CN107312827B - SSR molecular marker method for identifying authenticity of conventional cotton varieties - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses an SSR molecular marker method for identifying the authenticity of a conventional cotton variety, which comprises the following steps: preparing a sample; carrying out PCR amplification on the SSR core primer by using 26 percent of genome DNA as a template, and carrying out 8 percent non-denaturing polyacrylamide gel electrophoresis on a PCR product to obtain an electrophoresis pattern of a sample to be detected; and comparing the electropherogram of each sample to be detected with the electropherogram of the obtained standard sample, wherein if the band composition or band type of the sample to be detected is consistent with that of the standard sample, the seed to be detected and the standard seed are of the same variety, and the authenticity of the seed to be detected is obtained. The method has the advantages of stable and reliable result, simple operation, economy and high efficiency, has the advantages of high efficiency, accuracy, time and labor saving, no season limitation and the like compared with a morphological identification method, shows better application prospect, and better serves for identifying and evaluating the authenticity of the conventional cotton variety in production practice.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an SSR molecular marker method for identifying the authenticity of a conventional cotton variety.
Background
The method can accurately and quickly identify the varieties of the crops, and has important effects on the aspects of crop variety approval, variety protection, true and false variety identification, variety property dispute and the like. For a long time, the authenticity identification of cotton varieties at home and abroad is based on morphology, and specifically comprises seed morphology identification, seedling identification and field plot planting identification. The morphological identification of the seeds is to judge the authenticity of the varieties according to the morphological characteristics of the seeds, such as the fiber length and the uniformity of different cotton varieties, the quantity and the existence of short fibers, the shapes and the colors of the cotton seeds and the like; the seedling identification method mainly measures some quantitative characters, and determines the number of the variety and the seedlings of the hybrid plants according to the characteristics of the color of the leaves, the shape of the leaves, the veins, the villi on the leaves and the like of the cotton seedlings; when the authenticity of the variety is identified by the field plot planting identification method, seed cotton, ginned cotton and the like are observed and identified at the seedling stage, the bud stage, the boll opening stage and after harvesting, and the evaluation and judgment are carried out according to the characteristic characteristics of plants, leaves, flowers and bolls of the cotton and the fiber quality index.
In summary, the problems of the prior art are as follows:
the morphological identification method of the seeds is easily influenced by various production and cultivation conditions, the characters for identification are very limited, and the identification is difficult when the variety difference is small and the quantity is large. The method is limited in application and can only be used as a reference index; the seedling identification method has an expanded application range, but the method mainly measures certain quantitative characters, can only be used for identifying the authenticity of varieties and cannot be used for identifying heterotypic plants; when the authenticity of the variety is identified by the field plot planting identification method, the defects of long period, labor and time waste, influence of the investigation characters by the cultivation conditions and environmental factors, large artificial observation error and the like exist. And the detection result is relatively lagged, the time efficiency and the reliability of the detection result are limited to a certain extent, and the quality problem of seeds on the market and disputes such as variety infringement are difficult to monitor in time.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an SSR molecular marker method for identifying the authenticity of a conventional cotton variety.
The invention is realized in such a way that an SSR molecular marker method for identifying the authenticity of a conventional cotton variety comprises the following steps:
preparation of a sample: detecting 15 single plants in each sample, mixing DNA of 5 single plants in equal amount, and preparing 3 samples for SSR analysis;
performing PCR amplification on the SSR core primer by using 26 percent of extracted genome DNA as a template, and performing 8 percent non-denaturing polyacrylamide gel electrophoresis on the obtained PCR amplification product to obtain an electrophoresis pattern of a sample to be detected;
and comparing the electropherogram of each sample to be detected with the electropherogram of the obtained standard sample, wherein if the band composition or band type of the sample to be detected is consistent with that of the standard sample, the seed to be detected and the standard seed are of the same variety, and the authenticity of the seed to be detected is obtained.
For 3 groups of mixed samples with inconsistent spectral bands at the same site, further analyzing 15 individual plant spectrums forming the mixed samples, and determining non-product materials;
judging the result of the cotton variety authenticity identification: when the authenticity of the cotton variety is identified, the basic core marker is analyzed by using 26, and the authenticity of the detected sample is judged according to the difference and identity of the mixed sample electrophoretic band of the sample to be detected and the standard control sample.
Further, the modified CTAB method is combined with an automatic sample grinding machine to quickly grind the sample, and the genome DNA of each individual plant is extracted.
