Canine SNP-STR genetic marker site combination and application thereof

Abstract

The invention relates to a dog SNP-STR genetic marker site combination and application thereof. The dog genetic marker site combination comprises 24 STR (short tandem repeat) gene loci and 287 SNP (single nucleotide polymorphism) gene loci, and integrates the advantages of STR and SNP genetic markers. By simultaneously detecting the STR gene locus and the SNP gene locus, on the premise of ensuring the accuracy of the detection result, the detection quantity of the SNP gene locus can be greatly reduced, and the detection cost and the detection difficulty are greatly reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly relates to a canine SNP-STR genetic marker locus combination and its application. Background Art

[0002] At present, there are mainly two categories of commonly used gene loci for genetic determination of dogs. One category is to detect microsatellite markers (STR), and the second category is to detect single nucleotide polymorphism markers (SNP). STR is a simple repetitive sequence evenly distributed in the eukaryotic genome, consisting of tandem repetitive fragments of 2-6 nucleotides. Its advantages are high polymorphism and high resolution, so it is widely used in individual identification and paternity testing. SNP refers to the variation of a single nucleotide in the genome, including transition, transversion, deletion and insertion. Its advantage is high genetic information content, so it is widely used in complex disease research and breed analysis.

[0003] Both of these two gene markers can be used for dog breed analysis, but each has its own advantages and disadvantages: STR has high polymorphism and high resolution at a single locus, but generally the number of detectable loci is small, and it is difficult to increase the number of detected STR loci in experimental design; the number of detectable SNP loci is large, but its polymorphism and resolution at a single locus are low, and at least about 500 loci are required to ensure the accuracy of the analysis results, and the detection cost is relatively high. The two detection methods are limited in practical applications. Summary of the Invention

[0004] The purpose of the present invention is to disclose a canine SNP-STR genetic marker locus combination and its application, so as to solve one or more technical problems existing in the prior art, and provide at least one beneficial choice or creation condition.

[0005] To achieve the above purpose, the present invention is realized through the following technical solutions: The first aspect of the present invention is to provide a canine genetic marker locus combination.

[0006] The second aspect of the present invention is to provide the application direction of the canine genetic marker locus combination.

[0007] The third aspect of the present invention is to provide a canine breed analysis kit.

[0008] The fourth aspect of the present invention is to provide a solid-phase chip.

[0009] The fifth aspect of the present invention is to provide a liquid-phase chip.

[0010] The canine genetic marker locus combination described in the first aspect of the present invention includes an STR locus and an SNP locus. The information of the STR locus is shown in Table 1, the information of the SNP locus is shown in Table 2, and the canine genome used is the CanFam3.1 version.

[0011] Table 1

[0012] In Table 1, the first column is the STR locus name code, the second column is the chromosome and specific position of the locus, the third column is its core repeat sequence, and the fourth column is the allele genotype with the number of repeats of its core sequence as the typing.

[0013] Table 2

[0014] In Table 2, the first column is the SNP code, the second column is its chromosomal position, the third column is the specific position on the chromosome, and the fourth and fifth columns are its allele genotypes.

[0015] In some embodiments of the first aspect of the present invention, the canine genetic marker locus combination includes all 24 STR loci and 287 SNP loci.

[0016] The present invention combines the advantages of two genetic markers, STR and SNP. By simultaneously detecting STR loci and SNP loci, on the premise of ensuring the accuracy of the detection results, the detection amount of SNP loci can be greatly reduced, and the detection cost and detection difficulty can be greatly reduced.

[0017] The application described in the second aspect of the present invention refers to that the canine genetic marker locus combination can be used for canine breed analysis.

[0018] In some embodiments of the second aspect of the present invention, the canine breed analysis refers to the ability to identify Belgian Shepherd Dog, French Bulldog, German Shepherd Dog, Kunming Dog, Bloodhound, Labrador Retriever, Rottweiler and Springer Spaniel.

[0019] The canine breed analysis kit described in the third aspect of the present invention includes an SNP probe combination for capturing and identifying the canine genetic marker locus combination, and the nucleotide sequences of the probe combination are shown in SEQ ID No: 1 to SEQ ID No: 287.

