CN108823330B - Soybean HRM-SNP molecular marker point marking method and application thereof - Google Patents
Soybean HRM-SNP molecular marker point marking method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a soybean HRM-SNP molecular marker and application thereof, which are used for developing soybean SNP markers, extracting Zhejiang fresh nine-number genome DNA, carrying out high-resolution melting curve process and analysis, carrying out SNP typing on Zhejiang fresh nine-number based on an HRM technology, carrying out typing verification on Zhejiang fresh nine-number HRM, and constructing a Zhejiang fresh nine-number SNP fingerprint. The invention has high flux and short analysis time: 96 or 384 samples can be analyzed at one time, and the method is suitable for large-scale development and detection of SNP sites; the method is simple and convenient to operate, sequence specific probes and subsequent sequencing are not needed, PCR reaction is carried out only by designing PCR primers, and then HRM is directly carried out, so that genotype analysis of the sample can be completed; the invention has good specificity: the PCR product is completely closed without subsequent treatment.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a soybean HRM-SNP molecular marker point marking method and application thereof.
Background
Currently, the current state of the art commonly used in the industry is such that:
up to now, more than 20 methods have been carried out for SNP typing detection. The detection methods of SNP can be roughly divided into two major categories according to the need of Gel Electrophoresis and the degree of automation, one is the SNP detection method based on Gel Electrophoresis, such AS Single-Strand Conformational Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Cleaved Amplified Polymorphic sequence analysis (CAPS), Allele Specific PCR (AS-PCR), etc.; the second is a High-throughput and High-automation SNP detection method, such as direct sequencing, DNA chip, Denaturing High Performance Liquid Chromatography (DHPLC), mass spectrometry, High-resolution Melting (HRM) curve, and the like.
The above methods for detecting SNP have advantages and disadvantages, some methods can accurately detect SNP sites, such as DNA sequencing and DNA chip technology, but the cost is high and the methods cannot be generally applied; some methods are complex in operation, time-consuming and labor-consuming, such AS AS-PCR technology; some have high throughput, low cost and accurate result, but have higher requirements on equipment hardware, such as HRM technology. An ideal SNP detection method should have the following characteristics: the sensitivity and the accuracy are high; fast, simple and high flux; the cost is relatively low. However, no detection method which completely meets the above characteristics exists so far, so that a more appropriate method can be selected according to actual conditions.
Soybeans are main oil plants and economic crops in China, and have been planted for more than five thousand years. Because of rich nutrition, good flavor, high economic benefit and diversified forms of collection, processing, sale and the like, the Chinese and the Japanese are popular.
Although soybeans originate from China, the development is late, and early breeding mainly comprises screening or cross breeding by introducing excellent varieties from Taiwan province or Japan, so that the genetic basis of breeding resources is narrow, and the development of new varieties is not facilitated. At present, Simple Sequence Repeat (SSR) markers mainly used for various researches on soybeans also have the disadvantages of relatively small quantity, complex analysis operation, difficulty in determining the size of the markers, incapability of being used in the cross-research and the like.
The high-resolution melting (HRM) technique is a novel SNP detection technique. In recent years, with the continuous development and perfection of the technology, the technology has been successfully applied to genotyping, mutation scanning, methylation research and other aspects, and is successfully applied to the research of crops such as potatoes, alfalfa, oranges and the like. Therefore, the SNP novel molecular marker which has higher density, simpler analysis and cross-research use in genotype determination has very important application value.
In summary, the problems of the prior art are as follows:
(1) at present, Simple Sequence Repeat (SSR) markers mainly used for various researches on soybeans also have the disadvantages of relatively small quantity, complex analysis operation, difficulty in determining the size of the markers, incapability of being used in cross-research and the like.
(2) Because soybean genome is extremely complex, the HRM technology has high requirements on primer design, and the situation that SNP site sequences are not unique in the genome or designed primers are subjected to non-specific amplification is often encountered in the primer design process.
