CN114107545B - CAPS molecular marker of wax gourd fruit and flour wax powder gene and application thereof - Google Patents
CAPS molecular marker of wax gourd fruit and flour wax powder gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to a wax gourd fruit wheat flour gene CAPS molecular marker and application thereof, wherein the nucleotide sequence of the wax gourd fruit wheat flour gene CAPS molecular marker disclosed by the invention is shown as SEQ ID NO.1, the 1370 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP (single nucleotide polymorphism) site, and the base is G or A. Meanwhile, the CAPS molecular marker primer can simultaneously generate a specific marker of the fruit surface wax powder material and a specific marker of the fruit surface wax powder-free material, and has good repeatability and strong specificity. The wax gourd fruit flour wax powder gene CAPS molecular marker developed by the invention is used for identifying the fruit flour wax powder phenotype of wax gourd, especially for quickly identifying the fruit flour wax powder phenotype of wax gourd in seedling stage, has the advantages of accuracy, rapidness, low cost, short identification period, simple and convenient operation and the like, and can assist in breeding new varieties of wax gourd.
Description
Technical Field
The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to a CAPS molecular marker of wax gourd fruit flour wax powder genes and application thereof.
Background
Wax gourd (Benincasa hispida cogn.) is an annual herbaceous plant of the genus wax gourd of the family cucurbitaceae, native to south China and india, and cultivated all over the country. The white gourd is crisp and succulent, has rich nutrient components, is storage and transportation resistant, has strong heat resistance, is a good raw material suitable for modern agricultural product processing, also has the effects of diuresis, heat clearing, phlegm reduction, thirst quenching and the like, and has wide application in the field of medicine.
According to the existence of the aged white gourd fruit and flour wax powder, the white gourd can be divided into two phenotypes of white gourd with sheet jelly and green tangerine peel. The preference of different areas for wax gourd types is different, wherein the southern China is mainly green-peel wax gourd, and the provinces of Sichuan, jiangxi, hunan and the like are mainly pink-peel wax gourd. In addition, the wax gourd fruit flour wax powder plays an important role in the aspects of fruit storage resistance, disease resistance and the like. Therefore, the wax powder of the white gourd fruit and the flour is one of important characters in the white gourd breeding process.
However, since the wheat flour is an appearance character which is shown only in the mature period of the wax gourd fruit, in the traditional breeding process, the targeted breeding can be carried out only in the mature period of the fruit, and the breeding method is time-consuming and labor-consuming and has low efficiency. Therefore, there is a need to develop molecular markers closely linked with wax gourd wax powder characters so as to facilitate large-scale screening directly in the seedling raising period, thereby reducing breeding population and improving breeding efficiency.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a wax gourd fruit wheat flour gene CAPS molecular marker which can be used for identifying the wax gourd fruit wheat flour phenotype.
The second purpose of the invention is to provide a primer for amplifying the CAPS molecular marker, wherein the primer can generate a wax powder material specific marker and a wax powder-free material specific marker (co-dominant marker), and has good repeatability and strong specificity.
The third purpose of the invention is to provide the CAPS molecular marker or the application of the CAPS molecular marker primer in wax gourd fruit flour wax powder phenotype breeding.
The fourth purpose of the invention is to provide a method for identifying the existence of wax powder of white gourd fruit. The method has the advantages of accuracy, rapidness, low cost, short identification period, simple operation and the like.
The first object of the present invention is achieved by the following technical solutions:
a CAPS molecular marker of wax gourd fruit flour powder genes is disclosed, wherein the nucleotide sequence of the CAPS molecular marker is shown as SEQ ID NO.1, the 1370 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP locus, and the SNP locus is G or A.
The second object of the present invention is achieved by the following technical solutions:
and the primers are used for amplifying the CAPS molecular marker and comprise an upstream primer and a downstream primer.
Preferably, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
The third object of the present invention is achieved by the following technical solutions:
the CAPS molecular marker or the CAPS molecular marker primer is applied to wax gourd fruit flour wax powder phenotypic breeding.
