CN112961931B - Rapid identification method for purity of Yongtian No.5 melon seeds - Google Patents
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Abstract
The invention belongs to the technical field of seed purity identification, and provides a rapid identification method for the purity of Yongtian No.5 melon seeds, which comprises the steps of carrying out PCR amplification on seedling DNA of Yongtian No.5 melon single plants by using three pairs of specific primers including SSR1, SSR2 and SSR 3; the sequence information of the SSR1, SSR2 and SSR3 specific primers is as follows: SSR1: SEQ ID NO.1 and SEQ ID NO.2; SSR2: SEQ ID NO.3 and SEQ ID NO.4; SSR3: SEQ ID NO.5 and SEQ ID NO.6. The identification result of the method is accurate and reliable, is not influenced by the environment, and can better ensure the purity of the Cucumis melo No.5 seeds.
Description
Technical Field
The invention belongs to the technical field of seed purity identification, and particularly relates to a method for quickly identifying the purity of Yongtian No.5 melon seeds.
Background
Seed purity is an important index for evaluating the quality of crop seeds, and causes that the seed purity does not reach the standard mainly include low parent purity, mechanical mixing, insufficient pollination isolation and the like (Li Yilong. The research status and development of seed purity. Yangtze river vegetables. 2011.18. Once the seeds with the purity not up to the standard are sold and used, great economic loss can be caused to farmers, and disputes are brought to seed production and sales departments. Therefore, the seed purity test is an essential and important step in the seed production work, and is also a problem which needs to be highly regarded by the seed production department.
Generally, the identification method of seed purity is determined by observing phenotypic characters of seed, plant and fruit of crops (Li Shujuan. Summary of seed purity identification method. University of Qinghai. 2003.21 (1): 16-19). These methods, which rely on phenotyping for purity, are generally time consuming, labor intensive, environmentally sensitive and unreliable. Moreover, for phenotype characteristics with unobvious differences, the identification result is seriously interfered by human factors. The efficiency and the identification result of the existing seed purity identification method are still to be improved.
The melon has high economic benefit, the yield per mu is 5000-6000 yuan when the melon is planted in a climbing way, the heterogeneity of the melon is enhanced if the purity of seeds is reduced, the quality of fruits is reduced, the yield is reduced, and the disastrous economic loss in the production, storage and sale processes can be caused. Yongtian No.5 is a new melon variety with high quality and crisp meat cultivated by Ningbo city agricultural science research institute, and obtains new plant variety right (CNA 20121325.8), and the white skin orange meat has the advantages of fine taste, high quality, easy cultivation and wide adaptability. The method has been popularized to Zhejiang, jiangsu, shandong, guangdong, xinjiang and other places through radiation, has wide application prospect, and is continuously listed as the leading melon and vegetable variety in Zhejiang province. With the continuous expansion of planting area of Yongtian No.5, the seed sale and supply are not required. The conventional phenotypic seed identification method has low efficiency and poor accuracy, and is difficult to meet the requirement of seed purity detection in the rapid mass seed production and sale processes.
The SSR (Simple Sequence Repeat) molecular marker technology is Simple to operate, has good repeatability, meets the identification criteria of crop seed purity, and is already used on the melon, for example, saligla et al, the SSR marker is used for identifying the seed purity of melon hybrid seeds, molecular plant breeding 2006.32 (7): 19-22; the purity of muskmelon hybrid seeds is identified by utilizing SSR molecular markers, zhejiang agricultural science 2016.57 (2): 178-181, patent ZL2014107372664 discloses a rapid detection method for purity of cantaloupe seeds of a melon variety, but the types of cantaloupe are rich, genetic backgrounds are complex, and hybrid seeds prepared by parents with different genetic backgrounds need to be subjected to SSR molecular marker screening on parent materials to obtain purity identification of the hybrid seeds prepared by the parents.
In view of the above, the present invention is specifically proposed.
