CN107299146B - Soybean cyst nematode-resistant related CAPS (cleaved amplified polymorphic sequence) marker detection method and primers - Google Patents
Soybean cyst nematode-resistant related CAPS (cleaved amplified polymorphic sequence) marker detection method and primers Download PDFInfo
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Abstract
The invention discloses a CAPS marker detection method and primers related to soybean cyst nematode resistance. The invention provides the application of the single nucleotide polymorphism of the following SNP sites in a soybean genome or a substance for detecting the single nucleotide polymorphism of the following SNP sites in the soybean genome in identifying or assisting in identifying the resistance of soybeans to soybean cyst nematodes; the SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T. According to the invention, the soybean cyst nematode resistant material can be selected by detecting the genotype of the SNP locus, so that the time-consuming and labor-consuming phenotype identification can be avoided, and the soybean cyst nematode resistance screening is carried out on the breeding material through the genotype, so that the selection efficiency of disease-resistant varieties in the breeding process is greatly improved, the breeding time is shortened, and the cultivation effect of the soybean cyst nematode resistant varieties is remarkable.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method and a primer for detecting CAPS (cleaved amplified polymorphic sequence) markers related to soybean cyst nematode resistance.
Background
Soybean Cyst Nematode (SCN) is an obligate static parasitic Nematode in roots, causing great loss in Soybean production. In the united states alone, the damage caused by soybean cyst nematodes among various diseases on soybeans from 1996 to 2014, reached approximately 36%. The cultivation of resistant materials and the combination of crop rotation are one of the main methods for controlling diseases. Due to the genetic complexity of the SCN resistance gene and the diversity of the physiological races of the soybean cyst nematodes, the breeding of the soybean cyst nematode resistant variety by using the traditional breeding method not only needs a long time, but also is difficult to succeed, cannot cope with the toxicity variation of the nematodes, cannot meet the development of the current anti-nematode breeding, and urgently needs a more accurate and efficient method for identifying the SCN. With the development and use of Molecular markers, Molecular marker-assisted selection (MAS) becomes an effective method that can save manpower and material resources and accelerate breeding processes. The great advantage of MAS is that it can increase the efficiency and speed of breeding work by identifying resistant plants by confirming whether they carry the target gene without the need to evaluate phenotypic characteristics.
Disclosure of Invention
The first purpose of the invention is to provide the application of the single nucleotide polymorphism of the following SNP sites in the soybean genome (Glyma. Wm82.a2) or a substance for detecting the single nucleotide polymorphism of the following SNP sites in the soybean genome in identification or auxiliary identification of soybean cyst nematode resistance.
The SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T.
It is a second object of the present invention to provide an agent for identifying or aiding in the identification of soybean resistance to soybean cyst nematode.
The reagent for identifying or assisting in identifying the resistance of the soybean to the soybean cyst nematode is a substance for detecting the single nucleotide polymorphism of the following SNP sites in a soybean genome; the SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T.
Specifically, the "substance for detecting a single nucleotide polymorphism at a SNP site in soybean genome" may be the following a) or b):
a) the primer pair A is (a1) or (a 2):
(a1) a primer pair consisting of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table;
(a2) and (b) the primer pair which consists of two single-stranded DNAs shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequences 2 and 3 in the sequence table and has the same functions as the primer pair in (a 1).
b) The primer pair A and a restriction endonuclease AciI or an isoschizomer thereof.
In the primer pair A, the molar ratio of the single-stranded DNA shown in the sequence 2 in the sequence table to the single-stranded DNA shown in the sequence 3 in the sequence table can be 1: 1. In the primer pair A, two single-stranded DNAs can be independently packaged or mixed and packaged according to the molar ratio of 1: 1.
Kits containing the reagents are also within the scope of the invention.
The application of the reagent or the kit in identifying or assisting in identifying the resistance of the soybean to be detected to the soybean cyst nematode also belongs to the protection scope of the invention.
