CN117025786A

CN117025786A - Fine wool sheep 50K SNP liquid phase chip based on targeted capturing sequencing and application thereof

Info

Publication number: CN117025786A
Application number: CN202311043177.5A
Authority: CN
Inventors: 郭婷婷; 杨博辉; 袁超; 岳耀敬; 刘建斌; 卢曾奎; 牛春娥; 刘继刚; 王喜军; 朱韶华; 赵洪昌; 乔国艳
Original assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Current assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Priority date: 2023-08-18
Filing date: 2023-08-18
Publication date: 2023-11-10

Abstract

The application belongs to the field of biotechnology, and particularly relates to a nap 50K SNP liquid phase chip based on targeted capture sequencing and application thereof, wherein the application firstly provides 45213 SNP locus combinations which are obviously associated with important characteristics of the nap, and the application is suitable for application of resource evaluation, genetic identification and genome selection of Chinese nap varieties; the application 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

Fine wool sheep 50K SNP liquid phase chip based on targeted capturing sequencing and application thereof

Technical Field

The technology belongs to the technical field of gene chips, and particularly relates to a nap sheep 50K SNP liquid-phase chip based on targeted capturing sequencing and application thereof.

Background

Along with the rapid development of the sequencing technology and the reduction of the cost, more livestock and poultry samples are sequenced to generate massive sequencing data, so that a data source is provided for mining SNP loci related to important traits, and unprecedented convenience is brought for the development of SNP chips. SNP chip technology is used as a new generation molecular genetic variation detection tool, has been widely applied in livestock and poultry research and generates a large amount of livestock and poultry genetic variation data, and has very important significance for accurate inference of livestock and poultry evolution history, important character genetic evaluation and seed selection and matching. The technology can be used for comprehensively researching genome by taking varieties or differentiated populations with similar genetic relations as research objects, and can analyze tens of thousands of genetic markers simultaneously to accurately determine genetic variation of different species, varieties and populations. Therefore, compared with a sequencing technology, the SNP chip has the advantages of high efficiency and accuracy of SNP locus typing, less redundant data quantity, convenience in subsequent analysis and the like.

SNP chips are classified into solid-phase chips and liquid-phase chips according to the difference of SNP locus dependent carriers. The solid phase chip performs typing through fluorescent signals of the markers based on complementary hybridization of the probes and the DNA sequences, has the advantages of high typing accuracy, short period and the like, but also has the defects of high single-site typing cost, difficult customization and the like. The liquid phase chip is a novel molecular detection technology following gel electrophoresis, fluorescence detection and solid phase chip, and the technology is based on targeted sequencing genotype detection (Genotyping By Target Sequencing, GBTS), namely, after a specific target point is selected from a genome, a target probe and a target sequence are complementarily combined to perform fixed-point capture, so that a marker with a relatively fixed physical position is obtained. The kit capable of simultaneously and rapidly completing hybridization reactions of thousands of probes in a liquid phase is visually called as a liquid phase chip. Compared with the solid-phase SNP chip technology, the liquid-phase SNP chip technology has the advantages of platform universality, marking flexibility, high detection efficiency, information additivity, supporting convenience and broad application spectrum. Different mark forms (mSNP, SNP and haplotype) can be obtained by the same SNP chip site set, and various mark densities can be obtained by controlling the sequencing depth according to the requirements of application scenes. The liquid chip technology can be widely applied to the fields of biological evolution, genetic map construction, gene location cloning, marker character association detection (whole genome association analysis-GWAS and mixed sample analysis-BSA), descendant identification, gene introgression, gene accumulation, variety weight protection, variety quality monitoring, transgenic component/gene editing/associated biological detection and the like. The existing commercial sheep chip design data are derived from different varieties in the world, the number of each variety is small, the application direction and purpose are greatly different, and the method has the defects of low utilization rate, high cost and the like when being used for the genotyping of the fine wool sheep flocks in China. The high-density chip is suitable for scientific research, the research direction tends to species evolution, whole genome association analysis and the like, and the application and popularization are difficult due to the excessively high breeding cost when the high-density chip is applied to genome selection. The number of sites in the medium-low density chip is small, and the obtained sites for effective genotyping are greatly dependent on the coverage of the variety sources for designing the chip.

