Probe combination for yak breeding, kit and application thereof
By screening and designing SNP probe combinations suitable for yak breeding, the problem of insufficient accuracy and efficiency of targeted sequencing genotyping in yak breeding has been solved, enabling efficient parentage identification and pedigree reconstruction, and improving the accuracy and reliability of the breeding process.
Patent Information
- Application Number
- CN202511628723.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-01-02
AI Technical Summary
Existing technologies lack highly accurate and efficient targeted sequencing genotyping (GBTS) probe sets suitable for yak breeding, resulting in a high pedigree error rate, inaccurate breeding value estimation, and difficulty in achieving effective parentage identification and pedigree reconstruction during yak breeding.
A probe ensemble for yak breeding was designed, including specific SNP site probes. 175 functional SNP sites and 5873 background SNP sites were screened through GWAS analysis and resequencing data. The probe ensemble was synthesized for GBTS detection to achieve yak parentage identification and pedigree correction and reconstruction.
It improves the accuracy of parentage identification in yak breeding, reduces pedigree error rate, ensures the accuracy of breeding value estimation, supports genomic selection models, and enhances the reliability of early selection and genetic resource management.
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Figure CN121249900A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of targeted sequencing genotyping, and particularly relates to a probe combination for yak breeding, a kit and application thereof. BACKGROUND
[0002] The core goal of livestock breeding is to maximize the genetic progress of economic traits such as milk yield, daily weight gain, and feed conversion rate in the shortest time. To this end, the breeding value of individuals must be accurately estimated, and the calculation of breeding value depends on two premises: one is to know the exact parents of each offspring (parentage identification), and the other is to have a continuous and fault-free pedigree (pedigree correction and reconstruction).
[0003] In traditional pastures, pedigree error rates can reach 10%-30% due to multi-male and female mixed groups, missing records of artificial insemination, or confusion of embryo transfer. In yak breeding, the problem of pedigree loss is more serious, as most yaks are naturally mated in grassland grazing, and the pedigrees of many breeding farms are almost completely missing. Each 1% of pedigree error will cause a loss of about 2%-3% of genetic progress. Therefore, it is necessary to perform parentage identification within a few weeks after the birth of offspring in the breeding process to confirm the parents and reduce the error rate to below 1%. This can directly improve the accuracy of breeding value estimation and avoid the incorrect elimination of excellent individuals or the retention of inferior individuals.
[0004] Pedigree reconstruction integrates historical data with on-site genotypes to complete missing ancestral information, even tracing back to three or four generations ago. A complete pedigree not only serves for inbreeding monitoring to prevent the concentration of recessive harmful genes, but also supports genomic selection models to improve the reliability of early selection. For local conservation populations, pedigree reconstruction can clarify the genetic resource structure and guide hybrid combination design.
[0005] Genotyping By Target Sequencing (GBTS) technology is a low-cost method for obtaining high-density and high-accuracy genotypes by capturing several hundred to several thousand target SNP sites through probes or PCR, and reading them by second-generation sequencing. Its short fragments and flexible throughput are suitable for various samples such as blood, hair follicles, and ear tissue of livestock and poultry. In parentage identification, high polymorphic SNPs can be used to exclude non-parents, with an accuracy of >99%, and are not affected by multiple males and females in the same group. In pedigree reconstruction, the combination of GBTS data of historical and on-site individuals can complete missing ancestral information, correct record errors, and support genomic selection and inbreeding management.
[0006] However, in the development of GBTS technology, how to screen the set of target SNP sites is a technical difficulty. The number of GBTS probes is limited, and SNP with large information and compatible with multiple platforms must be screened out. There are three difficulties: ① The difference in population polymorphism is large, and high MAF (>0.3) sites need to be screened across varieties; ② The sites need to uniformly cover the whole genome, avoid linkage redundancy, and avoid repetitive sequences and difficult binding regions of probes; ③ Consider the future pedigree depth, and preferentially select SNP with low mutation rate and high typing stability. Traditional chip sites often have bias, and need to be combined with resequencing data, population LD decay curve and experimental verification to repeatedly iterate to construct a universal and efficient target set. At present, there is still a lack of GBTS probe set or primer set with high accuracy and high efficiency for yak breeding. Therefore, developing GBTS technology for yak breeding is still an important topic in the field. SUMMARY
[0007] In view of the problems of the prior art, the present application provides a probe combination, a kit and the use thereof for yak breeding.
