CN115058538B - SNP molecular marker closely linked with bitter gourd full female shape and application thereof - Google Patents
SNP molecular marker closely linked with bitter gourd full female shape and application thereof Download PDFInfo
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- 244000078912 Trichosanthes cucumerina Species 0.000 title 1
- 244000302512 Momordica charantia Species 0.000 claims abstract description 50
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Abstract
The invention discloses an SNP molecular marker closely linked with the full female shape of balsam pear and application thereof, wherein the molecular marker is subjected to PCR amplification by designing a primer, and the PCR amplification product is subjected to enzyme digestion treatment by using restriction endonuclease, so that the genotype of a balsam pear sample can be judged, and the sex of the balsam pear sample can be further judged. The SNP molecular marker provided by the invention is tightly linked with the full female state of the balsam pear, the overall fitness of molecular identification and phenotype identification is high, scientific basis and effective guidance can be provided for auxiliary breeding, manual selection is carried out only by means of scientific and technical judgment, and good economic benefit can be expected.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to an SNP molecular marker closely linked with the full female shape of balsam pear and application thereof.
Background
The balsam pear is an important characteristic melon vegetable crop in south China, and has higher economic and social values. The balsam pear plants have various types, such as full female plants, strong female plants, isogynic and androgenic isogynic plants, strong male plants, complete plants, full male plants and the like. Most bitter gourd plants are shown as isoflorid plants, flowers need to be clipped in advance or the male flowers need to be picked manually in the seed production process, time and labor are wasted, the seed purity is affected easily because the male flowers are not picked timely or are missed, parent materials are lost, and the development of the bitter gourd industry is severely restricted. Therefore, the method for producing hybrid seeds by using the full female plant of the balsam pear as a female parent is the most effective method for improving the seed purity and saving labor.
At present, people mainly judge the flowering condition of the balsam pear all-female plant through visual observation in the flowering period, and hysteresis exists. In addition, the sexual differentiation of balsam pear is greatly influenced by the environment such as temperature, illumination and the like, and is easy to cause misjudgment. With the rapid development of biotechnology, molecular marker assisted selection technology has been widely applied in the breeding process, and by developing molecular markers linked with the full female shape of balsam pear and screening plants in the seedling stage, the breeding efficiency is greatly improved, and the breeding process is shortened.
Disclosure of Invention
The invention aims to provide an SNP molecular marker closely linked with the full-female shape of balsam pear and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, SNP molecular markers closely linked to the female shape of momordica charantia are provided, whose sequences are:GC CGTCAATAGGCCGATCAAGATCAACAAAACCGCCAATAGGTCGATCAAGATCCACAAGTTGCCAATAGGCTGATCAAGATCAACAAAACCACCAAGATGCCGATCAAGATCAACAAAACCACCAAGATGCCGATCAAGATCATCAACAAACTGCCAATAGGCTGATCAAGAACCAACAATCTCCCCTAGCCACTCA(SEQ ID NO:1)。
in a second aspect of the invention, a primer pair is provided, wherein the primer pair is used for amplifying the molecular marker of the first aspect of the invention.
In some embodiments of the invention, the sequences of the primer pairs are:
Mc720-F：5’-GCCGTCAATAGGCCGATCAAGATCAACTTA-3’(SEQ ID NO:2)；
Mc720-R：5’-TGAGTGGCTAGGGGAGATTGTTGGT-3’(SEQ ID NO:3)。
in a third aspect of the invention, a kit is provided, which comprises the primer pair of the second aspect of the invention.
In some embodiments of the invention, the kit further comprises a restriction endonuclease.
In some embodiments of the invention, the restriction endonuclease is Mse i.
In a fourth aspect of the invention, there is provided the use of a molecular marker according to the first aspect of the invention in identifying or assisting in identifying the sex of momordica charantia and/or assisting in breeding.
In a fifth aspect of the invention, there is provided the use of a primer pair according to the second aspect of the invention in identifying or assisting in identifying the shape of momordica charantia and/or assisting in breeding.
According to a sixth aspect of the invention, there is provided use of a kit according to the third aspect of the invention in identifying or assisting in identifying a bitter melon shape and/or assisting in breeding bitter melon.
In a seventh aspect of the present invention, there is provided a method for identifying the sex of momordica charantia, comprising detecting the genotype of the SNP molecular marker of the first aspect of the present invention closely linked to the female shape of momordica charantia in the genome of a sample of momordica charantia to be tested.
In some preferred embodiments of the present invention, the PCR amplification of the genomic DNA of the sample of momordica charantia to be tested is performed by using the primers of the second aspect of the present invention or the kit of the third aspect of the present invention.