The third objective of the invention is to provide the following two methods for identifying soybean resistance to soybean cyst nematode.
The method I comprises the following steps: a method for identifying or assisting in identifying whether soybean to be detected is resistant to soybean cyst nematode specifically comprises the following steps: detecting nucleotides at the following SNP sites in the genome of the soybean to be detected so as to determine the genotype of the soybean to be detected; determining whether the soybean to be detected is resistant to soybean cyst nematode according to the genotype of the soybean to be detected as follows: if the soybean to be detected is T, namely T genotype, the soybean to be detected resists or is a candidate for resisting the soybean cyst nematode; and if the soybean to be detected is G: G genotype, the soybean to be detected does not resist or is not candidate to resist the soybean cyst nematode.
Method II: a method for identifying or assisting in identifying the strength of resistance of soybean to soybean cyst nematodes comprises the following steps: detecting nucleotides at the following SNP sites in the genome of the soybean to be detected so as to determine the genotype of the soybean to be detected; determining the strength of the resistance of the soybean to be detected to the soybean cyst nematode according to the genotype of the soybean to be detected as follows: the resistance of the soybean to be detected with the T gene type to the soybean cyst nematode is stronger than or is candidate to be stronger than that of the soybean to be detected with the G gene type.
In the two methods, the SNP site corresponds to the 113 th site of the nucleotide sequence shown in the sequence 1 in the sequence table in the soybean genome; the nucleotide at the SNP site is G or T. The T is homozygous at position 113 of the soybean genome corresponding to the nucleotide sequence shown in sequence 1 in the sequence table as T. The G gene type is homozygote with G at the 113 th site corresponding to the nucleotide sequence shown in the sequence 1 in the sequence table in the soybean genome.
In the above two methods, the method for detecting the nucleotides at the following SNP sites in the genome of the soybean to be detected may specifically include the following two steps: performing PCR amplification and complete enzyme digestion on a product obtained by the PCR amplification by using a restriction enzyme AciI or an isoschizomer thereof so as to determine the nucleotide at the SNP site; the primer pair used for PCR amplification meets the following conditions: and the sequence of an amplification product obtained by carrying out PCR amplification by using the genome DNA of the soybean to be detected as a template contains a sequence 1 in the sequence table.
Further, the primer pair may be the primer pair a described above.
More specifically, the method for detecting the nucleotides at the following SNP sites in the genome of the soybean to be detected comprises the following steps: performing PCR amplification on the A by using the genome DNA of the soybean to be detected as a template and adopting the primer to obtain an amplification product; and (2) completely digesting the amplification product by using a restriction enzyme AciI (the reaction condition can be that the digestion reaction is carried out for 40 minutes at 37 ℃), and determining the nucleotide at the SNP site in the soybean genome to be detected according to the digestion result as follows: if the obtained enzyme digestion product is a 283bp single DNA fragment, the nucleotide at the SNP site in the soybean genome to be detected is T; and if the obtained enzyme digestion product is two DNA fragments of 112bp and 171bp, the nucleotide at the SNP site in the soybean genome to be detected is G.
Wherein, the 283bp single DNA fragment is a DNA fragment shown in a sequence 1 in a sequence table (the 113 th site of the sequence 1 is T). The two DNA fragments of 112bp and 171bp are respectively the two DNA fragments shown in the 1 st-112 th site and the 113 th-283 th site of the sequence 1 in the sequence table (the 113 th site of the sequence 1 is G).
The application of the reagent or the kit or the method in soybean breeding also belongs to the protection scope of the invention.
The fourth purpose of the invention is to provide a method for cultivating a new soybean variety resisting soybean cyst nematode.
The method for cultivating the soybean variety resisting the soybean cyst nematode provided by the invention specifically comprises the steps of selecting the soybean with T: T genotype as a parent to breed; the T is homozygous at position 113 of the soybean genome corresponding to the nucleotide sequence shown in sequence 1 in the sequence table as T.
