CN109338009B - InDel molecular labeling and its application with pumpkin taro aroma characteristics close linkage - Google Patents
InDel molecular labeling and its application with pumpkin taro aroma characteristics close linkage Download PDFInfo
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- CN109338009B CN109338009B CN201811513265.6A CN201811513265A CN109338009B CN 109338009 B CN109338009 B CN 109338009B CN 201811513265 A CN201811513265 A CN 201811513265A CN 109338009 B CN109338009 B CN 109338009B
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Abstract
The present invention provides a kind of and pumpkin taro aroma characteristics close linkage InDel molecular labeling, and the InDel Molecular mapping is on the 10th article of chromosome of musky gourd genome, and nucleotide sequence is as shown in SEQ ID NO.1.The present invention also provides the special primers and method for detecting InDel label, and the sequence of the specific primer pair is as shown in SEQ ID NO.2-3.Utilize specific primer pair provided by the invention, the electrophoretic band that based on PCR and polyacrylamide gel electrophoresis obtain, the InDel molecular labeling is applied to the pumpkin that screening has taro aroma characteristics, be conducive to determine the objective judgment criteria of the pumpkin with taro fragrance, pumpkin taro aroma characteristics are quickly and accurately judged from genotype, to establish the molecule assisted selection technical system of pumpkin taro aroma characteristics.
Description
Technical field
The invention belongs to field of biology, in particular to a kind of InDel molecule with pumpkin taro aroma characteristics close linkage
Label and its application.
Background technique
Pumpkin taro aroma characteristics more special, aromatic flavour in the flavor character of pumpkin resource.With taro aroma characteristics
Pumpkin variety it is deep by market and consumers.But the judgement of taro aroma characteristics at present is main or relies on smelling for breeder
Feel judgement, lacks the standard of objective judgement.In addition, the performance of aroma characteristics is highly prone to the influence of environment, phenotype is relied solely on
Smell judgement Screening germplasm can be caused to perplex.Therefore, it is necessary to provide one kind, fast and accurately judgement is southern from genotype
The method of melon taro aroma characteristics finds the InDel molecule mark with taro fragrance close linkage by the assignment of genes gene mapping of pumpkin taro fragrance
Note, to establish the assisted selection technical system of pumpkin taro aroma characteristics.
Summary of the invention
Based on this, the purpose of the present invention is to provide with pumpkin taro aroma characteristics close linkage Indel molecular labeling and
It is applied.
To achieve the above object, the present invention provides the following technical scheme that
A kind of InDel molecular labeling with pumpkin taro aroma characteristics close linkage, the InDel Molecular mapping is in
On 10th article of chromosome of state's pumpkin genome, nucleotide sequence is as shown in SEQ ID NO.1.
Application of the above-mentioned InDel molecular labeling in the pumpkin that screening has taro aroma characteristics.
A method of screen have taro aroma characteristics pumpkin, comprising: detection as described above with pumpkin taro fragrance
The primer pair of the InDel molecular labeling of shape close linkage.
A kind of kit of pumpkin of the screening with taro aroma characteristics, which is characterized in that include for detecting nucleotide
The reagent of sequence InDel molecular labeling as shown in SEQ ID NO.1.
Based on the above-mentioned technical proposal, the invention has the following advantages:
The a large amount of creative works of the present inventor are found and taro fragrance by the assignment of genes gene mapping to pumpkin taro fragrance
The InDel molecular labeling of character close linkage, the InDel Molecular mapping is in the 10th article of dyeing of musky gourd genome
On body, nucleotide sequence is applied to sieve as shown in SEQ ID NO.1, by the InDel molecular labeling and molecular labeling amplimer
The pumpkin with taro aroma characteristics is selected, the objective judgment criteria for determining the pumpkin with taro fragrance is conducive to, it is fast from genotype
Speed accurately judges pumpkin taro aroma characteristics, to establish the assisted selection technical system of pumpkin taro aroma characteristics.
