CN109251986B - Molecular marker comprising SNP7-1 and application thereof in assisted breeding of Hu sheep - Google Patents

Abstract

The invention provides a plurality of SNP molecular markers related to the body weight traits of Hu sheep and application thereof in molecular marker assisted breeding of the Hu sheep. The invention also provides a primer pair for detecting a plurality of SNPs related to the body weight traits of the Hu sheep, a kit containing the primer pair and application of the primer pair and the kit in molecular marker assisted breeding of the Hu sheep. The invention further provides a method for screening the weight traits of the Hu sheep and application of the method in molecular marker assisted breeding of the Hu sheep. The method can be used for the molecular marker-assisted breeding of the characters for the Hu sheep meat and the early breeding of core group individuals of the Hu sheep meat line.

Description

Molecular marker comprising SNP7-1 and application thereof in assisted breeding of Hu sheep

Technical Field

The invention belongs to the field of molecular markers, and particularly relates to a Hu sheep SNP molecular marker, a related primer pair, a kit and application thereof in Hu sheep molecular marker assisted breeding, and further relates to a method for screening the weight character of Hu sheep and application thereof in Hu sheep molecular marker assisted breeding.

Background

Since the middle of the 20 th century, the genetic improvement of sheep in China is subjected to selection, cultivation and purification of local varieties, hybridization for improving the local varieties and cultivation of new varieties, and the production direction is also changed from 'meat with main feather and auxiliary feather' to 'meat with main feather and auxiliary feather'. At present, the development speed of sheep farming in domestic animal husbandry is second to that of poultry farming^[1]。

Although the breeding research work of various new species of meat sheep is developed in China, Bamei meat sheep are bred^[2]Shouwuda mutton sheep^[3]Hezhou haer sheep^[4]And the mutton sheep breeds with independent intellectual property rights. However, the phenomenon of 'heavy introduction and light breeding' still exists in the breeding work^[1]. Taking Hu sheep and small tailed han sheep as examples, the two varieties are famous multiparous sheep varieties in China, but the fur value of the varieties is not high, the meat production performance is low, and the requirements of mutton consumption markets can not be met. Therefore, the sheep breeding worker is to carry out genetic improvement and improve the production efficiency of sheep breedingThe problem to be solved in the process is an inexhaustible task in the presence of extensive animal genetic breeding researchers.

Under the big background of human haplotype map plan, continuous publishing of multi-species Genome information, appearance of commercial medium-high density SNP chips and maturity of gene data analysis methods, genetic variation research associated with important economic traits of animals is searched based on the whole Genome range, a new thought and a new approach are brought for molecular breeding research, and Genome-wide association analysis (GWAS) becomes one of analysis methods and means for identifying functional genes of complex traits. Many GWAS researches are carried out on economic traits of sheep and goats^[5-29]. The Ovine SNP50 BeadChip chip was developed by Illumina and International sheep genome Association experts, and contains 54241 SNP sites, with an average of one marker per 46kb, covering the entire sheep genome. The chip becomes an important tool for GWAS analysis of economic traits of cotton and goats, and most of the existing research results are obtained by using the chip.

GWAS is mainly divided into two types in the research of related trait genes of sheep: (1) contacts on an unrelated individual basis. (2) Contact profiling on a family basis. The case-control analysis method is mainly used for analyzing the distribution characteristics and the difference of genotypes in the whole genomes of the case group and the control group. Random population based assays are used primarily for quantitative trait analysis in animal applications. At present, researchers in various countries have carried out a plurality of research analyses of GWAS on complex diseases and economic traits, a plurality of SNPs related to the diseases or the economic traits are discovered, more than one thousand related genes or sites are proved, and the quantity of GWAS research results is published and reported in recent years and is in an increasing situation year by year^[30]。

Although GWAS analysis has achieved excellent results and effects in relevant research, there is also a question of the attitude of the scholars^[31]The research and analysis are also in the continuous repairing process^[32-34]. At present, research and analysis on GWAS need to be started from multiple aspects and scientific research needs to be transformed into practice.

Along with the improvement of living standard, people have more and more consumption requirements on mutton, meat sheep varieties such as Hu sheep, small tailed Han sheep and the like in some parts of China are deeply favored by consumers due to the advantages of strong fecundity, delicious meat quality and the like, but compared with meat varieties, the varieties have the advantages of low meat yield, long production period and the like and cannot meet the requirements of the consumers, Hu sheep is a unique precious sheep variety in China, and integrates the advantages of strong fecundity, fast growth in early stage, good meat quality and the like, but also has the defects of low meat production performance and unsatisfactory meat body types. Therefore, the breeding of the Hu sheep meat line is carried out to improve the production performance of the Hu sheep, and the method is an important problem to be solved urgently in the Hu sheep breeding work.

