CN117025786B - A 50K SNP liquid phase chip for fine wool sheep based on targeted capture sequencing and its application - Google Patents

Abstract

The invention belongs to the field of biotechnology, and particularly relates to a nap 50K SNP liquid chip based on targeted capture sequencing and application thereof, wherein the invention firstly provides 45213 SNP locus combinations which are obviously associated with important characteristics of the nap, and is suitable for the application of resource evaluation, genetic identification and genome selection of Chinese nap varieties; the invention further provides the nap 50K SNP liquid-phase chip, the nap 50K SNP liquid-phase chip can break through the key technical bottleneck restricting the genome selection breeding of the nap, promote the genetic improvement process of the nap, accelerate the high-quality development of the nap breeding industry and industry, lay a foundation for establishing a nap genome selection technical system and establishing a nap genome selection sharing platform, and accelerate the crossing development of the nap breeding from 'phenotype selection' to 'genome selection'.

Description

A 50K SNP liquid phase chip for fine wool sheep based on targeted capture sequencing and its application

Technical Field

The present technology belongs to the field of gene chip technology, and specifically relates to a fine-wool sheep 50KSNP liquid phase chip based on targeted capture sequencing and its application.

Background Art

With the rapid development of sequencing technology and the reduction of costs, more livestock and poultry samples are sequenced to generate massive sequencing data, which provides a data source for mining SNP sites related to important traits and brings unprecedented convenience to the development of SNP chips. As a new generation of molecular genetic variation detection tools, SNP chip technology has been widely used in livestock and poultry research and has generated a large amount of livestock and poultry genetic variation data. It is of great significance for the accurate inference of livestock and poultry evolutionary history, as well as the genetic evaluation of important traits and selection of breeds. This technology can not only be used for comprehensive genome research on breeds or differentiated populations with similar genetic relationships as research objects, but also can simultaneously analyze tens of thousands of genetic markers to accurately determine the genetic variation of different species, breeds and populations. Therefore, SNP chips are used for high-throughput SNP detection. Compared with sequencing technology, they have the advantages of efficient and accurate SNP site typing, less redundant data and convenient subsequent analysis.

SNP chips can be divided into solid-phase chips and liquid-phase chips according to the different SNP loci that rely on carriers. Solid-phase chips are based on complementary hybridization between probes and DNA sequences, and typing is performed through the fluorescent signal of the marker. They have the advantages of high typing accuracy and short cycle, but they also have disadvantages such as high cost of typing per unit point and difficulty in customization. Liquid-phase chips are a new type of molecular detection technology after gel electrophoresis, fluorescence detection, and solid-phase chips. This technology is based on targeted sequencing genotyping (Genotyping By Target Sequencing, GBTS), that is, after selecting specific targets from the genome, the target probes are complementary to the target sequences for fixed-point capture, thereby obtaining markers with relatively fixed physical positions. Because it can quickly complete thousands of probe hybridization reactions in the liquid phase at the same time, the kit is figuratively called a "liquid-phase chip". Compared with solid-phase SNP chip technology, liquid-phase SNP chip technology has platform adaptability, labeling flexibility, detection efficiency, information additivity, support convenience, and broad spectrum of application. The same SNP chip locus set can obtain different marker forms (mSNP, SNP and haplotype) and obtain a variety of different marker densities by controlling the sequencing depth according to the needs of the application scenario. Liquid phase chip technology can be widely used in biological evolution, genetic map construction, gene positioning cloning, marker trait association detection (whole genome association analysis-GWAS and mixed sample analysis-BSA), progeny identification, gene introgression, gene accumulation, variety right protection, variety quality monitoring, transgenic components/gene editing/companion organism detection and other fields. The existing commercial sheep chip design data comes from different breeds in the world. The number of each breed involved is small, and the application direction and purpose are very different. In addition, there are disadvantages such as low utilization rate and high cost for genotyping of fine wool sheep populations in my country. High-density chips are suitable for scientific research and the research direction tends to be species evolution, whole genome association analysis, etc. When used in genomic selection, the breeding cost is too high, which makes it difficult to apply and promote. Due to the small number of loci, the effective genotyping loci obtained by medium and low density chips depend largely on the coverage of the variety source for designing this chip.

