CN108660246B - InDel molecular markers of Ma (Citrus paradisi) pomelos and application of InDel molecular markers in early-stage differentiation of rough-peel Ma pomelos of citrus varieties - Google Patents
InDel molecular markers of Ma (Citrus paradisi) pomelos and application of InDel molecular markers in early-stage differentiation of rough-peel Ma pomelos of citrus varieties Download PDFInfo
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Abstract
The invention provides a group of Majia pomelo InDel molecular markers and application thereof in early-stage differentiation of rough-skinned Majia pomelos in citrus variety seedlings, belonging to the field of genetic breeding. The Majia shaddock InDel molecular marker comprises a segment obtained by amplifying a primer pair chr2.10-9584F/chr2.10-9584R and a segment obtained by amplifying a primer pair p-chr6.21-8689F/p-chr 6.21-8689R. The molecular marker provided by the invention provides a new molecular tool for identifying, breeding and early screening the Ma jia pomelo seedlings with stable heredity. The InDel molecular marker provided by the invention can be used for distinguishing the rough-skinned Ma pomelos in citrus varieties at the early stage of seedlings, and has important significance for identification of the citrus varieties and risk control in production of the Ma pomelos.
Description
Technical Field
The invention belongs to the field of genetic breeding, and particularly relates to an InDel molecular marker of Ma pomelo and application thereof in early-stage differentiation of rough-skinned Ma pomelo in citrus variety seedlings.
Background
Pomelos (Citrus grandis) are Citrus fruits of the Rutaceae family, and have a long history of cultivation in China. Pomelos are lucky fruits in traditional culture in China and are commonly used for wedding, sacrifice and other activities. Residents in south China prefer to plant the pomelos in front of and behind the house, and in most cases, abundant local varieties and strains are formed in the long-term cultivation process through seedling propagation. However, at present, the genetic background and the source of the pomelo are not clear, and there are cases of synonyms or homonyms. The Guangfeng county and the surrounding areas of Jiangxi province have the tradition of planting pomelos, and the households of the family have the planting in front of and behind the house. In 1991, Majia pomelos are discovered in general investigation of pomelo resources in Jiangxi province, have the characteristics of large fruit size, red pulp, high juice yield, sour and sweet palatability and the like, and are well favored by wide consumers. The fructus Citri Grandis pulp is rich in lycopene, carotenoid, mineral elements, etc.
However, with the development of the grapefruit industry and the intensive research and study, it is found that grapefruit has very different morphological characters and is not stable, moreover, people divide grapefruit into two categories, namely coarse peel (high acid type) and fine peel (moderate acidity) in production, fine peel is superior to coarse peel in flavor and taste, but is difficult to distinguish morphologically in seedling stage, particularly in the seedling breeding process of grapefruit, the types of coarse peel and fine peel are often easily mixed, and a seedling factory has no way to distinguish, thus bringing risks to the grapefruit industry. The Ma family pomelos generally begin to bear fruit after 4-5 years, if the seedlings are found to be impure after 5 years, great loss is caused, and the problem is brought to orchard management.
Disclosure of Invention
In view of the technical problems, the invention provides a group of Ma Jia shaddock InDel molecular markers and application thereof in early-stage differentiation of rough-skinned Ma shaddock seedlings of citrus varieties.
The invention provides a group of InDel molecular markers for Ma Piezou, which comprises a segment obtained by amplifying genomic DNA of Ma Piezou by a primer pair chr2.10-9584F/chr2.10-9584R and a segment obtained by amplifying genomic DNA of Ma Piezou by a primer pair p-chr6.21-8689F/p-chr 6.21-8689R;
the nucleotide sequences 5 '-3' of the primer pair chr2.10-9584F/chr2.10-9584R are respectively:
chr2.10-9584F：AGTGAAAGGGAGAGAAAGAAGC；
chr2.10-9584R：ATGGTGCCACATTGCGAGT；
the nucleotide sequences 5 '-3' of the primer pair p-chr6.21-8689F/p-chr6.21-8689R are respectively:
p-chr6.21-8689F：GGGAGGGAGCTTGCTTGATT；
p-chr6.21-8689R：ACCCGATAAAGTCACGTACACA。
the invention provides a kit for distinguishing Ma shaddock peel, which comprises a primer pair chr2.10-9584F/chr2.10-9584R and a primer pair p-chr6.21-8689F/p-chr 6.21-8689R.