The significance of solving the technical problems is as follows:
compared with the first two generations of DNA molecular markers, the SNP marker has the remarkable characteristics of higher genetic stability, high density, rapid detection, easy realization of automatic analysis and the like, has very high distribution frequency in a soybean genome, has 1 SNP per 273bp on average, and is an important means for encrypting a soybean genetic map, positioning important trait QTL and assisting in molecular marker selection.
Primer design using Primer-BLAST ensures amplification specificity and high success rate of Primer design. Since a large number of SNP sites are discarded and replaced in the preliminary primer design process, the final success rate of primer design is roughly estimated to be higher than 85%.
The 101 SNP markers are uniformly distributed on 20 chromosomes of the soybean, can be directly used in research, can also be used for continuously developing new markers between two adjacent SNP sites by using the method disclosed by the invention to improve the marker density, and can also be used as a reference for other crops with known genome information.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a soybean HRM-SNP molecular marker point marking method and application thereof.
The invention is realized in such a way that a soybean HRM-SNP molecular marker point marking method comprises the following steps:
the first step is as follows: development of soybean SNP marker: selecting an SNP marker from a Williams 82 physical map by using a Genome browser tool in a database at every 10Mb, selecting 4-6 SNPs on each chromosome, uniformly distributing the SNPs on 20 chromosomes in a soybean Genome, and acquiring detailed information of each SNP marker;
the second step is that: extracting genome DNA of soybean (Zhejiang fresh nine), extracting genome DNA of Zhejiang fresh nine young leaves according to CTAB method, and diluting to 25 ng. mu.L-1Storing in a refrigerator at-20 ℃ for subsequent HRM analysis;
the third step: high resolution melting curve procedure and analysis: performing PCR amplification and HRM analysis on a quantitative PCR instrument; high resolution melting curve analysis
480, a Gene Scanning module of software is used for obtaining a better standardized melting curve;
the fourth step: soybean (zhe xian jiu) SNP typing based on HRM technique: amplifying and HRM analyzing the Zhejiang fresh No. nine DNA in a quantitative PCR instrument;
the fifth step: soybean (Zhejiang fresh nine number) HRM typing verification: carrying out Sanger sequencing on PCR products marked by part of SNPs at random, and verifying the accuracy of HRM analysis;
and a sixth step: construction of soybean (Zhejiang fresh nine) SNP fingerprint:
and a sixth step: construction of soybean (Zhejiang fresh nine) SNP fingerprint: with Williams 82 as a contrast, HRM typing is carried out on vegetable soybean Zhejiang 9 by utilizing 101 developed SNP markers, and typing results are listed in sequence from small to large according to chromosome numbers-physical map distances. For example, the results of typing from chromosome 1 marker to chromosome 20 last marker are: "AGTTCC … … GGA", the string of letters is the DNA fingerprint of "Zhejiang Xian No. 9". Therefore, the SNP map of Zhejiang Uji is TCAACT AGCCA CTGCT CCTAG ATGAC CGCGA TGGGG TGGGC CACCA TCACT TCTT CTGGT TACCA TACCG TGGCG AGGTC TAAAC GATCTC CTCTG CTACT.
