quickness and low cost, and can be used for identifying whether burley tobacco exists in tobacco to be detected.
Description
Technical Field
The invention relates to the technical field of burley tobacco type identification, in particular to a codominant InDel marker for identifying burley tobacco types and application thereof.
Background
Tobacco is a leaf-used commercial crop, and can be classified into 6 types, such as flue-cured tobacco, burley tobacco, cigar tobacco, aromatic tobacco, yellow-scented tobacco, wild tobacco and the like, according to the quality characteristics, biological properties and cultivation and modulation methods of tobacco leaves.
Burley tobacco is a green-lacking mutant strain discovered in 1864 years in a broadleaf tobacco seedbed in Maryland in Brownshire, Ohio, and is proved to have special use value through systematic breeding and planting, so that the burley tobacco is developed into a new type of tobacco. The burley tobacco as one of 6 types of tobacco is mainly characterized in that the main veins of the leaves are milk white, the leaves in the seedling stage are yellow green, and the chlorophyll content is about one third of that of other normal green tobacco leaves. Thus, the milky white appearance of the tobacco lamina mainvein is a typical characteristic of the burley tobacco type to distinguish it from the other 5 tobacco types. In the seedling stage of tobacco and even in the whole growth period of the tobacco, the type of burley tobacco in the tobacco can be identified according to the color of the main vein of the tobacco leaf (whether the color of the main vein of the leaf is milky), but in the industrial production application, the tobacco leaf utilized in the cigarette industry is a tobacco-making raw material which is matured and is subjected to a series of complicated process flows such as baking (or airing), storage, fermentation, shredding, rolling and the like, so that the identification of the type of the burley tobacco in the tobacco-making raw material cannot be carried out according to the milky main vein. In view of the above, the present invention provides a marker and a method for identifying the type of burley tobacco in tobacco simply, rapidly and stably on a molecular level.
Disclosure of Invention
The invention aims to provide a codominant InDel marker for identifying burley tobacco types and application thereof, wherein the codominant InDel marker can realize rapid and stable identification of the burley tobacco types in tobacco on a molecular level.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the first aspect of the invention provides a co-dominant InDel marker for identifying the type of burley tobacco, wherein the nucleotide sequence of the co-dominant InDel marker is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The second aspect of the present invention is a primer pair for obtaining the above co-dominant InDel marker, the primer pair comprising an upstream primer and a downstream primer;
the sequence of the upstream primer is shown as SEQ ID NO: 3 is shown in the specification; the sequence of the downstream primer is shown as SEQ ID NO: 4, respectively.
In a third aspect, the invention provides the use of a co-dominant InDel marker as described above for identifying burley tobacco types.
The fourth aspect of the invention provides an application of the primer pair in identifying the type of burley tobacco.
A fifth aspect of the present invention provides a method of identifying a type of burley tobacco, the method comprising the steps of:
using SEQ ID NO: 3 and SEQ ID NO: 4, carrying out PCR amplification on the genome DNA of the tobacco to be detected by the primer pair shown in the specification, and then detecting the size of a PCR amplification product fragment;
if the PCR amplification product only contains the segment with the length of 169bp, the type of the tobacco to be detected is burley tobacco; if the PCR amplification product only contains a fragment with the length of 181bp, the type of the tobacco to be detected is a non-burley tobacco type; if the PCR amplification product contains the fragments with the lengths of 169bp and 181bp at the same time, the tobacco to be detected is a heterozygote of the burley tobacco type and other tobacco types.
Preferably, the PCR amplification product is subjected to 6% native polyacrylamide gel electrophoresis to detect fragment size.
The invention also provides a kit for identifying the type of the burley tobacco, which comprises the primer pair.