The invention also provides application of the molecular marker or the kit in distinguishing the Majia pomelo with other varieties of oranges, wherein the other varieties of oranges comprise any one or more of the Majia pomelo with thin peel, the MiYou xi pomelo, the Shatian pomelo or the local county in Guangfeng province in Jiangxi province.
Preferably, the application comprises the following steps:
(1) extracting the genome DNA of the material to be detected;
(2) carrying out PCR amplification on the genomic DNA of the material to be detected by using a primer pair chr2.10-9584F/chr2.10-9584R and a primer pair p-chr6.21-8689F/p-chr6.21-8689R respectively to obtain two groups of amplification products;
(3) performing agarose gel electrophoresis on the two groups of amplification products respectively to obtain an electrophoresis picture;
(4) judging according to the electrophoresis chart: and if the electrophoresis image of the amplification product of the primer pair chr2.10-9584F/chr2.10-9584R has a bright band, and the electrophoresis image of the amplification product of the primer pair p-chr6.21-8689F/p-chr6.21-8689R has two bright bands, judging that the material to be detected is the Ma pomelo.
Preferably, the reaction system for PCR amplification is: 7 μ L of Mix solution, 0.3 μ L of 10mmol/L forward primer, 0.3 μ L of 10mmol/L reverse primer, 1.5 μ L of 100ng/ul template DNA and 6 μ L of ddH2O。
Preferably, the PCR amplification step is: 95 ℃ for 5 min; 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; 72 ℃ for 10 min.
Preferably, the agarose gel electrophoresis is performed by 2.0-4.0% of agarose gel by mass percent, the voltage is 100-150V, and the electrophoresis is performed for 10-30 min.
Has the advantages that:
the invention provides a group of Ma Piao ethopanaeus InDel molecular markers, which comprise a segment obtained by amplifying genomic DNA of Ma Piao ethopanaeus by using a primer pair chr2.10-9584F/chr2.10-9584R and a segment obtained by amplifying genomic DNA of Ma Piao ethopanaeus by using a primer pair p-chr6.21-8689F/p-chr 6.21-8689R. The primer pair chr2.10-9584F/chr2.10-9584R is subjected to PCR amplification by using a raw material of Ma-jia pomelo or Mi-xi-GUAN pomelo as a template, and an amplification product is subjected to agarose gel electrophoresis to obtain a bright band; the primer pair chr2.10-9584F/chr2.10-9584R is subjected to PCR amplification by taking other citrus variety materials as templates, and two bright bands can be obtained by performing agarose gel electrophoresis on an amplification product; the primer pair chr2.10-9584F/chr2.10-9584R can be used for distinguishing rough-skinned Ma Ji pomelos and fine-skinned Ma Ji pomelos in the Ma Ji pomelos variety, and also can distinguish the rough-skinned Ma pomelos from other citrus varieties except for Mixi GU. The primer pair p-chr6.21-8689F/p-chr6.21-8689R is subjected to PCR amplification by taking a rough pomelo material as a template, and an amplification product is subjected to agarose gel electrophoresis to obtain two bright bands; the primer pair p-chr6.21-8689F/p-chr6.21-8689R is subjected to PCR amplification by using a Mixi GUAN material as a template, and an amplification product is subjected to agarose gel electrophoresis to obtain a bright band. The primers p-chr6.21-8689F/p-chr6.21-8689R can be used to distinguish between Ma-Guzu and MiYou-Mixi GUAN.
The primer pair chr2.10-9584F/chr2.10-9584R and the primer pair p-chr6.21-8689F/p-chr6.21-8689R are used in combination, so that the Ma-mei pomelo and other citrus varieties can be distinguished. The molecular marker is used for distinguishing the Majia pomelo superior line and the large-volume cultivated pomelo, so that the defect of selection depending on phenotype in the traditional breeding can be overcome, the breeding workload is reduced, the process of cultivating the pure superior line is accelerated, and the seedling guarantee is provided for the industrial development.