Further, in the extraction of soybean (Zhejiang fresh nine) genome DNA, the genome DNA of Zhejiang fresh nine young leaves is extracted according to the improved CTAB method and diluted to 25 ng. mu.L-1And storing the sample in a refrigerator at the temperature of-20 ℃ for subsequent HRM analysis, and specifically comprises the following steps:
(1) extraction buffer 2% CTAB, 100 mmol. multidot.L-1Tris-HCL，pH8.0；20mmol·L-1EDTA；2％PVP；1.4mol·L-1NaCl, adding 0.2% mercaptoethanol before use, and preheating to 65 ℃;
(2) taking 0.05g of fresh leaves, properly shearing the fresh leaves, putting the fresh leaves into a 2.0ml centrifuge tube, and adding 1 steel ball with the diameter of 5mm and 2-3 steel balls with the diameter of 2 mm;
(3) adding 1.0mL of preheated extraction buffer solution into a centrifuge tube containing a sample and a steel ball, covering a centrifuge tube cover, putting the centrifuge tube cover into an adapter, and grinding the centrifuge tube cover in a TissueLyser-192 sample grinder for 60 seconds;
(4) taking out the centrifuge tube, and carrying out water bath at 65 ℃ for 10-30 min. Then taking out the centrifuge tube, and centrifuging for 5-10min at 1200 rpm;
(5) taking 1mL of supernatant into a new 2mL centrifuge tube, adding 700 mu L of phenol, chloroform and isoamylol according to the volume ratio of 25:24:1, slowly reversing and uniformly mixing for several times; centrifuging at 1200rpm for 5-10min at room temperature;
(6) adding 800 mu L of supernatant into a new 2mL centrifuge tube, adding equal volume of chloroform isoamylol (24:1), slowly reversing and uniformly mixing for several times; centrifuging at 1200rpm for 5-10min at room temperature;
(7) taking 600 mu L of supernatant to a new 1.5mL centrifuge tube; adding 400 μ L of pre-cooled isopropanol, slowly reversing, mixing, and standing in a refrigerator at-20 deg.C for 30 min;
(8) centrifuging at room temperature and 1200rpm for 10min, and removing the supernatant;
(9) adding 1mL of precooled 70% ethanol to wash the DNA, centrifuging at 1200rpm for 10min, and removing the supernatant;
(10) the DNA is put into a superclean bench for blow-drying, and 100 and 200 mu L deionized water are added for dissolving the DNA;
(11) adding 21 mu L of 0mg/mL RNase in water bath at 37 ℃ for 30 min;
(12) 5 mu.L of DNA is taken, the quality of the DNA is detected by 1 percent agarose gel electrophoresis, and the concentration and the quality of the DNA are detected by a UVS-99 trace nucleic acid detector.
Further, in the third step, in the process and analysis of the high resolution melting curve,
the combination of 20 μ L reaction system was: 2 μ L of DNA50ng, 4 μ L of 5 × EVAGreen Realtime PCR Mix, 10 μmol. L-1The upstream primer and the downstream primer are respectively 0.4 mu L, and the rest is complemented with water; placing the mixed system in a 96-hole PCR plate, attaching a sealing plate film, performing instantaneous centrifugation, and placing in an instrument for reaction;
the reaction procedure is as follows: pre-denaturation at 95 ℃ for 15min, performing 45 conventional cycles, then melting with high resolution, and finally cooling the product to 40 ℃ and keeping the temperature for 10 s;
the cycle program was 95 ℃ for 15s, 60 ℃ for 20s, 72 ℃ for 20s, for a total of 45 cycles;
the high resolution melting process is: the melting curve is read at 95 ℃ for 1min, 40 ℃ for 1min, 65 ℃ for 1s and 65-95 ℃, the temperature resolution is 0.02 ℃, and fluorescence information is continuously acquired at the rate of 25 times per degree centigrade.
Further, in the fourth step, the SNP typing results of soybean (Zhejiang fresh nine #) based on HRM technique are represented by Melting Peaks (Melting Peaks), Normalized Melting Curves (Normalized Melized Melting Curves), Normalized temperature Difference views (Normalized & Temp-Shifted Difference Plots).
The invention also aims to provide a soybean variety DNA fingerprint atlas database constructed by the soybean HRM-SNP molecular marker point marking method.
In summary, the advantages and positive effects of the invention are:
the invention has high flux and short analysis time: can simultaneously complete the analysis of 96 or 384 samples at one time, and is suitable for large-scale development and detection of SNP sites.
The invention has simple and convenient operation: the genotype analysis of the sample can be completed by designing PCR primers to carry out PCR reaction and then directly carrying out HRM without sequence specific probes and subsequent sequencing.
The invention has good specificity: the PCR product is completely closed without subsequent treatment.