[0008] A probe combination for yak breeding, the probe combination comprising probes corresponding to SNP sites, the SNP sites being shown in Table 1 (see Example 1);
[0009] Wherein the yak reference genome used as a chromosome position reference is Maiwa_STV1.0, and the index number is GWHGRYZ00000000.1.
[0010] Preferably, the nucleotide sequences of the probes corresponding to the SNP sites are respectively corresponding to the chromosome positions shown in Table 2 (see Example 1).
[0011] Preferably, the probe combination is used for GBTS detection.
[0012] The present application also provides the use of the above-mentioned probe combination in the preparation of a yak breeding kit.
[0013] The present application also provides a yak breeding kit, which comprises the above-mentioned probe combination.
[0014] Preferably, the kit is a kit for targeted sequencing genotyping detection.
[0015] The present application also provides the use of the above-mentioned probe combination or yak breeding kit for yak parentage identification and / or pedigree correction and / or pedigree reconstruction.
[0016] In view of the application requirements of parentage identification, pedigree correction and pedigree reconstruction in yak breeding, the present application screens SNP sites and probe sets for GBTS detection. Specifically, the present application analyzes 114 yak 10X resequencing data and reference genome. First, 183 associated SNP sites are obtained according to the results of GWAS analysis; then, 5873 background SNP sites of 5K are screened. A probe is designed, and a set of panels is synthesized for the above 6056 target sites, and 10 test samples are tested (the test results of the 10 test samples are shown in Experimental Example 1). Through the analysis results of site distribution and uniform distribution of sites, the above SNP sites have very high average site capture efficiency and uniformity. According to the test results, the probes corresponding to the 12 sites obtained by association analysis have a great influence on the overall target sequence proportion and effective data rate. Through GWAS, 4 sites are retained and the other 8 sites are removed. Finally, the sites are composed of 175 sites obtained by association analysis and 5873 background sites.
[0017] The sites and probe sets obtained by the above screening can efficiently and accurately perform parentage identification, pedigree correction and pedigree reconstruction of yak, and have good application prospects in yak breeding work.
[0018] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.
[0019] The above content of the present application will be further described in detail through the specific embodiments below. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 Distribution of SNP markers on different chromosomes;
[0021] Figure 2 Uniform distribution of SNP markers on different chromosomes;
[0022] Figure 3 Gap analysis results of target sites on the genome;
[0023] Figure 4 MAF distribution statistics of SNP markers;
[0024] Figure 5 SNP marker type statistics;
[0025] Figure 6Distribution of SNP markers in gene structure;
[0026] Figure 7 Population PCA distribution based on resequencing data;
[0027] Figure 8 Population PCA distribution based on target sites;
[0028] Figure 9 Population phylogenetic tree analysis result based on resequencing data;
[0029] Figure 10 Population phylogenetic tree analysis result based on target sites;
[0030] Figure 11 Polymorphism statistics between samples based on resequencing data;
[0031] Figure 12 Polymorphism statistics between samples based on target SNPs;
[0032] Figure 13 Population LD decay plot based on resequencing data;
[0033] Figure 14 Population LD decay plot based on target sites. DETAILED DESCRIPTION
[0034] In the following examples and experimental examples, reagents and materials not specifically mentioned are commercially available.
[0035] Example 1 Yak Breeding Kit
[0036] This example provides a kit comprising probes corresponding to 6048 SNP sites, as shown in Table 1 below:
[0037] Table 1
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[0056] The nucleotide sequences of the probes corresponding to the SNP sites are shown in Table 2 below:
[0057] Table 2
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[0115] In Table 1 and Table 2, the position of the SNP site is represented in the form of "N_M", wherein N represents that the SNP site is located on the Nth chromosome of the yak reference genome, and M represents that the SNP site is located at the Mth position of the chromosome.
[0116] In Table 2, the chromosome position corresponding to the probe sequence is represented in the form of "N:A_B", wherein N represents that the chromosome corresponding to the probe sequence is the Nth chromosome of the yak reference genome, and A and B represent that the starting site of the chromosome position corresponding to the probe sequence is A and the ending site is B.