In order to overcome the technical problems, the application provides a nap sheep 50K SNP liquid phase chip based on targeted capture sequencing, which comprises 45213 SNP loci. The nap sheep 50K SNP liquid phase chip can be used for genome selective breeding, genetic diversity analysis, variety identification, genetic relationship identification and whole genome association analysis of important characteristics of nap sheep in China.

Disclosure of Invention

In order to overcome the technical problems, the application provides a nap 50K SNP liquid phase chip based on targeted capture sequencing and application thereof in important character genome selective breeding, genetic diversity analysis, variety identification, genetic relationship identification and whole genome association analysis of nap sheep in China, and the chip specifically comprises the following contents:

in a first aspect, the present application provides a combination of SNP sites related to economic traits of a fine wool sheep, the combination of SNP sites related to economic traits of fine wool sheep consisting of 45213 SNP sites, the positions of the 45213 SNP site combinations on sheep oar_v4.0 reference genome being as shown in No.1 to No.45213 in table 2 of the specification; 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.

In a second aspect, the present application provides a reagent for detecting a combination of SNP loci related to economic traits of a fine wool sheep according to the first aspect, wherein the reagent has any of the following uses:

(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.

In a third aspect, the application provides an application of a molecular probe for detecting the SNP locus combination related to the economic traits of the fine wool sheep in preparation of a liquid-phase chip/reagent/kit for analyzing the genome of the fine wool sheep.

In a fourth aspect, the present application provides a molecular probe set for analysis of the genome of a fine wool sheep, the molecular probe set detecting the SNP site set as set forth in the first aspect above in a sample to be tested.

In a fifth aspect, the present application provides a liquid phase chip for analyzing the genome of a fine wool sheep, the liquid phase chip being loaded with a molecular probe for detecting the SNP site combination described in the first aspect.

In a sixth aspect, the present application provides a kit for analyzing the genome of a fine wool sheep, the kit comprising a reagent for detecting the SNP site combination according to the first aspect, or the molecular probe combination according to the fourth aspect, or the liquid phase chip according to the fifth aspect.

In a seventh aspect, the present application provides a molecular probe set according to the fourth aspect, or a liquid phase chip according to the fifth aspect, or a kit according to the sixth aspect, for use as follows:

(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;

The beneficial effects of the application are as follows: compared with the prior art, the application provides a nap sheep 50K SNP liquid-phase chip based on targeted capturing sequencing, which has the following advantages: firstly, SNP loci contained in the liquid phase chip are derived from the whole genome re-sequencing of 4 Chinese representative fine wool sheep varieties, so that the liquid phase chip has rich polymorphism in fine wool sheep populations, and is more suitable for genetic improvement and resource evaluation of fine wool sheep varieties; secondly, SNP loci contained in the chip are mined aiming at important characters of the fine wool sheep, so that the chip is suitable for the works such as breeding improvement of the important characters of the fine wool sheep; thirdly, the liquid phase chip is based on a targeted capture sequencing technology, so that not only can the target site be typed, but also SNP (single nucleotide polymorphism) close to a certain range of the target site can be typed, and more typing information can be obtained; fourthly, the liquid phase chip can adjust the target SNP locus in a mode of directly increasing or decreasing probes, and has better flexibility compared with a solid phase chip; and fifthly, the liquid phase chip is supported on a second generation sequencing platform, so that the liquid phase chip has universality, lower parting cost and higher cost performance.

Drawings

FIG. 1 distribution of SNP sites on chromosomes;

FIG. 2 full genome association analysis results of resequencing sites and 50K chip sites for the net hair rate trait;

FIG. 3 phylogenetic tree.

