CN113930531B - Grape stock specificity molecular marker and screening method and application thereof - Google Patents

Grape stock specificity molecular marker and screening method and application thereof Download PDF

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CN113930531B
CN113930531B CN202110756537.0A CN202110756537A CN113930531B CN 113930531 B CN113930531 B CN 113930531B CN 202110756537 A CN202110756537 A CN 202110756537A CN 113930531 B CN113930531 B CN 113930531B
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snp
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CN113930531A (en
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吴永兴
谢魏魏
王晓菲
钟醒宇
黄晓毅
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Ni Xiumei
Shandong Dafengyuan Agricultural Co ltd
Zhou Yangyan
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Zhou Yangyan
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Abstract

The invention discloses 3 kinds of grape stock specific molecular markers and a screening method and application thereof, comprising the following steps: (1) extracting leaf genomic DNA; (2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and sequencing the library on a machine after quality inspection of the library is qualified; (3) Filtering the sequencing data, obtaining SNP loci by using GATK, and filtering the SNP to improve the accuracy of the SNP; (4) Screening at least one locus of the 3 grape stocks, which is different from other two varieties, according to the parting result of each variety; (5) Finally, 5842 SNP loci for identifying 3 grape stocks are selected, wherein, among the SNP loci, hundreds of SNP locus marker groups capable of identifying 3 grape stocks with high efficiency can be combined according to the categories of the stocks which can be distinguished by each locus. The invention develops two simple, convenient, rapid and reliable methods for identifying 3 grape stocks based on the locus marks, and lays a technical foundation for accurately controlling grape varieties from the source.

Description

Grape stock specificity molecular marker and screening method and application thereof
Technical Field
The invention relates to 3 grape stock specific molecular markers, a screening method and application thereof, and belongs to the technical field of biology.
Background
Grape root nodule aphids pass from the eastern united states to europe in the middle of the 19 th century, nearly destroying the legal, ideological and germanic grapes and the wine industry within 25 years. Since the wild grape species in America has resistance to Rhizoma aphid, gaston Bazille in 1870 proposed grafting European grape on America grape, thereby determining a technical route for controlling Rhizoma aphid by using a stock, which is the only example of grafting cultivation required for one pest in the fruit tree cultivation history. To date, the major areas of grapes in the world are mostly grafted and cultivated by resistant stocks.
The Chinese research on grape stocks does not take nematode resistance and root nodule aphid resistance as main research directions, the research focus is on cold resistance stocks, and then drought resistance, salt and plant diseases and insect pests and the like, the method enters the 21 st century, the Chinese grape industry is rapidly expanded under the drive of the wine industry and facility cultivation, particularly rain-shielding cultivation, the grape cultivation area is continuously developed to the west and the south, the current national grape cultivation area reaches 55.2 ten thousand hm <2 >, the total yield is 843 ten thousand tons, but most vineyards are built from root seedlings, and the proportion of grafting seedling cultivation by using the resistance stocks is still very low. With the occurrence of the multipoint of the world quarantine pest grape root nodule aphids in the north and south and the threat of cold and dry winter, soil saline alkali and other ecological adversity brought by the climate of the mainland of China, the grape grafting cultivation has wide application potential in China, but the research on grape rootstocks in China starts later and has less research results.
France and Germany are the earliest countries engaged in grape resistant stock breeding that crossed with river bank, sand and winter grapes as parents. There are mainly 3 hybrid combinations, which are markedly different in lime resistance, drought resistance and in the resulting vigor or yield, except for being resistant to Rhizoma. (1) River bank grapes are hybridized and combined with sand grapes. The production mainly uses two varieties of 101-14 Mgt and 3309C, which are more resistant to root cancer and cold and are more resistant to root knot nematodes, and the grafted varieties can be promoted to be early matured, the yield is low and the quality is good. (2) River bank grape and winter grape hybrid combinations. The bred varieties 420A, SO, 5BB, 5C, 8B and the like are the most used types in the current production. Moisture resistance, drought tolerance, root knot nematode resistance, strong cold resistance, shallow root system, moderate and vigorous tree vigor, promotion of scion growth vigor, medium or high yield. (3) Hybrid combinations of sandy grape and winter grape. The bred stock is also a stock which is widely used in the current production, particularly in arid areas, such as 99R, 110R, 140Ru, 1103P, 1447P, 225Ru and the like. Drought resistance, deep root system distribution, barren tolerance, vigorous grafting growth vigor and high yield, and can prolong the growing period of scion variety.
