A SNP locus combination, liquid-phase chip and application for sheep genotyping

Abstract

The present invention discloses a SNP locus combination for sheep genotyping, a liquid-phase chip and an application thereof, belonging to the technical field of molecular breeding. The present invention provides a SNP locus combination for sheep genotyping, and the SNP locus combination comprises 58,859 SNP loci. The SNP locus combination of the present invention covers multiple important economic traits, and encompasses important genetic loci related to the production performance, reproductive performance, disease resistance traits and adaptability of sheep, which helps to improve the breeding efficiency and production performance. By combining functional locus selection and liquid-phase chip capture sequencing technology, the present invention designs an efficient, low-cost and high-throughput 50K SNP liquid-phase chip for the whole genome of sheep, which can accurately screen genetic markers related to important economic traits of sheep, and provides a reliable molecular marker tool for the genome-wide association analysis, genomic selection, genetic improvement and phenotypic regulation of sheep.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding, and particularly relates to a SNP locus combination for sheep genotyping, a liquid-phase chip and applications thereof. Background Art

[0002] With the continuous development of molecular breeding technologies, genomics methods have been increasingly widely applied in animal genetic improvement and genomic selection, and genotype analysis has become a key technology for improving breeding efficiency. As the most widely distributed genetic marker in the genome, single nucleotide polymorphism (SNP) has high genetic stability and demonstrates powerful application potential in fields such as genome-wide association study (GWAS) and genomic selection (GS), becoming the most commonly used marker type in animal genetics research.

[0003] Although the application of SNP markers in genomics research has been extensive and in-depth, existing genotyping technologies, especially solid-phase chip-based technologies, have some limitations. Solid-phase chips detect SNP loci by immobilizing specific DNA probes on the chip surface and through hybridization reactions with sample DNA fragments. However, they have poor flexibility and can only detect the fixed SNP loci included in the design, and cannot add or delete loci at any time. In addition, the genotyping cost of solid-phase chips is relatively high and cannot meet the requirements of large-scale and diverse-sample genetic analysis. Moreover, existing sheep chips also have the defects of incomplete coverage of functional loci and low effectiveness. Therefore, it is particularly important to provide a SNP locus combination for sheep genotyping and a sheep whole-genome liquid-phase chip that comprehensively cover functional loci, have a wide application range, and enable efficient, low-cost and high-throughput detection. Summary of the Invention

[0004] In view of this, an object of the present invention is to provide a SNP locus combination for sheep genotyping, which covers important genetic loci related to sheep production performance, reproductive performance, disease resistance and adaptability, etc., and helps to comprehensively evaluate the growth, reproduction and adaptability of livestock, thereby improving breeding efficiency and production performance.

[0005] Another object of the present invention is to provide a probe combination for detecting the SNP locus combination for sheep genotyping.

[0006] Another object of the present invention is to provide a kit for detecting sheep genotyping.

[0007] Another object of the present invention is to provide an application of the SNP locus combination, the probe combination or the kit in the preparation of a sheep whole-genome chip.

[0008] Another object of the present invention is to provide a sheep whole-genome 50K SNP liquid-phase chip.

[0009] Another object of the present invention is to provide an application of the SNP locus combination, or the probe combination, or the kit, or the liquid chip in sheep breeding.

[0010] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0011] The present invention provides an SNP locus combination for sheep genotyping, the SNP locus combination contains 58,859 SNP loci, and the position information of the SNP loci is shown in Table 2; the SNP locus combination contains SNP loci located on the sheep reference genome Oar v4.0.

[0012] The present invention also provides a probe combination for detecting an SNP locus combination for sheep genotyping. Each SNP locus in the SNP locus combination is correspondingly provided with a set of probes for detecting the corresponding SNP locus to form a probe combination.

[0013] Preferably, the design parameters of the probe are: the probe length is 120 nt; the GC content is 30% - 70%; the copy number of the whole probe in the genome is 1; the copy number of any 25 bp kmer sequence in the genome does not exceed 5.