Detailed description of the invention
Fig. 1 is molecular labeling primer in embodiment 1 in parent and F1Middle PCR result figure；
Fig. 2 is molecular labeling primer in embodiment 2 in F2The PCR testing result of verifying is detected in single plant；
Fig. 3 detects the PCR inspection of verifying for the molecular labeling primer in embodiment 3 in different types of musky gourd resource
Survey result；
Fig. 4 is that the labeled primer in embodiment 4 is applied to the PCR testing result that breeding improvement material is screened in detection.
Specific embodiment
To facilitate the understanding of the present invention, it below with reference to embodiment to invention is more fully described, is given below
Presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to described herein
Embodiment.Purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.It should be understood that
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, for example (,) Sambrook et al., molecule gram
It is grand: condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989),
Or according to the normal condition proposed by manufacturer.Used various common agents, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Used term is intended merely to describe specific reality in the description of the invention
Apply the purpose of example, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more relevant institutes
Any and all combinations of list of items.
A kind of InDel molecular labeling of and pumpkin taro aroma characteristics close linkage of the invention, the InDel molecular labeling
It is positioned on the 10th article of chromosome of musky gourd genome, nucleotide sequence such as SEQ ID NO.1
(CGCTTTTTCCATCGATTTTGCTGGA) shown in.The InDel molecular labeling can be in pumpkin of the screening with taro aroma characteristics
Middle application.Inventors have found that being positioned on the 10th article of chromosome of musky gourd genome, nucleotide sequence such as SEQ ID
The InDel molecular labeling of NO.1 and pumpkin taro aroma characteristics close linkage can distinguish pumpkin with taro fragrance shape and not
Pumpkin with taro fragrance.
The present invention provides a kind of methods that screening has the pumpkin of taro aroma characteristics, it can by detecting above-mentioned core
InDel molecular labeling of the nucleotide sequence as shown in SEQ ID NO.1 with pumpkin taro aroma characteristics gene linkage, has to screen
The pumpkin of taro aroma characteristics, wherein detect containing nucleotide sequence sequence as shown in SEQ ID NO.1.Preferably, it designs
Sequence primer as shown in SEQ ID NO.2 and SEQ ID NO.3 is obtained, which is detected using PCR method.Tool
Body, screen the method with the pumpkin of taro aroma characteristics, comprising the following steps: (1) extract the DNA of pumpkin；(2) with step
(1) extracting and obtaining the DNA of pumpkin is template, and design obtains and using nucleotide sequence such as SEQ ID NO.2 and SEQ ID NO.3
Shown in primer carry out PCR amplification；(3) detected through gel electrophoresis: if only containing 229bp band, south to be measured in amplified production
Melon is the pumpkin with taro aroma characteristics.Wherein, the amplification system of the PCR method includes: Taq enzyme, template DNA, dNTP, nucleosides
Acid sequence primer as shown in SEQ ID NO.2 and SEQ ID NO.3, PCR buffer.The amplification system of specific PCR are as follows:
Comprising 1U Taq enzyme, 1 μ L template DNA, the dNTP of 1.5 μ L, 1 μ L primer, 10 × PCR buffer of 2 μ L adds ddH2O to 20 μ
L, amplification program are as follows: 94 DEG C of 3min, cyclic process be 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 30 circulation, it is last 72 DEG C extension
5min。
The present invention also provides the kits that a kind of screening has the pumpkin of taro aroma characteristics, include for detecting nucleosides
The reagent of acid sequence InDel molecular labeling as shown in SEQ ID NO.1.Preferably, described for detecting nucleotide sequence such as
The reagent of InDel molecular labeling shown in SEQ ID NO.1 includes: nucleotide sequence such as SEQ ID NO.2 and SEQ ID NO.3
Shown in primer.
The location test of 1 pumpkin taro aroma characteristics gene of embodiment and indel marker development
1. test material:
The number of the glycan pumpkin (female parent) and non-taro fragrance that are saved with Vegetables Inst., Guangdong Academy of Agricultural Sciences's breeding
Resource for BX (male parent) is parent, and bi-parental mating obtains F1, and F1 selfing obtains F2 segregating population.