The InDel molecular marker for Ma pomelo provided by the invention can accurately screen out Ma pomelo peel from varieties with complex genetic background, can assist variety selection in any growing period, is not influenced by environmental conditions, can eliminate interference caused by interaction of non-allelic genes, and has the advantages of rapidness, economy, efficiency, strong accuracy and the like; particularly, when the varieties of citrus varieties are difficult to distinguish through characters in the early stage of seedling, the Majia pomelo InDel molecular marker provided by the invention can assist in distinguishing the varieties of the Majia pomelos.
Drawings
FIG. 1 is an electrophoretogram showing the results of measurement according to example 1 of the present invention, wherein 1 represents a rough Majia shaddock, 2 represents a fine Majia shaddock (Yougai), 3 represents a local native shaddock in Guangfeng county, Jiangxi province, 4 represents a Mixi GUAN shaddock, 5 represents a Shatian shaddock, and M represents a Marker;
FIG. 2 is an electrophoretogram showing the results of measurement according to example 2 of the present invention, wherein 1 represents Majia rough grapefruit, 2 represents Mixi GUAN grapefruit, and M represents Marker;
FIG. 3 is a graph showing electrophoresis results of measurement according to the protocol described in example 3 of the present invention, wherein C represents Ma Piao ethopanaeus, X represents Ma Piao ethopanaeus, and 1-80 represent randomly selected seedling numbers in a nursery;
FIG. 4 shows the results of electrophoresis of the results of the protocol of example 4 of the present invention, wherein C represents Majia rough skin pomelo, G represents Mixi GUAN, and 1-38 represent randomly selected seedling numbers in the nursery.
Detailed Description
The invention provides a group of InDel molecular markers of Ma Piao ethopanam maxim, which comprise a segment obtained by amplifying genomic DNA of the Ma Piao ethopam maxim by using a primer pair chr2.10-9584F/chr2.10-9584R and a segment obtained by amplifying genomic DNA of the Ma Piao ethopam maxim by using a primer pair p-chr6.21-8689F/p-chr 6.21-8689R.
In the invention, the primer pair for amplifying the InDel marker is designed by the following method: (1) sequencing the DNA of the Ma Ji pomelo by using a double-end Sequencing method (Pair-end Sequencing); (2) the resulting insertion/deletion site (InDel) information was verified using SamTools and GATK software; (3) designing primers on two sides of the InDel locus by using Primer Premier 5.0 software, wherein the length of the primers is more than 20bp, and the Tm is 50-60 ℃; the GC content is 50 to 60 percent; the size of the PCR product is between 150bp and 500 bp. The primer length is optimally selected to be 25 bp. The Tm is optimally 55 ℃.
In the invention, the nucleotide sequences 5 '-3' of the primer pair chr2.10-9584F/chr2.10-9584R are respectively:
chr2.10-9584F：agtgaaagggagagaaagaagc(SEQ ID NO:1)；
chr2.10-9584R：atggtgccacattgcgagt(SEQ ID NO:2)。
in the invention, when the primer pair chr2.10-9584F/chr2.10-9584R is subjected to PCR amplification by using raw material of Ma Guaji shaddock or Mixi Guo shaddock as a template, an amplification product can obtain a bright band by agarose gel electrophoresis; when the primer pair chr2.10-9584F/chr2.10-9584R is used for PCR amplification by taking other citrus variety materials as templates, two bright bands can be obtained by agarose gel electrophoresis of an amplification product. The primer pair chr2.10-9584F/chr2.10-9584R can be used for distinguishing rough-skinned Ma Ji pomelos and fine-skinned Ma Ji pomelos in the Ma Ji pomelos variety, and also can distinguish the rough-skinned Ma pomelos from other citrus varieties except for Mixi GU.