Disclosure of Invention
The invention provides a method for rapidly identifying the purity of Yongtian No.5 melon seeds, which comprises the steps of carrying out PCR amplification on seedling DNA of Yongtian No.5 melon single plants by using three pairs of specific primers including SSR1, SSR2 and SSR 3;
the sequence information of the SSR1, SSR2 and SSR3 specific primers is respectively as follows:
SSR1: SEQ ID No.1 and SEQ ID No.2;
SSR2: SEQ ID NO.3 and SEQ ID NO.4;
SSR3: SEQ ID NO.5 and SEQ ID NO.6.
As an embodiment, the identification method further comprises the step of amplifying the identified impure parent by using the SSR1, SSR2 or SSR3 specific primer, and improving the purity of the parent.
In one embodiment, when the hybrid strain shows the polymorphic site of the female parent P1 and the corresponding site of the male parent P2 is not pure, the impure male parent P2 is amplified by SSR1, SSR2 or SSR3 primers, and a single strain different from the P2 polymorphic site is eliminated.
As an embodiment, the amplification product of each individual is subjected to agarose electrophoresis separation and ethidium bromide staining, and then polymorphism is detected; and judging whether the single plant is a hybrid or not by observing the difference characteristics of the amplified products at the marked polymorphic sites.
As an embodiment, the PCR amplification procedure is: pre-denaturation at 94 ℃ for 1min; denaturation at 93 deg.C for 1min, annealing at 50 deg.C for 1min, and extension at 72 deg.C for 2min (40 cycles); extension at 72 ℃ for 1min.
As one embodiment, the identification method comprises the steps of:
(1) Sampling
Sowing Yongtian No.5 seeds, collecting 1g of new leaves when cotyledons of plants are flat and a first new leaf is unfolded, and storing at-80 ℃;
(2) DNA extraction
Extracting the total genomic DNA of the fresh leaves by adopting a CTAB method;
(3) PCR amplification
The specific primers SSR1, SSR2 and SSR3 are respectively and independently mixed and used for PCR amplification;
the sample mixing system for each pair of SSR specific primers was 15. Mu.l, including 1.5. Mu.l dNTP (2.5 mM), 1.5. Mu.l 10 XBuffer, 1.8. Mu.l MgCl 2 (25 mM), 2ul of primers (5 mM; 1.0u1 added to each of the two sequences of each pair of SSR-specific primers), 5ul of DNA (10 ng/U1), 0.05ul of Taq (1U), and 3.145ul of ultrapure water; the PCR amplification procedure was: pre-denaturation at 94 ℃ for 1min; denaturation at 93 deg.C for 1min, annealing at 50 deg.C for 1min, and extension at 72 deg.C for 2min (40 cycles); extending at 72 ℃ for 1min;
(4) PCR product detection
Using 2.5% high-resolution standard gel agarose, adding 20 mul ethidium bromide for color development, adding 2 mul Loading Dye into a PCR product, carrying out electrophoresis for 2-3 hours at a voltage of 200v, and observing the result by using a gel imaging system after the electrophoresis is finished;
(5) Collecting and analyzing data;
(6) And calculating the purity of the seeds.
Further, as an embodiment, the identification method further includes the steps of:
(7) Parent purity enhancement
And comparing the amplification band type of the detected Yongtian No.5 muskmelon hybrid strain with the parent band type, and when the hybrid strain is judged to be expressed as a polymorphic site of the female parent P1 and the corresponding site of the male parent P2 is impure, amplifying the impure male parent P2 by using SSR1, SSR2 or SSR3 primers, removing a single strain with a polymorphic site different from the P2, and improving the parent purity.
Compared with the prior art, the technical scheme of the invention has the following advantages:
1. the purity identification method of the Yongtian No.5 melon seed simultaneously uses three pairs of specific primers including SSR1, SSR2 and SSR3 to carry out PCR amplification, obviously improves the detection rate of the mixed plants, and compared with the conventional morphological identification, the method can accurately identify the Yongtian No.5 melon seed, has reliable identification result and no environmental influence, and can better ensure the purity of the Yongtian No.5 melon seed.