In the invention, the soybean cyst nematode is specifically soybean cyst nematode No. 3 physiological race.
Experiments prove that SNP loci existing on a disease-resistant gene Glyma.11G234500 are detected and developed into CAPS marks, materials carrying disease-resistant allelic variation of the gene can be selected by detecting the genotype of the SNP loci, time-consuming and labor-consuming phenotype identification can be avoided, and the soybean cyst nematode No. 3 physiological race resistance screening of breeding materials is carried out through the genotype, so that the selection efficiency of disease-resistant varieties in the breeding process is greatly improved, the breeding time is shortened, and the breeding effect on the soybean cyst nematode-resistant varieties is remarkable.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis detection of the amplified product of primer pair A after digestion.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Soybean cyst nematode, physiological race No. 3: the CAPS/dCAPS marker of soybean cyst nematode major disease-resistant gene Rhg4(GmSHMT) is developed and utilized in the article of plant science 2015,41(10):1463-1471, which is available to the public from the applicant and can only be used in the experiments of the duplicated invention.
Example 1 development and application of CAPS markers associated with soybean cyst nematode resistance
First, primer design and marker development
Glyma.11G234500 gene sequences are downloaded from phytozome (https:// phytozome.jgi.doe.gov/pz/portal.html), and Glyma.11G234500 gene and a homologous gene Glyma.18G022500 gene have high similarity in nucleotide sequences, so that in order to ensure the specificity of primer sequences, sequence alignment of the two genes is firstly carried out, and primers are designed at different sites. Primer pair a was designed using primer5.0 software. SNP is developed into CAPS marker (the marker site information selected by the invention is shown in Table 1) by using online software dCAPSFinder2.0(http:// helix.wustl.edu/dcaps. html), and enzyme AciI (Beijing Bailingke biology company, Cat: R0551S) is selected for enzyme cutting analysis. The estimated length of the PCR amplification product of the primer pair A is 283bp (shown as a sequence 1 in a sequence table). The SNP locus selected by the invention corresponds to the 113 th site of the nucleotide sequence shown in the sequence 1 in the sequence table in the soybean genome; the nucleotide at the SNP site is G or T. When the nucleotide at the SNP site is G (corresponding to allelic variation genotype GG in Table 1), the PCR amplification product of the primer pair A can theoretically obtain two fragments of 171bp and 112bp after enzyme digestion by AciI. When the nucleotide at the SNP site is T (corresponding to the allelic variation genotype TT in the table 1), the PCR amplification product of the primer pair A cannot be digested by AciI, and the size of the product is 283 bp.
Primer pair A:
f: 5'-CAACTTCTTGTGACTGGACAGCTTA-3' (SEQ ID NO: 2);
r: 5'-CTAGTGAATCAGCAAACAAAATAGT-3' (SEQ ID NO: 3).
TABLE 1 marker site information
| Type of mark | Type of variation (SNP) | The gene of | Chromosome | Allelic variant genotype |
| CAPS | T/G | Glyma.11G234500 | Chromosome 11 | TT、GG |
Second, establishing method for detecting soybean cyst nematode resistance by using CAPS (cleaved amplified polymorphic sequence) marker by using soybean variety with known soybean cyst nematode resistance
The tested soybeans comprise 5 parts of soybean varieties with known soybean cyst nematode resistance, such as medium yellow 13 (susceptible disease), medium quality 03-5373 (disease-resistant), black soybean with branch gray skin (disease-resistant), L ee (susceptible disease) and Peking (disease-resistant).
1. Extraction of genomic DNA
The DNA of the soybean leaves is extracted according to the operation guide of a rapid DNA extraction kit (MBI Fermentas company, the product number: K0512), and the DNA of the soybean genome to be detected is extracted.
2. PCR amplification
And (3) performing PCR amplification on the A by using the soybean genomic DNA to be detected as a template and using the primer pair A to obtain a PCR amplification product.