In the invention, the nucleotide sequences 5 '-3' of the primer pair p-chr6.21-8689F/p-chr6.21-8689R are respectively:
chr6.21-8689F：gggagggagcttgcttgatt(SEQ ID NO:3)；
chr6.21-8689R：acccgataaagtcacgtacaca(SEQ ID NO:4)。
in the invention, when the primer pair p-chr6.21-8689F/p-chr6.21-8689R is subjected to PCR amplification by taking a raw grapefruit peel material as a template, an amplification product can obtain two bright bands through agarose gel electrophoresis; when the primer pair p-chr6.21-8689F/p-chr6.21-8689R is subjected to PCR amplification by using a material of the MiYou xi GUAN or the Fine Ma Jia GUAN as a template, an amplification product can obtain a bright band through agarose gel electrophoresis. The primers p-chr6.21-8689F/p-chr6.21-8689R can be used to distinguish between Ma-Guzu and MiYou-Mixi GUAN.
In the present invention, the sources of the two primer pairs are not limited at all, and the sequence synthesis company may be entrusted with. In the present embodiment, the two sets of primers were synthesized by Beijing Optimalaceae Biotechnology Ltd.
The invention provides a kit for distinguishing Ma shaddock peel, which comprises a primer pair chr2.10-9584F/chr2.10-9584R and a primer pair p-chr6.21-8689F/p-chr 6.21-8689R.
The invention also provides application of the molecular marker or the kit in distinguishing the Ma-jia pomelo from other varieties of citrus.
In the present invention, the other citrus species includes any one or more of thin-skinned horse-grapefruit, MiYou-xi-Mi-shaddock, Shatian-shaddock, or local native shaddock in Guangfeng county of Jiangxi province.
In the invention, the application of the molecular marker in distinguishing the rough-skinned Ma pomelos in the early seedling stage of the citrus varieties preferably comprises the following steps:
(1) extracting the genome DNA of the material to be detected;
(2) carrying out PCR amplification on the genomic DNA of the material to be detected by using a primer pair chr2.10-9584F/chr2.10-9584R and a primer pair p-chr6.21-8689F/p-chr6.21-8689R respectively to obtain two groups of amplification products;
(3) performing agarose gel electrophoresis on the two groups of amplification products respectively to obtain an electrophoresis picture;
(4) judging according to the electrophoresis chart: and if the electrophoresis image of the amplification product of the primer pair chr2.10-9584F/chr2.10-9584R has a bright band, and the electrophoresis image of the amplification product of the primer pair p-chr6.21-8689F/p-chr6.21-8689R has two bright bands, judging that the material to be detected is the Ma pomelo.
The invention extracts the genome DNA of the material to be detected. In the invention, the material to be detected is preferably early seedling leaves of which the variety is difficult to judge through phenotypic characteristics. After a material to be detected is obtained, the invention extracts the genome DNA of the material to be detected. The method for extracting the genomic DNA of the material to be tested is not particularly limited in the present invention, and any method can be used for preparing the genomic DNA conventionally in the art.
After obtaining the genomic DNA of the material to be detected, the invention takes the genomic DNA of the material to be detected as a template, and respectively performs PCR amplification on the genomic DNA of the material to be detected by using a primer pair chr2.10-9584F/chr2.10-9584R and a primer pair p-chr6.21-8689F/p-chr6.21-8689R to obtain two groups of amplification products. The present invention is not particularly limited to specific PCR amplification systems and methods, and any PCR amplification method can be used as is conventional in the art. In the present example, the reaction system for PCR amplification is 7. mu.l of Mix solution, 0.3. mu.l of 10mmol/L forward primer, 0.3. mu.l of 10mmol/L reverse primer, 1.5. mu.l of 100ng/ul template DNA and 6. mu.l of ddH2And O. The Mix is limited in Nanjing NuoZan biotechnology2 × Taq Plus Master Mix (Dye Plus) supplied by the company; the water is preferably sterilized ultrapure water. The reaction procedure of the PCR amplification is as follows: 95 ℃ for 5 min; 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; 72 ℃ for 10 min.