2. The identification method can further judge and know whether the purity of the parent is problematic according to the identification result of the molecular marker purity, and can purify the parent in a targeted manner by utilizing the specific primers SSR1, SSR2 and SSR3, thereby ensuring the purity of the prepared seed from the source.
3. The identification method can be used for carrying out identification work in the seedling stage and even the germination stage, the time consumption is short, the result can be obtained after about 1 week, the operation is simple, the method is convenient and quick, and manpower, material resources and land data are saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the amplification of 3 pairs of specific SSR primers in 80 Yongtian No.5 single strains and parents.
Wherein, (A) is the amplification of SSR1 in Yongtian No.5 single plant and parents, (B) is the amplification of SSR2 in Yongtian No.5 single plant and parents, and (C) is the amplification of SSR3 in Yongtian No.5 single plant and parents; m:50bp ladder;1-80: the Yongtian No.5 single plant is numbered; p1: yongtian No.5 female parent; p2: yongtian No.5 male parent.
FIG. 2 shows the amplification of SSR1 primers in 15 Yongtian No.5 male parents and homozygous male parent control (P2).
Wherein, M:50bp ladder;1-15: the Yongtian No.5 male parent is numbered individually; p2: yongtian No.5 homozygous male parent.
FIG. 3 shows the amplification of SSR2 specific primers in 15 Yongtian No.5 male parents and homozygous male parent control (P2).
Wherein, M:50bp ladder;1-15: the Yongtian No.5 male parent is numbered individually; p2: yongtian No.5 homozygous male parent; horizontal arrow: the position of the male parent differential locus and the size of the fragment; vertical arrow: and marking the detected hybrid plants which are inconsistent with the homozygous male parent banding pattern.
FIG. 4 shows the comparison amplification of SSR3 specific primers in 10 Yongtian No.5 female parents, 6 Yongtian No.5 male parents, homozygous female parent (P1) and homozygous male parent (P2).
Wherein, M:50bp ladder;1-10: "Yongtian No. 5" female parent single plant number; 11-16: "Yongtian No. 5" male parent is numbered individually; p1: yongtian No.5 homozygous female parent; p2: yongtian No.5 homozygous male parent;
black horizontal arrow: the position of the maternal differential site and the size of the fragment; gray horizontal arrow: the position of the male parent differential site and the size of the fragment; no hybrid plants that did not match the homozygous parental banding pattern were detected.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention.
The invention provides a method for rapidly identifying the purity of Yongtian No.5 melon seeds, which utilizes 3 pairs of specific SSR molecules to carry out marking identification, wherein the specific information of the 3 pairs of specific SSR molecules is shown in the following table 1.
TABLE 1
The rapid identification method mainly comprises the following steps: extracting young leaf DNA of each single plant of Yongtian No.5 commercial variety by adopting a CTAB method; performing PCR amplification on the specific SSR primers by using 3 pairs of primers shown in the table 1; the amplified products of each individual plant were separated by agarose electrophoresis and stained with ethidium bromide, and then polymorphisms were detected on an ultraviolet transilluminator. By observing the difference characteristics of the amplified products at the marked polymorphic sites, whether the single plant is a hybrid can be judged. Only a single plant having both parental polymorphic sites is determined to be a true hybrid, and only a single plant having only one parental polymorphic site is determined to be a hybrid.
When more single plants with one of the polymorphic sites of the parents appear in the seeds to be detected, the site of the other parent is not homozygous, and the heterozygous site of the hybrid cannot be effectively heterozygous. The 3 pairs of specific SSR primers can be used for amplifying impure parents and rejecting single plants different from the polymorphic sites of the parents, thereby ensuring the purity of the parents and finally being beneficial to improving the purity of hybrid seeds.
Reference will now be made to a specific embodiment of the present invention to facilitate a better understanding of the invention.