In order to overcome the above technical problems, the present invention provides a fine-wool sheep 50K SNP liquid phase chip based on targeted capture sequencing, including 45213 SNP sites. The fine-wool sheep 50K SNP liquid phase chip of the present invention can be used for genome selection breeding, genetic diversity analysis, variety identification, kinship identification and whole genome association analysis of important traits of fine-wool sheep in China.

Summary of the invention

In order to overcome the above technical problems, the present invention provides a 50K SNP liquid phase chip for fine wool sheep based on targeted capture sequencing and its application in genome selection breeding of important traits of fine wool sheep in my country, genetic diversity analysis, variety identification, kinship identification and whole genome association analysis, which specifically includes the following contents:

In the first aspect, the present invention provides a SNP site combination associated with the economic traits of fine-wool sheep, wherein the SNP site combination associated with the economic traits of fine-wool sheep consists of 45213 SNP sites, and the positions of the 45213 SNP site combinations on the sheep Oar_v4.0 reference genome are shown in NO.1 to NO.45213 in Table 2 of the specification; the economic traits include birth weight, weaning weight, weaning hair length, weight at one year old, hair length at one year old, hair fiber diameter at one year old, wool clipping amount at one year old, net wool rate at one year old, net wool weight at one year old, adult weight, adult hair length, adult wool clipping amount, adult net wool rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter, and wool fiber diameter variation coefficient.

In a second aspect, the present invention provides a reagent for detecting the SNP site combination related to the economic traits of fine wool sheep described in the first aspect, which has any of the following uses:

(1) Application in the analysis of economic traits of fine wool sheep;

(2) Application in identification of fine wool sheep breeds;

(3) Application in the identification of fine wool sheep kinship;

(4) Application in genomic selection breeding of fine wool sheep;

(5) Application in genetic diversity analysis of fine wool sheep;

(6) Application in genome-wide association analysis of fine-wool sheep;

The economic traits include birth weight, weaning weight, weaning hair length, one-year-old body weight, one-year-old hair length, one-year-old hair fiber diameter, one-year-old hair clipping amount, one-year-old net hair rate, one-year-old net hair weight, adult body weight, adult hair length, adult hair clipping amount, adult net hair rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter, and wool fiber diameter variation coefficient.

In a third aspect, the present invention provides a use of a molecular probe for detecting a combination of SNP sites associated with the economic traits of fine-wool sheep as described in the first aspect above in the preparation of a liquid phase chip/reagent/kit for analyzing the genome of fine-wool sheep.

In a fourth aspect, the present invention provides a molecular probe combination for analyzing the genome of fine-wool sheep, wherein the molecular probe combination detects the SNP site combination as described in the first aspect in the sample to be tested.

In a fifth aspect, the present invention provides a liquid phase chip for analyzing the genome of fine-wool sheep, wherein the liquid phase chip is loaded with molecular probes for detecting the SNP site combination described in the first aspect.

In a sixth aspect, the present invention provides a kit for analyzing the genome of fine-wool sheep, the kit comprising reagents for detecting the SNP site combination described in the first aspect, or the molecular probe combination described in the fourth aspect, or the liquid phase chip described in the fifth aspect.

In a seventh aspect, the present invention provides the molecular probe combination described in the fourth aspect, or the liquid phase chip described in the fifth aspect, or the kit described in the sixth aspect, having any of the following uses:

(1) Application in the analysis of economic traits of fine wool sheep;

(2) Application in identification of fine wool sheep breeds;

(3) Application in the identification of fine wool sheep kinship;

(4) Application in genomic selection breeding of fine wool sheep;

(5) Application in genetic diversity analysis of fine wool sheep;

(6) Application in genome-wide association analysis of fine-wool sheep;

The economic traits include birth weight, weaning weight, weaning hair length, one-year-old body weight, one-year-old hair length, one-year-old hair fiber diameter, one-year-old hair clipping amount, one-year-old net hair rate, one-year-old net hair weight, adult body weight, adult hair length, adult hair clipping amount, adult net hair rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter, and wool fiber diameter variation coefficient.

