SNP (Single Nucleotide Polymorphism) molecular marker combination for paternity test and individual recognition of dairy cow and application

Abstract

The invention provides an SNP molecular marker combination for paternity test and individual recognition of dairy cows and application, the SNP molecular marker combination comprises 300 SNP markers on 29 autosomes of the dairy cows, the marker combination can be applied to paternity test and individual recognition of the dairy cows, and an identification method comprises the step of preparing a liquid phase chip of the 300 SNP markers, constructing a high-throughput sequencing library by using the genome DNA of the dairy cow to be detected; mixing the liquid phase chip probe with a high-throughput sequencing library, capturing a DNA fragment containing a target SNP site in the dairy cow DNA high-throughput sequencing library, amplifying and purifying to obtain a genetic typing of a dairy cow individual to be detected, and performing paternity test inference according to the genetic typing. The invention provides a probe and a kit for identifying SNP (Single Nucleotide Polymorphism) site information. According to the invention, the SNP polymorphism is detected through targeted capture sequencing, through high-depth sequencing, the typing result is accurate and reliable, and the accuracy of paternity test and individual recognition is ensured.

Description

Technical Field

[0001] This application relates to the field of molecular genetic testing, specifically to a combination of SNP molecular markers for dairy cow paternity testing and individual identification based on targeted capture technology and its application. Background Technology

[0002] Pedigree information plays a crucial role in breeding. Pedigree errors can significantly reduce the accuracy of genetic assessments, impact selection and mating effectiveness, slow population genetic progress, and cause substantial economic losses for businesses. However, pedigree errors are unavoidable in actual production, influenced by various factors such as human error in mating records, calving records, and pedigree entry and processing. Currently, the methods used for determining kinship in dairy cows are based on microsatellite-based methods, such as the microsatellite method recommended by ISAG / FAO internationally.

[0003] SNPs, or Specialized Nucleotides (SNPs), are the third generation of molecular markers following RFLPs and STRs. They are the most widely distributed in biological genomes, accounting for over 90% of all known polymorphic markers. They possess advantages such as large quantity, wide distribution, and high stability. Furthermore, because SNPs are bimorphic genetic markers, they are more suitable for rapid, large-scale screening. While some SNP marker combinations exist for cattle breeds, the significant differences in breeding histories among different breeds prevent their direct use for determining kinship in dairy cattle. From a future market promotion perspective, the number of SNP marker combinations determines a product's market competitiveness; more concise combinations offer better cost-effectiveness. Given the importance of pedigree records for dairy cattle quality traceability systems, and considering the application situation in China's dairy industry, developing concise and effective SNP combinations is essential for the development and promotion of the entire dairy farming industry. This accurate traceability method will provide crucial technical protection for companies' breeding achievements and buyers' rights, and is a vital link in promoting the healthy development of the dairy cattle breeding industry. Summary of the Invention

[0004] In view of the foregoing, in a first aspect, this application provides a combination of SNP molecular markers for paternity testing and individual identification in dairy cows, characterized in that it comprises: 300 SNP markers on the 29 autosomes of cattle, the information of the 300 SNP sites being shown in Table 1: Table 1. SNP molecular markers used for paternity testing and individual identification in dairy cows.

[0005] The sites of each SNP molecular marker are based on the reference genome ARS-UCD1.2.

[0006] Furthermore, the present invention also provides a probe for identifying the above-mentioned SNP molecular marker combinations; Preferably, the probe information is shown in Table 2.

[0007] Furthermore, the present invention also provides a gene chip prepared based on the above-mentioned SNP molecular marker combination.

[0008] Preferably, the type of gene chip includes a liquid phase chip.

[0009] Furthermore, the present invention also provides a kit prepared based on the above-mentioned SNP molecular marker combination.

[0010] Preferably, the kit contains probes shown in Table 2 for identifying the above-mentioned SNP molecular marker combinations.

[0011] Furthermore, the present invention also provides the application of the above-mentioned SNP molecular marker combination, probe, gene chip, or kit in identifying the kinship of dairy cows.

[0012] Furthermore, the present invention also provides the application of the above-mentioned SNP molecular marker combination, probe, gene chip, or kit in the genetic improvement breeding of dairy cows.

[0013] Furthermore, the present invention also provides the application of the above-mentioned SNP molecular marker combination, probe, gene chip, or reagent in paternity testing and individual identification of cattle, wherein the cattle breed is dairy cow.