Further, PCR amplification of SSR core primers comprises:
selecting a reaction volume of 10 μ L; the method comprises the following steps:
60ng of the DNA template of μ L-1, 1 μ L,
4pmol of each of the forward and reverse primers, 0.4. mu.L/. mu.L-1,
10×Buffer 1μL，
2.5mmol of L-dNTP 0.8. mu.L,
5U. mu.L-1 of Taq enzyme 0.1. mu.L and ddH2O 6.3.3. mu.L were amplified on a PCR amplifier.
Further, the reaction procedure of PCR amplification includes:
pre-denaturation at 95 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 45s for 35 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
Further, the detection of the PCR amplification product comprises:
the SSR amplification products are detected by 8% non-denaturing polyacrylamide gel electrophoresis, and the SSR fragments separated by the electrophoresis are dyed by a silver staining method.
Further, comparing the electropherogram of each sample to be detected with the electropherogram of the obtained standard sample, specifically comprising:
when the authenticity of cotton varieties is identified, carrying out data recording by taking the whole banding patterns amplified by each pair of primers in different varieties as a unit; the most common and fixed combined type band types of 3 to 6 band types exist, namely, a plurality of bands of the band types always exist and disappear at the same time, and the difference of the overall band types of the standard control and the variety to be detected is compared according to the overall band types amplified by each pair of primers in the variety to be detected and the standard control.
Further, the result of the authenticity identification of the cotton variety specifically comprises the following steps:
when the authenticity of the cotton variety is identified by using 26 core markers, judging the cotton variety to be different when 3 or more than 3 difference markers exist between the standard sample and the sample map to be detected;
when the difference marks between the two varieties of mixed sample maps are less than or equal to 2, the two varieties of mixed sample maps are judged to be similar varieties or the same varieties.
Further, the 26 pairs of SSR core primers are subjected to preliminary screening on 586 pairs of polymorphic primers which are positioned on a chromosome by 8 parts of materials with larger genetic difference, and then 120 parts of widely representative germplasm resources are used for secondary screening, so that 26 pairs of core primers with high polymorphism and clear banding patterns are finally determined; 26 pairs of core primers are uniformly distributed on 26 chromosomes of cotton, 138 allelic variations are detected on 120 parts of rescreened materials, the average number of SSRs is 5.31, the variation range is 2-9, 129 sites with polymorphism are provided, and the polymorphism rate is 93.48%; the 26 pairs of core primers have good identification capability on the whole, and can be widely applied to identification of cotton varieties.
The invention has the advantages and positive effects that:
the invention provides a method for identifying the authenticity of a conventional cotton variety. Experiments prove that the method has the advantages of stable and reliable results, simple operation, economy and high efficiency, has the advantages of high efficiency, accuracy, time and labor saving, no seasonal limitation and the like compared with a morphological identification method, shows better application prospect, and better serves for identifying and evaluating the authenticity of conventional cotton varieties in production practice. Therefore, the SSR core marker and the conventional cotton variety authenticity identification method have wide application prospects in the field of cotton variety identification, and have reference function in identification research of other related crop varieties.
Drawings
FIG. 1 is a flow chart of an SSR molecular marker method for identifying the authenticity of a conventional cotton variety, which is provided by the embodiment of the invention.
FIG. 2 is an amplification map of primer NAU2083 provided by the embodiment of the invention in 3 sets of mixed samples of standard sample and 10 test samples.
FIG. 3 is a 15 individual maps of primer NAU2083, which constitutes B1, B2, B3, F1, F2 and F3, provided in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings
In the SSR molecular marker method for identifying the authenticity of the conventional cotton variety, provided by the embodiment of the invention, the screening and verification of the core primers are as follows:
the invention takes 8 cotton varieties with larger genetic difference and wide representativeness as sample materials, preliminarily screens 586 pairs of primers published in a cotton molecular marker database, verifies the primers obtained by preliminary screening by 120 germplasm resources, and finally establishes 26 pairs of primers uniformly distributed on a cotton chromosome as the first-choice core primers for identifying the cotton varieties by combining the distribution condition of SSR primers on the chromosome (attached table 1).