Compared with the prior art, the invention has the beneficial effects that at least:
the co-dominant InDel marker disclosed by the invention can realize accurate identification of the burley tobacco type in the tobacco raw materials, has the characteristics of stability, reliability, simplicity, convenience, quickness and low cost, and can be used for identifying whether the burley tobacco exists in the tobacco to be detected.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 shows the results of screening for molecular markers linked to tobacco white vein genes in example 1 of the present invention;
FIG. 2 shows the results of gel electrophoresis of 50 copies of the tobacco genome amplification products of Burley tobacco, P1 (cured tobacco type parent K326) and P2 (Burley tobacco type parent TN90) in example 2 of the present invention;
FIG. 3 shows the results of gel electrophoresis of 50 other tobacco varieties, P1 (flue-cured tobacco type parent K326) and P2 (burley tobacco type parent TN90) tobacco genome amplification products in example 2 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
The embodiment of the invention provides a codominant InDel marker for identifying burley tobacco types, wherein the nucleotide sequence of the codominant InDel marker is shown as SEQ ID NO: 1 and SEQ ID NO: 2, wherein the nucleotide sequence shown in SEQ ID NO: 1 the specific sequence is as follows:
CCTGTTCATGGTGGAAGAGCCTAACTGACCCACGTGGTGGGCCCAGAGTTAGCTTTGGTATGCTGCGGAAAGAAGTTTCTGAACCAGGTCCAACAACTCTCTGGCAATATGTAATTGCTTTTCTGTTGTTCCTTCTCACAATTGGTTCCTCTGTGGAGCTAGGAATTGC；
SEQ ID NO: 2 the specific sequence is as follows:
CCTGTTCATGGTGGAAGAGCCTAACTTCAGAGGGGCCAGACCCACGTGGTGGGCCCAGAGTTAGCTTTGGTATGCTGCGGAAAGAAGTTTCTGAACCAGGTCCAACAACTCTCTGGCAATATGTAATTGCTTTTCTGTTGTTCCTTCTCACAATTGGTTCCTCTGTGGAGCTAGGAATTGC。
the co-dominant InDel mark can realize the accurate identification of the burley tobacco type, has the characteristics of stability, reliability, simplicity, convenience, quickness and low cost, and can be used for identifying whether burley tobacco exists in tobacco to be detected.
The invention further provides a primer pair for obtaining the co-dominant InDel marker, which comprises an upstream primer and a downstream primer;
the sequence of the upstream primer is shown as SEQ ID NO: 3 is shown in the specification; the sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of the upstream primer is specifically as follows: 5'-CCTGTTCATGGTGGAAGAGCCTAAC-3', respectively;
the sequence of the downstream primer is specifically as follows: 5'-GCAATTCCTAGCTCCACAGAGGAAC-3' are provided.
The primer pair can amplify the codominant InDel marker, has high amplification efficiency and is beneficial to improving the identification accuracy of the burley tobacco types.
Another embodiment of the invention provides the use of the co-dominant InDel marker described above for identifying burley tobacco types.
The invention further provides an application of the primer pair in identifying the type of the burley tobacco.
The embodiment of the invention also provides a burley tobacco type identification method, which comprises the following steps:
using SEQ ID NO: 3 and SEQ ID NO: 4, carrying out PCR amplification on the genome DNA of the tobacco to be detected by the primer pair shown in the specification, and then detecting the size of a PCR amplification product fragment;
if the PCR amplification product only contains the segment with the length of 169bp, the type of the tobacco to be detected is burley tobacco; if the PCR amplification product only contains a fragment with the length of 181bp, the type of the tobacco to be detected is a non-burley tobacco type; if the PCR amplification product contains the fragments with the lengths of 169bp and 181bp at the same time, the tobacco to be detected is a heterozygote of the burley tobacco type and other tobacco types.
In some embodiments, the PCR amplification products are detected by gel electrophoresis for fragment size.
The invention further provides a kit for identifying the type of burley tobacco, which comprises the primer pair.
The technical solution of the present invention is further described in detail by the following specific examples.