[0014] Beneficial effects This application provides a combination of SNP molecular markers for dairy cow parentage identification and individual identification, and establishes a targeted capture sequencing method for detection to ensure the integrity and accuracy of pedigree in breeding. After detecting SNP polymorphisms through targeted capture sequencing and high-depth sequencing, the genotyping results are accurate and reliable, ensuring the precision of individual identification. Attached Figure Description

[0015] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the principle of targeted capture sequencing technology.

[0018] Figure 2 This is a site detection map of species-specific tests in the examples. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0020] This invention analyzes the genetic data of 3108 dairy cows and screens out 300 SNP loci on 29 autosomes (reference genome ARS-UCD1.2), which can be used for SNP marker combinations for dairy cow parentage testing and individual identification. The probe sequences of the 300 SNP markers are shown in Table 2.

[0021] Table 2 Information on 300 SNP tag combinations

[0022] This invention has been validated through targeted capture sequencing to detect SNP polymorphisms, as well as through paternity testing and individual identification experiments. The aforementioned 300 SNP markers were selected according to relatively strict quality control parameters, namely call rate (>0.95) and Hardy-Wenner balance (p>10). -6 The sample was selected using a combination of MAF>0.3 and LD filtering (r2>0.5), resulting in a very high confidence level for paternity testing and individual identification in dairy cows. Furthermore, case studies demonstrate that the sample information inferred using the 300 SNP markers of this invention is completely consistent with the actual information.

[0023] The specific embodiments of the present invention will be described in further detail below.

[0024] Example 1: Selection of SNP marker combinations; This invention analyzes the genetic data of 3108 dairy cows, removes closely related individuals, and uses the data of the remaining 2195 samples to screen for SNP loci. The loci screening principles are as follows: (1) SNP filtering: callrate>0.95, hwe>0.000001, MAF>0.3; (2) Bootstrap filtering: remove loci with a lower limit of MAF confidence interval less than 0.3; (3) Based on window length and MAF, only one locus with the highest MAF is retained in one window. The window length is continuously increased, and finally 300 paternity and individual identification loci (referencing the genome ARS-UCD1.2) are selected, which can be used for SNP marker combinations for paternity and individual identification of dairy cows.

[0025] Example 2: SNP polymorphism analysis of individual dairy cows based on targeted capture sequencing technology Specifically, the following steps are included: 1. Sample DNA extraction Samples were selected from dairy cows, and their blood and semen samples were taken to extract genomic DNA.

[0026] 2. Construction of liquid phase capture library 2.1 Genomic DNA fragmentation, end repair, and addition of A at the 3' end Take 300 ng of DNA sample and add 4 μL of Smearase Buffer and 2 μL of Smearase Enzymes, for a total volume of 24 μL. Then place the reaction plate in a PCR instrument and run the following program: 4℃ 1 min → 30℃ 10 min → 72℃ 20 min → 4℃ for storage.

[0027] 2.2 Fragment Filtering The fragment range was screened using magnetic beads to remove fragments that were too large or too small, so that the DNA fragments were concentrated in the range of 200-300 bp. The final fragment screening volume was 25 μL and stored in a 96-well PCR plate.

[0028] 2.3 Connector Connection and Accumulation Add 5 μL of CAGT Universal Adapters and 20 μL of LigationMaster Mix to a 96-well PCR plate as described above. Vortex to mix, then briefly centrifuge to collect the reaction solution at the bottom of the tube. Incubate at 20°C for 15 min in a PCR instrument to complete the sequencing adapter ligation. After purifying the ligation product, perform PCR amplification and enrichment. Take 15 μL of the purified ligation product, add 10 μL of CAGT UDI Primer and 25 μL of Equinox Library Amp Mix (2x), mix well, and then transfer 35 μL of the reaction solution to a PCR instrument for library amplification. After the whole genome library is constructed, quantify the library using the dsDNA HS Assay Kit for Qubit; simultaneously, check by electrophoresis whether the main peak of the library fragments is in the range of 300-500 bp.

[0029] 2.4 Liquid-phase chip hybridization capture Whole-genome libraries from each sample were pooled, resulting in a final hybridization capture library volume of 4 μg. Probe hybridization was performed on the libraries to capture the target region fragments from the whole-genome libraries. Excess probes, hybridization reagents, and other reagent components were removed through an elution step. Finally, the target region was enriched by post-hybridization PCR amplification to obtain the PCR-ready library.