In some embodiments of the present invention, the method further comprises sequencing or enzymatic electrophoresis of the PCR amplification product.
In some embodiments of the present invention, when performing enzyme digestion electrophoresis on the PCR amplification product, if the PCR product is subjected to enzyme digestion to obtain an enzyme digestion fragment, the bitter gourd to be detected is a hermaphrodite isofloral; and if the PCR amplification product cannot be subjected to enzyme digestion to obtain an enzyme digestion fragment, determining that the bitter gourd sample to be detected is a full female plant.
In some embodiments of the present invention, when sequencing the PCR amplification product, if the 31 st base at the 5' end is a, the bitter gourd to be tested is an isosexual flower of the same male plant, and if it is G, it is a full female plant.
The invention has the beneficial effects that:
the invention provides an SNP molecular marker tightly linked with the full-female shape of balsam pear, which is characterized in that PCR amplification is carried out on the molecular marker by designing a primer, and restriction enzyme is utilized to carry out enzyme digestion treatment on a PCR amplification product, so that the genotype of a balsam pear sample can be judged, and the sex of the balsam pear sample can be further judged.
The SNP molecular marker provided by the invention is tightly linked with the full female of the balsam pear, the overall fitness of molecular identification and phenotype identification is high, scientific basis and effective guidance can be provided for assisted breeding, manual selection is carried out only by means of scientific and technical judgment, and good economic benefit can be expected.
Drawings
FIG. 1: mc720 in female parent B07 (B), male parent A06 (A) and both F 1 Amplification results in generation hybrids.
FIG. 2: f 2 And (3) detecting SNP of partial plants in the population. Wherein, 1-6 are isosexual plants and isosexual plants; 7-12 are all female plants.
FIG. 3: from F 2 And randomly selecting 66 strains from the generation segregation population for carrying out SNP detection, wherein 1-33 are isosexual plants and 34-66 are all female strains.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1
1. Construction of genetic populations
F is constructed by taking the preserved full-female inbred line B07 as a female parent and taking a female and male isofloral inbred line A06 obtained by multi-generation inbreeding of collected local varieties as a male parent 2 And (4) a group. Obtaining F of 313 individuals 2 A population for whole female trait genetic analysis and mapping of the population.
2. Determination of female characters of test material and genetic rule analysis
F1 and F2 genetic segregation populations are constructed by using the all-female parent B07 and the amphoteric parent A06, and the phenotype identification result shows that: the isosexual strain 240 strains and the full female strain 73 strains in the 313F 2 groups, the segregation ratio of the isogynic and gynoecial isogynic plants meets 3. The analysis result shows that the bitter gourd full-female character is controlled by 1 single recessive gene.
3. Based on BSA technology, the balsam pear hologynic gene is finely positioned
The method utilizes a cluster separation analysis method (BSA) to locate the bitter gourd hologynic gene and locate a related area on the bitter gourd scaffold NW _ 019104490.1.
Development and amplification of dCAPs marker.
And downloading nucleotide sequences of 200bp upstream and downstream of the SNP site in the positioning interval, and converting the SNP site into a dCAPS marker by using online analysis software dCAPS Finder 2.0 (http:// helix.wustl.edu/dCAPS/dcaps.html). Utilizing the parents B07, A06 and F 1 Polymorphism screening is carried out on the developed SNP marker, and the marker Mc720 is found to have stable polymorphism between parents and is combined with F 1 As a result of the detection, mc720 is judged to be a codominant marker (FIG. 1), the nucleotide sequence of which is shown as SEQ ID NO.1, the 31 st base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP site, and the base is A or G; if the 31 th base from the 5 'end is A, the plant is a hermaphrodite heterozygote, and if the 31 th base from the 5' end is G, the plant is a full-female plant.
1 sequence of SEQ ID NO:GCCGTCAATAGGCCGATCAAGATCAACAAAACCGCCAATAGGTCGATCAAGATCCACAAGTTGCCAATAGGCTGATCAAGATCAACAAAACCACCAAGATGCCGATCAAGATCAACAAAACCACCAAGATGCCGATCAAGATCATCAACAAACTGCCAATAGGCTGATCAAGAACCAACAATCTCCCCTAGCCACTCA。
the sequences of the PCR amplification primer pairs aiming at the molecular marker Mc720 are shown as follows:
Mc720-F:5’-GCCGTCAATAGGCCGATCAAGATCAACTTA-3’(SEQ ID NO:2)；
Mc720-R:5’-TGAGTGGCTAGGGGAGATTGTTGGT-3’(SEQ ID NO:3)。
example 2
At F 2 Screening 6 plants of the full female plant and the isogynic plant from the generation group for detection, wherein, 1-6 are isosexual plants and isosexual plants; 7-12 are all female plants. Using the genomic DNA as a template, PCR amplification was performed using the PCR amplification primers designed as described in example 1 to obtain PCR amplification products:
PCR System (10. Mu.l):
DNA template: 0.5. Mu.l
Forward direction of the primer: 0.2. Mu.l
And (3) reversing the primers: 0.2. Mu.l
2×Taq PCR StarMix：5μl
ddH 2 0: make up to 10. Mu.l.