[0020] The canine breed analysis kit can be based on the principle of targeted sequencing by GenoPlexs multiplex capture. After the PCR products are purified using carboxyl magnetic beads, the sequencing primers with Barcodes and a high-fidelity PCR reaction system are added again for PCR amplification. Different Barcodes are used to distinguish different samples. After the amplified products are purified using carboxyl magnetic beads, multiplex PCR capture and library construction are completed.

[0021] In some embodiments of the third aspect of the present invention, the canine breed analysis kit further includes an ARMS-PCR system for amplifying the STR loci.

[0022] The solid-phase chip according to the fourth aspect of the present invention includes a chip carrier and an SNP probe combination for capturing and identifying the canine genetic marker locus combination. The probe combination is fixed on the surface of the chip carrier to form a microarray, and the nucleotide sequences of the probe combination are shown in SEQ ID No: 1 to SEQ ID No: 287.

[0023] In some embodiments of the fourth aspect of the present invention, the solid-phase chip further includes at least one of a blocking agent, a reporter group, a reaction buffer, a positive control, and / or a negative control.

[0024] The solid-phase chip is suitable for the standardized detection of large-scale samples and has the advantages of low cost and good repeatability.

[0025] The liquid-phase chip according to the fifth aspect of the present invention includes a microsphere carrier and an SNP probe combination for capturing and identifying the canine genetic marker locus combination. The nucleotide sequences of the probe combination are shown in SEQ ID No: 1 to SEQ ID No: 287.

[0026] In some embodiments of the fifth aspect of the present invention, the liquid-phase chip further includes at least one of a reporter molecule, a reaction buffer, a blocking agent, a positive control, and / or a negative control.

[0027] The advantages of the liquid-phase chip are that it can detect low-frequency mutations, and has a wide sample compatibility, and can also detect low-quality DNA samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a color-coded diagram of the detection results of the Belgian Shepherd dog sample in Example 1; Figure 2 It is a color-coded diagram of the detection results of the French Bulldog sample in Example 1; Figure 3 It is a color-coded diagram of the detection results of the German Shepherd dog sample in Example 1; Figure 4It is the color-separated diagram of the detection results of the Kunming dog samples in Example 1; Figure 5 It is the color-separated diagram of the detection results of the bloodhound samples in Example 1; Figure 6 It is the color-separated diagram of the detection results of the Labrador retriever samples in Example 1; Figure 7 It is the color-separated diagram of the detection results of the Rottweiler samples in Example 1; Figure 8 It is the color-separated diagram of the detection results of the Springer Spaniel samples in Example 1. Detailed implementation mode

[0029] For the molecular biology test methods not specifically described in the following examples, they are all referred to "Molecular Cloning: A Laboratory Manual" (Third Edition) or carried out according to the methods and product specifications; for the biological materials of the methods, unless otherwise specified, they can all be obtained from commercial channels.

[0030] Example 1: Analysis of 8 dog breeds Construct a dog breed analysis kit based on the principle of targeted sequencing of GenoPlexs multiplex capture, which can target and detect the 311 gene loci. Blood samples of 8 different dog breeds were provided by a third party for double-blind detection and analysis.

[0031] (1) DNA sample extraction and detection.

[0032] Use a high-throughput DNA extraction kit to extract the sample DNA. The extracted DNA samples are subjected to 2 kinds of detections: use 1% agarose gel electrophoresis method to analyze the purity and integrity of DNA; use Qubit to accurately quantify the DNA concentration.

[0033] (2) Based on the GenoPlexs experimental procedure.

[0034] Add multiplex PCR Panel mix and multiplex PCR amplification enzyme system to the quantitatively qualified DNA samples through quality inspection, and place them on a PCR instrument to complete the PCR reaction. After the PCR products are purified using carboxyl magnetic beads, add sequencing primers with Barcode and a high-fidelity PCR reaction system for PCR amplification again. Different Barcodes are used to distinguish different samples. After the amplified products are purified using carboxyl magnetic beads, the multiplex PCR capture and library construction are completed.

[0035] (3) Library construction and sequencing on the machine.

[0036] After the library construction is completed, first use Qubit2.0 for preliminary quantification, and use the qPCR method to accurately quantify the effective concentration of the library to ensure the library quality. After the library passes the inspection, it enters the stage of sequencing on the machine.

[0037] (4)Genotyping.