Detailed Description

The application will be further elucidated with reference to the following detailed description of embodiments, but the examples described hereinafter are merely illustrative of the technical solution of the application and are not limiting thereof. Unless specifically stated otherwise, all technical means used in the following examples are conventional means known to those skilled in the art, all bioinformatics software and products used are commercially available, experimental procedures and methods are conventional methods known in the art, all materials sources, trade names and components listed as necessary are indicated at the first occurrence, and all reagents used thereafter are the same as indicated at the first occurrence unless otherwise stated.

In addition, it should be noted that the combination and application of the sites provided by the present application are accomplished by the inventor of the present application through hard creative work and optimization work.

The economic traits of the fine sheep include 17 traits including birth weight, weaning hair length, weight of the whole year old, hair length of the whole year old, fiber diameter of the whole year old, hair shearing amount of the whole year old, net hair rate of the whole year old, net hair weight of the whole year old, body weight of the whole year old, adult hair length, adult hair shearing amount, net hair rate of the whole year old, breaking strength of wool bundle fibers, breaking elongation of wool bundle fibers, fiber diameter of the wool and variation coefficient of fiber diameter of the wool; wherein wool traits include Fiber Diameter (Fiber Diameter, FD) (GB/T10685-2007), fiber Diameter coefficient of variation (Coefficient of Variation of Fiber Diameter, FDCV) (GB/T10685-2007), tuft Length (SL) (GB/T6976-2007), net hair rate (Clean Fleece Weight Rate, CFWR) (GB/T6978-2007), strand Strength (SS) (GB/T13835.5-2009), and strand elongation at break (Fleece Extension Rate, FER) (GB/T13835.5-2009); the wool characters are objectively tested in the animal fur and product quality supervision and test center (China, lanzhou) of the agricultural rural area according to the related national standard requirements. Phenotypic data such as body weight and shearing amount are collected from the fine wool sheep production performance measuring site and shearing site by well known methods.

As used herein, SNP refers to single nucleotide polymorphism (Single Nucleotide Polymorphism), which refers mainly to DNA sequence polymorphism at the genomic level caused by single nucleotide variation including variation caused by single base transition, transversion, insertion or deletion.

The molecular marker is a heritable and detectable DNA sequence or protein, 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, and the like. The molecular marker provided by the application can be used for genome mapping and gene localization research, map-based gene cloning, species genetic relationship, system classification and the like.

The probe referred to in the present application is a nucleic acid sequence (DNA or RNA) with a detection label and known in sequence complementary to the target gene.

It should be noted that the kit of the present application is any one of the kits conventionally used in the art, which contains reagents used for detection or experiment, and is convenient for operators to get rid of the heavy reagent preparation and optimization process. In one embodiment of the application, the kit comprises a probe for amplifying the site information provided by the application, a molecular marker or a probe or a gene chip for detecting the site information provided by the application, an enzyme and a buffer used for amplification, or a fluorescent marker used for detection.

Example 1 design and preparation of a 50K SNP liquid phase chip of a Fine wool sheep

1. Screening of important character related functional sites of fine wool sheep

Screening and obtaining SNP loci related to important characteristics of the fine wool sheep by adopting a whole genome association analysis strategy:

the test population is derived from 460 individuals of 4 Chinese typical representative fine wool sheep varieties of mountain merino, chinese merino, aoshan fine wool sheep and Qinghai fine wool sheep. Phenotyping selected 17 traits, including birth weight, weaning hair length, age weight, age hair length, age hair fiber diameter, age hair cut, age net hair rate, age net hair weight, adult hair length, adult hair cut, adult net hair rate, wool bundle fiber breaking strength, wool bundle fiber breaking elongation, wool fiber diameter coefficient of variation.

The experimental sheep were collected by vein for genomic DNA extraction and were subjected to whole genome resequencing using a Hiseq X-ten sequencer (Illumina), with average genome coverage of each sample being above 5X. After quality control, performing whole genome association analysis by using a GLM model of PLINK software to obtain 516 SNP (single nucleotide polymorphism) with obvious correlation with a phenotype index.

