CN113355392B - Apple rootstock specific molecular marker locus, and screening method and application thereof - Google Patents

Apple rootstock specific molecular marker locus, and screening method and application thereof Download PDF

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CN113355392B
CN113355392B CN202110536824.0A CN202110536824A CN113355392B CN 113355392 B CN113355392 B CN 113355392B CN 202110536824 A CN202110536824 A CN 202110536824A CN 113355392 B CN113355392 B CN 113355392B
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尼秀媚
王彦娟
钟醒宇
薛其勤
李美芹
刘晓明
杨园园
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Shandong Dafengyuan Agricultural Co ltd
Weifang University of Science and Technology
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Abstract

The invention discloses 6 dwarf apple rootstock specific molecular marker loci, a screening method and application thereof, comprising the following steps: (1) taking leaves to extract genome DNA; (2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and performing on-machine sequencing after the quality of the library is qualified; (3) Filtering the sequencing data, screening and verifying to obtain 14096 SNP marker loci for accurately identifying 6 apple rootstocks; (4) In 14096 SNP marker loci, thousands of SNP marker loci which can efficiently identify 6 stocks can be combined according to the type of the stocks which can be distinguished by each locus; (5) Two simple, convenient, rapid and accurate methods for identifying 6 dwarf apple rootstocks are developed based on the locus marks, and a technical foundation is laid for ensuring accurate identification and control of apple rootstock varieties in scientific research departments, nursery stock breeding and orchard planting enterprises.

Description

Apple rootstock specific molecular marker locus, and screening method and application thereof
Technical Field
The invention relates to 6 dwarf apple rootstock specific molecular markers and a screening method and application thereof, belonging to the technical field of biology.
Background
The apple is a plant of Maloideae (Maloideae) Maloideae (Rosaceae) Maloideae (Maloideae), and is one of main fruit trees in China. The planting area and the yield of the apples in China account for 40 percent of the world, and the cultivation mode is mainly based on the traditional arbor or dwarfing interstock. Dwarf stock close planting cultivation is a main cultivation mode of apples in the world, and newly-built orchards in advanced apple producing countries almost adopt dwarf self-rooted stocks, and have the advantages of high yield, quick response, easy updating of new varieties, low management cost and labor saving. French has wide application to apple nutritional rootstocks, and more than 85% of apple trees are dwarf rootstocks and semi-dwarf rootstocks of M lines in China. The Netherlands are one of the more common countries in Europe where dwarfing stocks are used, and apple production by using dwarfing stocks is one of the major export countries. At present, more than 70% of apple trees nationwide are grafted by M-series rootstocks. Along with the increasingly outstanding contradiction of labor shortage in China and the rapid optimization and updating of new varieties, the dwarf stock close planting cultivation mode gradually replaces the traditional orchard planting mode. The dwarfing self-rooted rootstock introduced abroad is popularized through experimental demonstration for over ten years, is widely adopted in newly-built orchards at present, and has extremely obvious production advantages.
M-series rootstocks are bred by British woodland laboratory stations in 1912, then, M-series rootstocks and MM-series rootstocks are bred by hybridization of M-series rootstocks of other varieties in various countries, wherein a virus-free dwarf rootstock superior line M9-T337 (NAKB-T337) bred by a Dutch woody plant nursery detection service center is most successfully and widely applied in various countries in the world, and the virus-free dwarf rootstock superior line has the characteristics of short and small tree bodies, early fruiting, easy early high yield, salt and alkali resistance and relatively moisture resistance.
The B-series rootstock is a dwarf rootstock popularized as a severe winter cold resistant rootstock at Soviet Union Mizulene gardening university, and M8 is bred by hybridization with Red Standard. Wherein, the B9 is widely applied, has certain resistance to the neck rot and has moderate resistance to the powdery mildew and the apple scab. The most important characteristics are cold resistance, frost resistance and extreme low temperature resistance of-40 ℃.
The G-line rootstock is bred by a Geneva test station in New York, connel university, USA, and breeding targets are focused on the aspects of production capacity, cold resistance, disease and insect resistance (mainly fire blight and continuous cropping disease) and the like. The serial rootstocks are mainly characterized by high yield, disease resistance and continuous cropping resistance, and are the most prominent achievements of world apple dwarf rootstocks in the last decade.