The grape stock identification varieties developed by the invention mainly comprise river shore grape and sandy grape typical offspring 101-14, river shore grape and winter grape hybridization typical offspring 5BB and sandy grape and winter grape typical offspring 1103P, and the 3 varieties are the most applied varieties at present, and 3 common grape stocks can be accurately identified by the invention, so that technical support is provided for the accurate production of the grape stocks.
Disclosure of Invention
The invention provides a screening method of specific molecular markers of 3 grape stocks, and the method is applied to screen 5842 variety-specific SNP markers, and two simple, rapid and reliable methods for identifying 3 grape stocks are developed based on the markers, so that a technical foundation is laid for accurately controlling grape varieties from the source.
The invention is realized by adopting the following technical scheme:
first, the invention provides a method for screening specific molecular markers of 3 grape stocks by using a simplified genome sequencing technology Super-GBS, which comprises the following steps:
(1) Taking 6 strains 1103P, 6 strains 5BB and 5 strains 101-14 grape stocks with known and accurate varieties as samples, and taking leaves to extract genome DNA;
(2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and sequencing the library on a machine after quality inspection of the library is qualified;
(3) Filtering the sequencing data, and obtaining SNP loci by using GATK;
(4) The SNP is filtered under the following conditions: SNP sequencing depth is not less than 4; rejecting sites with MAF less than 0.01; removing loci with SNP typing deletion rate higher than 20%; removing sites with consistent typing in all samples;
(5) According to the parting result of each variety, screening all the individual parting completely consistent sites in each variety, wherein the parting consistent sites have no individual deletion and are different from other varieties; finally 5842 SNP loci were selected, see in particular Table 1.
(6) The 5842 SNP loci or part of them are used to construct the evolutionary tree and cluster analysis.
According to the 5842 SNP loci screened in the table 1, hundreds of SNP locus marker groups capable of accurately identifying 3 grape stock varieties can be screened, wherein three SNP locus marker group information and marker amplification primer information capable of accurately identifying 3 grape stock varieties are shown in the following table 2-table 7:
TABLE 2 SNP locus information set capable of accurately identifying 3 grape stock varieties
Chromosome CHROM Position POS 1103P 5BB 101-14
NC_012008.3 80704 AA AA GG
NC_012008.3 116979 CC CG CG
NC_012008.3 117210 GG AG GG
Table 3 information on SNP site identifying primers in Table 2
TABLE 4 SNP locus information set capable of accurately identifying 3 grape stock varieties
SNP site identification primer information in Table 4 described in Table 5
TABLE 6 SNP locus information set capable of accurately identifying 3 grape stock varieties
Chromosome CHROM Position POS 1103P 5BB 101-14
NC_012007.3 47129 TT TT CT
NC_012007.3 175994 AC CC AC
NC_012007.3 343319 GT GG GG
SNP site identification primer information in Table 6 described in Table 7
Secondly, the invention provides a method for identifying 3 grape stocks by using a PCR method, which comprises the following steps:
(1) Screening a plurality of SNP loci from SNP loci in table 1 to form locus groups capable of identifying 3 grape stock varieties;
(2) Designing a specific PCR amplification primer according to the position of the genome where the locus is located;
(3) Extracting 3 grape stock genome DNAs;
(4) Screening SNP marker set according to the SNP marker set screening method of claim 2, wherein three sets of the selected sets of the SNP marker set and the specific primers corresponding to the sets are shown in tables 2 to 7;
(5) PCR amplification is carried out by using a primer capable of amplifying SNP loci;
(6) Performing first-generation sequencing on the obtained PCR product;
(7) And (5) analyzing and identifying the variety of the grape stock according to the sequencing information and the SNP locus information.
The invention has the beneficial effects that:
the invention can accurately distinguish the tissue culture seedlings, grafted seedlings and finished seedlings of 3 grape stocks, ensures the control of seedling breeding enterprises on varieties, and reduces economic loss caused by errors in the breeding process.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 is a diagram showing that 3 grape stocks can be accurately distinguished by constructing evolutionary trees by using 5842 specific SNP markers of 1103P, 5BB and 101-14 3 grape stocks which are screened.