After the PCR amplification reaction is completed, the present invention preferably stores the amplification product after cooling. The cooling temperature is preferably 10-15 ℃, more preferably 12 ℃, the cooling time is preferably 25-40 min, more preferably 30min, and the preservation temperature is preferably 2-8 ℃, more preferably 4 ℃. The specific PCR reaction apparatus of the present invention is not particularly limited, and any apparatus commonly used in the art may be used. In an embodiment of the present invention, the PCR reaction apparatus is a PTC-200PCR instrument (mj.
After obtaining two groups of amplification products, the invention respectively carries out agarose gel electrophoresis on the two groups of amplification products to obtain an electrophoresis picture. The present invention is not particularly limited to a specific electrophoresis method, and any electrophoresis method can be used as is conventional in the art. In an embodiment of the present invention, the electrophoresis specifically includes: detecting the amplification product by agarose gel electrophoresis on a horizontal electrophoresis tank, wherein the agarose gel with the mass percent of 2.0-4.0% is adopted for electrophoresis, and the agarose gel with the mass percent of 3.0% is preferred; the voltage of the electrophoresis is 100-150V, preferably 120V; the electrophoresis time is 10-30 min, preferably 20 min. The electrophoresis buffer used in the present invention was 1 XTAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH 8.0). After electrophoresis, the present invention is preferably stored by photographing with a gel imaging system (UVP).
The invention judges whether the material to be detected is the coarse-skinned Ma Jia shaddock according to the electrophoresis result: and if the electrophoresis image of the amplification product of the primer pair chr2.10-9584F/chr2.10-9584R has a bright band, and the electrophoresis image of the amplification product of the primer pair p-chr6.21-8689F/p-chr6.21-8689R has two bright bands, judging that the material to be detected is the Ma pomelo.
The primer pair chr2.10-9584F/chr2.10-9584R and the primer pair p-chr6.21-8689F/p-chr6.21-8689R are used in combination, so that the Ma-jia pomelo can be distinguished from the citrus varieties. The InDel molecular marker provided by the invention can be used for distinguishing the rough-skinned Majiayou in citrus varieties at the early stage of seedlings, and has great significance for breeding of the citrus varieties and risk control in the production of the Majiayou. In addition, the molecular marker provided by the invention provides a new molecular tool for identifying, breeding and early screening the genetic stable Ma-jia pomelo seedlings.
The group of Ma Jia shaddock InDel molecular markers and the application thereof in early-stage differentiation of rough-skinned Ma shaddock of citrus varieties are clearly and completely described below by combining with the embodiment. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention
Example 1
(1) The modified CTAB method is adopted to extract and prepare genome DNA of fine-skinned Majia shaddock (Yougu), coarse-skinned Majia shaddock, MiYou xi shaddock, Shatian shaddock and local native shaddock in Guangfeng county of Jiangxi province.
(2) The genomic DNA of all the citrus fruits was analyzed by amplification using the primer pair chr2.10-9584F/chr 2.10-9584R. The amplification reaction was performed in a PTC-200PCR instrument (mj. The reaction system for PCR amplification was 7. mu.l of Mix solution, 0.3. mu.l of 10mmol/L forward primer, 0.3. mu.l of 10mmol/L reverse primer, 1.5. mu.l of 100ng/ul template DNA and 6. mu.l of ddH2And O. The method comprises the following specific steps: denaturation at 95 deg.C for 5 min; following 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; finally, the mixture is stored at 72 ℃, 10min, 12 ℃, 30min and 4 ℃.
(3) The amplification products were detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank using 1 XTAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH8.0), voltage 120V, and electrophoresis for 20 min. After electrophoresis, the gel imaging system (UVP) is used for photographing and storing.
The detection results are shown in FIG. 1: amplification products in the genomic DNA of the Majia pomelo peel are all single bright bands. There was no band of interest in his citrus variety other than MiYou Mixi GUAN, identical to this band pattern.
Example 2
(1) Extracting and preparing genome DNA of Majia shaddock and xi Mi shaddock with modified CTAB method.
