SNP (Single Nucleotide Polymorphism) site combination for cotton variety identification, gene chip and application of SNP site combination and gene chip

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site combination for cotton variety identification, a gene chip and application of the SNP site combination and the gene chip, and relates to the technical field of bioinformatics and molecular breeding. According to the invention, 128 SNPs are screened from 100 parts of cotton whole genome re-sequencing data and can be used as the SNP fingerprint spectrum of the cotton variety. When the SNP molecular marker and the liquid-phase probe developed by the invention are used for identifying cotton, the detection rate is 100%, the minimum allele frequency is greater than 0.05, any two cotton varieties can be distinguished, and the identification of the cotton varieties can be efficiently and quickly completed. In addition, due to the adoption of a new-generation sequencing technology, the procedure is simple and standard, errors caused by manual operation are reduced, the unit price of batch identification analysis is extremely low, and the method has the characteristics of economy and high efficiency, is particularly suitable for large sample analysis and identification, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of bioinformatics and molecular breeding, and more specifically to a SNP site combination, a gene chip and an application thereof for cotton variety identification. Background Art

[0002] The selection and promotion of excellent crop varieties is the premise and foundation for achieving high-yield and high-quality agriculture, and is an important link in ensuring my country's food security. Variety identification is an important guarantee for the selection and promotion of excellent varieties, and is the main content of variety approval and seed market supervision. It is of great significance in the process of new variety breeding, seed production, processing and operation. The detection method is the key to accurately identify the variety.

[0003] Currently, the marker technology for cotton variety identification mostly uses SSR markers. SSR markers have the disadvantages of limited number of loci, uneven distribution in the genome, low genotyping polymorphism, insufficient genome coverage, and difficulty in accurate integration and comparison of data across laboratories. Existing molecular markers such as ISSR, SRAP and SSR are complex to operate, have long experimental cycles and low throughput.

[0004] Therefore, providing a simple, efficient and accurate cotton variety identification method is an urgent problem to be solved by technicians in this field. Summary of the invention

[0005] In view of this, the present invention provides a SNP site combination, gene chip and application thereof for cotton variety identification. SNP molecular markers are richly distributed throughout the genome, which can better distinguish cotton materials and assist in the construction of cotton fingerprints. The SNP detection technology based on capture sequencing has a fast detection speed and a short detection cycle, and is more suitable for the identification of cotton molecular markers and cotton varieties.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The SNP site combination used for cotton variety identification, the site physical information of the SNP site combination is shown in Table 2, and the physical position information of the site combination in Table 2 is determined based on the cotton G. hirsutum_TM-1_ICR genome sequence alignment.

[0008] Another object of the present invention is to provide a molecular probe combination for cotton variety identification, wherein the molecular probe combination is used to detect the SNP site combination shown in Table 2.

[0009] Preferably, the nucleotide sequence of the molecular probe combination is shown as SEQ ID NO.1-SEQ ID NO.128.

[0010] Another object of the present invention is to provide a kit, which comprises the above-mentioned molecular probe combination.

[0011] Another object of the present invention is to provide a gene chip, wherein the gene chip is loaded with the above-mentioned molecular probe combination.

[0012] Another object of the present invention is to provide any of the following applications of the above-mentioned SNP site combination, or the above-mentioned molecular probe combination, or the above-mentioned kit, or the above-mentioned gene chip:

[0013] A. Identification of cotton varieties;

[0014] B. Cotton variety screening;

[0015] C. Cotton variety traceability;

[0016] D. Identification of cotton variety purity;

[0017] E. Construction of SNP fingerprint of cotton varieties;

[0018] F. Molecular marker-assisted breeding of cotton varieties;

[0019] G. Cotton genotyping;

[0020] H. Analysis of cotton genetic diversity;

[0021] I. Functional genomics of cotton.