[0014] The present invention also provides a kit for detecting sheep genotyping, and the kit includes the probe combination.

[0015] The present invention also provides an application of the SNP locus combination, or the probe combination, or the kit in the preparation of a sheep whole-genome chip.

[0016] Preferably, the chip includes a solid-phase chip or a liquid chip.

[0017] The present invention also provides a sheep whole-genome 50K SNP liquid chip, and the liquid chip includes the probe combination.

[0018] The present invention also provides an application of the SNP locus combination, or the probe combination, or the kit, or the liquid chip in sheep breeding.

[0019] Preferably, the sheep breeding includes one or more of sheep genomic selection, mapping of sheep trait-related genes, analysis of sheep genetic diversity, sheep genome-wide association analysis, sheep breed identification, sheep kinship identification, sheep population classification, sheep phenotype regulation, improvement of sheep germplasm resources, and protection of sheep germplasm resources.

[0020] Preferably, the traits in the mapping of the sheep trait-related genes include one or more of vision, wool, immunity, reproductive ability, fat deposition, horns, teat morphology, high-altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxic adaptability, daily weight gain, birth weight, cold tolerance adaptability, lactose content, blood, bones, ovarian development, fertilization, litter size, body weight, body slant length, abdominal circumference, chest circumference, chest depth, height at the cross, width at the cross, hip width, hip height, shoulder width, and body height.

[0021] Advantages of the present invention:

[0022] The SNP locus combination for sheep genotyping in the present invention is evenly distributed on each chromosome, and the minor allele frequency is greater than 0.1. The SNP locus combination of the present invention covers multiple important economic traits, covering important genetic loci related to sheep production performance, reproductive performance, disease resistance traits, and adaptability, etc., which helps to comprehensively evaluate the growth, reproduction, and adaptability of livestock, thereby improving breeding efficiency and production performance.

[0023] The present invention combines functional locus selection and liquid-phase chip capture sequencing technology to design a highly efficient, low-cost, and high-throughput sheep whole-genome 50K SNP liquid-phase chip. Compared with traditional solid-phase chips with the same number of probes, the liquid-phase chip of the present invention can detect more SNP loci. In addition, the chip design of the present invention is flexible and new breeding-related marker loci newly discovered can be added at any time. The chip of the present invention can accurately screen genetic markers related to important economic traits of sheep, and provide a reliable molecular marker tool for genome-wide association analysis, genomic selection, genetic improvement, and phenotypic regulation of sheep, and can be used for large-scale genotyping of sheep, having an obvious price advantage compared with whole-genome resequencing. Description of the drawings

[0024] Figure 1 It is the distribution density map of SNP loci on each chromosome in Example 1;

[0025] Figure 2 It is the statistics of the minor allele frequency (MAF) of SNP loci in Example 1;

[0026] Figure 3 It is the data alignment rate of the test samples of the sheep whole-genome 50K SNP liquid-phase chip in Example 2, where the abscissa numbers represent different sheep blood DNA sample numbers;

[0027] Figure 4 It is the capture efficiency of the test samples of the sheep whole-genome 50K SNP liquid-phase chip in Example 2, where the abscissa numbers represent different sheep blood DNA sample numbers;

[0028] Figure 5 For the redundancy rate of the test sample data of the 50K SNP liquid chip for the whole genome of sheep in Example 2, where the abscissa numbers represent the numbers of different sheep blood DNA samples;

[0029] Figure 6 For the SNP detection rate of the test samples of the 50K SNP liquid chip for the whole genome of sheep in Example 2, where the abscissa numbers represent the numbers of different sheep blood DNA samples;

[0030] Figure 7 For the genetic clustering tree in Example 3;

[0031] Figure 8 For the Manhattan plot of the genome-wide association analysis of sheep in Example 3, where A is body weight, B is abdominal circumference, C is shoulder width, D is hip height, E is hip width, F is withers height, G is body height, H is body slant length, I is withers width, J is chest depth, and K is chest circumference. Detailed implementation manners

[0032] The present invention provides an SNP locus combination for sheep genotyping. The SNP locus combination contains 58,859 SNP loci, and the position information of the SNP loci is shown in Table 2; the SNP locus combination contains SNP loci located on the sheep reference genome Oar v4.0.