The beneficial effects of the present invention are as follows: compared with the prior art, the present invention provides a 50K SNP liquid phase chip for fine-wool sheep based on targeted capture sequencing, which has the following advantages: first, the SNP sites contained in the liquid phase chip are derived from the whole genome resequencing of 4 representative Chinese fine-wool sheep breeds, so it has richer polymorphism in the fine-wool sheep population, and is therefore more suitable for genetic improvement and resource evaluation of fine-wool sheep breeds; second, the SNP sites contained in the chip are mined for important traits of fine-wool sheep, and are therefore suitable for breeding and improvement of important traits of fine-wool sheep; third, the liquid phase chip is based on targeted capture sequencing technology, which can not only type the target site, but also type the SNPs within a certain range near the target site, so more typing information can be obtained; fourth, the liquid phase chip can adjust the target SNP site by directly adding or subtracting probes, and has better flexibility compared with the solid phase chip; fifth, the liquid phase chip relies on the second-generation sequencing platform, has universality, lower typing cost, and has a higher cost performance.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Distribution of SNP sites on chromosomes;

Figure 2 Results of genome-wide association analysis of resequencing sites and 50K chip sites for the net gross rate trait;

Fig. 3 Phylogenetic tree.

DETAILED DESCRIPTION

The present invention is further explained with reference to the detailed description of the specific embodiments below, but the following embodiments are merely illustrative of the technical solutions of the present invention and do not limit the technical solutions of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, the bioinformatics software and products used are also commercially available, and the experimental processes and methods are also conventional methods known in the art. The sources of the materials used, the trade names, and the components thereof that need to be listed are all indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.

In addition, it should be noted that the site combination and application provided by the present invention are completed by the inventors of this application through arduous creative labor and optimization work.

It should be noted that the economic traits of fine wool sheep include birth weight, weaning weight, weaning wool length, one-year-old body weight, one-year-old wool length, one-year-old wool fiber diameter, one-year-old shearing amount, one-year-old net wool rate, one-year-old net wool weight, adult body weight, adult wool length, adult shearing amount, adult net wool rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter, wool fiber diameter variation coefficient, a total of 17 traits; among which wool traits include fiber diameter (Fiber Diameter, FD) (GB/T 10685-2007), fiber diameter variation coefficient (Coefficient of Variation of FiberDiameter, FDCV) (GB/T 10685-2007), staple length (Staple Length, SL) (GB/T 6976-2007), clean wool rate (Clean Fleece Weight Rate, CFWR) (GB/T 6978-2007), bundle fiber breaking strength (StapleStrength, SS) (GB/T 13835.5-2009) and Fleece Extension Rate (FER) (GB/T 13835.5-2009); the above wool traits were objectively tested in accordance with the relevant national standards at the Animal Fur and Products Quality Supervision and Inspection Center of the Ministry of Agriculture and Rural Affairs (Lanzhou, China). Phenotypic data such as weight and shearing amount were collected from the fine wool sheep production performance measurement site and shearing site using well-known methods.

The SNP referred to in this application refers to single nucleotide polymorphism (SNP), which mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. The variation of the single nucleotide includes variation caused by conversion, transversion, insertion or deletion of a single base.

It should be noted that the molecular markers referred to in the present invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, molecular markers based on PCR technology, DNA markers based on restriction enzyme digestion and PCR technology, molecular markers based on DNA chip technology, analytical marker technology based on EST database development, etc. The molecular markers provided by the present invention can be used for genome mapping and gene location research, map-based gene cloning, species relationship and systematic classification, etc.

It should be noted that the probe referred to in the present invention is a nucleic acid sequence (DNA or RNA) with a detection label, a known sequence, and complementary to the target gene.