[0022] Another object of the present invention is to provide a method for identifying cotton varieties, comprising the following steps:

[0023] (1) The genomic DNA of cotton leaf tissue was extracted by CTAB method. After the DNA quality inspection was qualified, the genomic DNA was randomly sheared (200-300 bp), and the ends of the DNA fragments were repaired and connected to the adapter Pre-PCR amplification library;

[0024] (2) The probe hybridizes to the target region;

[0025] (3) Capture of hybridization probes by streptavidin affinity-labeled magnetic beads;

[0026] (4) enriching the target fragments, eluting them and performing post-PCR amplification after capture;

[0027] (5) Next-generation sequencing of the target region; comparing the sequencing results with the above-mentioned SNP site combination to determine the cotton variety.

[0028] Preferably, the probe is the above-mentioned molecular probe combination.

[0029] Beneficial effects:

[0030] The present invention screened 128 SNP molecular markers based on the cotton genome SNP data set, and developed 128 liquid phase probes. The SNP molecular markers and liquid phase probes described in the present invention are used to identify cotton, with a detection rate of 100%, a minimum allele frequency greater than 0.05, and any two cotton varieties can be distinguished, and the identification of cotton varieties can be completed efficiently and quickly. In addition, the present invention adopts the technology of new generation sequencing, the procedure is simple and standardized, the error of human operation is reduced, the unit price of batch identification analysis is extremely low, it has the characteristics of economic efficiency, is particularly suitable for large sample analysis and identification, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 This is a distribution diagram of 128 SNP marker combinations of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] The method for screening SNP marker combinations (Table 2) and designing liquid phase probes (Table 3) based on the whole genome resequencing data of 100 cotton accessions (Table 1) specifically includes the following process:

[0036] (1) The genomic DNA of 100 cotton leaf tissue samples was extracted using the CTAB method. After the DNA quality inspection was qualified, a whole genome resequencing library was constructed and sequenced using BGI DNBSEQ-T7.

[0037] (2) The clean data obtained by sequencing in step (1) was used as the reference genome of cotton G. hirsutum_TM-1_ICR, and the clean data was aligned to the genome using bwa and GATK mutation detection to obtain 18,009,233 high-quality SNP sites.

[0038] (3) The SNP sites screened out in step (2) were further filtered using vcftools software under the conditions "--minDP 3--min-alleles2--max-alleles 2--max-missing 0.8--maf0.05" to obtain 100,699 SNP sites detected in all samples with high quality.

[0039] (4) The SNP sites screened out in step (3) were used to screen SNP marker combinations using a perl script, and finally 128 SNP marker combinations were obtained (their distribution on the chromosome is shown in the attached Figure 1 ), the DNA fingerprint map was successfully constructed.

[0040] The principle is: the SNP sites with the most differences between any two cotton varieties are evenly distributed on chromosomes; the SNP sites and their upstream and downstream 100bp sequences are extracted, and the extracted 201bp fragments are separated by bedtools software. The SNP sites with GC content between 40-60% and no N bases in the sequence are retained. The extracted sequences are aligned to the cotton G.hirsutum_TM-1_ICR genome using blastn software, and only single-copy SNP sites are retained.

[0041] (5) Designing liquid phase probe sequences for the 128 SNPs screened in step (4) for subsequent capture sequencing.

[0042] Table 1100 Cotton Material Information

[0043]

[0044]

[0045] Table 2 Information of 128 SNP markers used to identify cotton varieties

[0046]

[0047]

[0048]

[0049]

[0050] Table 3 Information of 128 SNP marker liquid phase probes used to identify cotton varieties

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The above 128 SNP marker combinations and the number of SNP marker differences between any two cotton varieties are shown in Table 4.

[0059] Table 4 Statistics of SNP marker differences between any two cotton varieties

[0060]

[0061]

[0062] Example 2

[0063] Method for detecting cotton varieties using liquid phase probes corresponding to 128 SNPs obtained in Example 1

[0064] 1. Extraction of Genomic DNA

[0065] Cotton leaves were collected, stored with ice packs and promptly transported back to the laboratory, and total cotton DNA was extracted using the CTAB method.

[0066] 2. Genomic DNA fragmentation and end repair

[0067] (1) Prepare the whole genome DNA fragmentation system (30 μL) on ice: X μL of input DNA (250 ng), 4.5 μL of buffer, 5 μL of enzymes, and 1×TE buffer up to 30 μL; X μL represents any volume not exceeding 20 μL.