[0033] In the present invention, the SNP locus combination contains SNP loci located on the sheep reference genome Oar v4.0 and sheep whole-genome SNP variation loci in the iSheep database. The SNP locus combination of the present invention is based on the re-sequencing data of various domestic and foreign sheep breeds, with comprehensive data and a wide application range. The SNP locus combination of the present invention is evenly distributed on each chromosome, and the minor allele frequency is greater than 0.1. The SNP locus combination of the present invention covers multiple important economic traits, including but not limited to: vision, wool, immunity, reproductive ability, fat deposition, horns, teat morphology, high-altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxic adaptation, daily weight gain, birth weight, cold tolerance adaptation, lactose content, blood, bones, ovarian development, fertilization, litter size, body weight, body slant length, abdominal circumference, chest circumference, chest depth, withers height, withers width, hip width, hip height, shoulder width, and body height, etc., covering important genetic loci related to sheep production performance, reproductive performance, disease resistance traits, and adaptability, etc., which helps to comprehensively evaluate the growth, reproduction, and adaptability of livestock, thereby improving breeding efficiency and production performance.

[0034] The present invention also provides a probe combination for detecting the SNP locus combination for sheep genotyping. For each SNP locus in the SNP locus combination, a set of probes for detecting the corresponding SNP locus is provided correspondingly to form a probe combination.

[0035] In the present invention, the design parameters of the probe are preferably as follows: the probe length is 120 nt; the GC content is 30% - 70%; the copy number of the whole probe in the genome is 1; the copy number of any 25-bp kmer sequence in the genome does not exceed 5. The probe combination of the present invention has high capture efficiency, low redundancy rate, and good effectiveness.

[0036] The present invention also provides a kit for detecting sheep genotyping, and the kit includes the probe combination.

[0037] The present invention also provides an application of the SNP locus combination or the probe combination or the kit in the preparation of a sheep whole-genome chip.

[0038] In the present invention, the sheep whole-genome chip is preferably a solid-phase chip or a liquid-phase chip, and more preferably a liquid-phase chip. The working principle of the liquid-phase chip is based on the complementary binding of specific probes to target sequences for site-specific capture, and the captured target sequences are eluted, amplified, library constructed, and sequenced, and finally the genomic genetic variation information of the target region is obtained. Compared with the solid-phase chip, the liquid-phase chip has the advantages of high throughput, low cost, and strong flexibility, can cover more SNP loci, and is suitable for large-scale and complex-trait genomic research.

[0039] The present invention also provides a sheep whole-genome 50K SNP liquid-phase chip, and the liquid-phase chip includes the probe combination.

[0040] The present invention combines functional site selection and liquid-phase chip capture sequencing technology to design an efficient, low-cost, and high-throughput gene liquid-phase chip. Compared with traditional solid-phase chips with the same number of probes, the liquid-phase chip of the present invention can detect more SNP loci. In addition, the chip design of the present invention is flexible and new discovered breeding-related marker loci can be added at any time. The liquid-phase chip of the present invention covers the above-mentioned multiple important economic traits, can accurately screen genetic markers related to important economic traits of sheep, and provides a reliable molecular marker tool for the genome-wide association analysis, genomic selection, genetic improvement, and phenotypic regulation of sheep. Compared with whole-genome resequencing, the liquid-phase chip of the present invention can accurately identify genetic variations in genomic functional regions with less sequencing volume, has a lower cost, can provide important support for the discovery of phenotypic function mutations regulating organisms, and provides a more efficient tool for the genetic improvement and genomic selection of animals and plants.