TABLE 126 SSR core primer information
| Numbering | Primer name | Chromosomal location | fwd_Sequence5'_3' | rev_Sequence5'_3' |
| 1 | NAU2083 | ch01 | AGAAGAGGTTGACGGTGAAG | TGAGTGAAGAACCTGCACAT |
| 2 | NAU2277 | ch02 | GAACTAGCCACATGATGCAC | TTGTTGAGGCATTAGTTTGC |
| 3 | NAU1071 | ch03 | ACCAACAATGGTGACCTCTT | CCCTCCATAACCAAAAGTTG |
| 4 | BNL530 | ch04 | CGTAGGATGGAAACGAAAGC | GCCACACTTTTCCCTCTCAA |
| 5 | NAU1200 | ch05 | CAACAGCAACAACCACAA | CTGCCTCGAGGACAAATAGT |
| 6 | CGR5651 | ch06 | TTTGGCTTAGCATTTGGAGG | CCGATCACTGTCCGTCTCTT |
| 7 | BNL1694 | ch07 | CGTTTGTTTTCGTGTAACAGG | TGGTGGATTCACATCCAAAG |
| 8 | DPL0111 | ch08 | CTTTCATAATACATACGCCTTGCC | TCACAGCATCCTATCAGGTATCAG |
| 9 | CGR5707 | ch09 | AAACCCGATATCCTTAGCCTTT | GGAAAGGAGGAAGAGGAGGA |
| 10 | NAU879 | ch10 | AGGAACCGATTCAAAGCTAA | TTTCCCCATTCTTGGTTAAG |
| 11 | BNL3442 | ch11 | CATTAGCGGATTTGTCGTGA | AACGAACAAAGCAAAGCGAT |
| 12 | BNL598 | ch12 | TATCTCCTTCACGATTCCATCAT | AAAAGAAAACAGGGTCAAAAGAA |
| 13 | CGR5576 | ch13 | CGGTTCAACCCGACTGTTT | GAGGAAAGAAAGGAAGAGAGGG |
| 14 | CIR246 | ch14 | TTAGGGTTTAGTTGAATGG | ATGAACACACGCACG |
| 15 | NAU3736 | ch15 | CATGTGCATTTCATCCTGTC | CCAAGTGAGAGGCATTTTCT |
| 16 | MUSS95 | ch16 | GCAACCATTAATTAAGCAAGTAACAA | CGAAGAATATGTGAACCTACAGAAAC |
| 17 | HAU1413 | ch17 | CTGACTTGGACCGAGAACTT | AACCAGGACCGATGAAATAA |
| 18 | TMB2295 | ch18 | TGAGTTCATGTTCCCCACTG | CTAAACATACTCTGTCAAACAC |
| 19 | BNL3977 | ch19 | ATCCAAACCAACCATGCAAT | GAAGGGGTTTTGCATTTCAA |
| 20 | JESPR190 | ch20 | GCCCGCCATCTTTGAGGATCCG | GGCAAAACTTGACAATTTTCTCGGC |
| 21 | JESPR158 | ch21 | CACCATTCGGCAGCTATTTC | CTGCAAACCCTAGCCTAGACG |
| 22 | NAU2026 | ch22 | GAATCTCGAAAACCCCATCT | ATTTGGAAGCGAAGTACCAG |
| 23 | JESPR13 | ch23 | GCTCTCAAATTGGCCTGTGT | GGTGGAGGCATTCCTGCTAAC |
| 24 | BNL3452 | ch24 | TGTAACTGAGCAGCCGTACG | GCCAAAGCAGAGTGAGATCC |
| 25 | BNL3937 | ch25 | ACATCAAACAAAGCAAGCCA | ATCTCTGTTTTCTCCCCCGT |
| 26 | NAU1042 | ch26 | CATGCAAATCCATGCTAGAG | GGTTTCTTTGGTGGTGAAAC |
Wherein, the Sequence of fwd _ Sequence5'_3' core primer of NAU2083 is recorded as: SED ID NO: 1.
as shown in fig. 1, the SSR molecular marker method for identifying the authenticity of a conventional cotton variety provided in the embodiment of the present invention includes the following steps:
s101, preparing a sample: 15 individuals were detected for each sample, and 5 individuals were mixed in equal amounts to prepare 3 samples (A1, A2, A3) for analysis;
s102, performing PCR amplification on the SSR core primer by using the extracted genome DNA as a template and 26 percent of non-denatured polyacrylamide gel electrophoresis on the obtained PCR amplification product to obtain an electrophoresis pattern of a sample to be detected;
s103, comparing the electropherogram of each sample to be detected with the electropherograms of the standard samples (X1, X2 and X3) obtained by the methods of S101 and S102, wherein if the band compositions (band types) of the sample to be detected and the standard samples are consistent, the seed to be detected and the standard seeds are of the same variety, and obtaining the authenticity of the seed to be detected.