It should be noted that the kit referred to in the present application is any box that contains reagents used for detection or experiment and is commonly used in the art, which is convenient for operators to get rid of the cumbersome reagent preparation and optimization process. In one embodiment of the present invention, it contains probes for amplifying the site information provided by the present invention, molecular markers or probes or gene chips for detecting the site information provided by the present invention, enzymes and buffers used for amplification, or fluorescent markers for detection.

Example 1 Design and preparation of 50K SNP liquid phase chip for fine wool sheep

1. Screening of functional loci related to important traits of fine-wool sheep

Step 1: Use the whole genome association analysis strategy to screen and obtain SNP loci associated with important traits of fine-wool sheep:

The experimental population was derived from 460 individuals of four typical Chinese fine-wool sheep breeds, namely, Alpine Merino, Chinese Merino, Aohan fine-wool sheep and Qinghai fine-wool sheep. A total of 17 traits were selected for phenotypic measurement, including birth weight, weaning weight, weaning hair length, weight at one year old, hair length at one year old, hair fiber diameter at one year old, hair clipping amount at one year old, net hair rate at one year old, net hair weight at one year old, adult weight, adult hair length, adult hair clipping amount, adult net hair rate, breaking strength of wool bundle fiber, breaking elongation of wool bundle fiber, wool fiber diameter, and coefficient of variation of wool fiber diameter.

The experimental sheep were venously blooded for genomic DNA extraction, and the whole genome was resequenced using the HiSeq X-ten sequencer (Illumina). The average genome coverage of each sample was above 5×. After quality control, the GLM model of PLINK software was used for whole genome association analysis, and a total of 516 SNPs significantly associated with phenotypic indicators were obtained.

Step 2: Use the selection signal analysis method to screen and obtain SNP sites related to important traits of fine-wool sheep:

Venous blood was collected from 32 individuals of three fine-wool sheep breeds, namely, Alpine Merino, Chinese Merino and Aohan fine-wool sheep, for genomic DNA extraction. Whole genome resequencing was performed using the Hiseq X-ten sequencer (Illumina), and the average genome coverage of each sample was above 30×. Four selection signal methods were used, namely, Tajima’D test based on site frequency spectrum, joint analysis of selection signals of genomic heterozygosity, population differentiation selection index (Fst) and genomic heterozygosity (θπ), and joint analysis of selection signals of population differentiation selection index (Fst) and heterozygosity rate (Hp). The sites obtained from each selected region were then linked using Haploview software. A SNP located in the longest haplotype and in the gene region was selected in each region as a candidate site for the region. Finally, a total of 523 SNPs related to important traits of fine-wool sheep were obtained.

Step 3: Based on the reported OMIA (https://www.omia.org/home/), GWAS and selection signal studies, 139 SNPs related to important traits such as disease resistance, reproduction and growth and development of fine-wool sheep were obtained.

The above three steps obtained a total of 1178 SNPs as functional sites and added them into the chip design.

2. Screening of 50K SNP loci in fine-wool sheep

The present invention uses a Hiseq X-ten sequencer (Illumina) to perform whole genome resequencing on 620 individuals of four representative Chinese fine-wool sheep breeds, namely, alpine merino, Chinese merino, Aohan fine-wool sheep and Qinghai fine-wool sheep, and the average genome coverage of each sample is above 5×. After preliminary quality control of the data off the machine, the sequencing data is aligned to the fine-wool sheep reference genome Oar_4.0 by BWA software, and all SNPs obtained by resequencing are filtered using PLINK software to obtain high-quality SNPs for chip design, and haplotype analysis is performed using Haploview software (version 4.2), and the sliding window size is set to 50,000 bp, and the haplotype groups in the window are respectively counted, and the threshold is set to R ² ≥0.8. The importance of genomic genetic variation is scored according to the scores in Table 1, the total score of all haplotype groups in the whole genome is calculated, and the variation with the highest score is marked as tagSNPs of the corresponding haplotype. Based on the probe density, a certain number of tagSNPs were selected in each window and known functional sites were added, and finally 46,178 SNPs were obtained.