M111, N9, B9, G11, M26 and M9 are main popularization and application varieties of apple rootstocks in China, are suitable for different apple planting areas in China, and are widely applied to China in recent years. Because the dwarfing stock line has a plurality of strains, small morphological difference and difficult identification, the accurate identification of the strains has great significance for the links of new variety breeding, resource nursery garden establishment, enterprise nursery stock breeding and selling, orchard nursery stock planting production and the like of scientific research departments.
Disclosure of Invention
In recent years, the development of sequencing technology has brought unique advantages in the work of variety-specific genetic marker screening and variety identification. The invention provides specific molecular marker loci of 6 apple rootstocks, a screening method and application thereof, wherein 14096 variety-specific SNP loci are screened, two simple, convenient, rapid and reliable methods for identifying 6 apple rootstocks are developed based on the loci, and a technical basis is laid for accurately controlling apple varieties from the source.
In order to solve the technical problems, the invention adopts the following technical scheme to realize:
firstly, the invention provides a method for screening specific molecular markers of 6 apple rootstocks by using a simplified genome sequencing technology Super-GBS, which comprises the following steps:
(1) Taking 4M 111, 3N 9, 4B 9, 4G 11, 5M 26 and 4M 9 apple 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 performing on-machine sequencing after the quality of the library is qualified;
(3) Filtering sequencing data, and then obtaining SNP loci by using GATK;
(4) Filtering the SNP under the following conditions: the SNP sequencing depth is not less than 4; eliminating sites with MAF less than 0.01; eliminating the sites with SNP typing deletion rate higher than 20%; removing the sites with consistent typing in all samples;
(5) Screening all sites which have completely consistent individual types and have no individual deletion and are different from other varieties in the consistent sites of the individual types according to the typing result of each variety; finally 14096 SNP sites are screened, and the specific results are shown in Table 1;
(6) And (3) constructing an evolutionary tree and clustering analysis by using 14096 SNP sites or partial SNP sites.
Furthermore, by using 14096 SNP sites in Table 1, thousands of SNP site marker groups which can accurately identify 6 apple stocks can be screened out, wherein three groups of SNP site marker group information and marker amplification primer information which can accurately identify 6 apple stocks are as follows:
table 2A set of SNP locus marker groups capable of accurately identifying 6 apple rootstocks
Figure BDA0003070000740000021
TABLE 3 SNP site identifying primer information in Table 2
Figure BDA0003070000740000022
Figure BDA0003070000740000031
TABLE 4A set of SNP locus marker groups capable of accurately identifying 6 apple rootstocks
Figure BDA0003070000740000032
TABLE 5 SNP site identifying primer information in Table 4
Figure BDA0003070000740000033
Figure BDA0003070000740000041
TABLE 6A set of SNP locus marker groups capable of accurately identifying 6 apple rootstocks
Figure BDA0003070000740000042
TABLE 7 SNP site identifying primer information in Table 6
Figure BDA0003070000740000043
Secondly, the invention provides a method for identifying 6 apple rootstocks by using a PCR method, which comprises the following steps:
(1) Screening SNP loci from the SNP loci in the table 1 to combine several locus groups capable of identifying 6 apple rootstock varieties;
(2) Designing a specific PCR amplification primer according to the genome position of the locus;
(3) Extracting 6 kinds of apple rootstock genome DNA;
(4) The SNP marker cluster screening method according to claim 2, wherein the three selected SNP marker clusters and the specific primers corresponding to the selected sites are listed in the following tables 2 to 7;
(5) Carrying out PCR amplification by using a primer capable of amplifying the SNP locus;
(6) Carrying out first-generation sequencing on the obtained PCR product;
(7) And analyzing and identifying the apple rootstock variety according to the sequencing information and the SNP locus information.
The invention has the beneficial effects that:
the apple rootstock variety gene difference is small, the gene difference of the same series varieties is smaller, and the apple rootstock variety can not be obviously distinguished in appearance morphology, a large number of individual difference sites need to be removed in the SNP screening process and scientifically and accurately verified, so that the SNP sites of 6 apple rootstocks can be accurately identified, therefore, the simplified genome sequencing technology Super-GBS method is utilized in the invention and combined with the repeated verification of a standard sample, a large number of individual difference SNP sites are filtered and removed, and 14096 screened SNP sites can be used for simply, conveniently, quickly and accurately identifying 6 apple rootstocks accounting for more than 95% of the market share.