Example 1
This example is a process of screening for co-dominant InDel markers linked to genes controlling white veins in burley tobacco types using the segregating population group analysis (BSA) method:
first, experimental material
A flue-cured tobacco type variety K326 with normal green veins in leaves is taken as a female parent, a burley tobacco type variety TN90 with white veins in leaves (the white veins in the leaves are the unique character of the burley tobacco type in tobacco) is taken as a male parent, and a first filial generation (F1) population and a second filial generation (F2) population are obtained through hybridization and selfing respectively.
Second, obtaining leaf vein color data of parent and F2 segregation population
And transplanting the test material to a field after the test material is grown, and counting the main vein color of the tobacco leaves when the tobacco plants start to enter the vigorous growth period, namely counting the number of green and white in the main vein color of the leaves.
Construction of genome pool
Extracting tobacco genome DNA: extracting by using a plant tissue DNA extraction kit, wherein the extraction method refers to the instruction in the kit;
a separation population grouping analysis method (BSA) is adopted, 20 extreme green vein single plants (the vein color is consistent with the green color of a flue-cured tobacco type variety K326) and 20 white vein single plants in an F2 separation population are selected to be mixed in an equal amount, 2 extreme pools, namely a green vein pool (B1) and a white vein pool (B2) are respectively constructed, and 5 genome materials of K326(P1), TN90(P2), an amphiphilic intercross first generation (F1), a green vein pool (B1) and a white vein pool (B2) are obtained in the seedling stage according to a plant genome DNA extraction method.
Four, molecular marker analysis
Carrying out PCR amplification on 5 parts of genome materials including multiple InDel, SSR and SNP (single nucleotide polymorphism) molecular marker amplification primer pairs P1, P2, F1, B1 and B2, and screening molecular markers linked with tobacco white vein genes; the results of the screening are shown in FIG. 1;
as can be seen from FIG. 1, the co-dominant InDel marker of the invention is linked with the white vein gene of tobacco, and the band types of the co-dominant InDel marker in the white vein pool (B2) and the white vein parent (burley tobacco type variety TN90, P2) are completely consistent, and only one specific band of 169bp (sequence as SEQ ID NO: 1) appears; the band pattern in the green vein pool (B1) is completely consistent with that in F1, and two specific bands which are codominant (the specific bands of green vein parents and white vein parents are simultaneously displayed, namely, the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are simultaneously displayed); wherein, SEQ ID NO: 2 is 181bp in length and is a specific PCR amplification band of the codominant InDel marker in a non-burley tobacco type variety without a white vein gene.
The result shows that the codominant InDel marker is linked with the white vein gene in the burley tobacco type, and the marker is the codominant marker; the PCR amplification product only contains the nucleotide sequence shown as SEQ ID NO: 1(169bp) indicates that the type of the tobacco to be detected is a burley tobacco type; PCR amplification products only contain the following components such as ID NO: 2(181bp) indicates that the type of the tobacco to be detected is a non-burley tobacco type; the PCR amplification product simultaneously contains the nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO: 2, the type of the tobacco to be detected is a heterozygote of a burley tobacco type and other tobacco types.
Example 2
This example is the validation of co-dominant InDel markers in different tobacco types
Tobacco material
The tested tobacco materials amounted to 100 parts, wherein 50 parts of burley tobacco type, flue-cured tobacco type, cigar type, aromatic tobacco type, yellow tobacco type and wild tobacco each 10 parts, and the detailed information of 100 parts of tobacco materials is shown in table 1; the main veins of the leaves of the tobacco materials are all normally green except that the main veins of the leaves of 50 parts of the burley tobacco type materials are white.