[0068] (2) Use a pipette to pipette up and down or shake to ensure that the system is fully mixed, then centrifuge briefly.

[0069] (3) Immediately place the PCR tube on a PCR instrument preheated to 32°C, and run the fragmentation and end-repair reaction program: 32°C for 20 min; 65°C for 30 min; 4°C (after the temperature drops to 4°C, immediately place it on ice for the next experiment, and do not keep it at 4°C). Set the thermal cover of the PCR instrument to 75°C.

[0070] 3. Connect the connector

[0071] (1) Prepare the ligation reaction system (55 μL) in an ice bath: End Prep Reaction Mix (reaction product from step 2)

[0072] 30μL, Ligation Buffer 15μL, ddH2O 2.5μL, Ligation Enzymes 5μL, TruncatedAdaptor 2.5μL.

[0073] (2) Place the PCR tube on a PCR instrument and set the reaction time to 22°C for 15 min without a heated cover. Store the tube at 4°C (proceed to the next step immediately after cooling to 4°C).

[0074] (3) DNA sample purification:

[0075] Add 44 μL (0.8×) DNA Clean Beads to each sample and mix well; incubate at room temperature for 5 min;

[0076] Place the PCR tube on the magnetic rack for 3 minutes, and remove the supernatant after the solution becomes clear;

[0077] Add 180 μL 80% ethanol to rinse the beads, incubate for 30 seconds, remove the supernatant, and repeat the operation once;

[0078] Keep the PCR tube on the magnetic stand, use a 10μL pipette to remove the ethanol remaining at the bottom of the tube, and dry until no ethanol remains;

[0079] Resuspend the magnetic beads in 21 μL ddH2O and let stand at room temperature for 1 min to fully release the DNA on the magnetic beads;

[0080] Place the PCR tube on the magnetic stand for 2 min, and transfer 20 μL of supernatant to a new PCR tube for library amplification.

[0081] 4. Library Amplification and Purification

[0082] (1) PCR amplification

[0083] PCR reaction system (35 μL): 20 μL of adapter-ligated DNA, 12.5 μL of 2×PCR Mix, 2.5 μL of PCR Index Primer, and 2.5 μL of Universal PCR Primer.

[0084] PCR amplification program, see Table 5.

[0085] Table 5

[0086]

[0087] (2) Add 35 μL (1×) DNA Clean Beads to the amplified product, mix well, and incubate at room temperature for 5 min;

[0088] (3) Place the sample on a magnetic rack for 2 min. After the solution becomes clear, remove the supernatant.

[0089] (4) Add 200 μL 80% ethanol to rinse the magnetic beads, incubate for 30 seconds, remove the supernatant, and repeat this step once;

[0090] (5) Keep the PCR tube on the magnetic rack, use a 10 μL pipette to remove the ethanol remaining at the bottom of the tube, open the tube cap and dry until no ethanol remains;

[0091] (6) Resuspend the magnetic beads in 31 μL ddH2O and let stand at room temperature for 1 min to fully release the DNA on the magnetic beads;

[0092] (7) Place the sample on the magnetic rack for 2 min, transfer 30 μL of the supernatant to a new PCR tube, and store the library at −20°C for subsequent library quality testing and sequencing.

[0093] 5. Library and Probe Hybridization

[0094] (1) Take 750 ng of the library constructed in step 4 and add it to a PCR tube and mark it;

[0095] (2) Add purified magnetic beads to the library and mix gently with a pipette;

[0096] (3) Incubate at room temperature for 5 min and place the PCR tube on a magnetic rack for 3 min to clarify the solution;

[0097] (4) Remove the supernatant, place the PCR tube on the magnetic stand, add 180 μL 80% ethanol, and let stand for 30 seconds;

[0098] (5) Remove the supernatant and add 180 μL of 80% ethanol to the PCR tube. Let it stand for 30 seconds and then completely remove the supernatant.

[0099] (6) Leave at room temperature for 5 min to allow the residual ethanol to evaporate completely;

[0100] (7) Prepare hybridization reaction system (28 μL): add 13 μL Hyb Buffer, 5 μL Hyb Human Block, 2 μL Adapter Blocker, 5 μL RNase Block, and 3 μL Nuclease Free Water into the reaction tube after ethanol evaporation in step (6).