[0117] The yak reference genome used as a chromosome position reference is Maiwa_STV1.0, and the index number is GWHGRYZ00000000.1.
[0118] The specific sequences of all probes are designed, the probe sequences are synthesized, and the detection is performed by GenoBaits technology of Shijiazhuang Boerdi Biotechnology Co., Ltd.
[0119] The SNP sites selected in the embodiment include 175 functional SNP sites identified by GWAS analysis and 5873 background sites. These sites comprehensively cover the genomic range of yak, are uniformly distributed, and have good site capture efficiency, uniformity and target sequence proportion of detection results, which can meet the needs of individual parentage identification, pedigree correction and pedigree reconstruction in the process of yak breeding.
[0120] The technical solutions of the present application are further described below through experimental examples:
[0121] Experimental Example 1: Screening process and results of SNP sites
[0122] I. Screening process
[0123] 1. Description of raw data
[0124] The raw data includes 114 resequencing samples, a total of 54098646 target sites with sequencing depth ≥5X are screened for target site selection and resequencing related analysis.
[0125] 2. Background site selection process
[0126] From the resequencing data, 40K sites with SNP site detection rate >70%, MAF >0.2%, heterozygosity <60% and uniform distribution are screened, 10K sites with uniform distribution are screened from the 40K sites, sites with larger blank area are selected from the resequencing data to fill in to ensure uniform distribution, and 5873 sites with uniform distribution are selected from the 10K sites after probe evaluation.
[0127] 3. GWAS analysis screening
[0128] 183 SNP sites obtained through GWAS analysis results.
[0129] 4. Probe design
[0130] The above-mentioned 183 SNP sites obtained through GWAS analysis results and 5873 background sites screened are designed for these sites.
[0131] 5. Site test results
[0132] The test of site detection performance shows that, when 183 sites obtained by the association analysis and 5873 background SNP sites are tested, the average site capture efficiency is 99.87%, the uniformity is 99.92%, and the target sequence proportion is 8.76%.
[0133] Ten test samples are tested, and the test results of 6056 sites are shown in the following table:
[0134] Table 3 Test results of 6056 sites
[0135]
[0136] According to the test results, the probes corresponding to the 12 sites obtained by the association analysis have a great influence on the overall target sequence proportion and effective data rate. According to the P value of the sites obtained by GWAS, 4 sites are retained and the other 8 sites are removed. Finally, the sites are composed of 175 sites obtained by the association analysis and 5873 background sites (the specific sites are described in Example 1).
[0137] II. Screening results
[0138] Figure 1 The number statistics of the 5873 SNP sites (target sites) on different chromosomes are shown in the following table, wherein the abscissa is the chromosome ID; the left example Count is the number of sites; and the right example Length is the length of the chromosome (unit: bp). The average distance of the 5873 SNP sites is 460204. Figure 2 The distribution of the target sites on the chromosomes is shown in the following table, and the drawing unit length (i.e. the length of the genome represented by each vertical line) is 300000bp. The results show that the 5873 SNP sites are uniformly distributed in the genome.
[0139] Figure 3 The Gap analysis of the target sites on the chromosomes is shown in the following table. If the region without sites is greater than the genome size / target site number*10, it is considered as a Gap. The results show that only small fragments of gaps appear on chromosomes 7 and 30, indicating that the chip has good coverage of the whole genome.
[0140] Figure 4 The MAF value statistics of the target sites are shown in the following table. The abscissa is the MAF range, and the ordinate Count is the number of target sites in the MAF range. The proportion of sites with minimum allele frequency (MAF) ≥0.3 is 92%. The results show that the target sites obtained by the present application have good polymorphism.
[0141] Figure 5Statistics of different marker types of target sites. The abscissa Type is the marker type classification, which is divided into C / G, A / C, G / T, A / T, A / G, C / T and InDel, a total of 7 types, and the ordinate Count is the number of target sites of the type marker. The results show that the target sites screened by the application are uniformly distributed in various SNP types.
[0142] Figure 6 Statistics of the distribution of target sites on the gene structure. The results show the distribution of sites in various regions of the genome. As can be seen from the figure, the intergenic region and the intron region, which account for the largest proportion of the genome, are also among the top two in site distribution. This shows that the sites are uniformly distributed in various regions of the genome.