[0038] Use the software BWA (in the mem alignment mode) to align the quality-controlled clean reads with the reference genome (Canine Genome CanFam3.1 version) sequence. Through the alignment, the positions of the clean reads on the reference genome can be located. Use Perl scripts to extract the genotyping information of STR and SNP loci from the above alignment results and variant results, and finally summarize to obtain the genotyping data of each sample. The breed of the dog from which the sample is analyzed can be determined through the obtained genotyping data.

[0039] (5)Conduct a comparative experiment with high-throughput data.

[0040] To verify the accuracy of the canine breed analysis kit, the Illumina Canine HD BeadChip high-throughput chip is used as a comparative experiment. Genotype the above 8 samples. After quality control, the genotyping results of 190,000 SNP loci are obtained for each sample. Conduct breed analysis using an analysis system and compare the results with the analysis results of the canine breed analysis kit.

[0041] Experimental results: The following results confirm that the combination of 311 STR-SNP loci of the present invention is consistent with the breed identification results using 190,000 SNP loci in breed analysis. As Figures 1 to 8 shown, the breed information of the dog is represented by colors, and the proportion of the main color represents the proportion value of its purebred.

[0042] Figure 1 The detection result for the Belgian Shepherd Dog is shown. B1 is the detection result of the canine breed analysis kit, showing that the proportion value of the purebred of sample B1 is 94%; B2 is the result of the high-throughput chip, also showing that the proportion value of the purebred of sample B2 is 94%.

[0043] Figure 2 The detection result for the French Bulldog is shown. F1 is the detection result of the canine breed analysis kit, showing that the proportion value of the purebred of sample F1 is 93%; F2 is the result of the high-throughput chip, also showing that the proportion value of the purebred of sample F2 is 93%.

[0044] Figure 3 The detection result for the German Shepherd Dog is shown. D1 is the detection result of the canine breed analysis kit, showing that the proportion value of the purebred of sample D1 is 95%; D2 is the result of the high-throughput chip, also showing that the proportion value of the purebred of sample D2 is 95%.

[0045] Figure 4The detection results for Kunming dogs. K1 is the detection result of the dog breed analysis kit, showing that the proportion value of purebred in the K1 sample is 91%; K2 is the high-throughput chip result, also showing that the proportion value of purebred in the K2 sample is 91%.

[0046] Figure 5 The detection results for Bloodhounds. X1 is the detection result of the dog breed analysis kit, showing that the proportion value of purebred in the X1 sample is 92%; X2 is the high-throughput chip result, also showing that the proportion value of purebred in the X2 sample is 92%.

[0047] Figure 6 The detection results for Labrador Retrievers. L1 is the detection result of the dog breed analysis kit, showing that the proportion value of purebred in the L1 sample is 94%; L2 is the high-throughput chip result, also showing that the proportion value of purebred in the L2 sample is 94%.

[0048] Figure 7 The detection results for Rottweilers. R1 is the detection result of the dog breed analysis kit, showing that the proportion value of purebred in the R1 sample is 94%; R2 is the high-throughput chip result, also showing that the proportion value of purebred in the R2 sample is 94%.

[0049] Figure 8 The detection results for Springer Spaniels. S1 is the detection result of the dog breed analysis kit, showing that the proportion value of purebred in the S1 sample is 98%; S2 is the high-throughput chip result, also showing that the proportion value of purebred in the S2 sample is 98%.

[0050] After analysis and taking an error threshold of ±5%, it is found that the results of the dog breed analysis kit are completely consistent with the analysis results of 190,000 SNPs on the right side and are consistent with the prior information of the known dogs to be tested.

[0051] Example 2: Actual test of unknown samples Use the method provided in Example 1 to conduct dog breed analysis on a stray dog with an appearance similar to a German Shepherd. The STR detection results are shown in Table 3, and the SNP detection results are shown in Table 4.

[0052] Table 3

[0053] Table 4

[0054] Based on the detection results, it can be determined that the dog providing the sample belongs to a German Shepherd.