FIG. 2 is a diagram showing that sequencing and SNP screening of grape stock samples are carried out by using the Super-GBS method, finally screened SNP loci are overlapped with more than 90% of 5842 effective SNP loci, 3 grape stocks are classified by using the overlapped loci, and 3 grape stocks can be accurately distinguished.
In fig. 1 and 2 of the present invention, the varieties corresponding to the sample numbers are shown in the following table:
sequence number Variety of species Number of samples Sample numbering
1 1103P 8 E-22,E-10,E-26,E-29,E-3,E-5,E-30,E-32
2 5BB 8 E-8,E-7,E-20,E-16,E-14,E-11,E-21,E-23
3 101-14 7 E-34,E-33,E-4,E-25,E-19,E-12,E-15
Detailed Description
Example 1
The embodiment provides a method for screening 1103P, 5BB and 101-14 total 3 grape stock specific SNP molecular markers by using a simplified genome sequencing technology Super-GBS, which comprises the following steps:
(1) Extracting genome DNA from leaves of 6 strains 1103P, 6 strains 5BB and 5 strains 101-14 grape stock samples with known varieties;
(2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and sequencing the library on a machine after quality inspection of the library is qualified;
(3) Filtering the sequencing data, and obtaining SNP loci by using GATK;
(4) The SNP is filtered under the following conditions: SNP sequencing depth is not less than 4; rejecting sites with MAF less than 0.01; removing loci with SNP typing deletion rate higher than 20%; sites of consistent typing in all samples were knocked out.
(5) According to the parting result of each variety, screening that all individual parting is completely consistent in each variety, the parting consistent site has no individual deletion, and is different from at least one variety site in other 2 grape stock varieties, and finally screening
5842 SNP sites.
(6) The evolution tree and cluster analysis were constructed using 5842 SNP sites.
The specific operation steps are as follows:
this example essentially comprises the steps of digestion, ligation, purification, amplification, pooling and analysis.
1. And (3) enzyme cutting:
6 strains 1103P, 6 strains 5BB and 5 strains 101-14 with accurate varieties provided by standard institutions purchased by the company are subjected to Super-GBS library building, and the specific process is as follows (the usage amount of enzyme digestion reagent for each sample is as follows):
DEPC water: 21.4. Mu.L; 10×cutsmart buffer:3 μL; pstI-HF (4 units): 0.2 μl; mspI (8 units): 0.4. Mu.L; DNA (50 ng/. Mu.L): 5. Mu.L.
All the components are evenly mixed and then are cut for 2 hours at 37 ℃, and then the temperature is kept at 75 ℃ for 20 minutes to inactivate the enzyme.
2. And (3) connection:
adapter, barcode and restriction enzyme fragments were ligated in a 40. Mu.L system.
DEPC water: 13 μL;10×t4livebuffer: 4. Mu.L; pstI adapter (0.1 μm): 1 μl; common adapter (10. Mu.M): 1.5. Mu.L; t4 DNA library (400U/. Mu.L): 0.5. Mu.L; cleavage product: 20. Mu.L.
All the components are evenly mixed and then are cut for 2 hours at 22 ℃, and then the temperature is kept at 65 ℃ for 20 minutes to inactivate the enzyme.
3. Purification
mu.L of the ligation product was added to 0.7-fold volume of Sera-Mag beads (GE Healthcare Life Sciences), and the mixture was allowed to stand at room temperature for 5 minutes to remove small fragments of 300bp or less. The beads were recovered from the supernatant and eluted 3 times with 200. Mu.L of 70% alcohol. Finally, 40. Mu.l of 10mM Tris-HCl (pH 8.0) was used to recover the DNA from the beads.
4. Amplification of
DEPC water: 16. Mu.L; taq5 XMaster Mix: 5. Mu.L; primer 1 (10. Mu.M: 0.5. Mu.L; primer 2 (10. Mu.M): 0.5. Mu.L; purified ligation product: 3. Mu.L).