The fourth object of the present invention is achieved by the following means:
a method for identifying the existence of wax powder on wax gourd fruit surface comprises the following steps:
s1, performing PCR amplification by using wax gourd genomic DNA as a template and using the CAPS molecular marker primer of claim 2 or 3, performing enzyme digestion on a PCR amplification product, and performing electrophoretic analysis:
s2, when only 140bp and 305bp of WAX powder-free fruit surface parent specific marker WAX is generated 140+305 When the wax gourd is a wax gourd with fruit surface without wax powder; when only 445bp of fruit surface is generated, the WAX powder parent specific marker WAX is arranged 445 In practice, the wax gourd is wax gourd with wax powder on fruit surface; when 445bp fruit surface WAX powder parent specific marker WAX is generated simultaneously 445 And 140bp and 305bp of WAX-free powder seed parent specificity marker WAX 140+305 In practice, the wax gourd is wax gourd with wax powder on its fruit surface.
By analyzing the electrophoresis result, the primer for amplifying the CAPS molecular marker can generate a 445bp fruit surface WAX powder parent P131 specific marker WAX 445 And 140bp and 305bp WAX powder-free parent W3 specific marker WAX 140+305 . Since the marker is a co-dominant marker, the single plant with the double parental specificity bands is a heterozygote.
Therefore, when the CAPS molecular marker primer pair only generates 445bp fruit surface waxy powder parent specific marker WAX 445 In practice, the wax gourd is wax gourd with wax powder on fruit surface; when the molecular marker primer pair can simultaneously generate 445bp fruit surface WAX powder parent specific marker WAX 445 And 140bp and 305bp fruit surface WAX powder-free parent specific marker WAX 140+305 When the wax gourd is a wax gourd with wax powder on the fruit surface; when the molecular marker primer pair only generates 140bp and 305bp unilateral seed parent specificity marker WAX 140+305 In practice, the wax gourd is a wax gourd with fruit surface without wax powder.
Preferably, in the case of PCR amplification, the PCR reaction system comprises 1. Mu.L of genomic DNA and 2. Mu.L of Mg-containing DNA 2+ 10 XPCR buffer, 1.5. Mu.L dNTPs, 1. Mu.L upstream primer, 1. Mu.L downstream primer, and 1U Taq enzyme of (1) and ddH is added 2 O to 20. Mu.L.
Further, the PCR reaction system contained 1. Mu.L of 200 ng. Mu.L -1 2. Mu.L of the genomic DNA containing Mg 2+ 10 XPCRBuffer, 1.5. Mu.L of dNTPs with the concentration of 2.5mM, 1. Mu.L of upstream primer WAXS4-CAPS1-F (SEQ ID NO. 2) with the concentration of 10mM, 1. Mu.L of downstream primer WAXS4-CAPS1-R (SEQ ID NO. 3) with the concentration of 10mM, 1U of Taq enzyme, and ddH 2 O to 20. Mu.L.
Preferably, in the case of PCR amplification, the amplification procedure is: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min, after 35 cycles, final extension at 72 deg.C for 7min, and storage at 4 deg.C.
Preferably, the enzyme is cleaved with the restriction enzyme Ava II.
Preferably, the enzyme digestion system comprises 10. Mu.L of PCR amplification product, 1. Mu.L of 10 XBuffer, 1UAva II restriction enzyme, and ddH 2 O to 20 μ L; the digestion conditions were 37 ℃ for 3 hours.
Preferably, the wax gourd genomic DNA is the wax gourd leaf genomic DNA.
Preferably, the electrophoresis is performed by using agarose gel, and the agarose gel is 3% by mass.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a CAPS molecular marker of wax gourd fruit surface wax powder gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 1370 th base from the 5' end of the sequence shown as SEQ ID NO.1 is SNP locus, and the base is G or A. Meanwhile, the invention also discloses a primer for amplifying the CAPS molecular marker, the CAPS molecular marker primer can simultaneously generate a specific marker of a fruit surface wax powder material and a specific marker of a fruit surface wax powder-free material, and the primer has good repeatability and strong specificity. In addition, the wax gourd fruit and flour wax powder gene CAPS molecular marker developed by the invention is used for identifying the wax gourd fruit and flour wax powder phenotype, especially for quickly identifying the wax gourd fruit and flour wax powder phenotype in the seedling stage, has the advantages of accuracy, rapidness, low cost, short identification period, simplicity and convenience in operation and the like, can assist in breeding new wax gourd varieties, and has wide application prospects.