[0030] 2.5 Library quality control and sequencing After the hybridization capture library was constructed, it was quantified using the dsDNA HS Assay Kit for Qubit; simultaneously, electrophoresis was used to check if the main peak size of the library was within the range of 300-500 bp. The constructed library was then sequenced using a DNBSEQ-T7 sequencer.

[0031] 3. Data quality control and genotyping 1) Data Filtering The raw sequencing data should be filtered to remove adapters, low-quality or undetectable bases and sequences. Recommended filtering items include (the following filtering parameters should be adjusted based on the genetic markers being detected or the next-generation sequencing platform being used): a) Detect the connector sequence and cut it; b) Remove sequences containing a certain number of N bases; c) Set a threshold for identifying low-quality bases and remove sequences containing a certain proportion or more of low-quality bases; d) Remove sequences that are shorter than a certain length after cutting; 2) Sequence alignment Candidate genetic markers in the filtered sequencing data were genotyped using commercially available analysis software recommended by the next-generation sequencing platform or other suitable analysis software. Sequencing depth, sequencing coverage of genetic markers, and capture-specific quality assessment metrics were statistically analyzed.

[0032] Implementation Case 3: Genotyping Quality Assessment of Gene Chips Sixty Holstein dairy cows were selected for genotyping at 300 loci using Implementation Case 2. Twelve samples were randomly selected for repeated testing, and their quality was evaluated as follows.

[0033] The detection rate of SNPs and the individual detection rate are important indicators for measuring chip quality. The detection rate of individual loci in all 60 test samples was 100%. Therefore, it can be seen that the liquid-phase chip genotype detection containing 300 loci described in this invention has very good quality.

[0034] Genotyping stability is generally measured by the genotyping consistency and correlation coefficient of replicate samples. In Example 2, genotyping of 12 Holstein cows was performed twice using the liquid-phase chip, and the genotyping consistency of the 12 replicate samples was 100%. Therefore, the genotyping stability of the liquid-phase chip containing 300 loci in this invention is very good.

[0035] The proportion of data that specifically falls within the target region out of the total data is the probe's capture specificity, or capture efficiency. High capture efficiency means high utilization of sequencing data. The maximum capture efficiency for 60 samples was 53.94%, the minimum was 46.39%, and the average was 50.73%. Therefore, this gene chip has high capture specificity.

[0036] The results show that the liquid-phase chip containing 300 sites described in this invention can significantly improve the accuracy and stability of large-scale genotyping detection in dairy cows.

[0037] Implementation Case 4: Species Specificity Analysis Eight pigeon samples, eight chicken samples, eight pig samples, and five dairy cow samples from Example 3 were selected. The detection results were examined according to the DNA extraction, library construction, sequencing, and data analysis methods described in Example 2. like Figure 2 As shown, all 300 loci were detected in bovine samples, 7-8 SNP loci were detected in pigs, and none were detected in pigeons and chickens. Therefore, this product has good specificity.

[0038] Implementation of Case 5: Marked Paternity Testing and Individual Identification Capabilities Paternity testing is based on Mendel's laws of segregation and independent assortment. Through genotype matching analysis, if the results conform to Mendel's laws of inheritance, a parent-child relationship cannot be ruled out; otherwise, the parent-child relationship can be excluded. The probability of parent exclusion (PE) is an important indicator for evaluating the practical value of genetic markers in paternity testing. It is calculated by using the allele frequencies at each locus to determine the probability that the allele of a certain marker in a candidate parent does not originate from the biological parent. The calculation of the probability of parent exclusion generally falls into the following three categories: (1) When the genotypes of the individual and one parent are known, but the genotype of the other parent is unknown, the probability of excluding the offspring from the assumed parent is PE1; (2) When the genotypes of the individual, the mother, and the assumed father are all known, the probability of excluding the offspring from the assumed parent is PE2; (3) When the genotypes of both parents are unknown, the probability of excluding the offspring from the assumed parents is PE3.

[0039]

[0040]

[0041]

[0042] ——No. The probability of excluding non-parents of each label; ——No. The first mark The frequency of each allele; —Number of markers.

[0043] In practical identification, multiple marker sites need to be included to improve the accuracy of the determination. When using k markers, the cumulative exclusion probability (CPE) of the system is calculated as follows:

[0044] In the formula: ——No. The probability of excluding non-parents of each marker.