All the components are uniformly mixed and placed in a PCR instrument, the reaction conditions are that the reaction is carried out for 30 seconds at 95 ℃, the reaction is carried out for 16 cycles, each cycle is that the reaction is carried out for 30 seconds at 95 ℃, the reaction is carried out for 20 seconds at 62 ℃ and the reaction is carried out for 15 seconds at 68 ℃, and finally the reaction is carried out for 5 minutes at 68 ℃ and the reaction is preserved at 4 ℃.
5. Mixed warehouse
Library concentrations were determined for each sample using Qubit, with samples greater than 5 ng/. Mu.l for pool sequencing. Primers and small fragments in the library were removed by adding 0.7 volumes of Sera-Mag beads, and then mixed-sample sequencing was performed according to the sequencing requirements, with a sequencing platform of Illumina Nova PE150. The adaptor and primer sequences used in the library construction are detailed in Table 8 below.
TABLE 8 construction of adaptors and primer sequences from Super-GBS sequencing library
Name of the name Sequence (5 '-3')
Common adaptor top GATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
Common adaptor bot CGAGATCGGAAGAGCGGGGACTTTAAGC
PstI adaptor top CACGACGCTCTTCCGATCTAACXXXXXXTGCA
PstI adaptor bot YYYYYYAGATCGGAAGAGCGTCGTG
Primer1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
Primer2 CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAA
6. Analysis
Super-GBS sequencing was performed on a total of 17 samples for 3 grape stock varieties, yielding a total of 47M high quality reads. High quality reads were aligned to the reference genome at a rate of 76.65% -86.17% and an average sequencing depth of 38.12 × for all samples. Obtaining SNP loci by using GATK (v 3.8-1) software, screening SNP loci of which at least one variety is different from other varieties among 3 varieties, finally obtaining 5842 SNP loci, analyzing by using treebest software, drawing by using R-bag ggtree, and accurately identifying 3 grape stock varieties, wherein the identification result is shown in figure 1.
Example 2
This example provides a method according to the invention for variety identification of 2 strains of each of 3 types of grape stocks randomly collected in the variety nursery of Shandong Dafeng agricultural limited company, while adding sequencing data of 3 types of grape stock samples of known variety (sample in example 1) as a control for test and verification. The method comprises the following steps:
(1) Randomly collecting 2 strains of 1103P, 5BB and 101-14 from variety nursery of Shandong Dafeng garden agriculture limited company, and extracting genome DNA;
(2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and sequencing the library on a machine after quality inspection of the library is qualified;
(3) Filtering the sequencing data, and obtaining SNP loci by using GATK;
(4) The SNP is filtered under the following conditions: SNP sequencing depth is not less than 4; rejecting sites with MAF less than 0.01; removing loci with SNP typing deletion rate higher than 20%; removing sites with consistent typing in all samples, reserving SNP sites coincident with 5842 sites in table 1, and finally obtaining 5608 SNP sites;
(5) And constructing a evolutionary tree by utilizing the finally obtained 5608 SNP loci, and determining the variety of the collected grape samples.
The specific operation steps are as follows:
this example essentially comprises the steps of digestion, ligation, purification, amplification, pooling and analysis.
1. And (3) enzyme cutting:
Super-GBS pooling was performed on 6 strains 1103P, 5BB and 101-14 randomly collected from Shandong Dafeng garden agriculture limited company variety nursery, and the specific procedure was as follows (the amount used for each sample):
DEPC water: 21.4. Mu.L; 10×cutsmart buffer:3 μL; pstI-HF (4 units): 0.2 μl; mspI (8 units): 0.4. Mu.L; DNA (50 ng/. Mu.L): 5. Mu.L.
All the components are evenly mixed and then are cut for 2 hours at 37 ℃, and then the temperature is kept at 75 ℃ for 20 minutes to inactivate the enzyme.
2. And (3) connection:
adapter, barcode and restriction enzyme fragments were ligated in a 40. Mu.L system.
DEPC water: 13 μL;10×t4livebuffer: 4. Mu.L; pstI adapter (0.1 μm): 1 μl; common adapter (10. Mu.M): 1.5. Mu.L; t4 DNA library (400U/. Mu.L): 0.5. Mu.L. Cleavage product: 20. Mu.L.
