CN106755387B - Method for rapidly identifying cucumber rootstock joint strength secondary purity by using molecular marker - Google Patents
Method for rapidly identifying cucumber rootstock joint strength secondary purity by using molecular marker Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to the field of molecular markers, and provides a method for rapidly identifying purity of cucumber stock joint force II by using a molecular marker, wherein the method adopts a specially designed EST-SSR primer, amplifies the genomic DNA of the joint force II as a template, and detects the purity of F1 generation seeds of the cucumber stock joint force II to be detected by using a specific spectral band of an amplification result, so that the identification can be carried out at any period of the growth of a cucumber stock joint force II plant, and hybrids, female parent self-bred seeds and male parent self-bred seeds can be distinguished; the method has high accuracy and wide application and popularization prospect, and provides scientific basis for developing stock seed production and timely selling of improved seeds.
Description
Technical Field
The invention relates to the field of molecular markers, and particularly provides a method for rapidly identifying the purity of cucumber rootstock joint force by using molecular markers.
Background
The cucumber stock combined force II is an excellent cucumber stock variety newly cultivated by Huasheng agriculture shares GmbH of Shandong province, the variety has good grafting affinity, and the grafted cucumber has vigorous growth and enhanced disease resistance. The grafted melon strips are black, bright and straight, and the commodity is improved. Is suitable for planting in spring, autumn and overwintering in protected areas. In the hybrid seed production process of the variety, the female parent needs to be manually castrated, male parent pollen is pollinated to complete the hybrid process, and the castration of the female parent is incomplete or missed, so that the seed production purity is greatly influenced. Therefore, the identification of the purity of the hybrid rootstock seeds is very important.
The traditional rootstock hybrid seed purity identification work is still mainly characterized by field planting, the appearance shape comparison identification of the grafted cucumber is adopted, the identification method generally needs 2 months, the time is long, the cost is high, the difficulty is high, the accuracy is poor, identification personnel have rich experience, the method is easily influenced by cultivation measures and environmental conditions, the efficiency and the accuracy of variety identification are seriously influenced, and the requirements of modern breeding work and production and operation cannot be met more and more.
The indoor purity detection usually adopts a molecular marker technology, the technology reveals genetic difference between filial generation and parents from the DNA level, is not influenced by environmental conditions and cultivation measures, has no tissue, organ and development specificity, can detect tiny variation, is not limited by plant growing seasons, has rich polymorphism and high stability, and can greatly shorten the identification time. In the prior art, the purity of the pumpkin is identified by adopting three pairs of primers and applying a multiplex PCR method, but the purity of the corresponding rootstock cannot be detected, the multiplex PCR system is complex, the required time is long, the identification result cannot be obtained in a short time, and meanwhile, the cost is high, so that the wide popularization and application in the field are difficult; therefore, how to better and more quickly identify the purity of the resultant force of the cucumber rootstocks becomes one of the problems to be solved urgently.
Disclosure of Invention
Aiming at the conditions of the prior art, the inventor of the invention provides a method for rapidly identifying the purity of the second joint of cucumber stocks by using molecular markers, the method adopts specially designed EST-SSR primers, uses the genomic DNA of the second joint as a template for amplification, and detects the purity of F1 generation seeds of the second joint of cucumber stocks to be detected by using a specific spectral band of an amplification result, the method can identify the seeds at any period of the growth of the second joint of cucumber stocks, and can distinguish hybrid seeds from female parent self-bred seeds and male parent self-bred seeds; the method has high accuracy and wide application and popularization prospect, and provides scientific basis for developing stock seed production and timely selling of improved seeds.
The cucumber stock variety aimed by the invention is a good cucumber stock variety which is bred by researching and selecting from Huasheng agriculture stocks Limited company in Shandong province for many years, the variety has good grafting affinity, and the grafted cucumber has strong growth and enhanced disease resistance. The grafted melon strips are black, bright and straight, and the commodity is improved. Is suitable for planting in spring, autumn and overwintering in protected areas.