2. pumpkin taro scent gene Primary Location:
Using 2 parents and 200 plants of F2 single plants, exploitation SNP marker is sequenced by GBS-Seq and constructs high density genetic linkage
Map, finally obtained map total figure is away from for 1955.34cM, including 20 linkage groups.And taro perfume is carried out to 169 plants of F2 groups
The identification of taste trait phenotypes.In conjunction with dense genetic map and F2 single plant phenotypic evaluation as a result, taro scent gene is navigated to the 10th
In number linkage group, the genetic distances of two labels are 1.73cM, and the label at the section both ends is compared to musky gourd genome,
It navigates on the 10th article of chromosome of musky gourd genome.
3.InDel marker development
Full-length genome is carried out using 2 parents and resurveys sequence, finds the site InDel in taro scent gene positioning section.Fixed
In the section of position, it was found that, the parent of taro fragrance has 1 InDel molecular labeling site, nucleotides sequence with non-taro fragrance parent
It is classified as CGCTTTTTCCATCGATTTTGCTGGA (SEQ ID NO.1), and according to the sequence of site upstream and downstream 200bp, design
InDel labeled primer.
InDel labeled primer sequence are as follows:
F1:TTCTTTACAATTGCCCGATG (SEQ ID NO.2)；
R1:AAGAAGATGTTCCGTAACGA (SEQ ID NO.3).
PCR amplification system uses 20 μ l systems, including, it include 1U Taq enzyme, 1 μ L template DNA, the dNTP of 1.5 μ L, 1 μ L
Primer, 10 × PCR buffer of 2 μ L, adds ddH2O to 20 μ L.PCR amplification program are as follows: 94 DEG C of 3min, cyclic process are 94 DEG C
30s, 52 DEG C of 30s, 72 DEG C of 30s, 30 circulations, last 72 DEG C of extensions 5min.According to above-mentioned system and program, InDel label draws
Object carries out PCR amplification in two parents and F1, is detected using polyacrylamide gel electrophoresis, and specific band is presented between parent, female
This stripe size 229bp, male parent amplified band size 205bp, parents' band 229bp and 205bp is presented in F1, as a result such as Fig. 1 institute
Show, wherein the first swimming lane from left to right is marker, and the second swimming lane is maternal PCR as a result, third swimming lane is male parent
PCR is as a result, the 4th swimming lane is the PCR result of F1.
Embodiment 2
Using InDel labeled primer in embodiment 1, verified by the random single plant of F2 group.Using in embodiment 1
InDel labeled primer is detected with carrying out PCR amplification using polyacrylamide gel electrophoresis, is showed between parent specifically, in F2
Single plant testing result is shown in Fig. 2.First swimming lane marker from left to right, the second swimming lane is maternal PCR as a result, third swimming lane is male parent
PCR is as a result, the 4th to 14 swimming lanes are F2 single plant PCR result
As it can be seen that existing in the random single plant of female parent band length 229bp, male parent amplified band length 205bp, F2 group
The three types of P1, P2 and F1.It is found in conjunction with the phenotypic analysis of F2 single plant, single plant can with maternal presentation same size band
It is presented taro fragrance, and single plant band and male parent, F1 band are consistent that taro fragrance cannot all be presented.Therefore, above-mentioned InDel label can
Taro fragrance and the single plant of non-taro aroma characteristics to be separated.
Embodiment 3
Using InDel labeled primer in embodiment 1, verified using 20 kinds of different types of musky gourd resources.Benefit
PCR amplification is carried out with the primer, is detected using polyacrylamide gel electrophoresis, is showed specifically, in different type between parent
Testing result in state's pumpkin resource is shown in Fig. 3.Wherein, the first swimming lane marker, the second swimming lane are male parent PCR knot from left to right
Fruit, third swimming lane is maternal PCR as a result, the 4th swimming lane is F1PCR result.5th to 24 swimming lanes are different musky gourd
Resource material PCR result.Maternal band length 229bp, male parent amplified band length two kinds of banding patterns of 205bp, F1, there are 205bp
With the segment of 229bp.There is banding pattern in the three of female parent, male parent and F1 in different types of musky gourd resource.In conjunction with each material
Phenotype discovery fragrance phenotype it is consistent with PCR result.As it can be seen that above-mentioned InDel label can be by taro fragrance and non-taro aroma characteristics
Single plant separate.