Screening and obtaining SNP loci related to important properties of the fine wool sheep by adopting a selection signal analysis method:

the 32 individual vein blood collection of 3 fine wool sheep varieties of mountain merino, chinese merino and Aoshan fine wool sheep is utilized for extracting genome DNA, a Hiseq X-ten sequencer (Illumina) is utilized for carrying out whole genome resequencing, and the average genome coverage of each sample is more than 30 multiplied. The loci obtained from each selected region are subjected to four selection signal methods based on Tajima' D test of locus frequency spectrum, selection signal combination analysis of genome heterozygosity, population differentiation selection index (Fst) and genome heterozygosity (θpi), and selection signal combination analysis of population differentiation selection index (Fst) and heterozygosity (Hp), and then linkage analysis is carried out by utilizing Haploview software, and each region selects a SNP which is located in the longest haplotype and located in a gene region as a candidate locus of the region. Finally obtaining 523 SNP related to the important character of the fine wool sheep.

And thirdly, obtaining 139 SNPs related to important characters such as disease resistance, propagation, growth and development of the fine wool sheep in researches such as reported OMIA (https:// www.omia.org/home /), GWAS and selection signals.

1178 SNPs obtained in the three steps are taken as functional sites and added into the chip design.

2. Screening of fine wool sheep 50K SNP locus

The application uses Hiseq X-ten sequencer (Illumina) to carry out 620 individuals on 4 Chinese representative fine wool sheep varieties of mountain merino, chinese merino, aoshan fine wool sheep and Qinghai fine wool sheepWhole genome resequencing, average genome coverage per sample was above 5 x. After preliminary quality control is carried out on the off-machine data, sequencing data is compared to a reference genome of a lano sheep, namely oar_4.0, all SNPs obtained by re-sequencing are filtered by using PLINK software for obtaining high-quality SNPs for chip design, haplotype analysis is carried out by using Haploview software (version 4.2), the size of a sliding window is set to be 50,000 bp, haplotype groups in the window are counted respectively, and a threshold value is set to R ² And more than or equal to 0.8. The genomic genetic variation was scored for importance according to the scores in table 1, the total score for all haplotype groups of the whole genome was calculated, and the highest scoring variation was marked as tagSNPs for the corresponding haplotype. According to the probe density, a certain number of tagSNPs are selected from each window and added into known functional sites, and 46178 SNPs are finally obtained.

TABLE 1 Whole genome genetic variation importance score table

The total of 46178 functional sites and background sites are selected through the steps, and the functional sites and the background sites are submitted to Shijia boridetia biotechnology limited company for evaluation, so that sites which cannot be uniquely compared on genome, sites containing repeated sequences in flanking sequences and sites with the tested deletion rate of more than 80% are removed. The physical positions of 45213 SNP loci evenly distributed on the genome of the fine-wool sheep, specifically on the sheep oar_v4.0 reference genome, were finally obtained together as shown in Table 2. The distribution of all SNP sites on each chromosome of the genome is shown in FIG. 1.

Table 2 45213 SNP molecular marker positions

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Design of 3.50K SNP liquid phase chip

The design chip adopts a GenoBaits technical system; the working principle is that a probe designed based on the target SNP marker of the nap sheep performs targeted sequence complementary combination and sequencing with DNA of a detection sample, thereby achieving the purpose of detecting the genotype of the target SNP marker of the detection sample. Firstly, constructing a library of samples to be tested, and simultaneously, according to the DNA complementation principle, covering each SNP marker to be tested with a target SNP marker probe, and marking the target probe by adopting Biotin (Biotin) markers. Then, the target probe labeled with biotin hybridizes to the target region of the genome in a liquid reaction system to form a double strand. The target probe carrying the biotin label is then subjected to molecular adsorption using streptavidin coated magnetic beads, thereby capturing the sequence containing the target SNP label of interest hybridized to the probe. And finally, eluting, amplifying, banking and sequencing the captured target sequence to finally obtain the genotype result of the target SNP. According to the position and two-side sequence information of 45213 SNP loci, primers are designed and probe synthesis is carried out by Shijia Boruidi biotechnology limited company by adopting a targeted capture sequencing technology, so that a fine wool sheep 50KSNP liquid-phase chip is obtained.