[0045] Paternity testing analysis requires simultaneous assessment of CPE and CPI. The formulas for the Paternity Index (PI) and Cumulative Paternity Index (CPI) are as follows:

[0046]

[0047] In the formula: —The probability that the controversial parent provides parental genes; —The random frequency of this gene in the population; ——No. Parental index of each marker.

[0048] The power of discrimination (DP) is the probability that two randomly selected individuals from a biological population will have different genetic marker phenotypes. If the genotype of the tested sample differs from the true sample, it can be ruled out that the two samples originated from the same individual (except in cases of mutation). If the genotypes of the two samples are the same, the coupling probability is calculated; if its value is less than 5 × 10⁻⁶, the probability is considered valid. -9 At that time, it was determined that the two samples originated from the same individual. The coupling probability is the product of the frequencies of a set of marker genotypes. The formula for calculating the coupling probability is: P M = P1×P2×P3×……×Pn In the formula: P M Coupling probability P — Genotype frequency of the i-th marker Based on the allele frequency of each SNP locus, the non-parent exclusion probability and individual exclusion rate of each locus were calculated. The non-parent exclusion probability and individual recognition ability of 300 markers are shown in Table 3.

[0049] By calculating their exclusion probabilities and cumulative exclusion probabilities, the results showed that the cumulative exclusion rate for a minimum of 45 markers reached 0.997543242, and the cumulative parentage index was 10685.38. The cumulative individual identification rate for a minimum of 20 markers reached 3.02 × 10⁻⁶. -09 This indicates that these 300 SNP markers have extremely high capabilities for paternity testing and individual identification.

[0050] Table 3300: Exclusion rate of non-parents and individual identification ability.

[0051] To verify the feasibility and accuracy of using the complete set of SNP markers for paternity testing in a real population, nine paternity pairs of dairy cows were randomly selected from the experimental population for actual paternity testing. Ten duplicate samples were randomly selected for individual identification testing. DNA was extracted from each blood sample and detected using the liquid phase chip of this invention. Paternity testing and individual identification analysis were performed respectively.

[0052] The paternity test results show that, as shown in Table 4, the cumulative paternity index of all nine cow mother-cattle pairs is higher than 2000, and as shown in Table 2, the cumulative exclusion rate of 300 markers reaches 1.0000, indicating that the probable maternal lineage of the nine offspring is completely identical to the actual maternal lineage. Individual identification results show that, as shown in Table 5, the genotyping of the 10 duplicate samples is completely consistent, and the coupling probability of each duplicate sample is less than 5 × 10⁻⁶. -9 This allows for the accurate identification of duplicate individuals, demonstrating the extremely high paternity testing and individual identification capabilities of these 300 SNP markers.

[0053] Table 4. Application of 4300 SNP marker combinations in paternity testing

[0054] Table 5. Application of 5300 SNP marker combinations in individual identification

[0055] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A combination of SNP molecular markers for paternity testing and individual identification in dairy cows, characterized in that, include: The 300 SNP markers on the 29 autosomes of cattle are shown in Table 1 of the specification.

2. The SNP molecular marker combination as described in claim 1, wherein, The sites of each SNP molecular marker were based on the reference genome ARS-UCD1.

2.

3. A probe for detecting the SNP molecular marker combination for paternity testing and individual identification of dairy cows as described in claim 1.

4. The probe as described in claim 3, characterized in that... The probe information is shown in Table 2.

5. A gene chip prepared based on the SNP molecular marker combination of claim 1 and / or the probe of claim 3.

6. A kit prepared based on the SNP molecular marker combination of claim 1 and / or the probe of claim 3.

7. The application of the SNP molecular marker combination of claim 1, and / or the probe of claim 3, and / or the gene chip of claim 5, and / or the kit of claim 6 in identifying the kinship of dairy cows.

8. The application of the SNP molecular marker combination of claim 1, and / or the probe of claim 3, and / or the gene chip of claim 5, and / or the kit of claim 6 in the genetic improvement breeding of dairy cows.

9. The application of the SNP molecular marker combination of claim 1, and / or the probe of claim 3, and / or the gene chip of claim 5, and / or the kit of claim 6 in the individual identification of cattle, wherein the breed of cattle is dairy cow.

10. A method for identifying kinship among dairy cows, characterized in that, The method includes the following steps: using the genomic DNA of the cattle to be tested as a template, identifying the genotype of the SNP molecular marker combination as described in claim 1 or 2, and identifying the kinship of the cattle to be tested based on the genotype of the individual cattle.

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