The invention has good popularization: the 101 SNP markers developed by the invention can be directly used for various researches such as soybean fingerprint map construction, germplasm identification, variety protection, genetic diversity analysis and the like; it can also be used for similar marker development of other crops with known genome sequence.
Drawings
FIG. 1 shows SNP markers and their chromosomal distribution map developed based on HRM technology according to the embodiment of the present invention.
In the figure: left side is dbSNP name; the position (bp) of the SNP on the chromosome is shown on the right.
FIG. 2 is a diagram showing two cases of SNP typing based on HRM technology according to the embodiment of the present invention.
In the figure: A-D: typing results for the non-polymorphic marker ss715590226 (Williams 82 and Zhejiang Jiu); E-H: typing results of the polymorphic marker ss 71562667;
A. e, melting peak; B. f: standardizing a melting curve; C. g: normalizing the temperature difference view; D. h: williams 82 and Zhejiang Xian Jiu. Three biological replicates per variety were performed.
FIG. 3 is a schematic diagram of a normalized melting curve and a portion of a sample from a PCR product sequence provided in an embodiment of the present invention.
In the figure: SNP typing of zhe xian No. nine and Williams 82 was sequenced and marked near the corresponding normalized melting curves, a-F: the SNP typing results of ss715626377, ss715627029, ss715585535, ss715599654, ss715612033 and ss715624203 respectively.
FIG. 4 is a fingerprint of Zhejiang Xian No. nine constructed based on SNP markers of HRM according to an embodiment of the present invention.
In the figure: the genotype of each SNP site is ordered by its chromosome and physical location. The polymorphic SNP sites of Williams 82 and Zhejiang Xian nine are marked in red.
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.
At present, Simple Sequence Repeat (SSR) markers mainly used for various researches on soybeans also have the disadvantages of relatively small quantity, complex analysis operation, difficulty in determining the size of the markers, incapability of being used in cross-research and the like.
The invention develops SNP molecular markers which are evenly distributed on 20 chromosomes of the soybean based on the HRM technology according to the whole gene sequence information of the soybean variety Williams 82, applies the developed markers to the genotyping and DNA fingerprint construction of the soybean variety Zhejiang fresh nine (Zhejiang agricultural academy of sciences crop and nuclear technology utilizes the fresh soybean variety bred by the institute, national censorship 2009023), and can verify the reliability of the developed SNP marking method and the availability of the developed SNP markers. (Zhejiang Xian Jiu is a good variety in the prior art, and the main innovation of the invention is the new molecular marking method.)
The soybean HRM-SNP molecular marker marking method provided by the embodiment of the invention comprises the following steps:
the first step is as follows: development of soybean SNP marker:
according to the latest genome sequence information (Glyma. Wm82.a2, Gmax2.0) of the SoyBase and the Soybean Breeder' Toolbox databases (https:// www.soybase.org /), the physical map length of Williams 82 is from 34,766,867bp to 58,018,742bp, and the average length is 47,459,169 bp. One SNP marker was selected approximately every 10Mb from the "Williams 82" physical map using the Genome browser tool in the database, with 4 to 6 SNPs per chromosome, evenly distributed on 20 chromosomes of the soybean Genome (Table 1). Detailed information of each SNP marker, including dbSNP name, SNP position, SNP allele and 121bp genomic sequence, can be obtained (Table 2). Using Primer-Blast (https:// www.ncbi.nlm.nih.gov/tools/Primer-Blast) from NCBI database, 121bp genomic sequence of each SNP was pasted into PCR template frame, relevant parameters were set, and desired specific primers were designed on-line. The parameters for designing the primers were as follows: the range of the forward primer is 1-60bp, the range of the reverse primer is 62-121bp, the Tm value is 59-63 ℃, the optimal temperature is 61 ℃, the length of a PCR product is 60-100bp, Refseq representation genes are selected from a database, and Glycine max (L.) Merr. (taxi: 3847) is selected as a species.