[0101] (8) Mix by gently pipetting, let stand at room temperature for 3 min, centrifuge briefly, place the PCR tube on a magnetic rack, and let stand for 3 min;

[0102] (9) Pipette 28 μL of supernatant into a new PCR tube, add 2 μL of Target Probe, gently pipette to mix, and centrifuge briefly;

[0103] (10) Set the parameters of the PCR instrument as follows: hot cover temperature: 85°C; 80°C for 5 min; hold at 50°C; place the PCR tube on the PCR instrument, run the above program, and incubate overnight.

[0104] 6. Capture target region DNA library

[0105] (1) Magnetic bead pretreatment

[0106] Remove the capture beads from 4°C, vortex and resuspend, and equilibrate at room temperature for 30 minutes;

[0107] Take 50 μL of magnetic beads and add them to a new PCR tube. Place it on a magnetic rack for 1 min to allow the solution to clarify and remove the supernatant.

[0108] Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette several times to mix, and resuspend the magnetic beads; place on the magnetic stand for 1 minute, remove the supernatant; repeat this step once;

[0109] Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, gently pipette and resuspend the magnetic beads for later use;

[0110] (2) Capture target region DNA library

[0111] Keep the hybridization product on the PCR instrument, add 180 μL of capture magnetic beads resuspended in step (1) to the hybridization product, mix with a pipette, and place on a rotating mixer for binding at room temperature for 30 minutes;

[0112] Place the PCR tube on a magnetic stand for 2 min to clarify the solution, and remove the supernatant;

[0113] Add 150 μL of 50°C preheated Wash Buffer, gently pipette to mix, then briefly centrifuge, and incubate at 50°C on a constant temperature shaker for 10 min;

[0114] Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes to clarify the solution, and remove the supernatant; repeat this step 2 times, washing the magnetic beads 3 times in total;

[0115] Keep the sample on the magnetic rack, add 150 μL of 80% ethanol to the PCR tube, let it stand for 30 seconds, then completely remove the ethanol solution and dry it at room temperature;

[0116] Add 24 μL Nuclease-free Water to the PCR tube, remove the PCR tube from the magnetic stand, and gently use a pipette to resuspend and mix the magnetic beads for later use.

[0117] 7. Post-capture PCR Amplification

[0118] (1) Take out PostPCR MasterMix and PostPCR Primer from the -20℃ refrigerator, place them on an ice box to thaw, mix well and place on ice or at 4℃ for later use.

[0119] (2) After capture, the DNA library is amplified by PCR.

[0120] PCR reaction system (50 μL): 24 μL of the DNA library of the target region captured in step 6, 25 μL of Post PCR Master Mix, and 1 μL of Post PCR Primer (selected according to the library type). Adjust the pipette to 40 μL, gently pipette and mix 6 times, and then immediately place it on the PCR instrument;

[0121] PCR instrument program: hot cover temperature: 105℃; program: 95℃1min; 98℃20s; 60℃30s N cycles; 72℃30s; 72℃5min; 4℃ hold;

[0122] (3) After PCR, add 55 μL of purified magnetic beads to the sample, vortex or pipette to mix, and let stand at room temperature for 5 min;

[0123] (4) Briefly centrifuge and place the PCR tube on a magnetic rack for 3 min to allow the solution to clarify;

[0124] (5) Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution into the PCR tube, and let it stand for 30 seconds;

[0125] (6) Keep the PCR tube on the magnetic rack, remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely remove the supernatant; let it stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely;

[0126] (7) Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic rack, shake or pipette to mix 10 times, and let stand at room temperature for 2 min;

[0127] (8) Briefly centrifuge and place the PCR tube on a magnetic rack for 2 min to allow the solution to clarify;

[0128] (9) Use a pipette to transfer 23 μL of supernatant to a 1.5 mL centrifuge tube and mark the sample information;

[0129] (10) Take 1 μL of the library and use the Qubit dsDNA HS Assay Kit for quantification. Record the library concentration, which is approximately 1-20 ng / μL.

