CN113637793A - DNA fingerprint construction of new cotton stone K28 based on SSR marker - Google Patents

DNA fingerprint construction of new cotton stone K28 based on SSR marker Download PDF

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CN113637793A
CN113637793A CN202111079910.XA CN202111079910A CN113637793A CN 113637793 A CN113637793 A CN 113637793A CN 202111079910 A CN202111079910 A CN 202111079910A CN 113637793 A CN113637793 A CN 113637793A
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杨永林
秦江鸿
李豪
杨名
徐媛媛
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Shihezi Institute Of Agricultural Sciences
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Abstract

The invention discloses a DNA fingerprint construction of a new cotton stone K28 based on SSR markers, which comprises the following steps: s1, taking 42 parts of upland cotton variety seeds, and accelerating germination of the cotton seeds; s2, when the cotton seeds germinate, selecting one tender true leaf per plant 7d, placing the young true leaf in a mortar, adding liquid nitrogen into the mortar, grinding the true leaf to obtain a mixture; s3, extracting cotton genome DNA in the true leaf mixture by a CTAB method; s4, comparing 42 groups of experimental data, performing genetic diversity analysis, and establishing a file; preferably: the SSR primers in the S1-S4 are provided by key laboratories of the Cotton institute of agricultural sciences, China. The invention has the beneficial effects that: the variety right of a breeder and the authenticity of the variety are effectively protected; can provide reference basis for the Xinjiang upland cotton breeding work in the future.

