CN111471791A - Soybean DNA fingerprint construction method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of soybean DNA fingerprints, in particular to a method for constructing a soybean DNA fingerprint, which comprises the following steps: s1, screening SSR primers: s2, genetic diversity analysis of soybean: extracting soybean genome DNA; PCR amplification and detection; carrying out a PCR reaction program; detecting by capillary electrophoresis; data statistics and analysis; s3, constructing a DNA fingerprint, analyzing soybean resources collected in Heilongjiang by using SSR molecular markers, constructing SSR molecular marker fingerprints of various quality resources, and providing a theoretical basis for preservation and screening of Heilongjiang soybean germplasm.
Description
Technical Field
The invention relates to the technical field of soybean DNA fingerprints, in particular to a soybean DNA fingerprint construction method and application thereof.
Background
At the present stage, in the production of the black longjiang soybeans, the problems of multiple and disordered soybean varieties, the phenomena of cross-area planting, mixed planting and the like often occur, the yield of the soybeans is directly influenced, and the phenomena of poor quality of the soybeans and the like also occur. From a certain variety, the special quality of fat, protein and the like is not inferior to that of foreign products, but the special quality is reduced by mixing multiple varieties. This indicates that the quality of soybean is improved in the future, and special varieties need to be selected for production, so that the problem of multiple varieties and impurities is solved.
In variety identification, although field planting identification has the advantages of wide application range, reliable identification result and the like, the method has long time, low efficiency, large workload and lagged result. In addition, the target characters to be identified are mostly quantitative characters, are greatly influenced by the environment, and people familiar with the characteristic characteristics of the varieties and having abundant field inspection experience are required to distinguish genetic variation from non-genetic variation, and the varieties are seriously homogenized, so that the genetic relationship is closer and closer along with the mutual permeation among the germplasms of the bred varieties, the characteristic difference among the varieties is smaller and smaller, certain difficulty is brought to the identification of the varieties, and the requirement of breeders on widening the genetic basis of the varieties cannot be met.
The widely applied DNA molecular markers include 3 generations, namely, restriction fragment length polymorphism (RF L P) of the 1 st generation, Random Amplification Polymorphic DNA (RAPD), amplification enzyme section length polymorphism (AF L P) of the 2 nd generation, simple sequence repeat length polymorphism (SSR), Single Nucleotide Polymorphism (SNP) of the 3 rd generation and the like.
Microsatellite DNA refers to a DNA Tandem Repeat sequence consisting of Short Repeat units (generally 1 to 6 bases) in a genome, and is also called Short Tandem Repeats (STRs) or simple Repeat sequences (SSRs). The microsatellites are widely distributed at different positions of various eukaryotic genomes and are uniformly distributed, and a microsatellite sequence appears in every 10kb DNA sequence on average, and the high polymorphism is presented by different repetition times and different repetition degrees of the SSR. Two ends of the SSR are provided with a section of conservative DNA sequence, and an oligonucleotide primer with a complementary sequence can be designed according to the section of conservative DNA sequence to carry out PCR amplification on the SSR. The Ministry of agriculture has developed a series of industry standards for identifying major crop varieties by using SSR molecular markers, which have no success in development of seed industry, market management and quality monitoring, but the judgment of the results still needs to be correctly known. Although the traditional plant variety field plot planting identification is time-consuming and labor-consuming, the method has the characteristics of legality, scientificity and accuracy, and is still the most reliable method for identifying main crop varieties. Although the existing standard collects a large amount of materials for DNA map analysis, the germplasm resources of China are rich, and the complete coverage of the germplasm resource materials of China is not realized. The existing standard selection core primer is greatly simplified for convenient detection, the proportion of the selected SSR marker in the plant genome is extremely small, and the requirement of authenticity identification of all varieties cannot be met along with the increase of the number of the varieties.