Example 2 application method of nap sheep 50K SNP liquid phase chip in detection of nap sheep DNA sample

1. Extraction of a fine wool sheep genomic DNA sample:

the nape sheep is subjected to jugular vein blood sampling, and a CTAB method is adopted to extract sample DNA.

DNA sample mass detection:

the DNA concentration of the test sample was determined by Qubit Fluorometric Quantitation (Thermo Fisher) and the integrity of the DNA was checked by 1% agarose gel electrophoresis. And placing the sample which is qualified in detection into a refrigerator at the temperature of 4 ℃ for storage and standby.

3. Sample DNA fragmentation:

and (3) placing 12 mu L of DNA qualified in quality inspection in a 0.2 mu L PCR tube, placing the tube in an ultrasonic breaker to randomly and physically break the DNA, and breaking the fragments to 200-400 bp.

4. Sample end repair:

add 4. Mu. L GenoBaits End Repair Buffer (Shi Jia Bo Rui Di Biotechnology Co., ltd.) and 2.7. Mu. L GenoBaits End Repair Enzyme to the tube, make up water to 20. Mu.L, place into ABI 9700PCR apparatus and incubate at 37℃for 20 minutes to complete the end repair and addition of the fragment fragments.

5. Sample sequencing linker ligation:

the vials were removed from the PCR apparatus, added with 2. Mu. L GenoBaits Ultra DNA Ligase, 8. Mu. L GenoBaits Ultra DNA Ligase Buffer and 2. Mu. L GenoBaits Adapter, filled with water to 40. Mu.L, and then placed on an ABI 9700PCR apparatus for reaction at 22℃for 30 minutes to complete the ligation of sequencing adaptors.

6. Sample DNA purification:

48. Mu.L of Beackman AMPure XP Beads (Beackman Co.) was added to the ligation product to purify the ligation product, and after purification, fragment screening was performed using magnetic beads, retaining the ligation product with an insert of 200-300 bp.

7. Sample library amplification:

mu.L of sequencing adapter with Barcode sequence, 1 mu L P adapter, 10 mu L GenoBaits PCR Master Mix are added into the PCR tube of the previous step, and the mixture is supplemented to 20 mu L by pure water; amplification was performed using an ABI 9700PCR instrument, the amplification procedure being: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 60℃for 30s, and extension at 72℃for 30s; repeating the steps 2-4 for 8 cycles; extending at 72℃for 5min. Different Barcode was used to distinguish between different samples.

8. Sample library purification:

to the second round of PCR products, 24. Mu. L Beckmen AMPure XP Beads (Beackman Co.) was added, and after pipetting up and down well, 0.2. Mu.L of PCR tubes were placed on a magnetic rack until the solution was clear, the supernatant was discarded and the beads were washed once with 75% ethanol, and the library DNA was eluted with Tris-HCl pH 8.0.

9. Determining the target individual locus genotype by utilizing a nap sheep 50K SNP liquid phase chip:

(1) DNA hybridization

500ng of the constructed genomic DNA sequencing library was taken, 5 mu L GenoBaits Block I and 2 mu L GenoBaits Block II were added, and the mixture was placed on a Eppendorf Concentrator Plus (Eppendorf company) vacuum concentrator and evaporated to a dry powder at a temperature of not more than 70 ℃. 8.5 mu L of GenoBaits 2 XHyb Buffer, 2.7 mu L GenoBaits Hyb Buffer Enhancer and 2.8 mu L of nucleic-Free Water are added into a dry powder tube, the mixture is sucked and beaten by a pipettor and placed on an ABI 9700PCR instrument for incubation for 10 minutes at 95 ℃, then 3 mu L of synthesized probe (the concentration of the probe is 60 ng/. Mu.L) is taken out of the PCR tube, and the mixture is placed on the ABI 9700PCR instrument for incubation for 2 hours at 65 ℃ after vortex shaking and mixing, so that the probe hybridization reaction is completed.