[0041] The present invention also provides an application of the SNP locus combination or the probe combination or the kit or the liquid-phase chip in sheep breeding.

[0042] In the present invention, the SNP locus combination covers the above-mentioned multiple important economic traits, including but not limited to: vision, wool, immunity, reproductive ability, fat deposition, horns, teat morphology, high-altitude adaptability, muscle development, nerves, growth performance, lactation performance, carcass traits, hypoxia adaptability, daily weight gain, birth weight, cold tolerance adaptability, lactose content, blood, bones, ovarian development, fertilization, litter size, body weight, body slant length, abdominal circumference, chest circumference, chest depth, rump height, rump width, hip width, hip height, shoulder width, body height, etc., which helps to comprehensively evaluate the growth, reproduction and adaptability of sheep, thereby improving breeding efficiency and production performance. The SNP locus combination of the present invention, or a probe combination, kit or the liquid chip for detecting the SNP locus combination can accurately screen genetic markers related to important economic traits of sheep, and provide a reliable molecular marker tool for sheep genomic selection, mapping of sheep trait-related genes, analysis of sheep genetic diversity, genome-wide association analysis of sheep, sheep breed identification, sheep kinship identification, sheep population classification, sheep phenotype regulation, improvement of sheep germplasm resources and protection of sheep germplasm resources.

[0043] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] In the following embodiments, unless otherwise specified, all are conventional methods.

[0045] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0046] Example 1 Design and Preparation of Sheep Whole Genome 50K SNP Liquid Chip

[0047] 1. Data Preparation and Integration

[0048] First, obtain the whole genome re-sequencing data of 10 East Friesian sheep (accession number: PRJNA624020) in the NCBI database BioProject for analysis, align it to the Oar v4.0 reference genome, and screen and obtain high-quality SNP data after quality control. Subsequently, merge these SNP data with the sheep whole genome SNP variant site information in the iSheep database (https: / / ngdc.cncb.ac.cn / isheep / ) to ensure the comprehensiveness and consistency of the data source.

[0049] 2. Definition of Functional Regions

[0050] Based on existing literature (Cao Y, Ai Y, Zhang X, Zhang J, Long X, Zhu Y, Wang L, Gu Q, Han H. Genome-wide epigenetic dynamics during postnatal skeletal muscle growth in Hu sheep. Commun Biol. 2023 Oct 23;6(1):1077. doi: 10.1038 / s42003-023-05439-0. PMID: 37872364; PMCID: PMC10593826), element information related to sheep epigenetic regulation was obtained, and this information was used to define functional site regions for further optimization of the chip.

[0051] 3. Screening of fixed sites

[0052] According to literature reports, several functional genes or variant sites related to the positioning of traits of interest were determined as the fixed sites of the chip. These fixed sites mainly cover important genetic sites known to be related to sheep production performance, reproductive performance, disease resistance traits, and adaptability. The functional gene information is shown in Table 1.

[0053] Table 1 Functional gene information

[0054]

[0055] 4. Probe design

[0056] Probe design was performed on all regions of the genome using the OligoMiner tool. The parameters for probe design were: the probe length was set to 120 nt; the GC content was controlled between 30% and 70%; the copy number of the entire probe in the genome was 1; the copy number of any 25-bp kmer sequence in the genome did not exceed 5.

[0057] Through screening and optimization, it was ensured that the probes had high specificity and coverage, suitable for the detection requirements of the liquid chip.