At present, several people in China adopt SSR to distinguish soybean varieties and construct fingerprints, for example, Xuhaifeng and other people utilize 6 pairs of SSR primers to construct fingerprints of 26 vegetable soybean varieties (series) in the south of Huaihe river so as to distinguish the 26 vegetable soybean varieties (series) one by one. The high-yield method can completely distinguish 83 tested soybean varieties by utilizing 9 pairs of primers and construct a set of molecular ID of the soybean variety in Heilongjiang province. The 6 primers of the tourmaline and the like can be used for distinguishing 45 tested soybean varieties in the Yangtze river basin slice national region test and obtaining unique molecular ID. The data lay the foundation for meeting the requirement of authenticity identification of all varieties.
Therefore, we propose a method for constructing soybean DNA fingerprint and the application thereof to solve the above problems.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a soybean DNA fingerprint map construction method and application thereof.
A soybean DNA fingerprint map construction method comprises the following steps:
s1, screening SSR primers:
adopting a literature research method, selecting SSR markers with good polymorphism on different chromosomes according to the SSR marker information reported by domestic researchers, sorting 24 pairs of SSR primers from the SSR markers, evenly distributing the SSR markers on 15 chromosomes, searching 24 pairs of SSR marker primer sequences on Soybase according to the names of the primers in the literature, synthesizing common primers, and diluting the primers into stock solution and working solution for later use;
primer screening, namely performing availability screening on the common primer in the previous step, randomly selecting 3 test samples, performing primer condition and system exploration, detecting the amplified product by using 2% agarose electrophoresis, and confirming the primer which meets the target length;
synthesizing fluorescent primers, namely synthesizing the primers with the lengths meeting the target in the previous step, and adding FAM/HEX/Tamra to mark 5' of 1 primer;
s2, genetic diversity analysis of soybean:
soybean genomic DNA extraction
Placing fresh and tender leaves in a 2m L centrifugal tube, grinding the leaves with a overnight grinding pestle at the temperature of minus 20 ℃, adding 1m L DNA extracting solution, grinding the leaves into slurry, carrying out water bath at the temperature of 60 ℃ for 1h, centrifuging the slurry for 5min at the rotating speed of 12000rpm, taking supernatant, adding chloroform with the same volume, shaking the mixture violently, centrifuging the supernatant for 5min, taking the supernatant, adding chloroform with the same volume, repeating the steps once, adding isopropanol with the same volume, standing the mixture for 1h at the temperature of minus 20 ℃, centrifuging the mixture for 5min, discarding the supernatant, adding 500 mu L70% ethanol, washing the mixture twice, air drying the mixture, and adding 100 mu L TE buffer solution;
PCR amplification and detection
Firstly, performing a preliminary experiment, selecting 6 samples to perform the preliminary experiment on the primers, determining whether a target amplification product is available or not through agarose electrophoresis, further synthesizing fluorescent primers on the basis of the preliminary experiment, adding FAM/HEX/Tamra markers to 5' of 1 primer, and carrying out a PCR amplification system of 10 mu L;
PCR reaction procedure
The specific reaction procedure of Touchdown PCR is as follows: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 30s, extension at 72 ℃ for 30s, 10 cycles, final denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 10min, wherein the cycles are 35, and final constant-temperature storage at 10 ℃;
capillary electrophoresis detection
The test is carried out by using a 3730X L sequencer, original data obtained by the sequencer is analyzed by Fragment analysis software in a Genemarker, and the position of the molecular weight internal standard in each lane is compared and analyzed with the position of the peak value of each sample to obtain the size of the Fragment;
data statistics and analysis
Utilizing POPGENE software to calculate the genetic distance, the genetic similarity coefficient, the polymorphism frequency, the allele factor, the effective allele factor, the Nielsen gene diversity and the Shannon diversity coefficient of the SSR primer;
polymorphism information content index of SSR primers according to the Smith method: PIC, calculation formula:
PIC＝1-∑FPi 2
in the formula: FPi, representing the allele frequency of the ith locus, clustering the genetic similarity by a non-weighted class average method by using statistical analysis software NTSYS-2.10 according to the genetic similarity coefficient GS, and drawing a tree diagram;
s3 construction of DNA fingerprint
According to the results of the 17-pair soybean primer amplification and capillary electrophoresis assay, the presence or absence of the polymorphic fragment was recorded in 0/1 format, and was assigned as "1" in the presence of this band and as "0" in the absence of this band, and DNA fingerprints were constructed for 68 varieties.