[0143] Figure 7 、 Figure 8 The population PCA distribution map based on resequencing data and the population PCA distribution map based on target sites (SNP) are shown in FIGS. 9 and 10, respectively. The distribution of individuals in the figures shows that the principal component difference between individuals based on chip sites is close to the principal component difference between individuals based on all sites in the first three principal components.
[0144] Figure 9 The population phylogenetic tree analysis result based on resequencing data is shown in FIG. 11. Figure 10 The population phylogenetic tree analysis result based on target sites (SNP) is shown in FIG. 12. From the constructed phylogenetic tree, the phylogenetic tree based on resequencing data and the phylogenetic tree based on chip sites reflect the evolutionary relationship between different individuals, respectively.
[0145] Figure 11 The sample polymorphism statistics result based on resequencing data is shown in FIG. 13. Figure 12 The sample polymorphism statistics result based on target SNP is shown in FIG. 14. The abscissa Diversity is the sample diversity, and the ordinate Count is the number of sample combinations in the section. The above results show that the polymorphism of the chip sites is mainly distributed in the interval [0.5, 0.6) according to the needs of site design.
[0146] Figure 13 The population LD decay map based on resequencing data is shown in FIG. 15. Figure 14 The population LD decay map based on target sites is shown in FIG. 16. The above results show that the correlation between target sites is higher than the correlation between sites obtained based on resequencing data.
[0147] In summary, through the above results, it can be seen that the target sites screened by the application are uniformly distributed in the genome, have good polymorphism, are uniformly distributed in various SNP types, have good coverage of various regions of the whole genome, and the multi-angle evaluation results do not appear abnormal that affects the use of the chip. These characteristics ensure that the target sites of the application can efficiently and accurately obtain detection results in yak breeding work, and realize parentage identification, pedigree correction and pedigree reconstruction work.
[0148] Experimental Example 2: Application example of pedigree reconstruction
[0149] In this experimental example, the kit of Example 1 was used to perform parentage identification and pedigree correction on a series of yak samples, and the results were as follows:
[0150] According to the pedigree relationship in the original sample data and the sample genotype data (tested by the kit of Example 1), the Mendelian error rate between samples in the core family was calculated, and whether the pedigree relationship was correct was determined according to the calculation results (the Mendelian error rate threshold was set to 1%, and the Mendelian error rate ≤ 1% was a suspected parent-child relationship, and > 1% was a non-parent-child relationship). The results of pedigree analysis are as follows, and the bold font indicates the biological parent individual found according to the genotype data after pedigree correction.
[0151] Table 4: Results of parentage identification and pedigree correction
[0152]
[0153] When NA appears in the corrected father name or the corrected mother name, it indicates that the corresponding parent of the individual is not in the population. When the original father name or the original mother name of an individual is NA, but there is a corrected father or mother name, it indicates that the individual previously lacked pedigree data, and the kit provided by the application successfully located its parents from the population.
[0154] As can be seen from this example, the kit provided by the application can efficiently and accurately complete the work of parentage identification and pedigree reconstruction, and has good application prospects in yak breeding.
Claims
1. A probe assembly for yak breeding, characterized in that, The probe combination includes probes corresponding to SNP sites, which are shown in Table 1. The yak reference genome used as a reference for chromosome location is Maiwa_STV1.0, with index number GWHGRYZ00000000.
1.
2. The probe assembly according to claim 1, characterized in that, The chromosomal locations corresponding to the nucleotide sequences of the probes at the SNP sites are shown in Table 2.
3. The probe assembly according to claim 1 or 2, characterized in that, The probe combination is used for GBTS detection.
4. Use of the probe combination according to any one of claims 1-3 in the preparation of a yak breeding kit.
5. A yak breeding kit, characterized in that, It includes the probe combination as described in any one of claims 1-3.
6. The yak breeding kit according to claim 5, characterized in that, The kit is for targeted sequencing genotyping detection.
7. The probe combination according to any one of claims 1-3, or the yak breeding kit according to claim 5 or 6, for use in yak parentage identification and / or pedigree correction and / or pedigree reconstruction.