[0055] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. Canine genetic marker locus combination, characterized in that, Including STR loci and SNP loci, the STR loci include at least one of PEZ1, PEZ2, FH2010, PEZ5, PEZ12, FH2328, PEZ21, PEZ3, PEZ6, PEZ8, VGL3112, FH2054, VWFX, FH2611, FH2132, PEZ20, PEZ15, FH2079, PEZ17, FH3313, FH2088, FH2001, FH2017, and FH2107, and the SNP loci include chr1_13481722, BICF2G630714052, BICF2S23251261, BICF2P431438, BICF2S2323172, BICF2P836026, chr1_30263156, BICF2P1169198, BICF2G630724107, BICF2P250912, chr1_115870556, BICF2G630677006, BICF2G630674715, BICF2S23237356, G143f35S34, chr2_21814910, chr2_33896872, BICF2S23320651, BICF2P422938, BICF2P445652, chr2_75474841, BICF2P963113, BICF2S23546324, BICF2P447529, TIGRP2P49655_rs8812824, BICF2P552620, BICF2P1126281, BICF2G630346906, BICF2G630347144, BICF2G630361702, BICF2G630361787, BICF2G630362033, BICF2P486992, BICF2S23426325, BICF2P412012, BICF2S22963284, BICF2P1300820, BICF2S2298496, chr4_87759944, BICF2P606521, BICF2P723782, BICF2P1249104, BICF2P1098115, BICF2G63034005, BICF2G63035039, BICF2S23034980, BICF2G63035590, BICF2G630182200, chr5_30527547, chr5_30677222, chr5_30677658, BICF2G630182360, BICF2P1270366, chr5_32060951, BICF2G630182966,chr5_32366524, BICF2P731728, BICF2P464907, BICF2G630183440, chr5_33528365, BICF2G630183554, BICF2P153003, BICF2G630183638, BICF2G630183652, BICF2G630183659, BICF2G630183701, BICF2G630183965, chr5_34780036, BICF2G630184432, BICF2P1356360, BICF2G630187785, BICF2P384624, BICF2P874797, chr5_54509543, BICF2S23051512, chr5_55717237, BICF2P260580, BICF2P906690, chr5_64519744, BICF2P1007652, chr5_75165546, chr6_14670247, TIGRP2P80570_rs9112951, BICF2S2455844, BICF2P1009202, BICF2P10251, chr6_42028574, BICF2P1398108, BICF2S23554167, BICF2P1166527, BICF2S22960558, BICF2P578798, BICF2P852692, BICF2P1276890, BICF2G630558018, chr7_47563534, BICF2G63083475, BICF2P1337011, BICF2P437510, BICF2P792154, BICF2P1124008, BICF2P783992, TIGRP2P105577_rs8614726, BICF2P800120, TIGRP2P115636_rs8948238, BICF2S23340261, BICF2S23421008, BICF2S2291323, BICF2P1231531, BICF2P410707, chr9_19191061, BICF2G630837373, BICF2G630840308, BICF2S24110485, chr10_6378351, chr10_7351411, BICF2P1348793, BICF2S24414279, BICF2G630668751, BICF2P373368, BICF2S23152609, BICF2P879518, BICF2S23554214,BICF2G630491746, BICF2P719913, BICF2P114915, chr11_46248163, BICF2S23650669, chr11_54743555, BICF2G630304307, BICF2P249476, BICF2P314220, BICF2P1467670, BICF2S23349333, G741f32S214, BICF2S23242450, BICF2S23013803, BICF2S22926285, TIGRP2P170982_rs9078347, BICF2S23726680, BICF2G630613749, BICF2S229669, BICF2S23420743, BICF2S23234775, BICF2S22928314, G795f39S172, BICF2G630747282, TIGRP2P187673_rs8738408, BICF2S23059962, BICF2G630526886, TIGRP2P192720_rs9193023, BICF2P195450, BICF2G630444279, BICF2G630441249, BICF2S23130543, BICF2P453375, BICF2P186458, BICF2P465575, chr15_41193134, chr15_41223494, chr15_42433048, BICF2P293585, BICF2S2302040, BICF2P291403, BICF2P943539, BICF2P125863, BICF2S23735815, BICF2G630821872, chr16_58964577, chr16_58964995, BICF2G630221423, BICF2S23533459, BICF2P775852, BICF2P701784, BICF2G63016830, chr17_58289619, TIGRP2P257443_rs8758924, chr18_55050494, BICF2G63048878, BICF2P529003, chr19_30435108, BICF2P1214928, BICF2S23251214, BICF2P511218, G1073f33S35, BICF2G630231592, BICF2S23028343, BICF2G630233693, BICF2P149501BICF2S2332575, TIGRP2P278109_rs8917987, BICF2P1309051, chr21_2616038, BICF2G630658267, BICF2P771898, BICF2G630656562, BICF2S23514069, TIGRP2P281262_rs9070324, chr21_32091465, BICF2G630640423, BICF2G630313766, BICF2P982193, BICF2P190095, BICF2P726365, BICF2P1472258, BICF2G630320610, BICF2G630334840, chr22_61281935, BICF2G630382580, chr23_44469937, BICF2S23051382, BICF2S23349129, BICF2P2436, chr24_22524131, BICF2P125931, BICF2P1434131, BICF2S23026288, TIGRP2P315149_rs8768077, BICF2P899631, chr24_33780522, BICF2S2376501, chr25_13355212, BICF2P268305, BICF2S23425698, BICF2P910432, chr26_969841, BICF2P603694, BICF2S23438429, BICF2P1439146, BICF2S23528097, BICF2G630806859, BICF2P392824, chr26_38700556, BICF2G630139588, TIGRP2P349754_rs8652808, BICF2S23764835, BICF2G630270231, BICF2S23345190, BICF2P245661, chr29_20178071, BICF2S23517012, BICF2G630627093, chr29_31754525, chr30_8411496, chr30_11546886, chr30_32348898, TIGRP2P372215_rs8974301, chr31_11203994, BICF2S23659818, BICF2S23240648, BICF2P110728, chr32_5231894, BICF2G630600200, BICF2G630600182chr32_6228658, BICF2G630597058, chr32_14565543, BICF2S23214760, BICF2S2347335, BICF2P1146448, BICF2P1284124, BICF2P610456, chr33_13860281, BICF2P962658, chr33_29055490, BICF2G63075180, TIGRP2P393312_rs9035803, TIGRP2P408388_rs8669882, BICF2G630775377, BICF2G630775400, BICF2S23340435, TIGRP2P416638_rs9137769, BICF2G630764278, chr36_5979991, BICF2G630763059, BICF2P636730, BICF2P587501, chr37_7829425, BICF2P281477, BICF2P175174, chr37_30070899, BICF2P58084, BICF2S22916972, BICF2G63073429, BICF2P645944, BICF2G630544447 and BICF2G630544415., 2. The canine genetic marker locus combination according to claim 1, wherein The STR loci include PEZ1, PEZ2, FH2010, PEZ5, PEZ12, FH2328, PEZ21, PEZ3, PEZ6, PEZ8, VGL3112, FH2054, VWFX, FH2611, FH2132, PEZ20, PEZ15, FH2079, PEZ17, FH3313, FH2088, FH2001, FH2017, and FH2107.