Finally, a total of 101 SNP sites evenly distributed on 20 chromosomes of soybean for HRM analysis were selected and successfully converted into SNP markers. Of these, 6 SNP markers were present on Gm01 and Gm18, 4 on Gm11 and 5 on the other chromosomes (FIG. 1). The total average spacing of the 101 SNP markers on the chromosome is 9,397,855 bp. The primer is synthesized by Hangzhou Optingke Catalph and Xi biotechnology limited company, and the primer concentration is 10 mu mol.L-1And storing at-20 ℃ for later use.
TABLE 1 genomic information of "Williams 82" and the number of SNPs picked on each chromosome
TABLE 2 SNP site and primer information based on SNP analysis by HRM technique
The second step is that: zhejiang fresh nine-number genome DNA extraction
Extracting genome DNA of 'Zhejiang fresh nine' young leaf according to CTAB method, and diluting to 25 ng. mu.L-1And stored in a refrigerator at-20 ℃ for subsequent HRM analysis. The method comprises the following specific steps:
(1) extraction buffer (2% CTAB, 100 mmol. multidot.L)-1Tris-HCL，pH8.0；20mmol·L-1EDTA；2％PVP；1.4mol·L-1NaCl), 0.2% mercaptoethanol was added before use, preheated to 65 ℃.
(2) Fresh leaves 0.05g (about 4cm2) are taken, cut into pieces (about 3-4mm in size) and placed into a 2.0ml centrifuge tube, and 1 steel ball with the diameter of 5mm and 2-3 steel balls with the diameter of 2mm are added. The scissors are cleaned in time to avoid cross contamination of samples.
(3) 1.0mL of pre-heated extraction buffer was added to the centrifuge tube containing the sample and steel ball, the centrifuge tube cap was capped, and the adapter was loaded and ground for 60 seconds (frequency 70) in a TissueLyser-192 sample grinder.
(4) Taking out the centrifuge tube, and carrying out water bath at 65 ℃ for 10-30 min. Then taking out the centrifuge tube, and centrifuging for 5-10min at 1200 rpm.
(5) 1mL of the supernatant was taken into a new 2mL centrifuge tube, and 700. mu.L of phenol, chloroform, isoamyl alcohol (25:24:1) was added thereto, and mixed by slow inversion several times. Centrifuging at 1200rpm for 5-10min at room temperature.
(6) mu.L of the supernatant was taken into a new 2mL centrifuge tube, an equal volume of chloroform/isoamyl alcohol (24:1) was added, and the mixture was gently inverted and mixed several times. Centrifuging at 1200rpm for 5-10min at room temperature. Care was taken not to suck up the intermediate layer in the liquid phase.
(7) Remove 600. mu.L of supernatant into a new 1.5mL centrifuge tube. Add 400. mu.L of pre-cooled isopropanol (in which case filamentous DNA appeared), reverse slowly and mix well, place in-20 ℃ freezer for 30 min. Care was taken not to suck up the intermediate layer in the liquid phase.
(8) Centrifuge at 1200rpm for 10min at room temperature and discard the supernatant.
(9) 1mL of pre-cooled 70% ethanol was added to wash the DNA, and the mixture was centrifuged at 1200rpm for 10min, and the supernatant was discarded.
(10) The DNA is dried in a clean bench and dissolved in 100-200. mu.L deionized water.
(11) Add 21. mu.L of 0mg/mL RNase in a 37 ℃ water bath for 30min (the water bath step can be omitted).
(12) mu.L of DNA was collected and subjected to electrophoresis on a 1% agarose gel to determine the quality of the DNA, and a UVS-99 mininucleic acid detector (ACTGene, USA) was used to determine the concentration and quality of the DNA.