Preferably, in S2, the reaction solution contains 0.6. mu. L F/R primers (. mu.M), 1. mu. L10 × PCR Buffer, 0.8 mmol/L dNTP (10mM), 0.12. mu. L Taq DNA polymerase, 1ng DNA template, and ddH2O to 10. mu. L.
Preferably, in S2, the annealing temperature of the primer is decreased by 1 ℃ according to 1 cycle, and is decreased from 60 ℃ to 50 ℃.
Preferably, in S3, the 0/1 fingerprint of the rice variety is converted into 32-system by using online software, the soybean species is represented by the first letter of the academic name, and then re-encoded by combining the sequence number, the Code128A in the barcode generator is used to generate the id card barcode of the variety according to the requirements of the national technical standards GB/T18347-2001128 barcode and GB/T18284-2000 quick response matrix Code, and the generation software of the two-dimensional Code is used to generate the corresponding id card two-dimensional Code, thereby forming the unique id information of the variety.
Preferably, according to the specified requirements of the national technical standards GB/T18347-2001128 bar code and GB/T18284-2000 quick response matrix code, the online bar code and two-dimensional code generation software is used for generating the unique bar code and the unique two-dimensional code for the identification cards of the 68 soybean varieties respectively.
Preferably, Gm represents soybean names, 68 varieties are coded, 2-system is converted into 32-system by using online software, soybeans are replaced by letters Gm, Code128A in a bar Code generator is used for generating corresponding identification card bar codes, and finally corresponding identification card two-dimensional codes are generated by using two-dimensional Code generation software.
The invention has the beneficial effects that: the invention utilizes SSR molecular markers to analyze soybean resources collected in Heilongjiang areas, constructs SSR molecular marker finger prints of various quality resources, and provides a theoretical basis for preservation and screening of Heilongjiang soybean germplasm.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
A soybean DNA fingerprint map construction method comprises the following steps:
s1 screening of SSR primers
And selecting SSR markers with good polymorphism on different chromosomes according to average distribution by adopting a literature research method and according to SSR marker information reported by domestic researchers. 24 pairs of SSR primers were arranged and evenly distributed on 15 chromosomes (Table 1). According to the primer names of the literature, 24 pairs of SSR labeled primer sequences are searched for in Soybase (https:// Soybase. org/dlpages /), common primers are synthesized by Beijing Yolbolan gene technology Co., Ltd and diluted into stock solution and working solution for later use, and the results show that 17 pairs of primers can be used in total in 24 pairs of primers, and the soybean genome distribution and the primer sequences of 17 SSR labels are shown in Table 1.
And (3) primer screening, namely performing availability screening on the common primer in the last step, randomly selecting 3 test samples, performing primer condition and system exploration, detecting the amplified product by using 2% agarose electrophoresis, and confirming the primer which meets the target length.
And (3) synthesizing a fluorescent primer, namely synthesizing the primer with the band obtained in the previous step by using the fluorescent primer, and adding marks such as FAM/HEX/Tamra and the like to 5' of 1 primer.
Table 117 SSR-labeled soybean genome distribution and primer sequences
S2 genetic diversity analysis of soybean
Soybean genomic DNA extraction
Placing fresh and tender leaves in a 2m L centrifuge tube, grinding with a grinding pestle overnight at-20 ℃, adding 1m L DNA extracting solution, grinding into slurry, carrying out water bath at 60 ℃ for 1h, centrifuging at 12000rpm for 5min, taking supernatant, adding chloroform with the same volume, shaking vigorously, centrifuging at 12000rpm for 5min, taking supernatant, repeating the steps once, adding isopropanol with the same volume, placing at-20 ℃ for 1h, centrifuging at 12000rpm for 5min, discarding supernatant, adding 500 mu L70% ethanol, washing twice, air drying, and adding 100 mu L TE buffer solution for dissolving.