The PCR reaction system is 20 mu L, and comprises 60ng of genomic DNA, 10 × PCR buffer 2 mu L, 2mmol L- 1dNTPs 1.5μL，2mmol·L-1Primer 2. mu. L and 1U Taq polymerase 02 μ L (Total gold Biotechnology Ltd., cat # AP112) by ddH2O make up the volume to 20 μ L.
The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, optimized annealing temperature of 57 ℃ for 30s, extension at 72 ℃ for 30s, and 36 cycles; finally, extending for 10min at 72 ℃; stored at 4 ℃. The PCR reaction was amplified on a PCR amplification thermal cycler from ABI (applied biosystems, USA).
The PCR product corresponding to the primer pair A is detected by 1% agarose gel electrophoresis, 5 parts of DNA of known genotypes of Zhonghuang 13, Zhongpin 03-5373, Botrytis cinerea, L ee and Peking soybean varieties are subjected to PCR amplification, and all 5 parts of materials obtain a single PCR product with 283bp and similar length and size with the target fragment.
3. Enzyme digestion analysis
The PCR product obtained by primer pair A was completely digested with AciI endonuclease (enzyme-sufficient).
Mu.l of the above digestion reaction system, 5. mu.l of PCR product, 0.2. mu.l of AciI endonuclease, 1.5. mu. L NEB buffer using ddH2And O, complementing the volume to 10 mu L, placing the enzyme digestion reaction system into a 37 ℃ heat preservation box or a water bath kettle, carrying out enzyme digestion for 40min, and taking out to obtain an enzyme digestion product, detecting the AciI enzyme digestion product by adopting 2% agarose gel electrophoresis, wherein if the enzyme digestion product is a 283bp target strip, the soybean to be detected carries the TT allele, and if the enzyme digestion product is two 171bp and 112bp target strips (sequencing detection size), the soybean to be detected carries the GG allele.
The results of the amplification and the enzyme digestion of 5 identified material primer pairs A are shown in FIG. 1;
the 283bp middle-quality 03-5373 enzyme digestion product (carrying TT allele) of the soybean cyst nematode resistant;
the enzyme digestion products of the yellow 13 enzyme which is not resistant to the soybean cyst nematode are 171bp and 112bp (namely carrying GG allele);
the L ee enzyme cutting products which are not resistant to the soybean cyst nematode are 171bp and 112bp (namely carrying GG allele);
the Peking enzyme digestion product for resisting soybean cyst nematode is 283bp (namely carrying TT allele);
the enzyme digestion product of the branch of the tonka-bean cyst nematode-resistant black bean of the grey skin is 283bp (namely carrying TT allele).
From the above results, the following can be concluded: if the soybean to be detected carries TT allele, the soybean to be detected resists soybean cyst nematode; and if the soybean to be detected carries GG allele, the soybean to be detected does not resist or candidate does not resist soybean cyst nematode.
Third, specific application example of detecting soybean cyst nematode resistance by CAPS marker
1. Resistance test in the field
The soybean varieties in the table 2 are identified as soybean cyst nematode resistance in Harbin land area of the institute of agriculture and sciences of Heilongjiang province infected with soybean cyst nematode No. 3 physiological races, and greenhouse potting disease soil, a complete random block test design scheme is adopted in the field, the field planting row width is 0.65 m, the row length is 1.5 m, the plant spacing is 0.05 m, the greenhouse is identified by potting, 3 pots are planted for each variety, 5 plants are planted for each pot, after emergence of the seedlings for 30 days, 10 plants with the same growth vigor are randomly selected from each material in the field and the greenhouse, the cyst number of each plant is counted, the average cyst number of each identified variety is calculated, and in order to detect the cyst Index (Femallndex, FI) difference, the genetic analysis and resistance analysis of the excellent strain medium strain 03-5373 series spectrum and the genetic analysis and resistance of the soybean cyst resistance related markers of the excellent strain are used as the genetic analysis and resistance markers of the soybean cyst resistance markers of the excellent strain 03-5373 (Zsylvestiges) in every 40 rows, and the genetic analysis and resistance markers of the soybean cyst resistance of the soybean cyst of the soybean varieties are calculated as the comparison formula No. 10, the most recent resistance Index (MR19) of the Resistant varieties of the soybean cyst resistance of the soybean cyst.