The third step: high resolution melting curve procedure and analysis:
in Roche
PCR amplification and HRM analysis were performed in a 480II quantitative PCR instrument (Roche). Through the previous test, a better reaction system is established. The combination of 20 μ L reaction system was: mu.L of DNA (50ng), 4. mu.L of 5 × EVA Green real PCR Mix (Gene Solution), and 0.4. mu.L (10. mu. mol. L) of each of the upstream and downstream primers-1) And the balance is made up by using water. The mixed system is placed in a 96-hole PCR plate, is instantly centrifuged by attaching a sealing plate film, and is placed in an instrument for reaction. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 15min, 45 regular cycles, followed by high resolution melting, and final product cooling to 40 ℃ for 10 s. The cycle program was 95 ℃ for 15s, 60 ℃ for 20s, 72 ℃ for 20s, for a total of 45 cycles. The high resolution melting process is: the melting curve is read at 95 ℃ for 1min, 40 ℃ for 1min, 65 ℃ for 1s and 65-95 ℃, the temperature resolution is 0.02 ℃, and fluorescence information is continuously acquired at the rate of 25 times per degree centigrade.
High resolution melting curve analysis
480 software (v1.5.0). The values of "Pre-melt Slider Settings" and "Post-melt Slider Settings" were adjusted to achieve better Normalized Melting Curves (Normalized Melting Curves) based on the actual conditions. The "Temperature Shift" threshold is generally set to 1 and in individual cases to 0 for better analysis.
The fourth step: "Zhejiang Xian Jiu" SNP typing based on HRM technique:
"Zhejiang Xian Jiu" DNA in Roche
Amplification and HRM analysis were performed in 480II quantitative PCR instrument (Roche). Theoretically, there are 3 types of SNP typing based on HRM: namely allele I (same as reference variety "Williams 82"), allele II (different from reference variety "Williams 82"), and heterozygote (the SNP site is in heterozygous state). However, due to the high genomic homozygosity of "Williams 82" and "Zhejiang Xian No. nine", only two cases were found in the present invention, and no heterozygosity occurred. The typing results can be obtained by Melting Peaks (Melting Peaks), Normalized Melting Curves (Normalized Melted Melting Curves), Normalized temperature difference views (Normalized temperature differences)&Temp-Shifted differences Plots).
The fifth step: zhejiang fresh nine-number HRM typing verification
In addition to the comparison of genotype grouping and melting curves, Sanger sequencing was performed randomly on PCR products of some SNP markers to verify the accuracy of HRM analysis.
And a sixth step: construction of 'Zhejiang Xian Jiu' SNP fingerprint:
after the accuracy of the method is confirmed by sequencing, 101 SNP markers which are developed and evenly distributed on soybean chromosomes and are based on the HRM technology are used for genotyping Zhenxian. After analysis, 34 (33.7%) SNP sites among these markers were found to show polymorphism between "Zhejiang Jiu" and "Williams 82" (FIG. 4). And (3) sequentially listing the typing results according to the sequence of chromosome numbers-physical map distances from small to large, namely the Zhejiang fresh nine-number SNP fingerprint.
The invention is further described below in connection with specific experiments.
Construction of vegetable soybean Zhejiang fresh nine SNP fingerprint
(1) SNP typing of vegetable soybean Zhejiang Jiu based on HRM technology
FIG. 2 lists two examples of SNP typing "Zhejiang Xian Jiu" based on HRM technique: and (4) typing results marked by ss715590226 and ss 715626667.
SNP typing of "Zhenxian No. nine" at site ss715590226 (G/A) showed that: neither the melting peak (fig. 2A) nor the normalized melting curve (fig. 2B) were significantly different, nor was there a regular grouping in the normalized temperature difference plot (fig. 2C), indicating that there was no difference in "zhe xian nine" at this analysis site. The genotype of the reference variety Williams 82 at the site is known as G, and it can be inferred that the genotype of the reference variety 'Zhe Xian Jiu' is the same as that of the reference variety and is G.