At present, apple seedlings in China are grafted by adopting rootstocks, the method can accurately distinguish tissue culture seedlings, grafted seedlings and finished seedlings of 6 apple rootstocks, ensure the control of breeding enterprises on varieties and reduce economic loss caused by errors in the breeding process. Practice proves that the SNP loci screened by the invention can accurately identify 6 apple rootstock varieties.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows that 14096 specific SNP markers (SNP markers are shown in Table 1) of M111, N9, B9, G11, M26 and M9 apple rootstocks obtained by screening are used for constructing an evolutionary tree, and 6 apple rootstocks can be accurately distinguished.
FIG. 2 shows that the Super-GBS method is used for carrying out sequencing and SNP screening on apple rootstock samples, the finally screened SNP sites are overlapped with over 90% of 14096 SNP sites which are determined to be effective, and 6 apple rootstocks can be accurately distinguished by classifying the 6 apple rootstocks by utilizing the overlapped sites.
Detailed Description
Example 1
The embodiment provides a method for screening 6 apple rootstock specific SNP molecular markers including M111, N9, B9, G11, M26 and M9 by using a simplified genome sequencing technology Super-GBS, which comprises the following steps:
(1) Using 4M 111, 3N 9, 4B 9, 4G 11, 5M 26 and 4M 9 apple rootstock samples with known varieties to extract genome DNA from leaves;
(2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and performing on-machine sequencing after the quality of the library is qualified;
(3) Filtering sequencing data, and then obtaining SNP loci by using GATK;
(4) Filtering the SNP under the following conditions: the SNP sequencing depth is not less than 4; eliminating sites with MAF less than 0.01; eliminating the sites with SNP typing deletion rate higher than 20%; removing the loci with consistent typing in all samples;
(5) Screening sites which have completely consistent individual types and have no individual deletion at the consistent typing sites and are different from at least one variety site in other 5 apple rootstock varieties according to the typing result of each variety, and finally screening 14096 SNP sites;
(6) And (3) constructing an evolutionary tree and clustering analysis by using 14096 SNP loci.
The specific operation steps are as follows:
this example mainly comprises the following steps, i.e., digestion, ligation, purification, amplification, pooling and analysis.
1. Enzyme digestion:
performing Super-GBS library construction on 4M 111, 3N 9, 4B 9, 4G 11, 5M 26 and 4M 9 apple stocks with accurate varieties provided by a standard institution purchased by the company, wherein the specific process is as follows (the using amount of enzyme digestion reagents of each sample is as follows):
DEPC water 21.4μL
10×CutSmart buffer 3μL
PstI-HF(4units) 0.2μL
MspI(8units) 0.4μL
DNA(50ng/μL) 5μL
Total 30μL
All the components are mixed evenly and then are subjected to enzyme digestion at 37 ℃ for 2h, and then the temperature is kept at 75 ℃ for 20min to inactivate the enzyme.
2. Connecting:
the adapter, barcode and the enzyme cutting fragment are connected in a 40 mu L system.
DEPC water 13μL
10×T4 ligase buffer 4μL
PstI Adaptor(0.1μM) 1μL
Common adaptor(10μM) 1.5μL
T4 DNA ligase(400U/μL) 0.5μL
Enzyme digestion product 20μL
Total 40μL
All the components are mixed evenly and then are cut by enzyme for 2h at the temperature of 22 ℃, and then the temperature is kept for 20min at the temperature of 65 ℃ to inactivate the enzyme.
3. Purification of
35 μ L of the ligation product was added to 0.7-fold volume of Sera-Mag beads (GE Healthcare Life Sciences) and allowed to stand at room temperature for 5min to remove small fragments of 300bp or less. The magnetic beads were recovered from the supernatant and eluted 3 times with 200. Mu.L of 70% ethanol. Finally, the DNA was recovered from the magnetic beads using 40. Mu.l of 10mM Tris.HCl (pH 8.0).