[0058] 5. Site screening and optimization

[0059] Taking into account multiple indicators such as evolutionary function scores, eQTL information, and minor allele frequency (MAF), the initially screened SNP sites were optimized, and finally a set of sites for the sheep whole-genome 50K SNP liquid chip was generated. The probes designed for each site were sent to Shadow Gene Corporation for probe synthesis and liquid chip preparation to obtain the sheep whole-genome 50K SNP liquid chip. The position information of the 58,859 SNP sites of the present invention is shown in Table 2; the distribution density map of the SNP sites on each chromosome is asFigure 1 As shown, it can be seen that 58,859 core loci are evenly distributed on each chromosome; the statistics of the minor allele frequency (MAF) of SNP loci are as Figure 2 shown, and it can be seen that the MAF is greater than 0.1 for all.

[0060] Table 2 Location information of SNP loci

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Example 2: Sheep Whole Genome 50K SNP Liquid Phase Chip Test Evaluation

[0135] 1. Sheep Blood DNA Extraction

[0136] Blood samples were collected from the jugular vein of 114 sheep (84 East Frisians, 19 Small-Tailed Han sheep, and 11 F1 hybrids of East Frisians and Small-Tailed Han sheep). DNA was extracted using a genomic DNA extraction kit and stored at -20°C. DNA quality was verified using a microspectrophotometer and agarose gel electrophoresis. Verification criteria included: total DNA ≥ 1.0 μg, concentration ≥ 20 ng / μL, volume ≥ 50 μL; OD 260 / OD 280 The ratio is between 1.8 and 2.0, OD 260 / OD 230 ≥1.8; and the main band of the sample is clear, with no degradation or slight degradation.

[0137] 2. Liquid-phase gene chip test evaluation

[0138] (1) Comparison rate

[0139] The alignment rate reflects the similarity between the sample sequencing data and the reference genome, and is an overall indicator for measuring the overall sequencing accuracy and the presence of contaminating DNA. The higher the alignment rate, the better, indicating high data validity. Ten sheep blood DNA samples were randomly selected for the chip alignment rate test, and the results are as follows: Figure 3 As shown. Figure 3 The results show that in this evaluation, based on the DNBSEQ-T7 sequencing platform, the sheep whole genome 50K SNP liquid phase chip showed an excellent matching rate between sheep blood samples, and the matching rate of each library exceeded 99.7%.

[0140] (2) Target area capture efficiency

[0141] Capture efficiency refers to the ratio of the measured sequences aligned to the target region (valid sequences) to the total number of sequences aligned to the reference genome. The capture efficiency does not affect the data quality and is mainly used to evaluate the effectiveness of the probe design. The higher the capture efficiency, the smaller the amount of data required to cover the same depth, the lower the sequencing cost, and the more effective the probe design. Nine sheep blood DNA samples were randomly selected for the chip capture efficiency test. The results are as follows: Figure 4 As shown. Figure 4 The results show that in this evaluation, based on the DNBSEQ-T7 sequencing platform, the sheep whole genome 50K SNP liquid phase chip showed excellent capture efficiency, and the capture efficiency of each library was higher than 77%.

[0142] (3) Data redundancy rate

[0143] During the sample library construction process, PCR amplification can lead to inconsistent amplification degrees of different regions of the target sequence, thereby introducing artificial biases and generating some duplicate data, which is data redundancy. Duplicate data not only causes data waste but also introduces sequencing errors. Therefore, the lower the data redundancy rate, the better. Nine sheep blood DNA samples were randomly selected for the data redundancy rate test of the chip, and the results are as follows Figure 5 shown. From Figure 5 the results, it can be seen that in this evaluation, on the DNBSEQ-T7 sequencing platform, the sheep whole-genome 50K SNP liquid-phase chip showed a very low redundancy rate, lower than 20%.

[0144] (4)SNP detection rate

[0145] The SNP detection rate is an important indicator to measure the quality of the chip, representing the proportion of core SNPs with high-quality genotyping among all core SNPs. Nine sheep blood DNA samples were randomly selected for the SNP detection rate test, and the results are as follows Figure 6 shown. From Figure 6 the results, it can be seen that in this evaluation, the SNP detection rate of each sample (genotyping accuracy not less than 99%) exceeded 95.98%, indicating that the sheep whole-genome 50K SNP liquid-phase chip of the present invention can obtain more effective and complete variant genotyping results.