In order to identify the purity of the hybrid II and obtain excellent hybrid varieties of a female parent and a male parent, the inventor firstly provides a pair of EST-SSR primers which are named as HE L I2, wherein the forward sequence is 5'-GAATCAGAGGAAGGTTTGCG-3', the nucleotide sequence is shown as SEQ ID No.1, and the reverse sequence is 5'-CACTTCCTTCATGCTTCTCG-3', the nucleotide sequence is shown as SEQ ID No. 2.
After the primers are obtained, the inventor utilizes the primers to amplify hybrid seeds of cucumber rootstock joint force II of a variety to be detected and radicle genome DNA of parents of the hybrid seeds, and the specific process is as follows:
(1) extracting genome DNA of cucumber rootstock hybrid two hybrid seeds and parent radicle thereof;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and EST-SSR primers HE L I2:
the PCR reaction system is 10 mu L, which comprises 10 × PCR Buffer 1 mu L, 0.2mM dNTPs, 100ng DNA template, 5 mu M primers, 0.5U Taq DNA polymerase and sterile ultrapure water which is filled to 10 mu L;
the amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 20s, and 35 cycles; final extension at 72 deg.C for 5 min; the PCR product was stored at 10 ℃;
after a plurality of experiments, the inventor finds that when the annealing temperature is set to 55 ℃ in the amplification procedure aiming at the invention purpose of the invention and the specially designed specific primer, the amplification effect is best, the final detection result is closer to the actual situation, so the annealing temperature of the invention is set to 55 ℃. In addition, compared with other similar amplification programs in the prior art, the method has the advantages that the requirement of performing variety purity identification by combining force II is met, the pre-denaturation temperature and the denaturation temperature are increased to 95 ℃, so that the denaturation effect is better, the 72 ℃ extension time is shortened, each cycle is shortened by 40s, the whole identification process is faster, and the result is not adversely affected.
(3) Detecting amplification products, namely mixing the amplification products of 5 mu L with a sample loading buffer solution of 2 mu L, carrying out non-denaturing polyacrylamide gel electrophoresis with the mass-volume ratio of acrylamide of 8 percent, carrying out 180v constant voltage electrophoresis for 1.5h, and carrying out silver staining and color development for band type statistics;
wherein the mass ratio of acrylamide to methylene bisacrylamide in the adopted non-denatured polyacrylamide gel is 29: 1;
the loading buffer solution is selected from 10 × glycerol gel;
(4) the purity of the hybrid of 'cucumber stock joint force two' is identified according to the amplification result of the specific spectral band, and the judgment standard is as follows:
the female parent has 177bp specific band, and the nucleotide sequence is shown as SEQ ID No. 4; the male parent has a 159bp specific band, and the nucleotide sequence of the band is shown as SEQ ID No. 3; the hybrid has two specific bands of 159bp and 177 bp;
according to the identification result, the purity (%) of the two varieties of the combined force of the cucumber stocks can be converted to × 100% which is (1-N/N), wherein N is the number of the stocks to be detected, and N is the number of plants which have the spectrum band characteristics of the male parent alone or the female parent alone or are different from the spectrum band characteristics of the 'two combined force of cucumber stocks' or the parental spectrum band characteristics.
According to the method, the inventor does not adopt a conventional mode of extracting the genome DNA by using the leaves, but selects the young root for extraction, so that the growth time of the material is greatly shortened, and the inventor finds that the young root can extract the genome DNA suitable for purity identification after a plurality of tests, so that the method is no different from the leaves and is even more accurate, but the young root can greatly shorten the preparation time of the material, the method shortens 7 days compared with the mode of extracting the genome DNA by using the leaves, and the young root is extracted more conveniently and quickly.
By utilizing the method, the purity of the two varieties of the combined force of the cucumber and the rootstocks can be rapidly detected, and the method can not be influenced by cultivation measures and environmental conditions; the method has no organ, tissue and development specificity, can identify the plant at any growth period, and simultaneously reduces the minimum detection time limit from the leaf period to the radicle period, thereby greatly shortening the cultivation time of the plant to be detected; polymorphism-rich, co-dominant inheritance: the hybrid seeds can be distinguished from female parent inbred seeds and male parent inbred seeds; the requirement on the quality of DNA is not high and the required quantity is less; the cost is low, and the operation is simple; the method can be used for identification in the seedling stage, so that a large amount of manpower, material resources and land resources are saved; the repeatability and the stability are better; fast and efficient: the seed purity identification can be completed within one week; the accuracy is high: the method reveals genetic difference between filial generation and parents from genome level, and overcomes errors brought by field phenotype identification; the application and popularization prospect is wide: the purity of the hybrid of 'cucumber stock joint force two' can be rapidly and accurately detected.