PCR amplification procedure:
pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 15s,30 cycles, holding at 72 ℃ for 5min, and storage at 16 ℃.
And (3) carrying out enzyme digestion on the PCR product by using an endonuclease Mse I:
enzyme digestion system:
endonuclease Mse I: 0.2. Mu.l
CutSmart buffer：1μl
Enzyme cutting conditions are as follows: bathing at 37 deg.C for 30min, and inactivating at 65 deg.C for 20min.
When the SNP base site is A, the SNP base site is recognized and cut by Mse I, and the amplified fragment is cut off by a 30bp fragment. When parent A06 or F appears 1 When the banding pattern of the parent B07 appears, the plant appears as a full female plant. The results are shown in FIG. 2, where the banding pattern is consistent with the plant phenotype, which indicates that the molecular markers of example 1 can be used to identify female traits in Momordica charantia plants.
Example 3
And randomly taking 66F 2 generation segregating population strains, wherein 1-33 are isogynic and isogynic strains, 34-66 are all female strains, extracting genome DNA after phenotype identification, and detecting and identifying the molecular marker. PCR amplification was performed by the method of example 2, and digestion was performed with endonuclease. As a result, the detection efficiency was 100% (fig. 3). The SNP molecular marker of the invention can be used for molecular marker assisted breeding.
The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> institute of agriculture and facility agriculture of academy of agricultural sciences of Guangdong province
<120> SNP molecular marker tightly linked with balsam pear full female shape and application thereof
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 198
<212> DNA
<213> Momordica charantia
<400> 1
gccgtcaata ggccgatcaa gatcaacaaa accgccaata ggtcgatcaa gatccacaag 60
ttgccaatag gctgatcaag atcaacaaaa ccaccaagat gccgatcaag atcaacaaaa 120
ccaccaagat gccgatcaag atcatcaaca aactgccaat aggctgatca agaaccaaca 180
atctccccta gccactca 198
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<400> 2
gccgtcaata ggccgatcaa gatcaactta 30
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence
<400> 3
tgagtggcta ggggagattg ttggt 25
Claims (11)
- The SNP molecular marker is an SNP molecular marker closely linked with the whole female shape of the balsam pear, the sequence of the SNP molecular marker is shown as SEQ ID NO.1, the 31 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP locus, and the base is A or G.
- 2. A primer for amplifying the molecular marker of claim 1.
- 3. The primer of claim 2, wherein the sequence of the primer is:Mc720-F:5’-GCCGTCAATAGGCCGATCAAGATCAACTTA-3’(SEQ ID NO:2)；Mc720-R:5’-TGAGTGGCTAGGGGAGATTGTTGGT(SEQ ID NO:3)。
- 4. a kit comprising the primer according to claim 2 or 3.
- 5. The kit according to claim 4, wherein the kit further comprises a restriction endonuclease.
- 6. Use of a primer according to claim 2 or 3 for identifying or assisting in identifying the sex of Momordica charantia and/or for assisting in breeding Momordica charantia.
- 7. Use of a kit according to claim 4 or 5 for the identification or assisted identification of the sex and/or assisted breeding of Momordica charantia.
- 8. A method for identifying the sex of balsam pear, which comprises detecting the genotype of the SNP molecular marker which is closely linked with the female shape of the balsam pear in the genome of a sample of the balsam pear to be detected according to claim 1.
- 9. A method according to claim 8, comprising performing PCR amplification of genomic DNA from a sample of Momordica Charantia to be tested using the primers of claim 2 or 3 or the kit of claim 4 or 5.
- 10. The method of claim 9, further comprising sequencing or enzymatic electrophoresis of the PCR amplification product.
- 11. The method of claim 10,when the PCR amplification product is subjected to enzyme digestion electrophoresis, if the PCR amplification product is subjected to enzyme digestion to obtain an enzyme digestion fragment, the bitter gourd to be detected is a hermaphrodite isofloral strain; if the PCR amplification product cannot be subjected to enzyme digestion to obtain an enzyme digestion fragment, the bitter gourd sample to be detected is a full female plant;when the PCR amplification product is sequenced, if the 31 st basic group at the 5' end is A, the bitter gourd to be detected is a heterosexual isofloral plant, and if the basic group is G, the bitter gourd to be detected is a full-female plant.
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