All the components are evenly mixed and then are cut for 2 hours at 22 ℃, and then the temperature is kept at 65 ℃ for 20 minutes to inactivate the enzyme.
3. Purification
mu.L of the ligation product was added to 0.7-fold volume of Sera-Mag beads (GE Healthcare Life Sciences), and the mixture was allowed to stand at room temperature for 5 minutes to remove small fragments of 300bp or less. The beads were recovered from the supernatant and eluted 3 times with 200. Mu.L of 70% alcohol. Finally, 40. Mu.l of 10mM Tris-HCl (pH 8.0) was used to recover the DNA from the beads.
4. Amplification of
DEPC water: 16. Mu.L; taq5 XMaster Mix: 5. Mu.L; primer 1 (10 μm): 0.5. Mu.L; primer 2 (10 μm): 0.5. Mu.L; purified ligation product: 3. Mu.L.
All the components are uniformly mixed and placed in a PCR instrument, the reaction conditions are that the reaction is carried out for 30 seconds at 95 ℃, the reaction is carried out for 16 cycles, each cycle is that the reaction is carried out for 30 seconds at 95 ℃, the reaction is carried out for 20 seconds at 62 ℃ and the reaction is carried out for 15 seconds at 68 ℃, and finally the reaction is carried out for 5 minutes at 68 ℃ and the reaction is preserved at 4 ℃.
5. Mixed warehouse
Library concentrations were determined for each sample using Qubit, with samples greater than 5 ng/. Mu.l for pool sequencing. Primers and small fragments in the library were removed by adding 0.7 volumes of Sera-Mag beads, and then mixed-sample sequencing was performed according to the sequencing requirements, with a sequencing platform of Illumina Nova PE150. The adaptor and primer sequences used in the pooling procedure are detailed in Table 8 of example 1.
6. Analysis
Super-GBS sequencing was performed on 12 samples of the grape rootstock, yielding a total of 16.4M high quality reads. High quality reads were aligned to the reference genome at an alignment rate of 75.25-84.43 and an average sequencing depth of 37.27 for all samples. A large number of SNP loci are obtained by using GATK (v 3.8-1) software, compared with 5842 SNP loci in table 1, 5608 SNP loci in 5842 SNP loci are finally obtained, treebest software is adopted for analysis, R-bag ggtree is used for drawing, and the loci can be used for accurately identifying 3 grape stock varieties, and the identification result is shown in figure 2.
Example 3
The embodiment provides a PCR detection primer designed according to SNP loci of the invention, which randomly collects a plurality of grape stock genomic DNAs for PCR amplification in a Shandong Dafeng Yuan agricultural limited company variety nursery, and performs variety identification of 3 grape stocks by a sequencing method, and simultaneously adds 3 grape stocks of known varieties as positive control for test and verification. The method comprises the following steps:
(1) 2 strains of 1103P, 5BB and 101-14 varieties which are provided by standard institutions purchased by the company and are accurate are respectively taken, and genome DNA is extracted;
(2) Randomly collecting 2 strains of 1103P, 5BB and 101-14 from variety nursery of Shandong Dafeng garden agriculture limited company, and extracting genome DNA;
(3) The composition of SNP locus groups capable of accurately identifying 3 grape stock varieties is shown in table 2 by selecting loci from 5842 SNP loci;
(4) Designing upstream and downstream primers for PCR amplification according to the genomic position of the selected SNP locus, wherein the upstream and downstream primers are shown in a table 3;
(5) PCR amplification was performed using the universal primers for the 3 grape stock varieties in Table 3;
(6) Performing a first generation sequencing of the amplified sequence;
(7) And referring to the SNP locus information, and carrying out typing interpretation of the grape stock variety according to the sequence of the corresponding locus in the sequencing result.
The specific operation steps are as follows:
this example essentially comprises the steps of PCR, sequencing, alignment and analysis.
1. PCR amplification
PCR amplification primers were designed based on the positions of the sites in Table 2, and the primer sequences are shown in Table 3. The amplification conditions were 94℃for 3min,94℃for 30sec,55℃for 45sec,72℃for 45sec,37 cycles, 72℃for 7min, and 12℃for 30min. The amplification system is as follows.
