CN112029893A - Molecular marker primer for identifying sex of persimmon tree and identification method - Google Patents
Molecular marker primer for identifying sex of persimmon tree and identification method Download PDFInfo
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Abstract
The invention discloses a molecular marker primer for identifying persimmon tree sex and an identification method, comprising a forward primer and a reverse primer, which are SEQ ID No.1 and SEQ ID No.2 in a sequence table. The identification method comprises the following steps: extracting DNA of persimmon tree leaves; carrying out PCR amplification on the extracted DNA by using a molecular marker primer; separating the PCR amplification product by agarose gel electrophoresis and taking a picture; and (4) carrying out sex identification on the persimmon trees according to the band condition in the electrophoretogram. The method can separate the seedlings with the male flowers in the seedling stage, does not need to remove the male germplasm in the hybrid individuals after the persimmon trees bloom for 5-8 years, can carry out early identification on the sex of persimmon hybrid filial generations in the seedling stage, eliminates male plants as early as possible, screens out the female plants of the hybrid filial generations with excellent properties, improves the early selection efficiency of the persimmon hybrid filial generations, reduces the breeding cost, is beneficial to the smooth development of the hybrid breeding work, promotes the breeding and the industrial development, and promotes the smooth development of the persimmon industry.
Description
Technical Field
The invention relates to the technical field of molecular identification. In particular to a molecular marker primer for identifying the sex of persimmon trees and an identification method.
Background
Persimmon (Diospyros kaki Thunb.) is a plant of the genus Diospyros (Ebenaceae) and is an important woody grain tree species originally produced in China. The persimmon fruits are rich in nutrition, the content of soluble solid and tannin is high, the leaves are rich in Vc, flavone, polyphenol and other active ingredients, the development value and the application potential in the fields of food, health care, chemical industry, medicine and the like are more and more emphasized, and the research on the persimmon breeding and cultivation technology can provide powerful scientific and technological support for the aspects of precision poverty relief, agricultural complex construction, national grain safety and the like.
It is noted that most of the persimmon cultivars are completely female plants (only open female flowers) and hermaphrodite plants (both female flowers and male flowers), and there are completely male plants (only open male flowers) in persimmon wild resources. In addition, persimmon usually takes 5-8 years from the sowing of the seeds to the first flowering and fruiting of the seedlings. The characteristic of long childhood period of the persimmon severely limits the early screening of hybrid offspring female plants with excellent characters, and simultaneously expends a large amount of manpower, material resources and financial resources in the processes of seedling culture and planting management, thereby reducing the working efficiency of cultivating the improved persimmon seeds. Therefore, there is a need to develop efficient and stable sex molecular markers, and early identification of the sex of persimmon seedlings in the seedling stage is of great significance for accelerating the breeding process and controlling the seedling cost.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a molecular marker primer and a method for identifying the sex of a persimmon tree, wherein the sex of the persimmon tree is identified early by using the molecular marker primer in a seedling stage, and the molecular marker primer has important significance for improving the early selection efficiency of female plants of persimmon hybrid progeny and reducing the management cost of hybrid gardens.
In order to solve the technical problems, the invention provides the following technical scheme:
the molecular marker primer for identifying the sex of the persimmon tree comprises a forward primer and a reverse primer, and the sequences of the primers are respectively the nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 in a sequence table.
The method for identifying the sex of the persimmon tree comprises the following steps:
(1) extracting DNA of persimmon tree leaves;
(2) performing PCR amplification on the extracted DNA using the forward primer and the reverse primer of claim 1;
(3) separating the PCR amplification product by agarose gel electrophoresis and taking a picture;
(4) and (4) carrying out sex identification on the persimmon trees according to the band condition in the electrophoretogram.