(2) DNA capture

100 mu L GenoBaits DNA Probe Beads is added into the reaction system of the hybridization completion in the last step, then the reaction system is sucked up and down for 10 times by a pipette, and the reaction system is put into an ABI 9700PCR instrument for incubation at 65 ℃ for 45 minutes, so that the magnetic beads are combined with the probes. The probe-bound beads were subjected to a 65℃heat wash with 100. Mu. L GenoBaits Wash Buffer I and 150. Mu. L GenoBaits Wash BufferII, respectively, followed by a 100. Mu. L GenoBaits Wash Buffer I and 150. Mu. L GenoBaits Wash Buffer II wash at room temperature (and 150. Mu. L GenoBaits Wash Buffer III wash, respectively), and the washed beads were resuspended in 20. Mu.L of nucleic-Free Water.

13 μl of resuspended DNA (with magnetic beads) was added to a new 0.2mL PCR tube, and then 15 μ L GenoBaits PCR Master Mix, 2 μ L GenoBaits Primer Mix were added to configure a post-PCR system, and library amplification was performed using an ABI 9700PCR apparatus with the following amplification procedure: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 60℃for 30s, and extension at 72℃for 30s; repeating the steps 2-4 for 15 cycles; extending at 72℃for 5min.

To the post-PCR product was added 45. Mu. L Beckmen AMPure XP Beads (Beackman Corp.) and then blotted up and down with a pipette and then a 0.2mL PCR tube was placed on a magnetic rack until the solution was clear, the supernatant was discarded and the beads were washed twice with 75% ethanol and the library DNA eluted with Tris-HCl pH 8.0. The hybridization capture work of the probe is completed.

(3) DNA hybridization capture library quality inspection

The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (Thermo Fisher) and then agarose gel electrophoresis was used to determine if the fragment size of the library DNA was between 300 and 400bp.

(4) Sequencing of DNA hybrid Capture library

The constructed DNA library was sequenced using a Huada MGISEQ2000 sequencer.

(5) Genotype data analysis

After the sequencing data is subjected to FastQC (www.bioinformatics.babraham.ac.uk/project) quality control, the sequencing data is compared to a reference genome by using a default parameter of BWA (bio-BWA. Sourceforge. Net), SNP identification is carried out on the sequencing data by using GATK (software. Broadinstall. Org/GATK) software, and the genotyping information of the sequencing is captured by using a self-coded Perl script to extract the genotyping information of the probe capture, so that a final genotyping file is formed.

The steps can successfully realize that the genotype data of the DNA sample of the nap sheep to be detected is obtained by utilizing the nap sheep 50K SNP liquid phase chip.

Example 3 full genome association analysis of Fine wool sheep 50K SNP liquid phase chip on wool traits

The application uses the nap 50K SNP liquid phase chip obtained in the example 1 to genotype the nap samples of 620 individuals (the specific operation method is shown in the example 1). And carrying out full genome association analysis on the net wool rate (CFWR) of one of important wool traits of the nap sheep by utilizing genotyping data and re-sequencing data, wherein the full genome association analysis result of the wool traits of the nap sheep by adopting a mixed linear model of rMVP (global motion vector) for the nap sheep 50K SNP liquid phase chip and the re-sequencing data is not distinguished (the result is shown in figure 2). The above results demonstrate that even without genotype filling, the same genomic correlation analysis results as with resequencing can be obtained using a lano 50K SNP liquid chip, and thus lano breeding and research work can be carried out at lower cost.

Example 4 application of nap sheep 50K SNP liquid phase chip in nap sheep genetic relationship identification

The accuracy of the family information of the livestock and poultry has very important significance for breeding progress or seed preservation effect. However, due to the introduction of unknown blood-related individuals, artificial recording errors, multiple mating, mixed insemination or embryo transfer, pedigree recording errors are common in the production, breeding and seed preservation processes. In the breeding work of livestock and poultry, the wrong pedigree not only can influence the determination of the parent-child relationship of the livestock and poultry, but also can slow down the genetic progress of the population, thereby causing great loss on the economic benefit of animal breeding and the like. Meanwhile, the false genealogy can also have negative influence on the genetic and breeding researches of livestock and poultry groups, so that the in-person identification has very important value and significance on the genetic breeding production and the researches of animals. Therefore, the genetic relationship identification of the fine wool sheep population is also an important link in the breeding work.