Drawings
FIG. 1 is a PCR electrophoresis pattern of a hybrid seed 'cucumber stock joint force two' seed detection characteristic primer of the invention,
wherein, P1 is the female parent of 'cucumber stock joint force two'; f1 is a plurality of groups of 'cucumber rootstock joint force two' F1 hybrid seeds; p2 is the male parent of 'two hybrid cucumber stocks',
the results show that: the cucumber rootstock joint two F1 hybrid has two bands of 177bp and 159bp from a father parent and a mother parent respectively;
FIG. 2 is an electrophoresis diagram showing the influence of different PCR annealing temperatures on the purity detection result of a 'cucumber stock joint force two' commodity seed sample;
wherein, P1 is the female parent of 'cucumber stock joint force two'; f1 is a hybrid of 'cucumber rootstock joint force two' F1; p2 is the male parent of 'two hybrid cucumber stocks',
the result shows that the purity of the cucumber rootstock hybrid is most easily distinguished when the optimal annealing temperature is 55 ℃;
FIG. 3 is an electrophoresis chart showing the influence of different DNA template amounts on the purity detection result of a 'cucumber stock joint force two' commodity seed sample,
wherein, P1 is the female parent of 'cucumber stock joint force two'; f1 is a hybrid of 'cucumber rootstock joint force two' F1; p2 is the male parent of 'two hybrid cucumber stocks',
the result shows that the DNA template amount can be used for detecting the purity of the 'cucumber rootstock joint two' commodity seed sample from 100ng to 1000 ng.
Detailed Description
The present invention is further defined in the following examples, from which one skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Except for special notes, the invention adopts the prior art in the field; the percentages in the following examples are by weight, unless otherwise indicated.
Example 1 acquisition of genomic DNA from rootstock of cucumber of two varieties of Combined force
The specific operation method comprises the following steps:
(1) putting the 'cucumber rootstock joint two' parent and F1 seeds into a culture dish paved with moist filter paper, culturing in the dark at 30 ℃ for 3d, and taking the radicles;
in the hybrid seed production process of the variety, the female parent needs to be manually castrated, male parent pollen is fertilized to complete the hybrid process, and finally F1 generation seeds on the female parent are harvested;
(2) placing a young root into each centrifuge tube, adding 500 μ L extraction buffer solution preheated at 60-65 deg.C, placing 4 steel balls, crushing the material on a crusher, and water-bathing at 60-65 deg.C for 20-30 min;
wherein the extraction buffer solution is 2 × CTAB extract, and the mixture ratio is as follows:
sterilizing at high temperature, cooling to room temperature, adding 2m L mercaptoethanol, storing at room temperature,
(3) centrifuging the liquid obtained in the previous step at 10000rpm/min for 10min, taking the supernatant in another centrifuge tube, and adding a mixed solution of phenol, chloroform and isoamylol (the volume ratio is 25:24:1) with the same volume (500 mu L);
(4) centrifuging the mixed solution in the previous step at 10000rpm/min for 10min, taking the supernatant into another centrifugal tube, and adding chloroform with the same volume: isoamyl alcohol (volume ratio is 24:1) mixed solution;
(5) centrifuging the mixed solution at 10000rpm/min for 10min, putting the supernatant solution into another centrifuge tube, adding 400 mu L isopropanol, and mixing;
(6) centrifuging the mixed solution at 12000rpm/min for 15min, discarding the supernatant, washing the solid substance with 70% ethanol, and air drying;
(7) to the above solid was added 200. mu. L ddH2O redissolving, adding 1 mu L RNase, water bath at 37 ℃ for 30min, and keeping at-20 ℃ for later use.