2. CAPS marker detection of the invention
And respectively carrying out PCR amplification and enzyme digestion tests on the soybean variety in the table 2 by adopting the methods in the first step and the second step.
3. Results
The soybean varieties in the table 2 are respectively subjected to PCR amplification and enzyme digestion tests by adopting the methods of the first step and the second step, and the enzyme digestion results are shown in the table 2.
And (3) respectively carrying out a soybean cyst nematode resistance test on each soybean variety in the table 2 by adopting the third step, wherein the resistance result is shown in the table 2.
The result further shows that the primer pair A and the method can be used for carrying out soybean cyst nematode resistance detection on the soybean to be detected, and the identification is accurate.
TABLE 2 SNP site genotypes of Soybean varieties
The varieties referred to in Table 2 above are described in the following documents, wherein the list of Chinese soybean variety resources from the King national gao, Beijing, Chinese agricultural Press, 1982, Chang Zhen, Sun Jian, China soybean variety resources, Sankyo, agricultural Press, 1991, Chang Zhen, Sun Jian, Qili, Chen Yi, Chinese soybean variety resources, Chang Di, Beijing, Chinese agricultural Press, 1996, Zusangqi, Li Yingjin, Li Jinying, Qiu, Excellent line 03-5373 series of varieties, Genetic analysis and identification of marker related to soybean cyst Nematode Resistance, Genencor scientific report, 2013,39(10): 6-1753.Chen, Y., D.Wang, P.Arelli, M.Ebrahimi and R. L, Nelson.Moversal = molecular dynamics, Ttgrasonic strain, Ttgt 6378, Ttgt 5. Ttgrast and Ttgt 5. Ttgt × 3. Ttgrast strain and Ttgt.
The results in Table 2 show that after the target amplification products of 57 varieties in 60 soybean disease-resistant varieties are subjected to enzyme digestion, only one 283bp fragment exists, and the allelic gene type is TT; the enzyme digestion products of 12 varieties in 14 soybean susceptible (including susceptible) varieties are 171 and 112 fragments, and the allele type is GG.
The identification efficiency of the resistant varieties or lines (resistant varieties carrying the TT allele/all resistant varieties) in Table 2 was calculated to be 95%.
The identification efficiency of the susceptible varieties or strains (susceptible varieties carrying the GG allele/all susceptible varieties) in Table 2 was calculated to be 85.7%.
The method can be used for identifying the resistance of the soybean to be tested to the soybean cyst nematode.
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Claims (12)
1. The application of the substance for detecting the single nucleotide polymorphism of the SNP sites in the soybean genome in identifying or assisting in identifying the soybean cyst nematode resistance;
the SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T.
2. The application of a substance for detecting the single nucleotide polymorphism of the following SNP sites in the soybean genome in preparing a product for identifying or assisting in identifying the soybean cyst nematode resistance; the SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T.
3. Use according to claim 1 or 2, characterized in that: the 'substance for detecting the single nucleotide polymorphism of the SNP sites in the soybean genome' is a primer pair A and a restriction endonuclease AciI or an isoschizomer thereof;
the primer pair A is a primer pair consisting of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table.
4. Use of a kit containing the "substance for detecting a single nucleotide polymorphism at a SNP site in a soybean genome" as set forth in claim 3 for the preparation of a product for identifying or assisting in identifying soybean cyst nematode resistance.