PCR amplification and detection
On the basis of the preliminary experiment, a fluorescent primer is further synthesized, and FAM/HEX/Tamra and other labels are added to 5' of 1 primer, wherein the PCR amplification system is 10 mu L, the reaction solution comprises 0.6 mu L F/Rpcr (mu M), 1 mu L10 × PCR Buffer, 0.8 mmol/L dNTP (10mM), 0.12 mu L Taq DNA polymerase and 1ngDNA template, and ddH2O is used for complementing to 10 mu L.
PCR reaction procedure the specific reaction procedure for Touchdown PCR was: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 30s (the annealing temperature of the primer is reduced from 60 ℃ to 50 ℃ according to 1 cycle), extension at 72 ℃ for 30s and 10 cycles, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s and extension at 72 ℃ for 10min for 35 cycles, and constant-temperature storage at 10 ℃ at last.
Capillary electrophoresis detection
The test was performed using a 3730X L sequencer (argon ion laser source, excitation wavelengths of 488nm and 514.5nm), the raw data obtained by the sequencer was analyzed using fragment (plant) fragment analysis software in Genemarker, and the position of the internal molecular weight standard in each lane was compared with the position of the peak of each sample to obtain the size of the fragment.
Data statistics and analysis
The Genetic Distance (GD), Genetic similarity coefficient (GS), polymorphism Frequency (FP), Number of alleles (Na) effective allele factor (Ne), Nielsen gene diversity (Nei's (1973) gene diversity, h), Shannon's diversity index (I) of SSR primers were calculated using POPGENE version 1.32 software.
According to the polymorphism information content index (PIC) of the SSR primer by the method of Smith and the like, the formula is calculated:
PIC＝1-∑FPi 2
in the formula: FPiIndicates the allele frequency at the i-th site. And (4) clustering the genetic similarity by using a non-weighted average class method (UPGMA) according to the genetic similarity coefficient GS by using statistical analysis software NTSYS-2.10, and drawing a dendrogram.
S3 construction of DNA fingerprint
According to the results of the 17-pair soybean primer amplification and capillary electrophoresis assay, the presence or absence of the polymorphic fragment was recorded in 0/1 format, and was assigned as "1" in the presence of this band and as "0" in the absence of this band, and DNA fingerprints were constructed for 68 varieties. The 0/1 fingerprint of the rice variety is converted into 32-system by using online software, the soybean species is represented by the first letter of the academic name, and then the soybean species is recoded by combining the sequence number of the rice variety in the table 2, the ID card bar Code of the variety is generated by using Code128A in a bar Code generator according to the specified requirements of the national technical standard GB/T18347-2001128 bar Code and GB/T18284 and 2000 quick response matrix Code, and the corresponding ID card two-dimensional Code is generated by using the generation software of the two-dimensional Code, so that the unique identity information of the variety is formed.
Table 268 shares soybean variety information table
In the present example, genetic diversity analysis was performed on soybean
SSR marker polymorphisms
68 soybean varieties are amplified by using 17 pairs of rice SSR standard primers respectively, amplification products are detected by using a capillary electrophoresis technology, the polymorphism information results of the 17 pairs of primers are shown in table 3, 98 alleles are detected by capillary electrophoresis, and each pair of primers is amplified to 6.125 alleles on average, so that the rice SSR standard primers have good polymorphism. The marker Sat-128 detected the highest number of alleles in all varieties, 13 alleles, and Sat-294 and Sat-387 detected the lowest number of alleles, 2 alleles. Markers Sat _084, Sat _222, and Sat _295 detected 3 alleles, markers Satt551 detected 4 alleles, Satt233, Satt175, and Satt453 detected 5 alleles, Satt216 and Satt156 detected 6 alleles, Satt514 and Satt239 detected 7 alleles, Satt138 detected 8 alleles, and Satt100 and Satt369 detected 10 alleles. A total of 98 alleles were detected with 17 primer pairs in 68 soybean varieties, with primers Satt216, Satt514 and Satt453 being polymorphic in the varieties.
The PIC value range of the polymorphism information amount of the SSR marker is 0.00203401-0.9236687, and the average value is 0.548955451. Satt453 had the largest PIC value (0.9236687), followed by chromosome Satt514(0.85616971), Satt138 had the smallest PIC value (0.00203401), and the SSR markers for 68 varieties were overall more polymorphic.