4. Amplification of
DEPC water 16μL
Taq5×Master Mix 5μL
Primer 1(10μM) 0.5μL
Primer 2(10μM) 0.5μL
Purified ligation product 3μL
Total 25μL
Mixing all the components, placing in a PCR instrument, performing pre-denaturation at 95 ℃ for 30s, amplifying for 16 cycles, performing denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 20s, extending at 68 ℃ for 15s, extending at 68 ℃ for 5min, and storing at 4 ℃.
5. Mixed warehouse
The library concentration of each sample was determined using Qubit, samples at concentrations greater than 5 ng/. Mu.l were used for pool sequencing. Primers and small fragments in the library are removed by adding 0.7-fold volume of Sera-Mag beads, and then mixed sample sequencing is carried out according to the sequencing quantity requirement, wherein the sequencing platform is Illumina Nova PE150. The linker and primer sequences used in the library construction process are detailed in Table 8 below.
TABLE 8 construction of linker and primer sequences for Super-GBS sequencing libraries
Name(s) 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 of
Performing Super-GBS sequencing on 24 samples of 6 apple rootstock varieties to obtain 66M high-quality reads. Aligning high quality reads to a reference genome with an alignment rate of 78.65-85.25% and an average sequencing depth of 37.52 x for all samples. SNP sites are obtained by utilizing GATK (v 3.8-1) software, then, at least one SNP site different from other varieties among 6 varieties is screened, 14096 SNP sites are finally obtained, treebest software is adopted for analysis, R package ggtree mapping is utilized, the sites can be used for accurately identifying 6 apple rootstock varieties, and identification results are shown in figure 1.
Example 2
This example provides the identification of varieties of 1 each of 6 apple rootstocks randomly collected from the variety nursery of agriculture limited corporation, da feng garden, shandong, with a portion of the 14096 SNP sites obtained according to the present invention, and simultaneously, the sequencing data of 6 apple rootstock samples of known varieties (samples in example 1) were added as controls for testing and validation. The method comprises the following steps:
(1) Randomly collecting leaves of 1M 111, 1N 9, 1B 9, 1G 11, 1M 26 and 1M 9 apple rootstock seedlings from a variety garden of Shandong Dafengyuan agriculture Limited company, and extracting genome DNA;
(2) Constructing a library according to a simplified genome sequencing technology Super-GBS method, and performing on-machine sequencing after the quality of the library is qualified;
(3) Filtering sequencing data, and then obtaining SNP loci by using GATK;
(4) Filtering the SNP under the following conditions: the SNP sequencing depth is not lower than 4; eliminating sites with MAF less than 0.01; eliminating the sites with SNP typing deletion rate higher than 20%; all samples were rejected for sites that were typed consistently. Reserving SNP sites which are coincided with 14096 sites in the table 1, and finally obtaining 13421 SNP sites;
(5) And constructing an evolutionary tree by utilizing the finally obtained 13421 SNP sites, and determining the varieties of the samples in the collected variety nursery.
The specific operation steps are as follows:
this example mainly comprises the following steps, i.e., digestion, ligation, purification, amplification, pooling and analysis.
1. Enzyme digestion:
performing Super-GBS library construction on 1M 111, 1N 9, 1B 9, 1G 11, 1M 26 and 1M 9 apple rootstocks randomly collected from variety gardens of agriculture Limited company in Dafeng Garden in Shandong, and the specific process is as follows (the usage amount of enzyme digestion reagent of each sample is as follows):
DEPC water 21.4μL
10×CutSmart buffer 3μL
PstI-HF(4units) 0.2μL
MspI(8units) 0.4μL
DNA(50ng/μL) 5μL
Total 30μL
All the components are mixed evenly and then cut for 2h at 37 ℃, and then the temperature is kept for 20min at 75 ℃ to inactivate the enzyme.
2. Connecting:
the adapter, barcode and the enzyme cutting fragment are connected in a 40 mu L system.
Figure BDA0003070000740000081
Figure BDA0003070000740000091
All the components are mixed evenly and then are cut by enzyme for 2h at the temperature of 22 ℃, and then the temperature is kept for 20min at the temperature of 65 ℃ to inactivate the enzyme.
3. Purification of
35 μ L of the ligation product was added to 0.7-fold volume of Sera-Mag beads (GE Healthcare Life Sciences) and allowed to stand at room temperature for 5min to remove small fragments of 300bp or less. The magnetic beads were recovered from the supernatant and eluted 3 times with 200. Mu.L of 70% ethanol. Finally, the DNA was recovered from the magnetic beads using 40. Mu.l of 10mM Tris.HCl (pH 8.0).