S104, further analyzing 15 individual maps forming the mixed sample for 3 groups of mixed samples with inconsistent spectral bands on the same site, and determining non-product materials.
S105, judging the result of the cotton variety authenticity identification: when the authenticity of the cotton variety is identified, the basic core marker is analyzed by using 26, and the authenticity of the detected sample is judged according to the difference and identity of the mixed sample electrophoretic band of the sample to be detected and the standard control sample.
If there are a total of 26 SSR markers:
a) the number of the difference marks between the detected sample and the standard control sample is more than 2, and the samples are judged to be different varieties;
b) the difference mark number of the detected sample and the standard control sample is less than or equal to 2, and the detected sample and the standard control sample are interpreted as similar varieties;
c) the difference mark number of the detected sample and the standard control sample is equal to 0, and the detected sample and the standard control sample are judged to be the same or very similar varieties.
According to the authenticity identification scheme, 10 upland cotton varieties (strains) mainly planted in Xinjiang production are identified, and the identification result is completely consistent with the field test result. Experiments prove that the SSR-DNA fingerprint technology can quickly and accurately identify the authenticity of cotton varieties.
The application of the principles of the present invention will now be described in further detail with reference to specific embodiments.
The PCR amplification and detection steps of the SSR core marker are as follows:
1.1 sample preparation
The test selects the land cotton variety mainly planted in production: the method comprises 10 varieties of source cotton No. 6, New Luzao No. 13, New Luzao No. 26, New Luzao No. 37, New Luzao No. 9, New Luzao No. 37, Lu cotton research No. 28, Zhongmiao No. 35, Zhongmiao No. 41 and Zhongmiao No. 43, wherein 15 individuals are randomly selected from each variety, and the leaf DNAs of 5 individuals are equivalently mixed for SSR analysis.
1.2 extraction of DNA
And (3) rapidly grinding by adopting an improved CTAB method and combining an automatic sample grinder, and extracting the genome DNA of each individual plant. Because the SSR marker has low requirement on the purity of the template DNA, PCR amplification can be directly carried out without purifying the DNA.
1.3 SSR-PCR amplification
1.3.1 reaction System
Reaction volume of 10. mu.L: amplification was performed on a PCR amplification apparatus using 1. mu.L of DNA template (60 ng. mu.L-1), 0.4. mu.L of each of the forward and reverse primers (4 pmol. mu.L-1), 1. mu.L of 10 XBuffer, 0.8. mu.L of dNTP (2.5mmol L-1), 0.1. mu.L of Taq enzyme (5U. mu.L-1) and 6.3. mu.L of ddH2O 6.3.
1.3.2 reaction sequence
Pre-denaturation at 95 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 45s for 35 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C. Since each pair of SSR primers has its specific annealing temperature, the annealing temperature in the amplification procedure is slightly changed according to the actual Tm values of the different primers, and the other procedures are not changed.
1.4 PCR product detection
The SSR amplification products are detected by 8% non-denaturing polyacrylamide gel electrophoresis, and the SSR fragments separated by the electrophoresis are dyed by a silver staining method.
1.5 interpretation of SSR amplification profiles
When the authenticity of cotton varieties is identified, data recording is carried out by taking the whole banding patterns amplified by each pair of primers in different varieties as a unit. The most common and most fixed combined type band types of 3-6 band types exist, namely, a plurality of bands of the band types always exist and disappear at the same time, the difference of the overall band types of the standard control and the variety to be detected can be compared according to the overall band types amplified by each pair of primers in the variety to be detected and the standard control, compared with the common '0' and '1' band recording method, the workload is effectively simplified, and meanwhile, the artificial error can be avoided.
1.6 identification of the authenticity of Cotton varieties
Selecting 10 cotton conventional varieties as objects, numbering the cotton conventional varieties (table 2), randomly selecting 15 original cotton No. 6 varieties, uniformly mixing 5 single plant DNAs into 1 group, and preparing 3 samples (A1, A2 and A3) as standard controls. Preparing 3 samples (X1, X2 and X3) from the sample to be detected according to the method, amplifying the standard sample and the sample to be detected by using 26 pairs of core markers, and judging the authenticity of the sample to be detected by analyzing the difference sites of the 10 samples to be detected and the standard control integral map.