Description

DNA fingerprint construction of new cotton stone K28 based on SSR marker
Technical Field
The invention relates to the technical field of cotton, in particular to a DNA fingerprint construction method of a novel cotton stone K28 based on SSR markers.
Background
Cotton is an important fiber crop, and Xinjiang is the largest cotton planting area in China. With the increase of the cotton breeding process in Xinjiang area, a few or single-character derived varieties of new cotton varieties are improved on the basis of original varieties, the shapes of the new cotton varieties are more and more similar, the genetic bases of parents are increasingly narrow, and the cotton varieties are more and more difficult to distinguish completely according to the morphological characters.
The authenticity and the purity of the traditional cotton variety are identified by a field planting method, the time consumption is long, the cost is high, the timeliness is poor, and the influence of environmental factors is easy to realize. Therefore, the DNA fingerprint construction of the novel cotton stone K28 based on SSR markers is provided by the technical personnel in the field to solve the problems in the background technology.
Disclosure of Invention
The invention aims to provide DNA fingerprint construction of the novel cotton stone K28 based on SSR markers, so as to solve the problems in the background technology.
The DNA fingerprint map construction of the new stone K28 of cotton based on SSR markers comprises the following steps:
s1, taking 42 parts of upland cotton variety seeds, and accelerating germination of the cotton seeds.
And S2, when the cotton seeds germinate, selecting one tender true leaf per plant 7d, placing the young true leaf in a mortar, adding liquid nitrogen into the mortar, grinding the true leaf to obtain a mixture.
S3, extracting cotton genome DNA in the true leaf mixture by a CTAB method.
S4, comparing 42 groups of experimental data, performing genetic diversity analysis, and establishing a file.
Preferably: the SSR primers in the S1-S4 are provided by key laboratories of the Cotton institute of agricultural sciences, China.
Preferably: the temperature of the cotton seeds in the S1 is 28 ℃ during pregermination, and the illumination L/D is 12h/12 h.
Preferably: the SSR-PCR amplification conditions are that the concentration of a cotton genome DNA solution extracted by a CTAB method is uniformly diluted to 50 ng.mu.L < -1 >, and a PCR amplification system comprises 1 mu.L of template DNA, 1 mu.L of Buffer (containing MgCl), 0.3 mu.L of dNTPs (10 mmol.L < -1 >), 0.5 mu.L of forward primer F (10 mu mol.L < -1 >), 0.5 mu.L of reverse primer R (10 mu mol.L < -1 >), 0.2 mu.L of Taq enzyme (2.5 U.mu.L < -1 >) and ddH2O6.5 mu.L. The PCR amplification procedure was: (1) pre-denaturation: 5min at 94 ℃; (2) amplification: denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and performing 32 cycles; (3) final extension: 72 ℃ for 10min, and (4) storing the amplification product at 4 ℃.
Preferably: carrying out electrophoresis detection on the amplification product on 8% non-denaturing polyacrylamide gel, loading 1.5 mu L of sample, carrying out 220V voltage electrophoresis for 1h, and carrying out silver staining by using a method such as Zhang Jun (14) after the electrophoresis is finished, wherein the method comprises the following steps of (1) carrying out silver staining (0.1% silver nitrate solution) for 12-15 min; (2) developing (2% sodium hydroxide and 1% formaldehyde) for 5-10 min; (3) rinsing with pure water for 2-3 times, silver staining and developing, and taking a picture by using a camera for recording.
Preferably: each primer is amplified corresponding to different genotypes, and the polymorphism information content of the calculated sites is calculated according to a formula PIC (positive identity) 1-sigma Pi 2And (4) calculating.
Preferably: the established file generates the two-dimension code according to a two-dimension code coding rule of a crop seed label printed by an office of the department of agriculture.
42 parts of terrestrial cotton are selected in experiments and provided by original variety breeding units, and the variety name, the test number and the pedigree information are detailed in the following table:
Figure BDA0003263560720000021
Figure BDA0003263560720000031
Figure BDA0003263560720000041
a plurality of seeds of each experimental variety are taken and placed in a cotton room to germinate in a greenhouse (the temperature is 28 ℃, and the illumination L/D is 12h/12h), 1 tender true leaf is selected from each plant after germination for 7D, the young true leaves are placed in a mortar and ground by liquid nitrogen, and the cotton genome DNA is extracted by using an improved CTAB method. The concentration of the cotton genome DNA solution extracted by the CTAB method is uniformly diluted to 50 ng.mu.L-1, and the PCR amplification system comprises 1 mu.L of template DNA, 1 mu.L of Buffer (containing MgCl), 0.3 mu.L of dNTPs (10 mmol.mu.L-1), 0.5 mu.L of forward primer F (10 mu mol.L-1), 0.5 mu.L of reverse primer R (10 mu mol.L-1), 0.2 mu.L of Taq enzyme (2.5 U.mu.L-1) and 6.5 mu.L ddH2O6. The PCR amplification procedure was: (1) pre-denaturation: 5min at 94 ℃; (2) amplification: denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and performing 32 cycles; (3) final extension: 72 ℃ for 10min, and (4) storing the amplification product at 4 ℃.
Carrying out electrophoresis detection on the amplification product on 8% non-denaturing polyacrylamide gel, loading 1.5 mu L of sample, carrying out 220V voltage electrophoresis for 1h, and carrying out silver staining by a method such as Zhang Jun and the like (14) after the electrophoresis is finished, wherein the steps comprise (1) carrying out silver staining (0.1% silver nitrate solution) for 12-15 min; (2) developing (2% sodium hydroxide and 1% formaldehyde) for 5-10 min; (3) rinsing with pure water for 2-3 times. Silver staining was developed and recorded using a camera.