Embodiment 4
Using InDel labeled primer in embodiment 1, it is applied in the improvement breeding material screening of taro aroma characteristics.Utilize this
Primer carries out PCR amplification, is detected using polyacrylamide gel electrophoresis.As a result as shown in Figure 4, wherein the first swimming from left to right
Road marker, the second swimming lane is maternal PCR as a result, third swimming lane is male parent PCR as a result, the 4th to 54 swimming lanes are that breeding changes
Good timber material PCR result.In 51 parts of pumpkin improved materials, maternal and F1 type band is only detected, in conjunction with each improvement material
The phenotype of material illustrates that the material of improvement is divided into and homozygous does not have taro fragrance list with taro aroma characteristics single plant and heterozygosis
Strain.As it can be seen that above-mentioned InDel label can be screened efficiently and accurately and have taro aroma characteristics single plant, improves taro aroma characteristics and change
Good efficiency.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to the above reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Vegetables Inst., Guangdong Academy of Agricultural Sciences
<120>with the InDel molecular labeling of pumpkin taro aroma characteristics close linkage and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cgctttttcc atcgattttg ctgga 25
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttctttacaa ttgcccgatg 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aagaagatgt tccgtaacga 20
Claims (8)
1. a kind of and pumpkin taro aroma characteristics close linkage InDel molecular labeling is in the pumpkin that screening has taro aroma characteristics
Application, which is characterized in that the InDel Molecular mapping is described on the 10th article of chromosome of musky gourd genome
The nucleotide sequence of InDel molecular labeling is as shown in SEQ ID NO.1.
2. a kind of method that screening has the pumpkin of taro aroma characteristics characterized by comprising detect described in claim 1
InDel molecular labeling of the nucleotide sequence as shown in SEQ ID NO.1 with pumpkin taro aroma characteristics close linkage.
3. the method that screening according to claim 2 has the pumpkin of taro aroma characteristics, which is characterized in that described to be detected as
PCR method.
4. the method that screening according to claim 3 has the pumpkin of taro aroma characteristics, which is characterized in that the PCR method
Used in detect InDel molecular labeling described in claim 1 primer sequence such as SEQ ID NO.2 and SEQ ID
Shown in NO.3.
5. the method that screening according to claim 4 has the pumpkin of taro aroma characteristics, which is characterized in that including following step
It is rapid:
(1) DNA of pumpkin is extracted；
(2) DNA for obtaining pumpkin is extracted as template, using nucleotide sequence such as SEQ ID NO.2 and SEQ ID using step (1)
Primer shown in NO.3 carries out PCR amplification；
(3) detected through gel electrophoresis: if only containing 229bp band in amplified production, pumpkin to be measured is with taro aroma characteristics
Pumpkin.
6. having the method for the pumpkin of taro aroma characteristics according to the described in any item screenings of claim 3 ~ 5, which is characterized in that institute
The amplification system for stating PCR method includes: Taq enzyme, template DNA, dNTP, nucleotide sequence such as SEQ ID NO.2 and SEQ ID
Primer shown in NO.3, PCR buffer.
7. the method that screening according to claim 6 has the pumpkin of taro aroma characteristics, which is characterized in that the PCR method
Amplification system include: 1U Taq enzyme, 1 μ L template DNA, the dNTP of 1.5 μ L, 1 μ L primer, 10 × PCR buffer of 2 μ L add
ddH2O to 20 μ L.
8. the kit that a kind of screening has the pumpkin of taro aroma characteristics, which is characterized in that include for detecting nucleotides sequence
Arrange the reagent of the InDel molecular labeling as shown in SEQ ID NO.1；It is described to be used to detect nucleotide sequence such as SEQ ID NO.1
Shown in the reagent of InDel molecular labeling include: nucleotide sequence primer as shown in SEQ ID NO.2 and SEQ ID NO.3.
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| CN110760611A (en) * | 2019-12-09 | 2020-02-07 | 武汉市农业科学院 | Molecular marker closely linked with taro meat fiber color and application thereof in taro breeding |
| CN116574828B (en) * | 2023-03-14 | 2023-11-28 | 广东省农业科学院蔬菜研究所 | KASP molecular marker linked with pumpkin soluble solid content major QTL and application thereof |
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