The effective allele factors Ne range from 1.000(Satt514) to 1.9935(Satt100), with an average value of 1.2374. The genetic diversity range of Nielsen is 0-0.4984, and the average value is 0.1485. The Shannon information index range is 0-0.6915, and the average value is 0.2435.
TABLE 317 polymorphism information for primers
Construction of soybean DNA fingerprint
Based on the results of the 17 primer amplification and capillary electrophoresis assays, which were the first choice, the presence of polymorphic fragments was recorded in the manner of 0/1, and DNA fingerprints were constructed for 68 varieties. For example, the fingerprint of crofton 17 is counted, the sizes of the amplified products of 17 SSR markers in the variety and the fragments directly read by capillary electrophoresis are counted, compared with the fragments read by 17 markers in all the tested varieties, the fragment reading is marked as 1, the fragment reading is not marked as 0, and the fingerprint 00001010010101100000100000100000000000011100010100000100100000010100000000000000000001000101000110 of the Longjing 21 variety is obtained because 98 alleles are detected by 17 pairs of SSR primers through capillary electrophoresis.
Construction of soybean variety identity card
According to the specified requirements of the national technical standards GB/T18347-2001128 bar code and GB/T18284-2000 quick response matrix code, the identification cards of 68 soybean varieties are respectively generated into unique bar codes and unique two-dimensional codes by using online bar code and two-dimensional code generation software. The soybean name is represented by Gm, and 68 varieties are encoded according to the variety ranking in table 2. (for example, constructing the bar Code and the two-dimensional Code of the variety of the Ningfeng 17 soybean, firstly, the fingerprint 0/1 Code 00001010010101100000100000100000000000011100010100000100100000010100000000000000000001000101000110 of the Ningfeng 17 is converted into 32-system (AAR4200E50J0M0004A6) by utilizing online software, soybeans are replaced by letters Gm, and the serial number of the Ningfeng 17 in the table 2 is 01, so that the variety Code is Gm01, the identity information of the variety is formed by adding the variety fingerprint Code (C01K 1GM5GOK Gm01 AAR4200E50J0M0004A6), then, the identity card bar Code of the variety of the Ningfeng 17 is obtained by utilizing Code128A in a bar Code generator, and finally, the corresponding identity card two-dimensional Code is generated by utilizing the generation software of the two-dimensional Code. The bar code and two-dimensional code information of all soybean variety identity cards in the study are shown in table 4 respectively.
468 soybean variety ID card information tables
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 the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. A soybean DNA fingerprint map construction method is characterized by comprising the following steps:
s1, screening SSR primers:
adopting a literature research method, selecting SSR markers with good polymorphism on different chromosomes according to the SSR marker information reported by domestic researchers, sorting 24 pairs of SSR primers from the SSR markers, evenly distributing the SSR markers on 15 chromosomes, searching 24 pairs of SSR marker primer sequences on Soybase according to the names of the primers in the literature, synthesizing common primers, and diluting the primers into stock solution and working solution for later use;
primer screening, namely performing availability screening on the common primer in the previous step, randomly selecting 3 test samples, performing primer condition and system exploration, detecting the amplified product by using 2% agarose electrophoresis, and confirming the primer which meets the target length;
synthesizing fluorescent primers, namely synthesizing the primers with the lengths meeting the target in the previous step, and adding FAM/HEX/Tamra to mark 5' of 1 primer;
s2, genetic diversity analysis of soybean:
soybean genomic DNA extraction
Placing fresh and tender leaves in a 2m L centrifugal tube, grinding the leaves with a overnight grinding pestle at the temperature of minus 20 ℃, adding 1m L DNA extracting solution, grinding the leaves into slurry, carrying out water bath at the temperature of 60 ℃ for 1h, centrifuging the slurry for 5min at the rotating speed of 12000rpm, taking supernatant, adding chloroform with the same volume, shaking the mixture violently, centrifuging the supernatant for 5min, adding chloroform with the same volume, repeating the steps once, adding isopropanol with the same volume, standing the mixture for 1h at the temperature of minus 20 ℃, centrifuging the mixture for 5min, discarding the supernatant, adding 500 mu L70% ethanol, washing the mixture twice, air drying the mixture, and adding 100 mu L TE buffer solution to dissolve the mixture;