Drawings
FIG. 1 is an agarose gel electrophoresis pattern of a PCR amplification product of a wax gourd fruit surface wax powder gene CAPS marker after enzyme digestion (M: 100bp ladder, P131: a fruit surface wax powder parent, W3: a fruit surface wax powder-free parent, F1: a first filial generation of P131 and W3;
FIG. 2 shows the amplification result of the wax gourd fruit powder gene CASP molecular Marker in the F2 family single plant constructed by 88 th P131 and W3 (M is DNA Marker, lane 1 is P131, lane 2 is W3, lane 3 is F1, and lanes 4-99 are 88F 2 single plants);
FIG. 3 shows the amplification results of molecular markers of wax gourd fruit flour wax powder gene CASP in different ecotype wax gourd resource germplasms (M is DNA Marker, lanes 1-30 are different ecotype wax gourd single plants).
Detailed Description
The following further describes embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, and is not intended to limit the present invention. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 development and amplification of molecular marker for wax gourd fruit powder Gene CASP
The development and verification processes of the CASP molecular marker of the wax gourd fruit flour wax powder gene and the primer pair for amplifying the CAPS molecular marker provided by the embodiment are as follows:
(1) Materials (provided by vegetable research institute of agricultural academy of sciences, guangdong province): the parent source is as follows: p131: the fruit surface has wax powder parent, W3: the fruit surface has no wax powder parent.
(2) The test method comprises the following steps: a6-generation segregating group is constructed by taking wax gourd P131 with wax powder on fruit surface and wax gourd W3 without wax powder on fruit surface as parents, the genetic rule of wax gourd seed types is researched, and the result shows that the wax powder on the fruit surface of the wax gourd and the wax powder without wax powder are quality characters controlled by a pair of nuclear genes, and the wax powder is dominant. Subsequently, using F 2 The gene of wax gourd fruit flour powder is positioned in the physical interval range of wax gourd chromosome 9, 46,164,000-46,668,969 by combining extreme character mixed pool re-sequencing (BSA-seq) and a genetic mapping-based gene positioning method.
Comprehensively comparing the positioning intervals to obtain a candidate gene WAXS4 gene, and naming the candidate gene WAXS4 gene as BhWAXS4. By comparing the sequences of BhYABBY4 in the genomes of the fruit surface wax powder material and the fruit surface wax powder-free material, the change of 1 SNP (single nucleotide polymorphism) between the two is found. The nucleotide sequence of the BhWAXS4 gene (SEQ ID NO. 1) is as follows:
TTATGATGAAGGTCTTTCGTTCTCTCCACATCAAATAGTTAAACAGATTCAATAATAATGTTAGGATAAGGTCGAAGGTTCAAAGTTCGACATACAAAAAATAAGGTCGACAAAATTCAATCTACTTAAAAAAAAAACAACAATATTTTTACAAATAATTTGAAAACAACAATATTTTTCTAAATAATTCCTAAAAGAACAATATCCTTTTAATTTTCTCTTTTAAAACTGTATAAAAAAAATCCATAAAATGGACTTTTTTCTTAACAGTTTGTGTGGGATGTCGAAATTTCAACCTGATTTACTTATCCATAGAAATCAACATTATTTCATAAAAGAACATTATAATATAATATTTTAATGGTTTCAAAGATGTTCATCTCCTTCATCTTCCCCGCCCTCTCACATTAAATATTCAGACACCAAAATCTCAACTAAAATGGCGGTTCACCATTCAACACTCGCACCAAACTCCGCCGCACCCACCGCCGCCGGAATCCAAATGGACGGCGGCGAATTCCACAGACTCGCCAAGGTATGGACAATCGCCATTGCATTGATTTCTTACTCATACTATATTGCTTCCAAAATTCATAAAGGACTCCCAAGGCTCATCGCTCTCTTCCCCGTTATCTTCATCTTCCTCCTCCTTCCTCTCAATCTCCATTCCTTCCATCTCTGCGGTCCGACTGCCTTCTTGCTCGCTTGGCTCGGCAATTTCAAACTCCTCCTCTTTGCCTTCGATCTCGGTCCTCTGTTCTCATCGCCGCTCCCTAGCCTTCTCCACTTCGCTACCATGCTCTGCCTTCCGATCAAGATCAACAATCAGAAATCCGAGATCCGGAGAACTGGAAATCAGATCGTTCTTTCTTTCAAGGTTTTGCTCCTGGCGATTGTCATTCGGCTCTACGATTATCGAAATCGGATGGATCCGGCGATCGCTTCCGTTCTGTATTGCCTCCATTTATACTTCGGCATCGAAATCGTCCTCGCTCTGTGTTCTCTTCCGGCAAAGGCCATCTTCGGAGCTCCGATCGAACCGCAGTTCGACGAACCCTATCTCTCCACTTCGCTTCAGGACTTCTGGGGGCATCGATGGAATCTCATGGTTCCGAGTATTCTACGCCCGGCCGTGTACAATCCCACCCGTTTCATAACCTCCCGTGTTTTTGGGCCTAGGTGGGCCCAGTTTCTCGCAATGGTAGCTACGTTTGCTGTCTCCGGCTTTATGCACGAGCTTATCTTTTATTACTTCACACGTGCCGCTCCCATGTGGGAGGTCACGTGGTTCTTTGTCCTACATGGCTTCTGCACGGCCGCGGAGGTGGCCGCCAAAGCTGCGGTGGCTAGCCGGAAGGTACGGTGGAAGGTCCACCGGATGGTGTCTGGGCCTATGACTTTGGTGTTTCTAATGGCGACGGCACGGTGGCTGATGTTCCCTCAGTTAATTCGAAATGGTGTTGTTGACCGGACGATTGGTGAGTATTATACTATGGCCCATTTCGTGAAGGGAAAACTTATTTAGTTAGTTGAACCTTGAAAGTAAGGAGTGAACTTGTGATGAAAAATTAGAGTGTCGTGAGATGTATAGTCTAAGGAGTTAAAATGGATGTTTTTTAGTCGTGACCAAACATCCTCTTCATGATTAAAAAAAATGTTTGATGTTTTTTTTTTGTTTTTTTTTTTAATGTGATCCATGTAATTTAGACCTTAGTTACTTTTTCTCAATAAACTATAGTTTGATCTTCTACTCACGATAATTGAATTCAGATTAAGAAGAAAACACGATTTAAATGGA
the SNP is located at 1370bp of a BhWAXS4 gene, the wax powder parent on the fruit surface is adenine nucleotide (A) (shown as a sequence table SEQ ID NO.1, except A at the 1370bp position), and the wax powder-free parent on the fruit surface is guanine nucleotide (G) (shown as a sequence table SEQ ID NO. 1).
A pair of specific primers is designed on both sides of the SNP locus, and comprises an upstream primer WAXS4-CAPS1-F and a downstream primer WAXS4-CAPS1-R.
The specific nucleotide sequences of the upstream primer and the downstream primer are respectively as follows:
WAXS4-CAPS1-F:5'-CCACTTCGCTTCAGGACTTC' (shown in SEQ ID NO. 2);
WAXS4-CAPS1-R:5'-TTCACGAAATGGGCCATAGT' (as shown in SEQ ID No. 3).
(3) Test verification: according to the earlier stage research results of WAX gourd fruit surface WAX powder gene positioning, candidate gene screening and the like, the pair of specific CAPS markers is used for amplifying and enzyme-cutting a fruit surface WAX powder parent P131 and a fruit surface WAX powder-free parent W3, and the CAPS markers can generate 445bp fruit surface WAX powder parent P131 specific markers WAX 445 And 140bp and 305bp fruit surface waxless powder parent W3 specific marker WAX 140+305 The result shows that the mark band has clear shape and good repeatability.