(2) The genomic DNA of all the citrus fruits was analyzed by amplification using the primer pair p-chr6.21-8689F/p-chr 6.21-8689R. The amplification reaction was performed in a PTC-200PCR instrument (mj. The reaction system for PCR amplification was 7. mu.l of Mix solution, 0.3. mu.l of 10mmol/L forward primer, 0.3. mu.l of 10mmol/L reverse primer, 1.5. mu.l of 100ng/ul template DNA and 6. mu.l of ddH2And O. The method comprises the following specific steps: denaturation at 95 deg.C for 5 min; following 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; finally, the mixture is stored at 72 ℃, 10min, 12 ℃, 30min and 4 ℃.
(3) The amplification products were detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank using 1 XTAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH8.0), voltage 120V, and electrophoresis for 20 min. After electrophoresis, the gel imaging system (UVP) is used for photographing and storing.
The detection results are shown in FIG. 2: the amplified products in the genomic DNA of Majia pomelo were both bright bands. And the amplification product in the genomic DNA of MiYou xi GUAN was a bright band.
Example 3
(1) A modified CTAB method is adopted to extract and prepare genome DNA of a detection material from 80 random seedling plants of fine-skinned Ma pomelo (superior line) nursery mixed with coarse-skinned Ma pomelo.
(2) The genomic DNA of all the citrus fruits was analyzed by amplification using the primer pair p-chr6.21-8689F/p-chr 6.21-8689R. The amplification reaction was performed in a PTC-200PCR instrument (mj. The reaction system for PCR amplification was 7. mu.l of Mix solution, 0.3. mu.l of 10mmol/L forward primer, 0.3. mu.l of 10mmol/L reverse primer, 1.5. mu.l of 100ng/ul template DNA and 6. mu.l of ddH2And O. The method comprises the following specific steps: denaturation at 95 deg.C for 5 min; following 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; finally, the mixture is stored at 72 ℃, 10min, 12 ℃, 30min and 4 ℃.
(3) The amplification products were detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank using 1 XTAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH8.0), voltage 120V, and electrophoresis for 20 min. After electrophoresis, the gel imaging system (UVP) is used for photographing and storing.
The results are shown in FIG. 3. FIG. 3 shows that the seedling plants numbered 24, 63, 66, 71, 72, 78 and 79 were pomelos with rough peel and the remaining seedling plants were judged to be pomelos with fine peel (top line).
Example 4
(1) 38 young seedlings are randomly selected from coarse-skinned Majiayou nursery gardens mixed with other varieties to serve as detection materials, and the genome DNA of the detection materials is extracted and prepared by adopting an improved CTAB method.
(2) The genomic DNA of all the citrus fruits was analyzed by amplification using the primer pair p-chr6.21-8689F/p-chr 6.21-8689R. The amplification reaction was performed in a PTC-200PCR instrument (mj. The reaction system for PCR amplification was 7. mu.l of Mix solution, 0.3. mu.l of 10mmol/L forward primer, 0.3. mu.l of 10mmol/L reverse primer, 1.5. mu.l of 100ng/ul template DNA and 6. mu.l of ddH2And O. The method comprises the following specific steps: denaturation at 95 deg.C for 5 min; following 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; finally, the mixture is stored at 72 ℃, 10min, 12 ℃, 30min and 4 ℃.
(3) The amplification products were detected by 3.0% agarose gel electrophoresis on a horizontal electrophoresis tank using 1 XTAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH8.0), voltage 120V, and electrophoresis for 20 min. After electrophoresis, the gel imaging system (UVP) is used for photographing and storing.
The results are shown in FIG. 4. FIG. 4 shows that all Majia Mi GUAN can amplify two bright bands, and all Mi GUAN can amplify one bright band.
The embodiments of the present invention have been described in detail above, but this is only an example for easy understanding and should not be construed as limiting the scope of the present invention. Also, various equivalent changes or substitutions are possible for those skilled in the art according to the technical solution of the present invention and the description of the preferred embodiment thereof, but all such changes or substitutions shall fall within the protection scope of the claims of the present invention.