Example 2 PCR amplification
And (3) performing PCR amplification by using the obtained RNA enzymolysis mixture as a template and using an HE L I2 primer:
the PCR reaction system is 10 mu L, which comprises 10 × PCR Buffer 1 mu L, 0.2mM dNTPs, primers of 5 mu M each, 0.5U Taq DNA polymerase, template of 100ng and sterile ultrapure water which is filled to 10 mu L;
the targeted amplification procedure according to the invention was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 20s, and 35 cycles; final extension at 72 deg.C for 5 min; the PCR product was stored at 10 ℃.
Example 3 detection of amplification products
Mixing the amplified product of 5 mu L with a sample loading buffer solution of 2 mu L, carrying out non-denaturing polyacrylamide gel electrophoresis with the mass-volume ratio of acrylamide of 8%, carrying out 180v constant voltage electrophoresis for 1.5h, and carrying out silver staining and color development for band type statistics;
the loading buffer solution is selected from 10 × glycerol gel;
wherein the 12% non-denaturing polyacrylamide gel formulation (100m L) is as follows:
the preparation process of the adopted 30 percent acrylamide/methylene bisacrylamide comprises the following steps:
29g of acrylamide and 1g of methylene bisacrylamide are subjected to constant volume until the volume is 100m, L4 ℃ and are stored in a dark place, and the quality guarantee period is one month;
the 10 gamma TBE configuration procedure used is as follows:
tris Base 27g, boric acid 13.75g, 0.5M/L EDTA 10M L, distilled water to reach 250M L and pH value to be 8.3;
the 10% Ammonium Persulfate (APS) used was prepared on site in a proportion of 0.1g ammonium persulfate to 1m L distilled water.
The electrophoresis buffer solution is 1 × TBE, and after electrophoresis is finished, a silver staining method is adopted for gel color development, and the specific process is as follows:
1. washing with water: ddH2Washing with water for 2 times (each time for 1 min);
2. dyeing: using 1% AgNO3Soaking, and shaking for 10-15 min;
3. washing with water: pouring off the developing solution, ddH2Washing with water for 2 times (each time for 1 min);
4. developing, namely adding developing solution (each liter of developing solution contains 16g of sodium hydroxide and 8m L of formaldehyde, and the balance is purified water) into a disc, and then shaking in parallel until the developing is clear;
5. and (4) terminating: soaking in stop solution (containing 7.5g sodium carbonate per liter and purified water for 2 min), and adding ddH2Cleaning with O water for 1-2 times; and finally, taking pictures and storing the pictures by using a camera.
Example 4 identification of purity of "cucumber rootstock syngamy two" hybrids
The purity of the hybrid of 'cucumber stock joint force two' is identified according to the electrophoresis result of the specific spectral band, and the judgment standard is as follows:
the female parent has 177bp specific bands; the male parent has a specific band of 159 bp; the hybrid has two specific bands of 159bp and 177 bp;
according to the identification result, the purity (%) of the two varieties of the combined force of the cucumber stocks can be converted to × 100% which is (1-N/N), wherein N is the number of the stocks to be detected, and N is the number of plants which have the spectrum band characteristics of the male parent alone or the female parent alone or are different from the spectrum band characteristics of the 'two combined force of cucumber stocks' or the parental spectrum band characteristics.
Examples of the experiments
Taking cucumber rootstock joint-combining two-seed produced in 2015 as an example, after F1 generation seeds are harvested, F1 generation seeds are immediately subjected to germination accelerating, rootstock genome DNA is extracted from radicles after about 2d, purity identification is carried out by utilizing the cucumber rootstock joint-combining two-molecule marker, and the seed purity result can be obtained after 4 d;
and (3) identifying by using a conventional identification method, namely field planting form: the generation-adding purity identification is carried out in Hainan, the growth cycle is about 60 days, the method is influenced by various severe weather, repeated seeding identification can be carried out for many times, and the identification time is not easy to estimate. The specific comparison is as follows:
compared with the conventional identification method, the molecular marking method is used for identifying the purity of the cucumber rootstock hybrid two-hybrid seeds, and has the advantages of rapidness, high efficiency, simple operation, good repeatability and lower cost.