The specific process of the verification is as follows: PCR amplification was performed using the above CAPS molecular marker primer using wax gourd genomic DNA as a template, and the amplification product was subjected to Ava II enzyme digestion, followed by agarose gel electrophoresis of the enzyme digestion product, and the results are shown in FIG. 1. Analysis of the results in FIG. 1 revealed that this marker produced a 445bp specific marker WAX for the fruit surface waxy powder parent P131 445 And 140bp and 305bp fruit surface WAX powder-free parent W3 specific marker WAX 140+305 。
Example 2 identification method of wax gourd fruit and flour wax powder
The method for identifying wax powder of wax gourd fruit surface provided by the embodiment comprises the following steps:
(1) Extraction of DNA of white gourd
The experimental material is fresh leaves of P131, W3 and filial generation F1 of the P131 and the W3, and the steps of extracting genome DNA are as follows:
(1) placing a small amount of fresh leaves into a 2mL centrifuge tube, adding 5mm steel balls and 700uL 2% CTAB extracting solution, placing the mixture into a tissue disruptor to grind a sample, uniformly mixing, and performing water bath at 65 ℃ for 30min;
(2) after standing to room temperature, 700 μ L of chloroform: isoamyl alcohol (24;
(3) adding 500uL of precooled isopropanol, gently mixing uniformly, and standing at-20 ℃ for 30min;
(4) centrifuging at 12000rmp for 10min, and discarding the supernatant;
(5) adding 700uL of precooled 75% ethanol into a centrifugal tube, washing DNA precipitate for 2 times, and drying on an ultraclean workbench;
(6) add 50. Mu.L of ddH 2 And dissolving the DNA precipitate by using O, and extracting to obtain the wax gourd genome DNA for later use.
(2) PCR amplification was performed using the CAPS molecular marker primers designed in example 1, using the wax gourd genomic DNA as a template. The PCR reaction system comprises: the concentration of 1 μ L is 200ng μ L -1 2. Mu.L of the genomic DNA of (1), containing Mg 2+ 10 XPCR buffer, 1.5. Mu.L dNTPs with the concentration of 2.5mM, 1. Mu.L upstream primer WAXS4-CAPS1-F with the concentration of 10mM, 1. Mu.L downstream primer WAXS4-CAPS1-R with the concentration of 10mM, 1U Taq enzyme, and ddH addition 2 O to 20. Mu.L. The amplification procedure was: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min,35 cycles, final extension at 72 deg.C for 7min, and storage at 4 deg.C.
(3) The PCR amplification product was digested by digestion, 10. Mu.L of LPCR product, 1. Mu.L of 10 XBuffer, 1UAvaII restriction enzyme, and ddH was added 2 O to 20 μ L; the enzyme was cleaved at 37 ℃ for 3 hours.
(4) Carrying out agarose gel electrophoresis detection on the enzyme digestion product
Adding the enzyme digestion product into 4 mu L of 6 multiplied Loading buffer, throwing off, and then, uniformly mixing in a vortex manner; 10 mu L of the mixed solution is added with 3 percent (mass percent) of GelStain agarose gel for electrophoresis, and the mixture is subjected to 120V stable pressure electrophoresis for 30min and then is subjected to photographic analysis on a gel imager.
(5) Amplification results
The CAPS molecular marker primer pair generates 455bp bands in the parent P131 with wax powder on fruit surface, generates 140bp and 305bp bands in the parent W3 without wax powder on fruit surface, and F1 can simultaneously generate 445bp parent bands with wax powder on fruit surface and 140bp and 305bp parent bands without wax powder on fruit surface (see figure 1).
Example 3 method for identifying wax gourd Single plants
The 88 individuals (Nos. 3 to 91) of the F2 family groups of P131 and W3 were verified by the method of example 2. Numbering each individual plant respectively, and extracting DNA of the individual plants for detection; only the individual plants producing 140bp and 305bp bands were wax powder-free, the individual plants producing 455bp bands were wax powder-coated, and the individual plants producing 445bp, 140bp and 305bp bands were wax powder-coated.