Sequence listing
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<120> a group of Ma Jia shaddock InDel molecular markers and application thereof in distinguishing coarse-skinned Ma shaddock in early seedling stage of citrus variety
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Claims (7)
1. A group of InDel molecular markers of Ma Piao ethnic shaddock is characterized by consisting of a segment obtained by amplifying genomic DNA of the Ma Piao ethnic shaddock by a primer pair chr2.10-9584F/chr2.10-9584R and a segment obtained by amplifying genomic DNA of the Ma Piao ethnic shaddock by a primer pair p-chr6.21-8689F/p-chr 6.21-8689R;
the nucleotide sequences 5 '-3' of the primer pair chr2.10-9584F/chr2.10-9584R are respectively:
chr2.10-9584F：AGTGAAAGGGAGAGAAAGAAGC；
chr2.10-9584R：ATGGTGCCACATTGCGAGT；
the nucleotide sequences 5 '-3' of the primer pair p-chr6.21-8689F/p-chr6.21-8689R are respectively:
p-chr6.21-8689F：GGGAGGGAGCTTGCTTGATT；
p-chr6.21-8689R：ACCCGATAAAGTCACGTACACA。
2. a kit for identifying Ma Piao pomelo, comprising the primer pair chr2.10-9584F/chr2.10-9584R and the primer pair p-chr6.21-8689F/p-chr6.21-8689R according to claim 1.
3. Use of a primer set or the kit of claim 2 for distinguishing between Majia pomelo squash and other citrus species, said other citrus species being any one or more of Majia pomelo squash, Mixi pomelo squash, Shatian pomelo squash; the primer set comprises the primer pair chr2.10-9584F/chr2.10-9584R and p-chr6.21-8689F/p-chr6.21-8689R of claim 1.
4. Use according to claim 3, characterized in that it comprises the following steps:
(1) extracting the genome DNA of the material to be detected;
(2) carrying out PCR amplification on the genomic DNA of the material to be detected by using the primer pair chr2.10-9584F/chr2.10-9584R and the primer pair p-chr6.21-8689F/p-chr6.21-8689R respectively as described in claim 1 to obtain two groups of amplification products;
(3) performing agarose gel electrophoresis on the two groups of amplification products respectively to obtain an electrophoresis picture;
(4) judging according to the electrophoresis chart: and if the electrophoresis image of the amplification product of the primer pair chr2.10-9584F/chr2.10-9584R has a bright band, and the electrophoresis image of the amplification product of the primer pair p-chr6.21-8689F/p-chr6.21-8689R has two bright bands, judging that the material to be detected is the Ma pomelo.
5. The use according to claim 4, wherein the reaction system of the PCR amplification is: 7 μ L of Mix solution, 0.3 μ L of 10mmol/L0.3. mu.l of 10mmol/L reverse primer, 1.5. mu.l of 100ng/ul template DNA and 6. mu.l of ddH2O。
6. The use according to claim 5, wherein the PCR amplification step is: 95 ℃ for 5 min; 35 cycles: 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 35 s; 72 ℃ for 10 min.
7. The application of claim 4, wherein the agarose gel electrophoresis is performed by using 2.0-4.0% of agarose gel by mass percent, and the voltage is 100-150V, and the electrophoresis is performed for 10-30 min.
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| CN110699477B (en) * | 2019-10-21 | 2021-06-11 | 华中农业大学 | Molecular marker for distinguishing raw material plants of exocarpium citri grandis and light exocarpium citri grandis and application of molecular marker |
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| CN111286556A (en) * | 2020-04-03 | 2020-06-16 | 江西省农业科学院园艺研究所 | Method for identifying variety of golden orchid pomelo based on whole genome InDel marker |
| CN113355448B (en) * | 2021-07-09 | 2022-04-08 | 华中农业大学 | InDel molecular marker for identifying phyllanthus emblica and application thereof |
| CN116287364B (en) * | 2022-02-22 | 2023-11-28 | 江西省农业科学院园艺研究所 | Primer group, kit and method for identifying cultivation type of Jiangxi local pomelo |
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