4. Amplification of
DEPC water 16μL
Taq5×Master Mix 5μL
Primer 1(10μM) 0.5μL
Primer 2(10μM) 0.5μL
Purified ligation product 3μL
Total 25μL
Mixing all the components, placing in a PCR instrument, performing pre-denaturation at 95 ℃ for 30s, amplifying for 16 cycles, performing denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 20s, extending at 68 ℃ for 15s, extending at 68 ℃ for 5min, and storing at 4 ℃.
5. Mixed warehouse
The library concentration of each sample was determined using Qubit, samples at concentrations greater than 5 ng/. Mu.l were used for pool sequencing. Primers and small fragments in the library are removed by adding 0.7-fold volume of Sera-Mag beads, and then mixed sample sequencing is carried out according to the sequencing quantity requirement, wherein the sequencing platform is Illumina Nova PE150. The linker and primer sequences used in the library construction process are detailed in Table 8 of example 1.
6. Analysis of
Performing Super-GBS sequencing on 6 samples of 6 apple rootstock varieties to obtain 16.65M high-quality reads. High quality reads were aligned to the reference genome at 77.45% -84.25% and the average sequencing depth for all samples was 37.52 x. SNP loci are obtained by utilizing GATK (v 3.8-1) software, and are compared with 14096 SNP loci in a table 1, 13421 SNP loci in the 14096 SNP loci are finally obtained, treebest software is adopted for analysis, R package ggtree mapping is utilized, the loci can be used for accurate identification of 6 apple rootstock varieties, and identification results are shown in a figure 2.
Example 3
This example provides 14096 SNP sites according to the present invention, a few SNP sites are screened, corresponding PCR detection primers are designed according to the positions of the genomes of the sites, PCR amplification is performed on multiple apple rootstock genomic DNAs randomly collected from the variety garden of santong, yoyo agricultural limited, shandong, and 6 apple rootstock varieties are identified by a first-generation sequencing method, and simultaneously 6 apple rootstocks of known varieties are added as positive controls for testing and verification. The method comprises the following steps:
(1) 2 strains of accurate M111, N9, B9, G11, M26 and M9 apple rootstocks provided by a standard institution purchased by the company are taken respectively, and genome DNA is extracted;
(2) Randomly collecting 2 plants of M111, N9, B9, G11, M26 and M9 apple stocks from a variety garden of agriculture Limited company in Dafeng garden in Shandong, and extracting genome DNA;
(3) Selecting sites from 14096 SNP sites to form a group of SNP site groups which can accurately identify 6 apple rootstocks, and the group is shown in Table 2;
(4) Designing upstream and downstream primers for PCR amplification according to the genomic position of the selected SNP locus, as shown in Table 3;
(5) Performing PCR amplification by using universal primers of 6 apple rootstocks in the table 3;
(6) Performing a first-generation sequencing of the amplified sequence;
(7) And (4) referring to the SNP locus information, and performing typing interpretation on the apple rootstock variety according to the sequence of the corresponding locus in the sequencing result.
The specific operation steps are as follows:
this example comprises mainly the following steps, i.e. PCR, sequencing, alignment and analysis.
1. PCR amplification
PCR amplification primers were designed according to the positions of the sites in Table 2, and the sequences of the primers 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
ddH2O 21μL
Total 50μL
2. Sequencing
The obtained PCR amplification product is detected by using 1% agarose gel electrophoresis, and a sample of which a specific amplification band is obtained at a position with an expected size is sent to Shanghai Biometrics Ltd for sequencing.
3. Sequence alignment
The sequencing result utilizes DNAMAN software or SnapGene to carry out sequence comparison, and utilizes a group of SNP locus marker groups (shown in table 2) screened from 14096 SNP loci of the invention to carry out the typing of 6 apple rootstocks.
4. Analysis of
The sequence alignment identification result shows that the identification results of 24 strains in total of 6 apple rootstocks M111, N9, B9, G11, M26 and M9 are consistent with the actual variety condition.