DNA:2μL;Taq2×Master Mix:25μL;Primer F(10μM):1μL;Primer R(10μM):1μL; ddH 2 O:21μL。
2. Sequencing
The PCR amplified product obtained was detected by 1% agarose gel electrophoresis, and a sample having a specific amplified band obtained at the expected position was sent to Shanghai BioLimited for sequencing.
3. Sequence alignment
The sequencing results were aligned using DNAMAN software or SnapGene, and 3 grape stock varieties were typed using a set of SNP locus marker sets (see Table 2) screened at 5842 loci according to the invention.
4. Analysis
The sequence comparison and identification result shows that the identification result of 6 strains of 3 grape stock varieties 1103P, 5BB and 101-14 in total accords with the actual variety condition.
Example 4
The embodiment provides a PCR detection primer designed according to SNP loci of the invention, which randomly collects a plurality of grape stock sapling genome DNAs for PCR amplification in a Shandong Dafeng garden agriculture limited company variety nursery, and performs 3 kinds of grape stock variety identification by a sequencing method, and simultaneously adds 3 kinds of grape stocks of known varieties as positive control for test and verification. The method comprises the following steps:
(1) 2 strains of 1103P, 5BB and 101-14 varieties which are provided by standard institutions purchased by the company and are accurate are respectively taken, and genome DNA is extracted;
(2) Randomly collecting 2 strains of 1103P, 5BB and 101-14 from variety nursery of Shandong Dafeng garden agriculture limited company, and extracting genome DNA;
(3) The composition of SNP locus groups capable of accurately identifying 3 grape stock varieties is shown in table 4 by selecting loci from 5842 SNP loci;
(4) Designing upstream and downstream primers for PCR amplification according to the genomic position of the selected SNP locus, wherein the upstream and downstream primers are shown in Table 5;
(5) PCR amplification was performed using the universal primers for the 3 grape stock varieties in Table 5;
(6) Performing a first generation sequencing of the amplified sequence;
(7) And referring to the SNP locus information, and carrying out typing interpretation of the grape stock variety according to the sequence of the corresponding locus in the sequencing result.
The specific operation steps are as follows:
this example essentially comprises the steps of PCR, sequencing, alignment and analysis.
1. PCR amplification
PCR amplification primers were designed based on the positions of the sites in Table 4, and the primer sequences are shown in Table 5. The amplification conditions were 94℃for 3min,94℃for 30sec,55℃for 45sec,72℃for 45sec,37 cycles, 72℃for 7min, and 12℃for 30min. The amplification system is as follows.
DNA:2μL;Taq2×Master Mix:25μL;Primer F(10μM):1μL;Primer R(10μM):1μL; ddH 2 O:21μL。
2. Sequencing
The PCR amplified product obtained was detected by 1% agarose gel electrophoresis, and a sample having a specific amplified band obtained at the expected position was sent to Shanghai BioLimited for sequencing.
3. Sequence alignment
The sequencing results were aligned using DNAMAN software or SnapGene, and 3 grape stock varieties were typed using a set of SNP locus marker sets (see Table 4) screened at 5842 loci according to the invention.
4. Analysis
The sequence comparison and identification result shows that the identification result of 6 strains of 3 grape stock varieties 1103P, 5BB and 101-14 in total accords with the actual variety condition.
Example 5
The embodiment provides a PCR detection primer designed according to SNP loci of the invention, which randomly collects a plurality of grape stock sapling genome DNAs for PCR amplification in a Shandong Dafeng garden agriculture limited company variety nursery, and performs 3 kinds of grape stock variety identification by a sequencing method, and simultaneously adds 3 kinds of grape stocks of known varieties as positive control for test and verification. The method comprises the following steps:
(1) 2 strains of 1103P, 5BB and 101-14 varieties which are provided by standard institutions purchased by the company and are accurate are respectively taken, and genome DNA is extracted;
(2) Randomly collecting 2 strains of 1103P, 5BB and 101-14 from variety nursery of Shandong Dafeng garden agriculture limited company, and extracting genome DNA;
(3) The SNP locus group which can accurately identify 3 grape stock varieties is formed by selecting loci from 5842 SNP loci is shown in table 6;
(4) Designing upstream and downstream primers for PCR amplification according to the genomic position of the selected SNP locus, wherein the upstream and downstream primers are shown in Table 7;
(5) PCR amplification was performed using the universal primers for the 3 grape stock varieties in Table 7;
(6) Performing a first generation sequencing of the amplified sequence;
(7) And referring to the SNP locus information, and carrying out typing interpretation of the grape stock variety according to the sequence of the corresponding locus in the sequencing result.