<110> Lixing Sheng
<120> method for rapidly identifying cucumber rootstock joint force secondary purity by using molecular marker
<160>4
<210>1
<211>20
<212>DNA
<213> Artificial sequence
<400>1
gaatcagagg aaggtttgcg 20
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<400>2
cacttccttc atgcttctcg 20
<210>3
<211>159
<212>DNA
<213> Artificial sequence
<400>3
gaatcagagg aaggtttgcg aagactgaag aatgttagat gaggaacatc aggaaaagca 60
tgaatctgca gttagaagga tagttgattt gtaaatattg tttgctcaat aagaacgaga 120
acgagaacga gaacgagaac gagaagcatg aaggaagtg 159
<210>4
<211>177
<212>DNA
<213> Artificial sequence
<400>4
gaatcagagg aaggtttgcg aagactgaag aatgttagat gaggaacatc aggaaaagca 60
tgaatctgca gttagaagga tagttgattt gtaaatattg tttgcttaat aagaacgaga 120
acgggaacga gaacgagaag aagaacgaga agaagaacga gaagcatgaa ggaagtg 177
Claims (3)
1. A method for rapidly identifying the purity of cucumber rootstock joint force by using molecular markers is characterized in that an EST-SSR primer is used for identification, the EST-SSR primer is named as HE L I2, the forward sequence of the EST-SSR primer is 5'-GAATCAGAGGAAGGTTTGCG-3', the nucleotide sequence of the EST-SSR primer is shown as SEQ ID No.1, the reverse sequence of the EST-SSR primer is 5'-CACTTCCTTCATGCTTCTCG-3', and the nucleotide sequence of the reverse sequence of the EST-SSR primer is shown as SEQ ID No. 2.
2. The method for rapidly identifying the cucumber rootstock joint strength secondary purity by using the molecular marker as claimed in claim 1, which is characterized in that: the method comprises the following specific steps:
(1) extracting genome DNA of cucumber rootstock hybrid two hybrid seeds and parent radicle thereof;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and EST-SSR primers HE L I2:
the PCR reaction system is 10 mu L, which comprises 10 × PCR Buffer 1 mu L, 0.2mM dNTPs, 100ng DNA template, 5 mu M primers, 0.5U Taq DNA polymerase and sterile ultrapure water which is filled to 10 mu L;
the amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 20s, and 35 cycles; final extension at 72 deg.C for 5 min; the PCR product was stored at 10 ℃;
(3) detecting amplification products, namely mixing the amplification products of 5 mu L with a sample loading buffer solution of 2 mu L, carrying out non-denaturing polyacrylamide gel electrophoresis with the mass-volume ratio of acrylamide of 8 percent, carrying out 180v constant voltage electrophoresis for 1.5h, and carrying out silver staining and color development for band type statistics;
wherein the mass ratio of acrylamide to methylene bisacrylamide in the adopted non-denatured polyacrylamide gel is 29: 1;
(4) the purity of the hybrid of 'cucumber stock joint force two' is identified according to the amplification result of the specific spectral band, and the judgment standard is as follows:
the female parent has 177bp specific band, and the nucleotide sequence is shown as SEQ ID No. 4; the male parent has a 159bp specific band, and the nucleotide sequence of the band is shown as SEQ ID No. 3; the hybrid has two specific bands of 159bp and 177 bp;
according to the identification result, the purity (%) of the two varieties of the combined force of the cucumber stocks can be converted to × 100% which is (1-N/N), wherein N is the number of the stocks to be detected, and N is the number of plants which have the spectrum band characteristics of the male parent alone or the female parent alone or are different from the spectrum band characteristics of the 'two combined force of cucumber stocks' or the parental spectrum band characteristics.
3. The method for rapidly identifying the cucumber rootstock joint strength secondary purity by using the molecular marker as claimed in claim 2, which is characterized in that:
the loading buffer solution adopted in the step (3) is selected from 10 × glycerol gel sample solution.
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Denomination of invention: A method for rapidly identifying the purity of Heli II of Cucumber Rootstock by molecular marker Effective date of registration: 20211224 Granted publication date: 20200721 Pledgee: Weifang Bank Co.,Ltd. Qingzhou Qicheng sub branch Pledgor: Huasheng Agricultural Group Co.,Ltd. Registration number: Y2021980016501 |
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