The method for identifying the sex of the persimmon tree comprises the following steps of (1):
(1-1) rapidly weighing about 0.5g of young persimmon tree leaves in a mortar by using an electronic balance, fully grinding a sample into powder by using liquid nitrogen, rapidly transferring the powder into a 2ml centrifuge tube containing 800 mu L of 2 x cetyltrimethylammonium bromide CTAB buffer solution preheated to 65 ℃, and extracting for 30min in a water bath at 65 ℃;
(1-2) adding a chloroform and isoamyl alcohol mixed solution with the same volume ratio, wherein the chloroform to the isoamyl alcohol volume ratio is 24: 1, after vortex mixing, centrifuging at the rotating speed of 12000rpm for 10min, transferring the supernatant into a new centrifuge tube, adding the chloroform and isoamylol mixed solution with the same volume again, and after vortex mixing, centrifuging at the rotating speed of 12000rpm for 10 min; taking the supernatant fluid in a new centrifuge tube, adding 2 times of ethanol with volume and precooling to-20 ℃, and slowly shaking up until DNA flocculent precipitate is separated out;
(1-3) centrifuging at 10000rpm for 10min, and pouring out the supernatant; washing the precipitate with 70% ethanol for 2-3 times; standing at room temperature for 5min to allow the precipitate to air dry naturally, adding50 μ L of ddH preheated to 65 ℃2O, obtaining persimmon genome DNA;
(1-4) detecting the purity and concentration of the DNA sample by using a 1.0% agarose gel electrophoresis method and an ultraviolet spectrophotometer method, and storing the qualified sample in a refrigerator at the temperature of-20 ℃ for later use.
In the method for identifying the sex of the persimmon tree, in the step (2), the PCR amplification process comprises the following steps: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 1min, and repeated cycling for 30 times; extension at 72 ℃ for 5 min.
In the method for identifying the sex of the persimmon tree, in the step (2), a PCR amplification system comprises the following steps: 12.5 mu L of Taq DNA polymerase; 10 μ M of the forward primer 1 μ L; 10 μ M of the downstream primer 1 μ L; 1 μ L of template DNA; addition of ddH2The amount of O was made up to 25. mu.L.
In the method for identifying the sex of the persimmon tree, in the step (3), the PCR amplification product is detected by 1.0 wt% of agarose gel electrophoresis; the electrophoresis conditions were 150V and 400mA, and the electrophoresis time was 20 min.
In the method for identifying the sex of the persimmon tree, a complete male plant or a complete female and male plant with 1 strip is displayed on 268bp of an electrophoresis gel, and a complete female plant without strips is not shown.
The technical scheme of the invention achieves the following beneficial technical effects:
crossbreeding is an important means for germplasm innovation and improved variety cultivation. Persimmon single plants are diversified in types, common plants are completely female plants and hermaphrodite plants, and a few are completely male plants. In addition, persimmon usually takes 5-8 years from the sowing of the seeds to the first flowering and fruiting of the seedlings. The characteristics of complicated nature and long childhood period of the persimmon flowers seriously limit the screening of male and female hybrid offspring individuals, and the current situation is not favorable for the management of hybrid gardens, and simultaneously, the working efficiency of germplasm innovation and fine variety cultivation by screening the offspring with excellent characters through hybrid breeding is further reduced.
The invention adopts the molecular marker technology to identify the sex of the persimmon tree, firstly extracts the DNA of the persimmon tree, then carries out PCR amplification by using the molecular marker provided by the invention, and the amplified product is separated by agarose gel electrophoresis and photographed, thus identifying whether the persimmon germplasm blooms or not according to the existence of target strips. The method can separate the seedlings (including hermaphrodite and complete male plants) of the opened male flowers in the seedling stage, the accuracy of the identification result is more than 94%, the male germplasm in the hybrid individual is not required to be removed after the persimmon trees bloom for 5-8 years, and the sex of the persimmon hybrid progeny can be identified in the early stage of seedling, so that the male plants are eliminated as soon as possible, and the female plants of the hybrid progeny with excellent characters are selected, thereby improving the early selection efficiency of the persimmon hybrid progeny, reducing the breeding cost, being beneficial to the smooth development of the hybrid breeding work, promoting the breeding and industrial development, and promoting the smooth development of the persimmon industry.
Drawings
FIG. 1 is a PCR amplification electrophoretogram for detecting 36 individuals of fully female persimmon trees;
FIG. 2 is a PCR amplification electrophoretogram for detecting 33 fully male individuals of persimmon tree;
FIG. 3 is a PCR amplification electrophoresis chart for detecting 17 hermaphrodite individuals of persimmon tree.