The specific operation steps are as follows:
this example essentially comprises the steps of PCR, sequencing, alignment and analysis.
1. PCR amplification
PCR amplification primers were designed based on the positions of the sites in Table 6, and the primer sequences are shown in Table 7. The amplification conditions were 94℃for 3min,94℃for 30sec,55℃for 45sec,72℃for 45sec,37 cycles, 72℃for 7min, and 12℃for 30min. The amplification system is as follows.
DNA:2μL;Taq2×Master Mix:25μL;Primer F(10μM):1μL;Primer R(10μM):1μL; ddH 2 O:21μL。
2. Sequencing
The PCR amplified product obtained was detected by 1% agarose gel electrophoresis, and a sample having a specific amplified band obtained at the expected position was sent to Shanghai BioLimited for sequencing.
3. Sequence alignment
The sequencing results were aligned using DNAMAN software or SnapGene, and 3 grape stock varieties were typed using a set of SNP locus marker sets (see Table 6) screened at 5842 loci according to the invention.
4. Analysis
The sequence comparison and identification result shows that the identification result of 6 strains of 3 grape stock varieties 1103P, 5BB and 101-14 in total accords with the actual variety condition.
Other SNP locus marker groups are selected from 5842 SNP loci, and 3 grape stock varieties of 1103P, 5BB and 101-14 can be accurately identified.
In Table 1, the 11 grape varieties represented by the numbers 1-3 are in order: 1103P, 5BB, 101-14.
Table 1 is as follows:
sequence listing
<110> Shandong Dafeng Yuan agricultural Co Ltd
<120> grape stock specific molecular marker and screening method and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gatccatgcg tcggagaagt 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
acagggcata ccagaactgc 20
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
aagatgctcc tcagccttca g 21
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aaaactgatg cattggcggc 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
cctgcatcca acccaagtga 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gcttccgaat tggcgctatc 20

Claims (3)

1. The application of the primer pair combination for detecting SNP loci in table 2 in accurately identifying 3 grape stocks is characterized in that the 3 grape stocks are 103P, 5BB and 101-14;
the SNP locus information is shown in Table 2, and the primer pair information is shown in Table 3:
TABLE 2 SNP locus information set capable of accurately identifying 3 grape stocks
Chromosome CHROM Position POS 1103P 5BB 101-14 NC_012008.3 80704 AA AA GG NC_012008.3 116979 CC CG CG NC_012008.3 117210 GG AG GG
Table 3 information on SNP site identifying primers in Table 2
2. The application of the primer pair combination for detecting SNP loci in table 4 in accurately identifying 3 grape stocks, wherein the 3 grape stocks are 103P, 5BB and 101-14;
the SNP locus information is shown in Table 4, and the primer pair information is shown in Table 5:
TABLE 4 SNP locus information set capable of accurately identifying 3 grape stocks
Chromosome CHROM Position POS 1103P 5BB 101-14 NC_012009.3 38889 CT CC CC NC_012009.3 41444 CT CT TT NC_012009.3 302576 TT AT TT
SNP site identification primer information in Table 4 described in Table 5
3. The application of the primer pair combination for detecting SNP loci in table 6 in accurately identifying 3 grape stocks, wherein the 3 grape stocks are 103P, 5BB and 101-14;
the SNP locus information is shown in Table 6, and the primer pair information is shown in Table 7:
TABLE 6 SNP locus information set capable of accurately identifying 3 grape stocks
Chromosome CHROM Position POS 1103P 5BB 101-14 NC_012007.3 47129 TT TT CT NC_012007.3 175994 AC CC AC NC_012007.3 343319 GT GG GG
SNP site identification primer information in Table 6 described in Table 7
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CN103937885A (en) * 2014-04-02 2014-07-23 西北农林科技大学 DNA (Deoxyribonucleic Acid) molecular marker for rapidly identifying phylloxera resistance of grape stock and application thereof
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