Example 4
This example provides that 14096 SNP sites according to the present invention are screened for a few SNP sites, and corresponding PCR detection primers are designed according to the positions of the genomes of the sites, PCR amplification is performed on genomic DNAs of multiple apple rootstock seedlings randomly collected from the variety garden of santong fengyuan agriculture ltd, and 6 apple rootstock varieties are identified by a first-generation sequencing method, and simultaneously 6 apple rootstocks of known varieties are added as positive controls for testing and verification. The method comprises the following steps:
(1) 2 strains of accurate M111, N9, B9, G11, M26 and M9 apple stocks which are provided by a standard institution and purchased by the company are taken respectively, and genome DNA is extracted;
(2) Randomly collecting 2 plants of each of M111, N9, B9, G11, M26 and M9 apple rootstocks from a variety garden of agriculture Limited company in Dafeng garden in Shandong, and extracting genome DNA;
(3) Selecting loci from 14096 SNP loci to form SNP locus groups capable of accurately identifying 6 apple rootstocks, which is shown in Table 4;
(4) Designing upstream and downstream primers for PCR amplification according to the genomic position of the selected SNP locus, see Table 5;
(5) Performing PCR amplification by using universal primers of 6 apple rootstocks in the table 5;
(6) Performing first generation sequencing on the amplified sequence;
(7) And (4) referring to the SNP locus information, and performing typing interpretation on the apple rootstock variety according to the sequence of the corresponding locus in the sequencing result.
The specific operation steps are as follows:
this example comprises mainly the following steps, i.e. PCR, sequencing, alignment and analysis.
1. PCR amplification
PCR amplification primers were designed according to the positions of the sites in Table 4, and the sequences of the primers 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
ddH2O 21μL
Total 50μL
2. Sequencing
The obtained PCR amplification product is detected by using 1% agarose gel electrophoresis, and a sample of which a specific amplification band is obtained at a predicted position is sent to Shanghai Biometrics, inc. for sequencing.
3. Sequence alignment
The sequencing result is subjected to sequence comparison by using DNMAN software or SnapGene, and 6 apple rootstocks are subjected to typing by using a group of SNP site marker groups (shown in Table 4) screened from 14096 SNP sites.
4. Analysis of
The sequence alignment identification result shows that the identification results of 24 strains in total of 6 apple rootstocks M111, N9, B9, G11, M26 and M9 are consistent with the actual variety condition.
Example 5
This example provides that 14096 SNP sites according to the present invention are screened for a few SNP sites, and corresponding PCR detection primers are designed according to the positions of the genomes of the sites, PCR amplification is performed on genomic DNAs of multiple apple rootstock seedlings randomly collected from the variety garden of santong fengyuan agriculture ltd, and 6 apple rootstock varieties are identified by a first-generation sequencing method, and simultaneously 6 apple rootstocks of known varieties are added as positive controls for testing and verification. The method comprises the following steps:
(1) 2 strains of accurate M111, N9, B9, G11, M26 and M9 apple stocks which are provided by a standard institution and purchased by the company are taken respectively, and genome DNA is extracted;
(2) Randomly collecting 2 plants of M111, N9, B9, G11, M26 and M9 apple stocks from a variety garden of agriculture Limited company in Dafeng garden in Shandong, and extracting genome DNA;
(3) Selecting sites from 14096 SNP sites to form an SNP site group capable of accurately identifying 6 apple rootstocks, which is shown in Table 6;
(4) Designing upstream and downstream primers for PCR amplification according to the genomic position of the selected SNP locus, see Table 7;
(5) Performing PCR amplification by using universal primers of 6 apple rootstocks in the table 7;
(6) Performing first generation sequencing on the amplified sequence;
(7) And (4) referring to the SNP locus information, and performing typing interpretation on the apple rootstock variety according to the sequence of the corresponding locus in the sequencing result.
The specific operation steps are as follows:
this example comprises mainly the following steps, i.e. 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
ddH2O 21μL
Total 50μL
2. Sequencing
The obtained PCR amplification product was detected by 1% agarose gel electrophoresis, and a sample in which a specific amplification band was obtained at a predetermined position was sent to Shanghai Biotech Ltd for sequencing.
3. Sequence alignment
The results obtained by sequencing were subjected to sequence alignment using DNMAN software or SnapGene, and 6 apple rootstocks were typed using a panel of SNP site marker groups (see Table 6) selected among 14096 SNP sites according to the present invention.