Detailed Description
The method for identifying the sex of the persimmon tree in the embodiment comprises the following steps:
(1) extracting DNA of persimmon tree leaves;
(1-1) rapidly weighing about 0.5g of young persimmon tree leaves in a mortar by using an electronic balance, fully grinding a sample into powder by using liquid nitrogen, rapidly transferring the powder into a 2ml centrifuge tube containing 800 mu L of 2 x cetyltrimethylammonium bromide CTAB buffer solution preheated to 65 ℃, and extracting for 30min in a water bath at 65 ℃;
(1-2) adding a chloroform and isoamyl alcohol mixed solution with the same volume ratio, wherein the chloroform to the isoamyl alcohol volume ratio is 24: 1, after vortex mixing, centrifuging at the rotating speed of 12000rpm for 10min, transferring the supernatant into a new centrifuge tube, adding the chloroform and isoamylol mixed solution with the same volume again, and after vortex mixing, centrifuging at the rotating speed of 12000rpm for 10 min; taking the supernatant fluid in a new centrifuge tube, adding 2 times of ethanol with volume and precooling to-20 ℃, and slowly shaking up until DNA flocculent precipitate is separated out;
(1-3) centrifuging at 10000rpm for 10min, and pouring out the supernatant; washing the precipitate with 70% ethanol for 2-3 times; standing at room temperature for 5min to allow the precipitate to air dry naturallyAdd 50. mu.L of ddH preheated to 65 ℃2O, obtaining persimmon genome DNA;
(1-4) detecting the purity and concentration of the DNA sample by using a 1.0% agarose gel electrophoresis method and an ultraviolet spectrophotometer method, and storing the qualified sample in a refrigerator at the temperature of-20 ℃ for later use.
(2) Performing PCR amplification on the extracted DNA by using a forward primer and a reverse primer;
a molecular marker primer for identifying the sex of a persimmon tree is disclosed, wherein the sequence of a forward primer is as follows: TTTATCACAACTGCCCAGGG (5'-3'), the reverse primer sequence being: TGTGATAAATTTTTTTTTTTTATTATTATGCTCCAAC (5'-3') which are the nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 in the sequence Listing, respectively.
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 1min, and repeated cycling for 30 times; extension at 72 ℃ for 5 min.
The PCR amplification system is as follows:
| taq DNA polymerase | 12.5μL |
| Upstream primer with concentration of 1. mu.M | 1μL |
| Downstream primer at a concentration of 1. mu.M | 1μL |
| Template DNA | 1μL |
| ddH2O | 9.5μL |
(3) Separating the PCR amplification product by agarose gel electrophoresis and taking a picture;
detecting the PCR amplification product by using 1.0% agarose gel electrophoresis; the electrophoresis conditions were 150V and 400mA, and the electrophoresis time was 20 min.
(4) And (4) carrying out sex identification on the persimmon trees according to the band condition in the electrophoretogram.
On the electrophoresis gel, 268bp shows that 1 strip is a complete male plant or a hermaphrodite plant with a male flower, and a strip-free complete female plant without a male flower.
In order to prove the identification effect of the identification method, 36 parts of a completely female adult persimmon plant, 33 parts of a completely male adult persimmon plant and 17 parts of a hermaphrodite persimmon plant which blossoms or fruits are adopted to verify the identification effect.
Experimental materials: 36 parts of a complete female plant, 33 parts of a complete male plant and 17 parts of a hermaphrodite plant of the adult persimmon which blooms or fruits.
The experimental method comprises the following steps: the above-mentioned method for discriminating the sex of persimmon trees was used (leaf DNA was extracted, PCR amplification was performed, and then agarose gel electrophoresis was used for separation and photographing).
The experimental results are as follows: and (4) identifying the sex of the persimmon tree according to the band condition in the electrophoretogram (shown in figures 1-3). The results are shown in table 1:
TABLE 1 identification of persimmon tree sex
The result shows that the accuracy of the identification result of the method is more than 94 percent; therefore, the sex of the persimmon tree can be accurately identified through DNA.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are possible which remain within the scope of the appended claims.
Sequence listing
<110> research and development center of paulownia of national forestry and grassland bureau
<120> molecular marker primer for identifying sex of persimmon tree and identification method
<141> 2020-09-23
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tttatcacaa ctgcccaggg 20
<210> 2
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgtgataaat tttttttttt tattattatg ctccaac 37
Claims (7)
1. The molecular marker primer for identifying the sex of the persimmon tree is characterized by comprising a forward primer and a reverse primer, wherein the sequences of the forward primer and the reverse primer are respectively the nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 in a sequence table.