The marking detection result shows that 27 single plants with 445bp band patterns (the fruit surface is homozygous with wax powder), 15 single plants with 140bp and 305bp band patterns (the fruit surface is homozygous without wax powder), and 46 single plants with heterozygous band patterns (the fruit surface is heterozygous with wax powder) (see figure 2).
The field character identification result is highly consistent with the mark detection result, and the accuracy rate reaches 97.44%.
Example 4 screening of wax gourd germplasm resources for the presence or absence of wax powder varieties
30 wax gourd germplasm resources (provided by vegetable research institute of academy of agricultural sciences, guangdong province) with different ecotypes were verified by the method of example 2 (Table 1). Mixing and sampling each resource, and extracting DNA for detection; as a result of detection, 10 strains with 445bp band patterns (homozygous with wax powder on fruit surface), 18 strains with 140bp and 305bp band patterns (homozygous without wax powder on fruit surface) and 2 heterozygous band patterns (heterozygous with wax powder on fruit surface) are found (see figure 3).
The field character identification result is highly consistent with the mark detection result, and the accuracy rate reaches 90 percent.
TABLE 1 wax gourd resource information of different ecotypes
The embodiment is integrated, so that the CASP molecular marker developed by the invention can effectively distinguish wax powder materials on the surfaces of white gourd fruits from wax powder-free materials on the surfaces of the white gourd fruits, and the wax powder phenotype of the white gourd fruits can be accurately and quickly detected in the seedling stage.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> vegetable institute of academy of agricultural sciences, guangdong province
<120> CAPS molecular marker of wax gourd fruit surface wax powder gene and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1798
<212> DNA/RNA
<213> BhWAXS4(Benincasa hispida Cogn.)
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atgaaaaatt agagtgtcgt gagatgtata gtctaaggag ttaaaatgga tgttttttag 1620
tcgtgaccaa acatcctctt catgattaaa aaaaatgttt gatgtttttt ttttgttttt 1680
ttttttaatg tgatccatgt aatttagacc ttagttactt tttctcaata aactatagtt 1740
tgatcttcta ctcacgataa ttgaattcag attaagaaga aaacacgatt taaatgga 1798
<210> 2
<211> 20
<212> DNA/RNA
<213> WAXS4-CAPS1-F(Artificial Sequence)
<400> 2
ccacttcgct tcaggacttc 20
<210> 3
<211> 20
<212> DNA/RNA
<213> WAXS4-CAPS1-R(Artificial Sequence)
<400> 3
Claims (6)
1. The application of the reagent for detecting CAPS molecular markers of wax gourd fruit wheat flour genes in wax gourd fruit wheat flour phenotype breeding is characterized in that the reagent comprises a primer for amplifying CAPS molecular markers and a restriction endonuclease Ava II, the nucleotide sequence of an upstream primer of the primer is shown as SEQ ID NO.2, the nucleotide sequence of a downstream primer of the primer is shown as SEQ ID NO.3, the nucleotide sequence of the CAPS molecular markers is shown as SEQ ID NO.1, the 1370 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP site, the SNP site is G or A, the primer is adopted for PCR amplification, the restriction endonuclease Ava II is adopted for carrying out enzyme digestion on PCR products, and when only bands of 140bp and bp are generated, the wax gourd fruit wheat flour is free; when only 445bp strips are generated, wax powder white gourds are coated on fruit surfaces; when 445bp, 140bp and 305bp bands are generated simultaneously, the wax gourd is a fruit with wax powder on the surface.
2. A method for identifying the existence of wax powder on wax gourd fruit surface is characterized by comprising the following steps:
s1, performing PCR amplification by using wax gourd genomic DNA as a template and the primer for amplifying the CAPS molecular marker in claim 1, performing enzyme digestion on a PCR amplification product by using restriction enzyme Ava II, and performing electrophoretic analysis:
s2, when only strips of 140bp and 305bp are produced, the wax gourd is white gourd without wax powder on fruit surface; when only 445bp strips are generated, wax powder white gourds are coated on fruit surfaces; when 445bp, 140bp and 305bp bands are generated simultaneously, the wax powder white gourd is in the fruit surface;
the nucleotide sequence of the CAPS molecular marker is shown as SEQ ID NO.1, the 1370 th base from the 5' end of the sequence shown as the SEQ ID NO.1 is an SNP locus, and the SNP locus is G or A.