TABLE 2 10 conventional cotton varieties for authenticity identification
Table2 List of 10conventional cotton varieties for authenticity identification
1.6.1 analysis of the differential sites between 3 sets of mixed samples of the same sample
The 3 groups of mixed samples of the same sample are compared with the electrophoresis pattern, wherein the bands of the 3 groups of mixed samples of source cotton No. 6 and middle cotton No. 43 are consistent in 26 marks; there was a 4-13 site band inconsistency between the 3 pools of the remaining 8 varieties (FIG. 2). It was found that 3 pools of each variety showed two amplified banding patterns in sites with inconsistent bands. For 3 mixed samples with inconsistent bands at the same site, 15 individual spectra (FIG. 3) constituting the mixed sample were further analyzed to determine non-product material.
1.6.2 differential site analysis between test and Standard samples
Different cotton variety definitions were set experimentally with 3 difference markers as limits: when 26 core markers are used for identifying the authenticity of cotton varieties, when 3 or more than 3 difference markers exist between the standard sample and the sample map to be detected, the cotton varieties can be identified; when the difference marks between the two varieties of mixed sample maps are less than or equal to 2, the two varieties of mixed sample maps can be interpreted as similar varieties or the same varieties. In 10 test samples, only the sample A1-A3 and the standard sample have the amplification band types completely consistent on 26 sites, and the rest samples have 8-18 difference sites with the standard sample, so that the sample A1-A3 is actually 3 samples of the variety No. 6 of the source cotton, and the identification result is completely consistent with the field test result.
The invention not only realizes the identification of the test sample and the standard sample, but also can distinguish 10 detected samples from each other. The authenticity of the detected sample is detected by using 26 pairs of core primers in a laboratory, the detection can be completed in 1-2 days, and the result is quick and accurate
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A method for identifying the authenticity of a conventional cotton sample by utilizing an SSR molecular marker, which is characterized by comprising the following steps:
preparation of a sample: selecting 15 individual plants from each variety, extracting genome DNA of each individual plant, and mixing 5 individual plant genome DNAs in equal amount to prepare 3 groups of samples for SSR analysis;
performing PCR amplification on the SSR core primer by using 26 percent of extracted genome DNA as a template, and performing 8 percent non-denaturing polyacrylamide gel electrophoresis on the obtained PCR amplification product to obtain an electrophoresis pattern of a sample to be detected; the 26 pairs of SSR core primer information is shown in the following table:
For 3 groups of samples with inconsistent bands at the same site of each variety, further analyzing the electrophoretic spectrum bands of 15 single plants forming the samples, wherein the inconsistent bands are non-product materials;
comparing the electropherogram of each sample to be detected with the electropherogram of the obtained standard control sample, and judging the sample to be detected as a different variety if the difference mark number of the sample to be detected and the standard control sample is more than 2; if the number of the difference markers between the sample to be detected and the standard control sample is more than 0 and less than or equal to 2, judging the sample to be detected to be an approximate variety; if the number of the difference marks between the sample to be detected and the standard control sample is equal to 0, judging the sample to be detected to be the same or extremely similar variety; the standard control sample is source cotton No. 6;
extracting the genome DNA of each individual plant, and quickly grinding the sample by adopting an improved CTAB method and combining an automatic sample grinder;
the total volume of the PCR amplification system is 10 mu L, and the PCR amplification system comprises the following components:
1. mu.L of 60 ng/. mu.L DNA template,
0.4. mu.L each of the forward and reverse primers at 4 pmol/. mu.L,
10×Buffer 1μL，
0.8. mu.L of 2.5mmol/L dNTP,
5U/. mu.L Taq enzyme 0.1. mu.L and ddH2O 6.3μL；
The reaction procedure for PCR amplification was: pre-denaturation at 95 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 45s for 35 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
2. The method for identifying the authenticity of a conventional cotton sample using SSR molecular markers according to claim 1, wherein the PCR amplification product is detected by 8% native polyacrylamide gel electrophoresis, and the electrophoretically separated SSR fragments are stained by silver staining.
3. The method for identifying the authenticity of a conventional cotton sample using SSR molecular markers according to claim 1 wherein said specific step of comparing the electropherogram of each sample to be tested with the electropherogram of the standard control sample obtained comprises:
when the authenticity of the cotton sample is identified, carrying out data recording by taking the whole banding pattern amplified by each pair of primers in different samples as a unit; and comparing the difference of the overall banding patterns of the standard control sample and the sample to be detected according to the overall banding patterns amplified by each pair of primers in the sample to be detected and the standard control sample, wherein the standard control sample is source cotton No. 6.
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