2. The method for identifying the sex of the persimmon tree is characterized by comprising the following steps:
(1) extracting DNA of persimmon tree leaves;
(2) performing PCR amplification on the extracted DNA using the forward primer and the reverse primer of claim 1;
(3) separating the PCR amplification product by agarose gel electrophoresis and taking a picture;
(4) and (4) carrying out sex identification on the persimmon trees according to the band condition in the electrophoretogram.
3. The method for discriminating the sex of a persimmon tree as set forth in claim 2, wherein in the step (1):
(1-1) rapidly weighing about 0.5g of young persimmon tree leaves in a mortar by using an electronic balance, fully grinding a sample into powder by using liquid nitrogen, rapidly transferring the powder into a 2ml centrifuge tube containing 800 mu L of 2 x cetyltrimethylammonium bromide CTAB buffer solution preheated to 65 ℃, and extracting for 30min in a water bath at 65 ℃;
(1-2) adding a chloroform and isoamyl alcohol mixed solution with the same volume ratio, wherein the chloroform to the isoamyl alcohol volume ratio is 24: 1, after vortex mixing, centrifuging at the rotating speed of 12000rpm for 10min, transferring the supernatant into a new centrifuge tube, adding the chloroform and isoamylol mixed solution with the same volume again, and after vortex mixing, centrifuging at the rotating speed of 12000rpm for 10 min; taking the supernatant fluid in a new centrifuge tube, adding 2 times of ethanol with volume and precooling to-20 ℃, and slowly shaking up until DNA flocculent precipitate is separated out;
(1-3) centrifuging at 10000rpm for 10min, and pouring out the supernatant; washing the precipitate with 70% ethanol for 2-3 times; standing at room temperature for 5min to allow the precipitate to air dry naturally, adding 50 μ L ddH preheated to 65 deg.C2O, obtaining persimmon genome DNA;
(1-4) detecting the purity and concentration of the DNA sample by using a 1.0% agarose gel electrophoresis method and an ultraviolet spectrophotometer method, and storing the qualified sample in a refrigerator at the temperature of-20 ℃ for later use.
4. The method for sex discrimination of persimmon trees according to claim 2, wherein in the step (2), the PCR amplification process is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 1min, and repeated cycling for 30 times; extension at 72 ℃ for 5 min.
5. The method for discriminating the sex of persimmon trees as set forth in claim 2, wherein in the step (2), the PCR amplification system is: 12.5 mu L of Taq DNA polymerase; 10 μ M of the forward primer 1 μ L; 10 μ M of the downstream primer 1 μ L; 1 μ L of template DNA; addition of ddH2The amount of O was made up to 25. mu.L.
6. The method for discriminating the sex of persimmon trees as set forth in claim 2, wherein in the step (3), the PCR amplification product is detected by 1.0 wt% agarose gel electrophoresis; the electrophoresis conditions were 150V and 400mA, and the electrophoresis time was 20 min.
7. The method for sex discrimination of persimmon trees according to claim 2, wherein 1 band of the band is completely male or hermaphrodite with blossoming flowers at 268bp on the electrophoresis gel, and the band-free band is completely female without blossoming flowers.
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Cited By (3)
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| CN113136450A (en) * | 2021-05-25 | 2021-07-20 | 沈阳农业大学 | Molecular marker for identifying sex of vitis amurensis and identification method thereof |
| CN113234807A (en) * | 2021-05-17 | 2021-08-10 | 中国农业大学 | Sex identification method suitable for fig seedlings |
| CN114303669A (en) * | 2021-12-30 | 2022-04-12 | 湖北省农业科学院果树茶叶研究所 | Method for shortening childhood and childhood period of persimmon seedlings |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113234807A (en) * | 2021-05-17 | 2021-08-10 | 中国农业大学 | Sex identification method suitable for fig seedlings |
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| CN113136450B (en) * | 2021-05-25 | 2023-06-27 | 沈阳农业大学 | Molecular marker for identifying sex of amur grape and identification method thereof |
| CN114303669A (en) * | 2021-12-30 | 2022-04-12 | 湖北省农业科学院果树茶叶研究所 | Method for shortening childhood and childhood period of persimmon seedlings |
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