3. Use of the combination of canine genetic marker loci described in claim 1 in the analysis of canine breeds.

4. The application according to claim 3, wherein The canine breeds that can be identified by the said canine breed analysis include Belgian Shepherd Dog, French Bulldog, German Shepherd Dog, Kunming Dog, Bloodhound, Labrador Retriever, Rottweiler, and Springer Spaniel.

5. A canine breed analysis kit, characterized in that, It includes an SNP probe combination for identifying the canine genetic marker locus combination as described in claim 1 or 2, and the nucleotide sequences of the probe combination are shown as SEQ ID No: 1 to SEQ ID No:

287.

6. The canine breed analysis kit according to claim 5, characterized in that, It also includes an ARMS-PCR system for amplifying the STR loci.

7. A solid-phase chip, characterized in that, It includes a chip carrier and an SNP probe combination for identifying the canine genetic marker locus combination as described in claim 1 or 2. The probe combination is fixed on the surface of the chip carrier to form a microarray, and the nucleotide sequences of the probe combination are shown as SEQ ID No: 1 to SEQ ID No:

287.

8. The solid-phase chip according to claim 7, wherein It also includes at least one of a blocking agent, a reporter group, a reaction buffer, a positive control product, and / or a negative control product.

9. A liquid-phase chip, characterized in that, It includes a microsphere carrier and an SNP probe combination for identifying the canine genetic marker locus combination as described in claim 1 or 2, and the nucleotide sequences of the probe combination are shown as SEQ ID No: 1 to SEQ ID No:

287.

10. The liquid-phase chip according to claim 9, wherein It also includes at least one of a reporter molecule, a reaction buffer, a blocking agent, a positive control product, and / or a negative control product.

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