PCR amplification and detection
Firstly, performing a preliminary experiment, selecting 6 samples to perform the preliminary experiment on the primers, determining whether a target amplification product is available or not through agarose electrophoresis, further synthesizing fluorescent primers on the basis of the preliminary experiment, adding FAM/HEX/Tamra markers to 5' of 1 primer, and carrying out a PCR amplification system of 10 mu L;
PCR reaction procedure
The specific reaction procedure of Touchdown PCR is as follows: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 30s, extension at 72 ℃ for 30s, 10 cycles, final denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 10min, wherein the cycles are 35, and final constant-temperature storage at 10 ℃;
capillary electrophoresis detection
The test is carried out by using a 3730X L sequencer, original data obtained by the sequencer is analyzed by Fragment analysis software in a Genemarker, and the position of the molecular weight internal standard in each lane is compared and analyzed with the position of the peak value of each sample to obtain the size of the Fragment;
data statistics and analysis
Utilizing POPGENE software to calculate the genetic distance, the genetic similarity coefficient, the polymorphism frequency, the allele factor, the effective allele factor, the Nielsen gene diversity and the Shannon diversity coefficient of the SSR primer;
polymorphism information content index of SSR primers according to the Smith method: PIC, calculation formula:
PIC＝1-∑FPi 2
in the formula: FPi, representing the allele frequency of the ith locus, clustering the genetic similarity by a non-weighted class average method by using statistical analysis software NTSYS-2.10 according to the genetic similarity coefficient GS, and drawing a tree diagram;
s3 construction of DNA fingerprint
According to the results of the 17-pair soybean primer amplification and capillary electrophoresis assay, the presence or absence of the polymorphic fragment was recorded in 0/1 format, and was assigned as "1" in the presence of this band and as "0" in the absence of this band, and DNA fingerprints were constructed for 68 varieties.
2. The method for constructing soybean DNA fingerprint of claim 1, wherein in S2, the reaction solution contains 0.6 μ L F/R primers (μ M), 1 μ L10 × PCR Buffer, 0.8 mmol/L dNTP (10mM), 0.12 μ L Taq DNA polymerase, 1ng DNA template, and ddH is used2O to 10. mu. L.
3. The method for constructing a soybean DNA fingerprint as claimed in claim 1, wherein in S2, the annealing temperature of the primer is decreased by 1 ℃ according to 1 cycle, and is decreased from 60 ℃ to 50 ℃.
4. The method as claimed in claim 1, wherein in S3, 0/1 fingerprints of rice varieties are converted into 32-system data by online software, the soybean species are represented by the initials of the academic name, and then are recoded by combining the sequence number, the Code128A in the barcode generator is used for generating the ID card barcode of the varieties according to the requirements of national technical standards GB/T18347-.
5. The application of the soybean DNA fingerprint map construction method as claimed in claim 1, characterized in that, referring to the specified requirements of the national technical standards GB/T18347-2001128 bar code and GB/T18284-2000 quick response matrix code, the generation software of the online bar code and the two-dimensional code is used to respectively generate the unique bar code and the two-dimensional code for the identification cards of 68 soybean varieties.
6. The application of the soybean DNA fingerprint construction method according to claim 1, characterized in that Gm represents soybean names, 68 varieties are encoded, 2-system is converted into 32-system by online software, soybeans are replaced by letters Gm, corresponding ID card bar codes are generated by Code128A in a bar Code generator, and finally corresponding ID card two-dimensional codes are generated by two-dimensional Code generation software.
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| CN115631796A (en) * | 2022-10-13 | 2023-01-20 | 济宁市农业科学研究院 | Garlic biological fingerprint spectrum construction and identification method, terminal equipment and storage medium |
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| CN117757979B (en) * | 2023-12-27 | 2024-05-14 | 江汉大学 | Primer group, kit and identification method for identifying soybean varieties |
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