Table 1. Genome-wide genetic variation importance score table

Through the above steps, a total of 46,178 functional sites and background sites were selected and submitted to Shijiazhuang Boridi Biotechnology Co., Ltd. for evaluation, and sites that could not be uniquely aligned on the genome, sites containing repeated sequences in the flanking sequences, and sites with a deletion rate of more than 80% were removed. Finally, a total of 45,213 SNP sites were obtained that were evenly distributed in the fine-wool sheep genome, and their specific physical locations on the sheep Oar_v4.0 reference genome are shown in Table 2. The distribution of all SNP sites on each chromosome of the genome is shown in Figure 1.

Table 2 Locations of 45213 SNP molecular markers

3. Design of 50K SNP HPLC Array

The chip designed by the present invention adopts the GenoBaits technology system; the working principle is that the probe designed based on the target SNP marker of fine-wool sheep is complementary to the DNA of the test sample for targeted sequence binding and sequencing, so as to achieve the purpose of detecting the genotype of the target SNP marker of the test sample. First, the sample to be tested is constructed with a library, and at the same time, according to the principle of DNA complementarity, each SNP marker to be tested covers the probe of the target SNP marker, and the target probe is labeled with biotin. Then, the target probe labeled with biotin is hybridized with the target region of the genome in a liquid reaction system to form a double chain. Subsequently, the target probe carrying the biotin marker is molecularly adsorbed by streptavidin-coated magnetic beads, so as to capture the sequence containing the target targeted SNP marker hybridized with the probe. Finally, the captured target sequence is eluted, amplified, library built and sequenced, and the genotype result of the target SNP is finally obtained. According to the position of 45213 SNP sites and the sequence information on both sides, primers are designed and probes are synthesized by Shijiazhuang Boridi Biotechnology Co., Ltd. using targeted capture sequencing technology, so as to obtain a fine-wool sheep 50KSNP liquid phase chip.

Example 2 Application of fine-wool sheep 50K SNP liquid phase chip in fine-wool sheep DNA sample detection

1. Extraction of fine wool sheep genomic DNA samples:

Blood was collected from the jugular vein of fine-wool sheep, and DNA was extracted from the samples using the CTAB method.

2. DNA sample quality testing:

The DNA concentration of the test sample DNA was measured by Qubit Fluorometric Quantitation (Thermo Fisher), and the integrity of the DNA was detected by 1% agarose gel electrophoresis. The qualified samples were placed in a 4°C refrigerator for storage and standby use.

3. Sample DNA fragmentation:

Take 12 μL of DNA that has passed the quality inspection and place it in a 0.2 μL PCR tube. Place the tube in an ultrasonic crusher to randomly physically crush the DNA into fragments of 200 to 400 bp.

4. Sample end repair:

Add 4 μL GenoBaits End Repair Buffer (Shijiazhuang Boridi Biotechnology Co., Ltd.) and 2.7 μL GenoBaits End Repair Enzyme to the tube, add water to 20 μL, and incubate at 37°C for 20 minutes in an ABI 9700 PCR instrument to complete the end repair and A addition process of the broken fragments.

5. Sample sequencing adapter connection:

Remove the small tube from the PCR instrument, add 2μL GenoBaits Ultra DNA Ligase, 8μL GenoBaitsUltra DNA Ligase Buffer and 2μL GenoBaits Adapter, add water to 40μL, and then place it on the ABI9700 PCR instrument at 22℃ for 30 minutes to complete the connection of the sequencing adapter.

6. Sample DNA purification:

48 μL of Beackman AMPure XP Beads (Beackman) were added to the ligation product to purify it. After purification, magnetic beads were used to screen the fragments, and the ligation product with an insert fragment of 200 to 300 bp was retained.