TABLE 1100 parts of information on the type of tobacco material tested
Detection of burley tobacco type in tobacco material by codominant InDel mark
The detection method comprises the following steps:
obtaining the genome DNA of tobacco to be detected;
using SEQ ID NO: 3 and SEQ ID NO: 4, carrying out PCR amplification on the genome DNA of the tobacco to be detected, and carrying out gel electrophoresis on the PCR amplification product by adopting a gel electrophoresis method, wherein the gel electrophoresis results of 50 parts of burley tobacco, P1, P2 and Mark are shown in figure 2; the electrophoresis results of 50 other types of tobacco varieties, P1, P2 and Mark gel are shown in FIG. 3;
since 100 tobacco materials tested were all stable varieties or lines, i.e., NO heterozygotes, NO PCR amplification test using the co-dominant InDel marker occurred as shown in SEQ ID NO: 1 and SEQ ID NO: 2 occur simultaneously in one material; as can be seen from FIG. 2, only the PCR amplification products of the 50 burley tobacco types have the sequences shown in SEQ ID NO: 1, and the sequence shown in 1; and the PCR amplification products of the remaining 50 tobacco varieties of other types are only shown in SEQ ID NO: 2, as shown in fig. 3.
Analysis of PCR amplification products of 100 different types of tobacco materials by the co-dominant InDel marker shows that: the co-dominant InDel marker provided by the invention has specificity of burley tobacco types, can scientifically and accurately realize the identification and detection of the burley tobacco types and other tobacco types in tobacco, and has the characteristics of stability, reliability, simplicity, convenience, quickness and low cost.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
SEQUENCE LISTING
<110> Yunnan province tobacco quality supervision and detection station, Yunnan province tobacco agricultural science research institute
<120> co-dominant InDel marker for identifying burley tobacco types and application thereof
<130> 2021
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Claims (7)
1. A co-dominant InDel marker for identifying burley tobacco types, wherein the nucleotide sequence of the co-dominant InDel marker is as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
2. A primer pair for obtaining the co-dominant InDel marker of claim 1, wherein the primer pair comprises an upstream primer and a downstream primer;
the sequence of the upstream primer is shown as SEQ ID NO: 3 is shown in the specification; the sequence of the downstream primer is shown as SEQ ID NO: 4, respectively.
3. Use of the co-dominant InDel marker of claim 1 for identifying burley tobacco types.
4. Use of the primer pair of claim 2 for identifying a type of burley tobacco.
5. A method of identifying the type of burley tobacco, comprising the steps of:
using SEQ ID NO: 3 and SEQ ID NO: 4, carrying out PCR amplification on the genome DNA of the tobacco to be detected by the primer pair shown in the specification, and then detecting the size of a PCR amplification product fragment;
if the PCR amplification product only contains the segment with the length of 169bp, the type of the tobacco to be detected is burley tobacco; if the PCR amplification product only contains a fragment with the length of 181bp, the type of the tobacco to be detected is a non-burley tobacco type; if the PCR amplification product contains the fragments with the lengths of 169bp and 181bp at the same time, the tobacco to be detected is a heterozygote of the burley tobacco type and other tobacco types.
6. The method of claim 5, wherein the PCR amplification product is subjected to gel electrophoresis to detect fragment size.
7. A kit for identifying the type of burley tobacco, comprising the primer pair of claim 2.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210013453.2A CN114480703A (en) | 2022-01-06 | 2022-01-06 | Co-dominant InDel marker for identifying burley tobacco types and application thereof |
| ZA2022/02195A ZA202202195B (en) | 2022-01-06 | 2022-02-22 | Co-dominant indel markers for identifying burley tobacco and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210013453.2A CN114480703A (en) | 2022-01-06 | 2022-01-06 | Co-dominant InDel marker for identifying burley tobacco types and application thereof |
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| CN114480703A true CN114480703A (en) | 2022-05-13 |
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| CN202210013453.2A Pending CN114480703A (en) | 2022-01-06 | 2022-01-06 | Co-dominant InDel marker for identifying burley tobacco types and application thereof |
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| CN (1) | CN114480703A (en) |
| ZA (1) | ZA202202195B (en) |
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2022
- 2022-01-06 CN CN202210013453.2A patent/CN114480703A/en active Pending
- 2022-02-22 ZA ZA2022/02195A patent/ZA202202195B/en unknown
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| Title |
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| 耿歆淇 等: "基于不同烟草类型基因组重测序的 烟草SSR标记的开发和应用", 作物杂志, vol. 2, pages 84 - 89 * |
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