And numbering the bands amplified by each primer according to the relative position of the SSR primer amplification products on the gel chart. The cotton variety identification reading band standard is that for bands amplified in 53 varieties by the same primer, the band type with most band types is recorded as 1, and the rest are recorded as 2, 3, 4 and the like. The band pattern recorded as 1 was the main band pattern, and the rest were the irregular bands. Sites with a banding pattern different from 1 on a single plant are all non-pure SSR sites. The individual plant containing the non-pure SSR locus is a heterotypic individual plant. Root of herbaceous plant
The 60 SSR primers are uniformly distributed on 26 chromosomes of the genome of the cotton A, D, and 30 pairs of core primers which are clear in amplification band type, easy to distinguish, good in polymorphism and stable are screened out from the chromosomes, and are detailed in the following table. 30 pairs of primers detect 96 kinds of amplification band types in 42 parts of Xinjiang upland cotton varieties, the number of the amplification band types of each pair of primers is between 2 and 7, and the average value is 3.2. And 13 pairs of primers with the polymorphism information content value larger than 0.500 in 30 pairs of SSR primers are used in total, the amplitude variation is 0.260-0.624, and part of molecular markers obtained by screening have higher polymorphism but still have primers with smaller PIC values. In the researches of cotton purity and authenticity detection, variety identification and the like, molecular markers with high polymorphic information content can be preferentially used, as shown in figure 1, polymorphic markers CCRI036 show various different amplification banding patterns in 42 Xinjiang upland cotton varieties, and test results show that the 42 Xinjiang upland cotton varieties have abundant genetic variation.
Among 30 pairs of primers (see the following table), 15 pairs of differential primers exist between Xinshi K28 and Xinluzao No. 46 varieties in the amplified band, 12 pairs of differential primers exist between A1, A8, A11, A12, D2, D5, D8, D11, D12 and D13 varieties respectively, 12 pairs of differential primers exist between Xinshi K28 and Xinluzao No. 7 varieties respectively, chromosome A5, A8, D2, D4, D5, D6, D8, D11 and D13 respectively, the differential primers common to the three materials are shown in FIG. 2, different polymorphic position information among the varieties can be distinguished by selecting the differential combined primers, and a fingerprint of the Xinshi K28 can be constructed according to the information.
Figure BDA0003263560720000051
Figure BDA0003263560720000061
Figure BDA0003263560720000071
Compared with the prior art, the invention has the beneficial effects that:
1. the SSR molecular marker has the advantages of good repeatability, simplicity in operation, co-dominant property and the like, the SSR molecular marker is rich in quantity, covers the whole genome, and is high in revealed polymorphism, and a plurality of crops adopt the SSR molecular marker to construct a DNA fingerprint, so that for example, library construction work is continuously carried out on dozens of crops such as corn, rice, cotton, wheat, soybean and the like, an identification standard based on the SSR marker is established, and the variety right and the variety authenticity of a breeder are effectively protected.
2. In the invention, 30 pairs of primers are screened out as core primers according to the difference of varieties, the primers have high repeatability and good polymorphism, and have more difference bands among different varieties, so that Xinshi K28 can be distinguished, reliable cotton DNA fingerprints can be established according to different primer combinations, and the authenticity and the purity of the K28 variety can be identified. The method has the advantages of stable and reliable detection result, simple and convenient operation and low cost, facilitates the standardization of the technology when the QR two-dimensional code marks of the crop seeds are popularized and used in production, has quick, accurate, stable and reliable detection result, and has great significance for ensuring the long-term large-area popularization and application of the new stone K28 in production. The genetic diversity analysis of the Xinjiang upland cotton variety can provide reference for the Xinjiang upland cotton breeding work in the future.
Drawings
FIG. 1 is a schematic diagram showing the amplification results of upland cotton according to the present invention;
FIG. 2 is a schematic diagram of the electrophoretic amplification of a core primer of part of the novel stone K28 according to the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The DNA fingerprint map construction of the new stone K28 of cotton based on SSR markers comprises the following steps:
s1, taking 42 parts of upland cotton variety seeds, and accelerating germination of the cotton seeds.
And S2, when the cotton seeds germinate, selecting one tender true leaf per plant 7d, placing the young true leaf in a mortar, adding liquid nitrogen into the mortar, grinding the true leaf to obtain a mixture.
S3, extracting cotton genome DNA in the true leaf mixture by a CTAB method.
S4, comparing 42 groups of experimental data, performing genetic diversity analysis, and establishing a file.
The first embodiment is as follows:
selecting Xinshi K28 and Xinluzao No. 46 seeds, accelerating germination of the seeds, then accelerating germination of the seeds, selecting a tender true leaf after accelerating germination and placing the young true leaf in a mortar, adding liquid nitrogen for grinding, extracting cotton genomic DNA in a grinding mixture through CTAB, mixing the cotton DNA with CCRI009 primers, carrying out PCR amplification and recording test information:
example two:
selecting Xinshi K28 and Xinluzao No. 46 seeds, then accelerating germination of the seeds, selecting a tender true leaf after accelerating germination and placing the young true leaf in a mortar, adding liquid nitrogen for grinding, extracting cotton genome DNA in a grinding mixture through CTAB, mixing the cotton genome DNA with a CCRI010 primer, carrying out PCR amplification and recording test information:
in both the first and second embodiments, the DNA fingerprint map of the Xinshi K28 is constructed by SSR markers by the following process method:
s1, taking 42 parts of upland cotton variety seeds, and accelerating germination of the cotton seeds.
S2, when the cotton seeds germinate, selecting one tender true leaf per plant 7d, placing the tender true leaf in a mortar, adding liquid nitrogen into the mortar,
grinding true leaves to obtain a mixture.
S3, extracting cotton genome DNA in the true leaf mixture by a CTAB method.
S4, comparing 42 groups of experimental data, performing genetic diversity analysis, and establishing a file.
Comparative example one:
selecting seeds of Xinluzao No. 46, accelerating germination of the seeds, selecting a young true leaf 7 days after the seeds germinate, putting the young true leaf into a mortar, adding liquid nitrogen for grinding, grinding to obtain a mixture, extracting cotton genome DNA of the mixture by a CTAB method, and mixing the cotton genome DNA with a CCRI009 primer;
there are 15 pairs of differential primers between Xinshi K28 and Xinluzao No. 46 cultivars, which are chromosome A1, chromosome A8, chromosome A11, chromosome A12, chromosome D2, chromosome D5, chromosome D8, chromosome D11, chromosome D12, and chromosome D13.
Figure BDA0003263560720000091
Comparative example two:
selecting No. 7 Xinluzao seeds, accelerating germination of the seeds, selecting a young true leaf 7 days after the seeds germinate, putting the young true leaf into a mortar, adding liquid nitrogen for grinding, grinding to obtain a mixture, extracting cotton genome DNA of the mixture by a CTAB method, and mixing the cotton genome DNA with a CCRI010 primer.
There are 12 pairs of primers for differentiation between Xinshi K28 and Xinluaozao No. 7, chromosome A5, chromosome A8, chromosome D2, chromosome D4, chromosome D5, chromosome D6, chromosome D8, chromosome D11, and chromosome D13, respectively
Figure BDA0003263560720000101
In the first and second comparative examples, SSR markers are carried out by the following process methods to construct cotton DNA fingerprints:
s1, taking 42 parts of upland cotton variety seeds, and accelerating germination of the cotton seeds.
And S2, when the cotton seeds germinate, selecting one tender true leaf per plant 7d, placing the young true leaf in a mortar, adding liquid nitrogen into the mortar, grinding the true leaf to obtain a mixture.
S3, extracting cotton genome DNA in the true leaf mixture by a CTAB method.
S4, comparing 42 groups of experimental data, performing genetic diversity analysis, and establishing a file.
Screening 60 SSR primers which are uniformly distributed on 26 chromosomes of a cotton A, D genome, screening 30 pairs of core primers which are clear in amplification band type, easy to distinguish, good in polymorphism and stable, and recording primer information in the following table. 30 pairs of primers detect 96 kinds of amplification band types in 42 parts of Xinjiang upland cotton varieties, the number of the amplification band types of each pair of primers is between 2 and 7, and the average value is 3.2. And 13 pairs of primers with polymorphism information content value larger than 0.500 in 30 pairs of SSR primers are selected, the amplitude is 0.260-0.624, and the screened partial molecular markers have higher polymorphism but still have primers with smaller PIC values. In the researches of cotton purity and authenticity detection, variety identification and the like, molecular markers with high polymorphic information content can be preferentially used, as shown in figure 1, polymorphic markers CCRI036 show various different amplification banding patterns in 42 Xinjiang upland cotton varieties, and test results show that the 42 Xinjiang upland cotton varieties have abundant genetic variation;
15 pairs of differential primers of the amplified band of 30 pairs of selected primers exist between Xinshi K28 and Xinluzao No. 46 varieties, 12 pairs of differential primers respectively exist between A1, A8, A11, A12, D2, D5, D8, D11, D12 and D13 chromosomes of the Xinshi K28 and the Xinluzao No. 7 varieties, 12 pairs of differential primers respectively exist between A5, A8, D2, D4, D5, D6, D8, D11 and D13 chromosomes of the Xinluzao No. 7 varieties, the differential primers common to the three materials are selected as shown in figure 2, the different position information among the varieties can be distinguished by selecting the differential combined polymorphic primers, and the fingerprint of the Xinshi K28 can be constructed according to the information:
different primers are combined by 30 pairs of primers, so that the discrimination capability is improved, the materials are further completely distinguished, the core primer with a large PIC value is preferentially selected, and the characteristic primers are combined to carry out primer combination, so that the distinguishing of all varieties is completed, and the fingerprint spectrum of the new stone K28 and other materials is constructed. The analysis result shows that the new stone series materials can be completely distinguished from other materials by using 20 pairs of core primers CCRI036, CCRI029, CCRI009, CCRI027, CCRI035, CCRI021, CCRI060, CCRI030, CCRI014, CCRI049, CCRI023, CCRI051, CCRI025, CCRI050, CCRI013, CCRI053, CCRI054, CCRI024, CCRI056 and CCRI031, the DNA fingerprint spectrum (shown in the following table) of the new stone series varieties is constructed by using the 20 pairs of core primers, and a decimal numeric string can be obtained by using a numeric description band type and can be used as a unique identification code of the corresponding Xinjiang land cotton variety. And constructing a special two-dimensional code for the variety according to a crop seed label two-dimensional code coding rule printed by an office hall of the Ministry of agriculture.
Figure BDA0003263560720000122
Figure BDA0003263560720000131
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and equivalent substitutions or changes according to the technical solution and the inventive concept of the present invention should be covered by the scope of the present invention.