7. Sample library amplification:

Add 5 μL of sequencing adapter with barcode sequence, 1 μL of P5 adapter, 10 μL of GenoBaits PCR Master Mix to the PCR tube in the previous step, and fill to 20 μL with pure water; amplify using ABI 9700 PCR instrument, the amplification program is: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s; repeat steps 2 to 4 for a total of 8 cycles; 72℃ extension for 5 min. Different barcodes are used to distinguish different samples.

8. Sample library purification:

Add 24 μL Beckmen AMPure XP Beads (Beackman) to the second-round PCR product, pipette up and down to mix evenly, place 0.2 μL of the PCR tube on a magnetic rack until the solution is clear, discard the supernatant and wash the magnetic beads once with 75% ethanol, and elute the library DNA with Tris-HCl at pH 8.0.

9. Determine the genotype of the target individual locus using the fine-wool sheep 50K SNP liquid phase chip:

(1) DNA hybridization

Take 500ng of the constructed genomic DNA sequencing library, add 5μL GenoBaits Block I and 2μL GenoBaits Block II, place it on an Eppendorf Concentrator Plus (Eppendorf) vacuum concentrator, and evaporate it to dry powder at a temperature of ≤70°C. Add 8.5μL GenoBaits 2×Hyb Buffer, 2.7μL GenoBaits Hyb Buffer Enhancer, and 2.8μL Nuclease-Free Water to the dry powder tube, mix it with a pipette, and place it on an ABI 9700PCR instrument at 95°C for incubation for 10 minutes, then take out the PCR tube and add 3μL of the synthesized probe (the concentration of the probe is 60ng/μL), vortex and shake to mix it, and place it on an ABI 9700PCR instrument at 65°C for incubation for 2 hours to complete the probe hybridization reaction.

(2) DNA capture

Add 100μL GenoBaits DNA Probe Beads to the reaction system after hybridization in the previous step, then pipette up and down 10 times, and incubate at 65℃ for 45 minutes in the ABI 9700PCR instrument to allow the magnetic beads to bind to the probe. Use 100μL GenoBaits Wash Buffer I and 150μL GenoBaits Wash Buffer II to heat wash the magnetic beads after binding to the probe at 65℃, and then use 100μL GenoBaits Wash Buffer I, 150μL GenoBaits Wash Buffer II (and 150μL GenoBaits Wash Buffer III to wash the magnetic beads at room temperature. Resuspend the washed magnetic beads with 20μL Nuclease-Free Water.

Take 13 μL of resuspended DNA (with magnetic beads) and add it to a new 0.2 mL PCR tube, then add 15 μL GenoBaits PCR Master Mix and 2 μL GenoBaits Primer Mix to configure the post-PCR system, and use ABI9700 PCR instrument to amplify the library. The amplification program is: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; repeat steps 2-4 for a total of 15 cycles; extend at 72°C for 5 min.

Add 45 μL Beckmen AMPure XP Beads (Beackman) to the post-PCR product and pipette up and down to mix evenly. Then place the 0.2 mL PCR tube on a magnetic stand until the solution is clear, discard the supernatant and wash the magnetic beads twice with 75% ethanol, and elute the library DNA with Tris-HCl at pH 8.0 to complete the hybridization capture of the probe.

(3) DNA hybridization capture library quality inspection

The DNA concentration of the library was measured by Qubit Fluorometric Quantitation (Thermo Fisher), and then agarose gel electrophoresis was used to detect whether the fragment size of the library DNA was between 300 and 400 bp.

(4) DNA hybridization capture library sequencing

The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

(5) Genotype data analysis

After FastQC ( www.bioinformatics.babraham.ac.uk/project ) quality control, the sequencing data were aligned to the reference genome using the default parameters of BWA ( bio-bwa.sourceforge.net ), SNPs were identified using GATK ( software.broadinstitute.org/gatk ) software, and the genotyping information of probe capture sequencing was extracted using a self-written Perl script to form the final genotyping file.

The above steps can successfully achieve the use of fine-wool sheep 50K SNP liquid phase chip to obtain the genotype data of the fine-wool sheep DNA sample to be tested.