Example 1 method for rapidly identifying purity of Yongtian No.5 melon seeds
(1) Sampling
Sowing 80 seeds of Yongtian No.5 commercial variety, collecting 1g of new leaves when the cotyledon of the plant is flat and the first new leaf is unfolded, and storing at-80 ℃.
(2) DNA extraction
With reference to the CTAB method (Murray HG, thompson wf. Rapid isolation of high light DNA. Nucleic Acids Res,1980,8 4321), fresh leaf total genomic DNA was extracted.
(3) PCR amplification
The specific primers SSR1, SSR2 and SSR3 are respectively and independently mixed and used for PCR amplification;
the sample mixing system for each pair of SSR specific primers was 15. Mu.l, and included 1.5. Mu. LdNTP (2.5 mM), 1.5. Mu.l 10 XBuffer, 1.8. Mu.l MgCl 2 (25 mM), 2ul of primers (5 mM; 1.0u1 added to each of the two sequences of each SSR-specific primer), 5ul of DNA (10 ng/U1), 0.05ul of Taq (1U), and 3.145ul of ultrapure water.
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 1min; denaturation at 93 deg.C for 1min, annealing at 50 deg.C for 1min, and extension at 72 deg.C for 2min (40 cycles); extension at 72 ℃ for 1min.
(4) PCR product detection
PCR products obtained by amplifying SSR1, SSR2 and SSR3 can be electrophoresed together.
2.5% high resolution standard gel agarose (DNA fragment separation range 40-1000 bp) was used. Add 20. Mu.l EB (ethidium bromide ) for color development. Adding 2 mu l of loading Dye into PCR products obtained after the SSR1, the SSR2 and the SSR3 are amplified, carrying out electrophoresis for 2-3 hours, and carrying out voltage 200v. After electrophoresis, the results were observed using a gel imaging system.
(5) Data collection and analysis
Only a single plant with both parents 'polymorphic sites is judged as a true hybrid (pure Yongtian No. 5), and a single plant with only one parents' polymorphic sites is judged as a hybrid, and the analysis of the results is shown in FIG. 1 and Table 2.
FIG. 1 shows the amplification of 3 pairs of specific SSR primers in 80 Yongtian No.5 single strains and parents.
Wherein (A) is the amplification of SSR1 in Yongtian No.5 single plant and parents, (B) is the amplification of SSR2 in Yongtian No.5 single plant and parents, and (C) is the amplification of SSR3 in Yongtian No.5 single plant and parents.
The relevant labels in the above figures are illustrated as follows:
horizontal arrow at 270bp in FIG. 1 (A): the position of the male parent differential site and the size of the fragment.
Horizontal arrow at 180bp in FIG. 1 (B): the position of the male parent differential locus and the size of the fragment.
Horizontal arrow at 120bp in FIG. 1 (C): the position of the maternal difference site and the size of the fragment.
Horizontal arrow at 180bp in FIG. 1 (C): the position of the male parent differential locus and the size of the fragment.
Black vertical arrow: and marking the detected hybrid strains consistent with the maternal banding patterns.
Gray vertical arrow: and marking the detected hybrid plants with the same paternal banding patterns.
Table 2 shows the number and the number of the selected hybrid strains of 3 pairs of specific SSR primers in 80 Yongtian No.5 single strains.
TABLE 2
(6) Calculation of purity
The seed purity of the batch of 'Yongtian No. 5' is calculated according to the formula purity = [1- (number of hybrid plants/total number of tests) ] multiplied by 100%.
In the test, 15 mixed plants are detected, and the purity = [1- (15/80) ] × 100% is calculated through a formula, so that the purity of the sweet 5 seed in the batch is 81.25%, and the rate of the mixed plants is 18.75%.