[0146] Example 3 Application of the sheep whole-genome 50K SNP liquid-phase chip

[0147] 1. Application in genotyping

[0148] The 114 sheep blood DNA samples in Example 2 were processed through complete processes such as library construction, target region capture, Illumina sequencing, and bioinformatics analysis to complete the analysis of the variant information in the target interval of the test samples and perform genotyping. Quality control was performed on the obtained genotyping results, and only the SNP sites on chromosomes numbered 1 to 26 and the X chromosome were retained, and the SNP sites with a genotype missing rate greater than 1% and a minor allele frequency less than 1% were removed.

[0149] 2. Application in population classification

[0150] Cluster analysis is a method for classifying populations according to the degree of correlation or similarity between different individuals. We used the neighbor-joining method (NJ) to construct a genetic clustering tree for the 114 sheep in Example 2 through MEGA software, aggregating individuals with close relationships into a small classification unit and individuals with relatively distant relationships into a larger classification unit until all samples were aggregated. The results are as follows Figure 7 shown. Figure 7The results clearly demonstrate the clustering effects of different breeds of sheep (East Friesian, Small Tail Han sheep, and their hybrid F1 generation). This study further verifies that the liquid-phase chip of the present invention can distinguish the genetic backgrounds of different breeds of sheep, providing strong support for population classification and genetic research.

[0151] 3. Application in Genome-Wide Association Study of Sheep

[0152] Perform a genome-wide association study using the SNP loci obtained by screening and the growth trait data of the 114 sheep in Example 2 measured. The FarmCPU model is used for analysis, and the results are as Figure 8 shown. From Figure 8 the results, it can be seen that the SNP locus most significantly correlated with body weight is 1_52649252 ( Figure 8 A in Figure 8 ), the SNP locus most significantly correlated with body length is 13_64811000 ( Figure 8 H in Figure 8 ), the SNP locus most significantly correlated with abdominal circumference is 2_21355515 ( Figure 8 B in Figure 8 ), the SNP locus most significantly correlated with chest circumference is 18_20856334 ( Figure 8 K in Figure 8 ), the SNP locus most significantly correlated with chest depth is 8_19584881 ( Figure 8 J in Figure 8 ), the SNP locus most significantly correlated with rump height is 4_49499000 ( Figure 8 F in

[0153] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a probe combination, kit or liquid-phase chip for detecting a SNP locus combination for sheep genotyping in sheep breeding, characterized in that, Each SNP locus in the SNP locus combination is correspondingly provided with a set of probes for detecting the corresponding SNP locus, constituting a probe combination; the SNP locus combination contains 58,859 SNP loci, and the position information of the SNP loci is shown in Table 2 of the specification; the SNP locus combination contains SNP loci located on the sheep reference genome Oar v4.0; The kit includes the probe combination; the liquid chip includes the probe combination; The sheep are East Friesian, Small Tail Han sheep or the F1 hybrid of East Friesian and Small Tail Han sheep; The design parameters of the probe are: the probe length is 120 nt; the GC content is 30% - 70%; the copy number of the whole probe in the genome is 1; the copy number of any 25 bp kmer sequence in the genome does not exceed 5; The sheep breeding includes one or more of sheep genomic selection, localization of sheep trait-related genes, analysis of sheep genetic diversity, genome-wide association analysis of sheep, sheep breed identification, sheep kinship identification, sheep population classification, sheep phenotype regulation, improvement of sheep germplasm resources, and protection of sheep germplasm resources; The traits in the localization of the sheep trait-related genes include one or more of body weight, body slant length, abdominal circumference, chest circumference, chest depth, withers height, withers width, hip width, hip height, shoulder width, and body height.

Images