Example 3 Genome-wide association analysis of wool traits using 50K SNP liquid phase microarray in fine wool sheep

The present invention uses the fine wool sheep 50K SNP liquid phase chip obtained in Example 1 to perform genotyping on 620 individual fine wool sheep samples (see Example 1 for the specific operation method). The genotyping data and resequencing data are used to simultaneously perform a genome-wide association analysis on the clean wool rate (CFWR), one of the important wool traits of fine wool sheep, indicating that the results of the genome-wide association analysis of wool traits using the fine wool sheep 50K SNP liquid phase chip and the resequencing data using the mixed linear model of rMVP are comparable (the results are shown in Figure 2). The above results show that even without genotype filling, the use of the fine wool sheep 50K SNP liquid phase chip can obtain the same genome association analysis results as resequencing, so fine wool sheep breeding and research work can be carried out at a lower cost.

Example 4 Application of 50K SNP liquid phase chip in fine wool sheep kinship identification

The accuracy of livestock and poultry pedigree information is of great significance to breeding progress or seed conservation. However, due to the introduction of individuals with unknown blood, human recording errors, multiple matings, mixed insemination or embryo transplantation, pedigree recording errors are common in the production, breeding and seed conservation process. In livestock and poultry breeding, incorrect pedigrees will not only affect the determination of the parent-offspring relationship of breeding animals, but also slow down the genetic progress of the group, thereby causing significant losses to the economic benefits of animal breeding. At the same time, incorrect pedigrees will also have a negative impact on livestock and poultry population genetics and breeding research, so personal identification is of great value and significance for animal genetic breeding production and research. Therefore, kinship identification of fine-wool sheep groups is also one of the important links in breeding work.

This embodiment targets 152 fine-wool sheep ram individuals from a certain group of alpine Merino sheep, and uses the fine-wool sheep 50K SNP liquid phase chip prepared in Example 1 to perform kinship analysis and construct a molecular pedigree. Through the genomic kinship analysis and cluster analysis based on the G matrix, it is found that the existing ram samples can be divided into 7 families (as shown in Figure 3), and the rams are divided into different families according to the kinship of their father and mother. Based on the Mendel's genetic law, compared with the original pedigree, it is found that the Mendel error rate of the original pedigree of 5 samples is greater than the threshold, and samples with a Mendel error rate less than the threshold and the smallest Mendel error are found in other samples; the original pedigrees of 20 samples have no parent-child relationship, and samples with a Mendel error rate less than the threshold and the smallest Mendel error are found in the samples.

The above results suggest that the use of fine-wool sheep 50K SNP liquid phase chip can obtain more detailed population relationships at a lower cost, and at the same time can correct pedigree errors and provide support for improving genetic progress.

Example 5 Application of fine-wool sheep 50K SNP liquid phase chip in fine-wool sheep genome selection

Genomic selection is a method of predicting the genetic value of animals based on the genotyping data of genetic markers distributed on the genome, thereby selecting ideal individuals. Breeders can increase the speed of genetic improvement through early selection. With the continuous improvement of reference genomes, the cost reduction of sequencing technology and the continuous upgrading of commercial SNP chips, genomic selection is the mainstream method of animal breeding at present and has been widely used in livestock and poultry. Compared with conventional methods, genomic selection adds genomic data on the basis of using pedigree and phenotypic data, greatly increasing the accuracy of seed selection. In this embodiment, 2178 adult ewes of alpine Merino sheep were used as the reference group. All of them were genotyped and wool fiber diameter was measured by the fine wool sheep 50K SNP liquid phase chip in Example 1. The genomic breeding value (GEBV) was estimated by the SSGBLUP method. The results showed that the GEBV prediction accuracy of wool fiber diameter was 0.5806, which was significantly improved compared with the accuracy of 0.4983 estimated by the conventional breeding value of BLUP before.