Aiming at 152 nap ram individuals in a certain group of mountain merino sheep, the embodiment applies the nap 50K SNP liquid phase chip prepared in the embodiment one to carry out genetic relationship analysis and construct a molecular genealogy. Through genomic genetic relationship analysis and cluster analysis based on the G matrix, the existing ram sample can be divided into 7 families (shown in figure 3), and the ram is divided into different families according to the genetic relationship between the male parent and the female parent. Based on the Mendelian genetic rule, compared with the original family, the result is that the Mendelian error rate of the original family is larger than a threshold value by 5 samples, and samples with the Mendelian error rate smaller than the threshold value and the minimum Mendelian error are found in other samples; the 20 sample genealogy has no parent-child relationship, and a sample with the Mendelian error rate smaller than the threshold value and the minimum Mendelian error is found in the samples.

The result shows that the application of the nap sheep 50K SNP liquid phase chip can obtain finer genetic relationship of the population at lower cost, and can correct pedigree errors and provide support for improving genetic progress.

Example 5 application of a liquid phase chip of a 50K SNP in a Fine wool sheep genome selection

Genome selection is a method of predicting the genetic value of animals based on genotyping data of genetic markers distributed on the genome, so that ideal individual selection is performed, and breeders can increase the speed of genetic improvement by early selection. With the continuous perfection of a reference genome, the cost reduction of a sequencing technology and the continuous upgrading of a commercial SNP chip, genome selection is a mainstream method of animal breeding at present, and has been widely applied to livestock and poultry. Compared with the conventional method, the genome selection is based on the utilization of pedigree and phenotype data, and the genome data is increased, so that the accuracy of seed selection is greatly improved. In the embodiment, 2178 adult ewes of merino sheep in alpine are taken as a reference group, all the liquid phase chip of the 50K SNP of the fine wool sheep in the first embodiment is used for genotyping and measuring the diameter of wool fibers, and the result of estimating a genome breeding value (GEBV) by using an SSGBLUP method finds that the prediction accuracy of the diameter of the wool fibers is 0.5806, and the estimation accuracy of 0.4983 is obviously improved compared with that of the conventional breeding value of BLUP.

Claims

1. A combination of SNP sites related to economic traits of a fine wool sheep, characterized in that the combination of SNP sites related to economic traits of a fine wool sheep consists of 45213 SNP sites, and the positions of the 45213 SNP sites on sheep oar_v4.0 reference genome are shown as No.1 to No.45213 in table 2 of the specification; 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. The reagent for detecting the combination of SNP loci related to economic traits of the fine wool sheep according to claim 1 has the use as set forth 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;

3. Use of a molecular probe for detecting a combination of SNP sites associated with the economic traits of a fine wool sheep according to claim 1 in the preparation of a liquid phase chip/reagent/kit for analysing the genome of a fine wool sheep.

4. A molecular probe combination for analyzing the genome of a fine wool sheep, wherein the molecular probe combination detects the SNP locus combination according to claim 1 in a sample to be tested.

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

(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;

6. A liquid phase chip for analyzing the genome of a fine wool sheep, wherein the liquid phase chip is loaded with a molecular probe for detecting the SNP locus combination as set forth in claim 1.

7. The liquid-phase chip according to claim 6, wherein the liquid-phase chip has any of the following uses:

(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;

8. A kit for analyzing the genome of a fine wool sheep, comprising a reagent for detecting the SNP site combination as set forth in claim 1, or the molecular probe combination as set forth in claim 4, or the liquid phase chip as set forth in claim 6.

9. The kit of claim 8 having any one of the following uses:

(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;