Claims (7)

1. The DNA fingerprint construction of the new stone K28 of cotton based on SSR markers is characterized by comprising the following steps:
s1, taking 42 parts of upland cotton variety seeds, and accelerating germination of the cotton seeds.
And S2, when the cotton seeds germinate, selecting one tender true leaf per plant 7d, placing the young true leaf in a mortar, adding liquid nitrogen into the mortar, grinding the true leaf to obtain a mixture.
S3, extracting cotton genome DNA in the true leaf mixture by a CTAB method.
S4, comparing 42 groups of experimental data, performing genetic diversity analysis, and establishing a file.
2. The DNA fingerprinting construction of SSR-marker-based cotton neolith K28 according to claim 1, characterized in that: the SSR primers in the S1-S4 are provided by key laboratories of the Cotton institute of agricultural sciences, China.
3. The DNA fingerprinting construction of SSR-marker-based cotton neolith K28 according to claim 1, characterized in that: the temperature of the cotton seeds in the S1 is 28 ℃ during pregermination, and the illumination L/D is 12h/12 h.
4. The DNA fingerprinting construction of SSR-marker-based cotton neolith K28 according to claim 1, characterized in that: the SSR-PCR amplification conditions are that the concentration of a cotton genome DNA solution extracted by a CTAB method is uniformly diluted to 50 ng.mu.L < -1 >, a PCR amplification system comprises 1 mu.L of template DNA, 1 mu.L of Buffer (containing MgCl), 0.3 mu.L of dNTPs (10 mmol.L < -1 >), 0.5 mu.L of forward primer F (10 mu mol.L < -1 >), 0.5 mu.L of reverse primer R (10 mu mol.L < -1 >), 0.2 mu.L of Taq enzyme (2.5 U.mu.L < -1 >), and 6.5 mu.L of ddH2O 6.5. The PCR amplification procedure was: (1) pre-denaturation: 5min at 94 ℃; (2) amplification: denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and performing 32 cycles; (3) final extension: 72 ℃ for 10min, and (4) storing the amplification product at 4 ℃.
5. The DNA fingerprinting construction of SSR-marker-based cotton neolith K28 according to claim 1, characterized in that: carrying out electrophoresis detection on the amplification product on 8% non-denaturing polyacrylamide gel, loading 1.5 mu L of sample, carrying out 220V voltage electrophoresis for 1h, and carrying out silver staining by using a method such as Zhang Jun (14) after the electrophoresis is finished, wherein the method comprises the following steps of (1) carrying out silver staining (0.1% silver nitrate solution) for 12-15 min; (2) developing (2% sodium hydroxide and 1% formaldehyde) for 5-10 min; (3) rinsing with pure water for 2-3 times, silver staining and developing, and taking a picture by using a camera for recording.
6. The DNA fingerprinting construction of SSR-marker-based cotton neolith K28 according to claim 1, characterized in that: each primer is amplified corresponding to different genotypes, and the polymorphism information content of the calculated sites is calculated according to a formula PIC (positive identity) 1-sigma Pi 2And (4) calculating.
7. The DNA fingerprinting construction of SSR-marker-based cotton neolith K28 according to claim 1, characterized in that: the established file generates the two-dimension code according to a two-dimension code coding rule of a crop seed label printed by an office of the department of agriculture.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
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Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN102146448A (en) * 2011-01-13 2011-08-10 河南科技学院 DNA finger prints of 30 main cultivars of upland cotton
CN103614456A (en) * 2013-09-02 2014-03-05 石河子农业科技开发研究中心 Microsatellite marker specific primers for identification of Sinkiang colored cotton series No.1 to23 varieties and applications thereof
CN104862387A (en) * 2015-04-14 2015-08-26 新疆农垦科学院 Method for establishing cotton variety fingerprint and digital identity card

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Title
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王希文: "棉花优异种质遗传多样性分析及其SSR数字指纹图谱构建", 中国优秀硕士学位论文全文数据库农业科技辑, pages 047 - 194 *
王欣怡等: "基于SSR标记新疆陆地棉的DNA指纹图谱构建及遗传多样性分析", 棉花学报, vol. 30, no. 4, pages 308 - 315 *

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