3. The method for identifying the presence of wax gourd fruit and flour as claimed in claim 2, wherein the PCR reaction system comprises 1 μ L of genomic DNA and 2 μ L of Mg-containing DNA during PCR amplification 2+ The PCR buffer (10 XPCR buffer), 1.5. Mu.L dNTPs, 1. Mu.L upstream primer, 1. Mu.L downstream primer, and 1U Taq enzyme (1 XPCR buffer) were added with ddH 2 O to 20. Mu.L.
4. The method for identifying the presence of wax gourd fruit facial wax powder as claimed in claim 2, wherein the PCR amplification comprises the following steps: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min, after 35 cycles, final extension at 72 deg.C for 7min, and storage at 4 deg.C.
5. The method for identifying the presence of wax gourd fruit flour as claimed in claim 2, wherein the enzyme digestion system comprises 10 μ L PCR amplification product, 1 μ L10 XBuffer, 1U Ava II restriction enzyme, and ddH 2 O to 20 μ L; the digestion conditions were 37 ℃ for 3 hours.
6. The method of claim 2, wherein the genomic DNA of wax gourd is the genomic DNA of wax gourd leaves.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111461951.5A CN114107545B (en) | 2021-12-02 | 2021-12-02 | CAPS molecular marker of wax gourd fruit and flour wax powder gene and application thereof |
| ZA2022/09639A ZA202209639B (en) | 2021-12-02 | 2022-08-30 | Cleaved amplified polymorphic sequence (caps) molecular marker for gene of epicuticular wax biosynthesis on mature fruit surface of wax gourd and use thereof |
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| CN202111461951.5A CN114107545B (en) | 2021-12-02 | 2021-12-02 | CAPS molecular marker of wax gourd fruit and flour wax powder gene and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107484644A (en) * | 2017-08-09 | 2017-12-19 | 广东日可威食品原料有限公司 | A kind of selenium-rich bean sheet jelly wax gourd implantation methods |
| CN109234433A (en) * | 2018-10-19 | 2019-01-18 | 广东省农业科学院蔬菜研究所 | A kind of SNP marker and its application of wax gourd seed type gene |
| CN112226529A (en) * | 2020-09-09 | 2021-01-15 | 广西壮族自治区农业科学院 | SNP molecular marker of wax gourd blight-resistant gene and application |
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| US11591611B2 (en) * | 2016-07-12 | 2023-02-28 | Nunhems B.V. | TolCNDV resistant melon plants |
| CN109418683A (en) * | 2017-09-01 | 2019-03-05 | 莲创药业股份有限公司 | Wax gourd wax is as the purposes in preservative and antimicrobial |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107484644A (en) * | 2017-08-09 | 2017-12-19 | 广东日可威食品原料有限公司 | A kind of selenium-rich bean sheet jelly wax gourd implantation methods |
| CN109234433A (en) * | 2018-10-19 | 2019-01-18 | 广东省农业科学院蔬菜研究所 | A kind of SNP marker and its application of wax gourd seed type gene |
| CN112226529A (en) * | 2020-09-09 | 2021-01-15 | 广西壮族自治区农业科学院 | SNP molecular marker of wax gourd blight-resistant gene and application |
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| High-density genetic map construction and gene mapping of pericarp color in wax gourd using specific-locus amplified fragment (SLAF) sequencing.;Biao等;《BMC Genomics》;20151231;第16卷;第1-10页 * |
| The wax gourd genomes offer insights into the genetic diversity and ancestral cucurbit karyotype;Dasen Xie等;《NATURE COMMUNICATIONS》;20191231;第10卷(第1期);第1-12页 * |
| 与冬瓜枯萎病抗性连锁的RAMP标记的筛选及其运用;谢大森等;《分子植物育种》;20090928;第7卷(第05期);第928-933页 * |
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| CN114107545A (en) | 2022-03-01 |
| ZA202209639B (en) | 2022-11-30 |
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