[0130] (11) Take 1 μL of sample and use Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit) to measure the fragment length.

[0131] 8. High-throughput sequencing

[0132] The library obtained after capture and amplification in step 7 was subjected to high-throughput sequencing using the MGI DNBSEQ-T7 sequencing platform to obtain the sequencing results of cotton genomic DNA, and the obtained data was basically cleaned.

[0133] 9. Liquid Phase Probe Capture Rate Evaluation

[0134] (1) The cleaned sequencing data were aligned to the cotton reference sequence using bwa software, and the SNP genotyping data of cotton varieties were obtained using GATK software;

[0135] (2) Use SNP genotyping data to complete cotton variety fingerprint analysis.

[0136] Example 3

[0137] Liquid phase probe capture efficiency verification

[0138] The method described in Example 2 was used to perform 128 SNP liquid phase probe hybridization and genotyping on individual plants of the 12 cotton varieties in Table 6 to obtain typing data for each individual plant, as shown in Table 7.

[0139] Table 6 12 cotton samples

[0140] Serial number variety Serial number variety Serial number variety 1 Handan 5158 2 Handan Cotton 802 3 JI668 4 Hebei Cotton 20 5 Lumianyan 21 6 JI228 7 Hebei Cotton 616 8 Guoxin Cotton No. 3 9 Fast breeding 66 10 Hanwu23 11 Jifeng 914 12 Hebei Cotton 298

[0141] Table 7 Genotyping results in 12 cotton samples

[0142]

[0143]

[0144]

[0145]

[0146] The 12 cotton samples tested by the liquid phase probe designed based on the 128 SNP marker combination obtained the number of SNP genotyping. From the results, it can be seen that the cotton material detection rate is 100%. Using the 128 SNP genotyping results, the SNP fingerprints of the 12 tested varieties can be constructed for variety authenticity identification, and the genetic diversity analysis of the 12 varieties can also be performed. It shows that the liquid phase probe group developed using the 128 SNP marker combination of the present invention can efficiently and quickly complete the identification of cotton varieties.

[0147] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0148] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SNP locus combination for cotton variety identification, characterized in that: The site physical information of the SNP site combination is shown in Table 2. The physical position information of the site combination in Table 2 is determined based on the cotton G. hirsutum_TM-1_ICR genome sequence alignment. Table 2 is as follows:

2. A molecular probe combination for cotton variety identification, characterized in that: The molecular probe combination is used to detect the SNP site combination described in claim 1.

3. The molecular probe combination according to claim 2, characterized in that: The nucleotide sequences of the molecular probe combination are shown as SEQ ID NO.1-SEQ ID NO.

128.

4. A kit, characterized in that The kit comprises the molecular probe combination according to claim 2 or 3.

5. A gene chip, characterized in that The gene chip is loaded with the molecular probe combination according to claim 2 or 3.

6. Any of the following uses of the SNP site combination according to claim 1, or the molecular probe combination according to claim 2 or 3, or the kit according to claim 4, or the gene chip according to claim 5: A. Identification of cotton varieties; B. Cotton variety screening; C. Cotton variety traceability; D. Identification of cotton variety purity; E. Construction of SNP fingerprint of cotton varieties; F. Molecular marker-assisted breeding of cotton varieties; G. Cotton genotyping; H. Analysis of cotton genetic diversity; I. Functional genomics of cotton.

7. A method for identifying cotton varieties, characterized in that: The following steps are involved: (1) The genomic DNA of cotton leaf tissue was extracted by CTAB method. After the DNA quality inspection was qualified, the genomic DNA was randomly sheared (200-300 bp), and the ends of the DNA fragments were repaired and connected to the adapter Pre-PCR amplification library; (2) The probe hybridizes to the target region; (3) Capture of hybridization probes by streptavidin affinity-labeled magnetic beads; (4) enriching the target fragments, eluting them and performing post-PCR amplification after capture; (5) Next generation sequencing of the target region; comparing the sequencing results with the SNP site combination described in claim 1 to determine the cotton variety.

8. The cotton variety identification method according to claim 7, characterized in that: The probe is the molecular probe combination according to claim 2 or 3.

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