Claims (9)

1. Use of a reagent for detecting a combination of SNP loci associated with economic traits in a fine wool sheep, the use being as described in any one of the following:

(1) The application in the economic character analysis of the fine wool sheep;

(2) Application in the identification of fine wool sheep variety;

(3) Application in the paternity test of fine wool sheep;

(4) Application in selective breeding of fine wool sheep genome;

(5) The application in the analysis of the genetic diversity of the fine wool sheep;

(6) Application in the whole genome association analysis of the fine wool sheep;

the SNP locus combination related to the economic characters of the fine wool sheep consists of 45213 SNP loci, and the positions of the 45213 SNP locus combinations on the sheep oar_v4.0 reference genome are shown as NO.1 to NO.45213 in the specification table 2;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

2. Application of molecular probes for detecting SNP locus combinations related to economic traits of fine wool sheep in preparation of liquid phase chips for analyzing genome of fine wool sheep;

the SNP locus combination related to the economic characters of the fine wool sheep consists of 45213 SNP loci, and the positions of the 45213 SNP locus combinations on the sheep oar_v4.0 reference genome are shown as NO.1 to NO.45213 in the specification table 2;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

3. Application of molecular probes for detecting SNP locus combinations related to economic traits of fine wool sheep in preparation of kits for analyzing genome of fine wool sheep;

the SNP locus combination related to the economic characters of the fine wool sheep consists of 45213 SNP loci, and the positions of the 45213 SNP locus combinations on the sheep oar_v4.0 reference genome are shown as NO.1 to NO.45213 in the specification table 2;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

4. A molecular probe combination for analyzing the genome of a fine wool sheep, which is characterized in that the molecular probe combination detects SNP locus combination related to economic characters of the fine wool sheep in a sample to be detected;

the SNP locus combination related to the economic characters of the fine wool sheep consists of 45213 SNP loci, and the positions of the 45213 SNP locus combinations on the sheep oar_v4.0 reference genome are shown as NO.1 to NO.45213 in the specification table 2;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

5. The use of the molecular probe combination according to claim 4, wherein the use is any one of the following:

(1) The application in the economic character analysis of the fine wool sheep;

(2) Application in the identification of fine wool sheep variety;

(3) Application in the paternity test of fine wool sheep;

(4) Application in selective breeding of fine wool sheep genome;

(5) The application in the analysis of the genetic diversity of the fine wool sheep;

(6) Application in the whole genome association analysis of the fine wool sheep;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

6. A liquid phase chip for analyzing the genome of a fine wool sheep, characterized in that the liquid phase chip is loaded with a molecular probe for detecting SNP locus combinations related to economic traits of the fine wool sheep;

the SNP locus combination related to the economic characters of the fine wool sheep consists of 45213 SNP loci, and the positions of the 45213 SNP locus combinations on the sheep oar_v4.0 reference genome are shown as NO.1 to NO.45213 in the specification table 2;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

7. Use of the liquid phase chip of claim 6, said use being any one of the following:

(1) The application in the economic character analysis of the fine wool sheep;

(2) Application in the identification of fine wool sheep variety;

(3) Application in the paternity test of fine wool sheep;

(4) Application in selective breeding of fine wool sheep genome;

(5) The application in the analysis of the genetic diversity of the fine wool sheep;

(6) Application in the whole genome association analysis of the fine wool sheep;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.

8. A kit for analyzing the genome of a fine wool sheep, comprising a reagent for detecting a SNP site combination associated with economic traits of a fine wool sheep, or a molecular probe combination according to claim 4, or a liquid phase chip according to claim 6.

9. Use of the kit of claim 8, for any of the following:

(1) The application in the economic character analysis of the fine wool sheep;

(2) Application in the identification of fine wool sheep variety;

(3) Application in the paternity test of fine wool sheep;

(4) Application in selective breeding of fine wool sheep genome;

(5) The application in the analysis of the genetic diversity of the fine wool sheep;

(6) Application in the whole genome association analysis of the fine wool sheep;

The economic characters comprise birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair shearing amount, age hair cleaning rate, age hair cleaning weight, adult hair length, adult hair shearing amount, adult hair cleaning rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter and wool fiber diameter variation coefficient.