4. Analysis of
The sequence alignment identification result shows that the identification results of 24 strains in total of 6 apple rootstocks M111, N9, B9, G11, M26 and M9 are consistent with the actual variety condition.
Other SNP locus marker groups are picked from 14096 SNP loci, products obtained by a PCR method are sequenced, and 6 apple rootstocks of M111, N9, B9, G11, M26 and M9 can be accurately identified.
Table 1 is as follows:
Figure BDA0003070000740000141
Figure BDA0003070000740000151
Figure BDA0003070000740000161
Figure BDA0003070000740000171
Figure BDA0003070000740000181
Figure BDA0003070000740000191
Figure BDA0003070000740000201
Figure BDA0003070000740000211
Figure BDA0003070000740000221
Figure BDA0003070000740000231
Figure BDA0003070000740000241
Figure BDA0003070000740000251
Figure BDA0003070000740000261
Figure BDA0003070000740000271
Figure BDA0003070000740000281
Figure BDA0003070000740000291
Figure BDA0003070000740000301
Figure BDA0003070000740000311
Figure BDA0003070000740000321
Figure BDA0003070000740000331
Figure BDA0003070000740000341
Figure BDA0003070000740000351
Figure BDA0003070000740000361
Figure BDA0003070000740000371
Figure BDA0003070000740000381
Figure BDA0003070000740000391
Figure BDA0003070000740000401
Figure BDA0003070000740000411
Figure BDA0003070000740000421
Figure BDA0003070000740000431
Figure BDA0003070000740000441
Figure BDA0003070000740000451
Figure BDA0003070000740000461
Figure BDA0003070000740000471
Figure BDA0003070000740000481
Figure BDA0003070000740000491
Figure BDA0003070000740000501
Figure BDA0003070000740000511
Figure BDA0003070000740000521
sequence listing
<110> agriculture Co Ltd in Dafeng Yuan Shandong
Weifang Institute of science and technology
<120> apple rootstock specific molecular marker locus, and screening method and application thereof
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aaaccaccgg gtgatccaaa 20
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atcactccct tcccctcgaa 20
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acccgggttc tgggaataga 20
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acggcaagga cgacattgat 20
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tttggcgttt gaagctctgc 20
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caaatgggct gaccacagga 20
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<400> 14
gggcaacacc tctgttggta 20
<210> 15
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<213> Artificial Sequence (Artificial Sequence)
<400> 15
tagggcattt gccccagaga 20
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<211> 20
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<400> 16
ctggcagcac actgtggtat 20
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<211> 20
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<400> 17
agctgcatat tggggttccg 20
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gattttgagg tgctcggcag 20
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gattcggaga ccaggcgtag 20
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cggccacgta gcatatcagt 20
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<400> 21
agagcatttc cggtgcaaga 20
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<211> 20
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tcggaacaat tgtcggggag 20
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aactaggcct gctcaccaac 20
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<400> 24
tgtcccgaaa gctggttctc 20
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<212> DNA
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<400> 25
cccgaaagta ttgcctgcac 20
<210> 26
<211> 20
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<400> 26
tctaggtgag aagccggagt 20
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<400> 27
ccgggaacat cgaagtagca 20
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agcctcgcag aagacgtaag 20
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<212> DNA
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<400> 29
gtggaaacca gtgcatcagc 20
<210> 30
<211> 20
<212> DNA
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<400> 30
acgaaatctg cttgccaacg 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acaagtttgg ggttggtcgt 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cggggcactt tttgaagcaa 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acccgagctt tttcaatgcg 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tcccgtacga ttcaaggtgc 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atgccttaaa ccatgcgtgc 20
<210> 36
<211> 20
<212> DNA
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ggcgagcaag ttcgtccata 20

Claims (1)

1. The application of the primers for detecting the SNP loci in the table 2 in accurately identifying 6 apple rootstocks is characterized in that the 6 apple rootstocks are M111, N9, B9, G11, M26 and M9;
the SNP site information is shown in Table 2, and the primer information is shown in Table 3:
table 2A set of SNP locus marker groups capable of accurately identifying 6 apple rootstocks
Figure FDA0003860167380000011
SNP site identifying primer information in Table 2 described in Table 3
Figure FDA0003860167380000012
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