The SNP typing result of "Zhe Xian Jiu" at site ss715626667 (C/A) shows: the melting peak (fig. 2E) and normalized melting curve (fig. 2F) and normalized temperature difference plot (fig. 3) both show distinct sets of specific curves, indicating that there is a difference at this site between the analyzed germplasm. The genotype of the reference variety "Williams 82" at this site is known as C, and the "Zhe Xian Jiu" and "Williams 82" have different curves, so that the genotype of "Zhe Xian Jiu" can be inferred as A.
(2) HRM typing verification of vegetable soybean Zhejiang Jiu
Agreement between HRM-based SNP genotyping and Sanger sequencing was observed at all these sequenced SNP sites, indicating that the HRM-based SNP analysis system established in this study is accurate and can be used for soybean SNP genotyping.
FIG. 3 shows a sample of partial HRM genotyping and PCR sequencing.
(3) Construction of vegetable soybean Zhejiang fresh nine SNP fingerprint
After the accuracy of the method is confirmed through sequencing, 101 SNP markers which are developed and evenly distributed on soybean chromosomes and are based on the HRM technology are used for genotyping a vegetable soybean variety 'Zhenxian'. After analysis, 34 (33.7%) SNP sites among these markers were found to show polymorphism between "Zhejiang Jiu" and "Williams 82" (FIG. 4). And (3) sequentially listing the typing results according to the sequence of the chromosome numbers and the physical map distances from small to large, so that the SNP map of Zhejiang fresh No. nine is SEQ ID NO: 1: "TCAACT AGCCA CTGCT CCTAG ATGAC CGCGA TGGGG TGGGC CACCA TCACT TCTT CTGGT TACCA TACCG TGGCG AGGTC TAAAC GATCTC CTCTG CTACT".
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.
Sequence listing
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<120> soybean HRM-SNP molecular marker point marking method and application thereof
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tcaactagcc actgctccta gatgaccgcg atggggtggg ccaccatcac ttcttctggt 60
taccataccg tggcgaggtc taaacgatct cctctgctac t 101
Claims (2)
1. A soybean HRM-SNP molecular marker marking method is characterized by comprising the following steps:
development of soybean SNP marker: selecting an SNP marker from Williams 82 physical map by using a Genome browser tool in a database at every 10Mb, selecting 4-6 SNPs on each chromosome, uniformly distributing the SNPs on 20 chromosomes of a soybean Genome, and acquiring detailed information of each SNP marker, including dbSNP names, SNP positions, SNP alleles and upstream and downstream 121bp Genome sequences;
the SNP marker screening is as follows:
the dbSNP name and the PCR primer sequence of the SNP marker are as follows:
extracting soybean genome DNA, namely extracting the genome DNA of young soybean leaves according to an improved CTAB method, and diluting to 25 ng. mu.L-1Storing in a refrigerator at-20 ℃ for subsequent HRM analysis;
high resolution melting curve procedure and analysis: carrying out PCR amplification and HRM analysis on a quantitative PCR instrument by using the PCR primer marked by the SNP; high resolution melting curve analysis
480, a Gene Scanning module of software is used for obtaining a better standardized melting curve;
SNP typing of soybean based on HRM technology: soybean DNA is amplified and HRM analyzed in a quantitative PCR instrument;
soybean HRM typing verification: carrying out Sanger sequencing on PCR products marked by part of SNPs at random, and verifying the accuracy of HRM analysis;
constructing soybean SNP fingerprint: performing HRM typing on vegetable soybeans by using 101 developed SNP markers with Williams 82 as a control, and sequentially listing typing results according to the sequence of chromosome numbers and physical map distances from small to large;