Example 2 method for rapidly identifying purity of Yongtian No.5 melon seeds
The embodiment further includes a step 7 based on embodiment 1, which is specifically as follows:
(7) Parent purity detection
Comparing the amplification banding pattern of the detected Yongtian No.5 hybrid plant with the banding pattern of the parent plant, and according to the comparison result, the hybrid plant shows the polymorphic locus of the female parent P1, which shows that the locus of the male parent P2 is not homozygous, and the heterozygous locus of the hybrid can not be effectively heterozygous.
The SSR1 primers are used for amplifying impure male parent P2 (15 strains), single strains different from P2 polymorphic sites are removed, the parent purity is ensured, and the hybrid purity is improved, which is shown in figure 2.
FIG. 2 is the amplification of SSR1 primers in 1 "Yongtian No. 5" male parent and homozygous male parent control (P2); wherein, M:50bp ladder;1-15: "Yongtian No. 5" male parent is numbered individually; p2: yongtian No.5 homozygous male parent.
The relevant labels in fig. 2 are illustrated below: horizontal arrow at 270 bp: the position of the male parent differential site and the size of the fragment.
Vertical arrow: and marking the detected hybrid plants which are inconsistent with the homozygous male parent banding pattern.
Three specific primers SSR1, SSR2 and SSR3 can be used for identifying the parent purity, and the above embodiment only takes SSR1 as an example and is not limited only.
Referring to the operation process of example 2, the PCR amplification results of using SSR2 and SSR3 to identify parent purity are shown in fig. 3 and 4.
FIG. 3 is the amplification of SSR2 specific primers in 15 Yongtian No.5 male parents and homozygous male parent control (P2).
Wherein, M:50bp ladder;1-15: the Yongtian No.5 male parent is numbered individually; p2: yongtian No.5 homozygous male parent; horizontal arrow: the position of the male parent differential locus and the size of the fragment; vertical arrow: and marking the detected hybrid plants which are inconsistent with the homozygous male parent banding pattern.
FIG. 4 shows the comparison amplification of SSR3 specific primers in 10 Yongtian No.5 female parents, 6 Yongtian No.5 male parents, homozygous female parent (P1) and homozygous male parent (P2).
Wherein, M:50bp ladder;1-10: "Yongtian No. 5" female parent single plant number; 11-16: the Yongtian No.5 male parent is numbered individually; p1: yongtian No.5 homozygous female parent; p2: yongtian No.5 homozygous male parent; black horizontal arrow: the position of the maternal differential site and the size of the fragment; grey horizontal arrow: the position of the male parent differential site and the size of the fragment; no hybrid plants that did not match the homozygous parental banding pattern were detected.
In conclusion, the embodiment of the invention utilizes the pair 3 of specific SSR molecules to realize the effect of rapidly identifying the seed purity, and can further solve the problem of how to improve the seed purity from the source, thereby achieving multiple purposes and having higher practical application value.
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 derived therefrom are intended to be within the scope of the invention.
Sequence listing
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Rapid identification method for purity of Yongtian No.5 melon seeds
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Claims (5)
1. The method for rapidly identifying the purity of the Yongtian No.5 melon seed is characterized by comprising the steps of carrying out PCR amplification on seedling DNA of a single Yongtian No.5 melon plant by using three pairs of specific primers including SSR1, SSR2 and SSR 3;
the sequence information of the SSR1, SSR2 and SSR3 specific primers is respectively as follows:
SSR1: SEQ ID NO.1 and SEQ ID NO.2;
SSR2: SEQ ID NO.3 and SEQ ID NO.4;
SSR3: SEQ ID NO.5 and SEQ ID NO.6;
the method comprises the following steps:
(1) Sampling
Sowing Yongtian No.5 seeds, collecting 1g of new leaves when the cotyledon of the plant is flat and the first new leaf is unfolded, and storing at-80 ℃;
(2) DNA extraction
Extracting fresh leaf total genome DNA by a CTAB method;
(3) PCR amplification
The specific primers SSR1, SSR2 and SSR3 are respectively and independently mixed and used for PCR amplification;
the mixed sample system of each pair of SSR specific primers is 15 mu l and comprises 1.5 mu ll dNTP at a concentration of 2.5mM, 1.5ul 10 XBuffer, 1.8ul MgCl at a concentration of 25mM 2 2ul of 5mM primers, wherein 1.0ul of DNA with a concentration of 10ng/ul, 1U of Taq 0.05ul and 3.145ul of ultrapure water are added to the two sequences of each pair of SSR specific primers;
the PCR amplification procedure was: pre-denaturation at 94 ℃ for 1min; denaturation at 93 deg.C for 1min, annealing at 50 deg.C for 1min, extension at 72 deg.C for 2min, and performing 40 cycles; extending at 72 ℃ for 1min;
(4) PCR product detection
Adding 20 mu l of ethidium bromide into 2.5 percent high-resolution standard gel agarose for color development, adding 2 mu l of loading Dye into a PCR product, carrying out electrophoresis for 2-3 hours at a voltage of 200v, and observing a result by using a gel imaging system after the electrophoresis is finished;
(5) Collecting and analyzing data;
(6) And calculating the purity of the seeds.