5. Use of a kit containing the "substance for detecting a single nucleotide polymorphism at a SNP site in soybean genome" as set forth in claim 3 for identification or auxiliary identification of soybean cyst nematode resistance.
6. A method for identifying or assisting in identifying whether soybeans to be detected are resistant to soybean cyst nematodes comprises the following steps: detecting nucleotides at the following SNP sites in the genome of the soybean to be detected so as to determine the genotype of the soybean to be detected; determining whether the soybean to be detected is resistant to soybean cyst nematode according to the genotype of the soybean to be detected as follows: if the soybean to be detected is T, namely T genotype, the soybean to be detected resists or is a candidate for resisting the soybean cyst nematode; if the soybean to be detected is G, G genotype, the soybean to be detected does not resist or candidate does not resist the soybean cyst nematode;
the SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T;
the T genotype is homozygote with T at the 113 th site corresponding to the nucleotide sequence shown as the sequence 1 in the sequence table in the soybean genome;
the G gene type is homozygote with G at the 113 th site corresponding to the nucleotide sequence shown in the sequence 1 in the sequence table in the soybean genome.
7. A method for identifying or assisting in identifying the strength of resistance of soybeans to soybean cyst nematodes to be detected comprises the following steps: detecting nucleotides at the following SNP sites in the genome of the soybean to be detected so as to determine the genotype of the soybean to be detected; determining the strength of the resistance of the soybean to be detected to the soybean cyst nematode according to the genotype of the soybean to be detected as follows: the resistance of the soybean to be detected with the T genotype to the soybean cyst nematode is stronger than or is candidate to be stronger than that of the soybean to be detected with the G genotype;
the SNP site corresponds to the 113 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in a soybean genome; the nucleotide at the SNP site is G or T;
the T genotype is homozygote with T at the 113 th site corresponding to the nucleotide sequence shown as the sequence 1 in the sequence table in the soybean genome;
the G gene type is homozygote with G at the 113 th site corresponding to the nucleotide sequence shown in the sequence 1 in the sequence table in the soybean genome.
8. The method according to claim 6 or 7, characterized in that: the method for detecting the nucleotides at the following SNP sites in the genome of the soybean to be detected comprises the following two steps: performing PCR amplification and complete enzyme digestion on a product obtained by the PCR amplification by using a restriction enzyme AciI or an isoschizomer thereof so as to determine the nucleotide at the SNP site; the primer pair used for PCR amplification meets the following conditions: and the sequence of an amplification product obtained by carrying out PCR amplification by using the genome DNA of the soybean to be detected as a template contains a sequence 1 in the sequence table.
9. The method of claim 8, wherein: the primer pair is the primer pair A described in claim 3.
10. The method of claim 9, wherein: the method for detecting the nucleotides at the following SNP sites in the genome of the soybean to be detected comprises the following steps: performing PCR amplification on the A by using the genome DNA of the soybean to be detected as a template and adopting the primer to obtain an amplification product; and (3) completely digesting the amplification product by using a restriction enzyme AciI, and determining the nucleotide at the SNP site in the soybean genome to be detected according to the digestion result as follows: if the obtained enzyme digestion product is a 283bp single DNA fragment, the nucleotide at the SNP site in the soybean genome to be detected is T; and if the obtained enzyme digestion product is two DNA fragments of 112bp and 171bp, the nucleotide at the SNP site in the soybean genome to be detected is G.
11. The "substance for detecting a single nucleotide polymorphism at a SNP site in soybean genome" as set forth in claim 3 or a kit containing the "substance for detecting a single nucleotide polymorphism at a SNP site in soybean genome" as set forth in claim 3 or the use of the method as set forth in any one of claims 6 to 10 for breeding soybean.
12. A method for breeding soybean variety resisting soybean cyst nematode comprises the steps of selecting soybean with T: T genotype as parent to breed; the T is homozygous at position 113 of the soybean genome corresponding to the nucleotide sequence shown in sequence 1 in the sequence table as T.
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