in the extraction of soybean genome DNA, soybean is Zhejiang fresh nine, genome DNA of Zhejiang fresh nine young leaves is extracted according to an improved CTAB method and diluted to 25 ng. mu.L-1And storing the sample in a refrigerator at the temperature of-20 ℃ for subsequent HRM analysis, and specifically comprises the following steps:
(1) extraction buffer 2% CTAB, 100 mmol. multidot.L-1Tris-HCL，pH8.0；20mmol·L-1EDTA；2％PVP；1.4mol·L-1NaCl, makeAdding 0.2% mercaptoethanol before use, and preheating to 65 ℃;
(2) taking 0.05g of fresh leaves, properly shearing the fresh leaves, putting the fresh leaves into a 2.0ml centrifuge tube, and adding 1 steel ball with the diameter of 5mm and 2-3 steel balls with the diameter of 2 mm;
(3) adding 1.0mL of preheated extraction buffer solution into a centrifuge tube containing a sample and a steel ball, covering a centrifuge tube cover, putting the centrifuge tube cover into an adapter, and grinding the centrifuge tube cover in a TissueLyser-192 sample grinder for 60 seconds;
(4) taking out the centrifugal tube, carrying out water bath at 65 ℃ for 10-30min, then taking out the centrifugal tube, and centrifuging at 1200rpm for 5-10 min;
(5) taking 1mL of supernatant into a new 2mL centrifuge tube, adding 700 mu L of phenol, chloroform and isoamylol according to the volume ratio of 25:24:1, slowly reversing and uniformly mixing for several times; centrifuging at 1200rpm for 5-10min at room temperature;
(6) adding 800 mu L of supernatant into a new 2mL centrifuge tube, adding equal volume of chloroform isoamylol (24:1), slowly reversing and uniformly mixing for several times; centrifuging at 1200rpm for 5-10min at room temperature;
(7) taking 600 mu L of supernatant to a new 1.5mL centrifuge tube; adding 400 μ L of pre-cooled isopropanol, slowly reversing, mixing, and standing in a refrigerator at-20 deg.C for 30 min;
(8) centrifuging at room temperature and 1200rpm for 10min, and removing the supernatant;
(9) adding 1mL of precooled 70% ethanol to wash the DNA, centrifuging at 1200rpm for 10min, and removing the supernatant;
(10) the DNA is put into a superclean bench for blow-drying, and 100 and 200 mu L deionized water are added for dissolving the DNA;
(11) adding 21 mu L of 0mg/mL RNase in water bath at 37 ℃ for 30 min;
(12) taking 5 mu L of DNA, detecting the quality of the DNA by using 1% agarose gel electrophoresis, and detecting the concentration and the quality of the DNA by using a UVS-99 trace nucleic acid detector;
in the process and analysis of the high-resolution melting curve,
the combination of 20 μ L reaction system was: 2 μ L of DNA50ng, 4 μ L of 5 × EVAGreen Realtime PCR Mix, 10 μmol. L-1The upstream primer and the downstream primer are respectively 0.4 mu L, and the rest is complemented with water; placing the mixed system in a 96-hole PCR plate, attaching a sealing plate film, performing instantaneous centrifugation, and placing in an instrument for reaction;
the reaction procedure is as follows: pre-denaturation at 95 ℃ for 15min, performing 45 conventional cycles, then melting with high resolution, and finally cooling the product to 40 ℃ and keeping the temperature for 10 s;
the cycle program was 95 ℃ for 15s, 60 ℃ for 20s, 72 ℃ for 20s, for a total of 45 cycles;
the high resolution melting process is: reading a melting curve at 95 ℃ for 1min, 40 ℃ for 1min, 65 ℃ for 1s, 65-95 ℃, continuously acquiring fluorescence information at the rate of 25 times per degree centigrade, wherein the temperature resolution is 0.02 ℃;
the SNP map of Zhejiang Uxian No. nine is SEQ ID NO: 1: "TCAACT AGCCA CTGCT CCTAG ATGAC CGCGA TGGGG TGGGC CACCA TCACT TCTT CTGGT TACCA TACCG TGGCG AGGTC TAAAC GATCTC CTCTG CTACT".
2. The soybean HRM-SNP molecular marker labeling method according to claim 1,
SNP typing results of soybean based on HRM technique are represented by Melting peak fusing Peaks, Normalized Melting curve Normalized fusing Curves, Normalized temperature Difference view Normalized and Temp-Shifted Difference Plots.
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