2. The method for rapidly identifying the purity of the Cucumis melo seed as claimed in claim 1, which is characterized in that the method further comprises the step of amplifying the identified impure parent by using the SSR1, SSR2 or SSR3 specific primer and improving the purity of the parent.
3. The method for rapidly identifying the purity of the Cucumis melo No.5 seed according to claim 2, wherein when the impure plant is expressed as the polymorphic site of the female parent P1 and the corresponding site of the male parent P2 is impure, the impure male parent P2 is amplified by the SSR1, SSR2 or SSR3 primer, and a single plant different from the polymorphic site of P2 is removed.
4. The method for rapidly identifying the purity of the Cucumis melo No.5 seeds according to claim 1, wherein the polymorphism is detected after the amplification product of each individual plant is subjected to agarose electrophoresis separation and ethidium bromide staining; and judging whether the single plant is a hybrid or not by observing the difference characteristics of the amplified products at the marked polymorphic sites.
5. The method for rapidly identifying the purity of Cucumis melo No.5 seeds according to claim 1, wherein the identification method further comprises the following steps:
(7) Parent purity enhancement
And comparing the amplification band type of the detected hybrid Cucumis melo No.5 with the band type of the parent, and when the hybrid shows the polymorphic site of the female parent P1 and the corresponding site of the male parent P2 is impure, amplifying the impure male parent P2 by using SSR1, SSR2 or SSR3 primers, and removing the single plant different from the P2 polymorphic site to improve the purity of the parent.
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| CN115812593B (en) * | 2022-12-26 | 2023-10-24 | 宁波市农业科学研究院 | Method for creating anti-premature senility melon germplasm resources |
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| CN105154550A (en) * | 2015-09-11 | 2015-12-16 | 山东省华盛农业股份有限公司 | Method for quickly identifying purity of watermelon variety Zhentian 1217 by using EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker |
| CN109337999A (en) * | 2018-10-31 | 2019-02-15 | 宁夏泰金种业股份有限公司 | A kind of identification method of the muskmelon hybrid seed purity based on SSR marker method |
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| CN108531637B (en) * | 2018-03-13 | 2022-08-30 | 新疆农业科学院哈密瓜研究中心 | Method for high-throughput identification of Xinjiang muskmelon yellow skin 9818 hybrid purity based on SSR molecular marker |
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| CN110331226B (en) * | 2019-06-28 | 2021-02-02 | 北京市农林科学院 | Method for identifying authenticity of melon variety and special SSR primer combination thereof |
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| CN105154550A (en) * | 2015-09-11 | 2015-12-16 | 山东省华盛农业股份有限公司 | Method for quickly identifying purity of watermelon variety Zhentian 1217 by using EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker |
| CN109337999A (en) * | 2018-10-31 | 2019-02-15 | 宁夏泰金种业股份有限公司 | A kind of identification method of the muskmelon hybrid seed purity based on SSR marker method |
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