Disclosure of Invention

In order to solve the technical defects and the practical requirements in life in the prior art, the Hu sheep is taken as a research object, an ovine SNP50 Genotyping method is used for Genotyping a parent population (G1) and a child population (G2) of a new population core group for the Hu sheep meat, whole genome correlation analysis is carried out on body size properties such as the length, the height, the chest circumference, the tail length and the tail width of the meat core group, 11 sites which are obviously related to the body height and 1 site which is obviously related to the chest circumference are obtained, SNP population verification of G3 generation individuals of the Hu sheep meat system core group is carried out on the sites, and the analysis result of a general linear model shows that all SNPs do not influence the body size properties of G3 generation individuals of the Hu sheep meat system core group and are obviously related to the body weight properties of G3 generation individuals.

The method is used for researching the body size character GWAS of the Hu sheep for the first time, screening candidate functional genes and SNPs of the body size character of the Hu sheep by utilizing the GWAS technology, positioning the candidate genes of the body size character of the sheep and providing important theoretical basis and reference for exploring the functional genes of the body size character of the sheep. SNPs which can be used for molecular marker assisted breeding are obtained through group verification of the SNPs, and can be used for character molecular marker assisted breeding for Hu sheep meat and early breeding of core group individuals of the Hu sheep meat line.

The invention aims to provide an SNP molecular marker related to the body weight character of Hu sheep and application thereof in screening the body weight character of Hu sheep or assisting breeding of the molecular marker of Hu sheep. In one embodiment, the molecular marker includes SNP3 with the position located at the 209bp position of SEQ ID NO.1, which is G or A. In one embodiment, the molecular marker includes SNP5 with the position at the 129bp position of SEQ ID NO.2, which is either T or C. In one embodiment, the molecular marker includes SNP7-1 with the position located at the 303bp position of SEQ ID NO.3, which is either T or C. In one embodiment, the molecular marker includes SNP7-2 with position 373bp of SEQ ID NO.3, which is either T or C. In one embodiment, the molecular marker includes SNP10-1 with the position located at the 87bp position of SEQ ID NO.4, which is G or A. In one embodiment, the molecular marker includes SNP10-2 with the position located at the 207bp position of SEQ ID NO.4, which is either T or C. In one embodiment, the molecular marker includes SNP10-3 with the position located at the 211bp position of SEQ ID NO.4, which is C or A.

The invention also aims to provide the SNP molecular marker related to the body weight character of the Hu sheep and the application thereof in screening the body weight character of the Hu sheep or assisting breeding of the molecular marker of the Hu sheep. In one embodiment, the sequence of the molecular marker is shown as SEQ ID NO.1, wherein the base of the 209bp (gene positioning is OAR6_95218086.1 upstream 44bp) site is G or A. In another embodiment, the sequence of the molecular marker is shown in SEQ ID NO.2, wherein the base of the 129bp (gene positioning is 16bp downstream of s 10476.1) site is T or C. In another embodiment, the sequence of the molecular marker is shown in SEQ ID NO.3, wherein the base of the 303bp (gene location is 192bp downstream of OAR1_ 164254640.1) site is T or C. In another embodiment, the sequence of the molecular marker is shown in SEQ ID NO.3, wherein the base of the 373bp (gene location is OAR1_16425464 downstream 235bp) site is T or C. In another embodiment, the sequence of the molecular marker is shown as SEQ ID NO.3, wherein the base of the 303bp (gene positioning is 192bp downstream of OAR1_ 164254640.1) site is T or C; and the base of 373bp (gene positioning is 235bp downstream of OAR1_ 16425464) site is T or C. In another embodiment, the sequence of the molecular marker is shown in SEQ ID NO.4, wherein the base of the 87bp (gene location is 41bp upstream of OAR6_ 90337552.1) site is G or A. In another embodiment, the sequence of the molecular marker is shown in SEQ ID NO.4, wherein the base of the 207bp (gene location is 79bp downstream of OAR6_ 90337552.1) site is T or C. In another embodiment, the sequence of the molecular marker is shown in SEQ ID NO.4, wherein the base of the 211bp (gene location is 83bp downstream of OAR6_ 90337552.1) site is C or A.

The invention also aims to provide a primer pair for detecting the SNP3, the SNP5, the SNP7-1, the SNP7-2, the SNP10-1, the SNP10-2 and the SNP10-3 related to the weight traits of the Hu sheep, a kit containing the primer pair and application of the primer pair in screening the weight traits of the Hu sheep or molecular marker assisted breeding of the Hu sheep. In one embodiment, the nucleotide sequences of the primer pairs are set forth in Table 3-1. In one embodiment, the primer pair is 3F +3R in Table 3-1, SEQ ID NO.5 and SEQ ID NO. 6. In another embodiment, the primer pair is 5F +5R in Table 3-1, SEQ ID NO.7 and SEQ ID NO. 8. In another embodiment, the primer pair is 7F +7R in Table 3-1, SEQ ID NO.9 and SEQ ID NO. 10. In another embodiment, the primer pair is 10F +10R in Table 3-1, SEQ ID NO.11 and SEQ ID NO. 12.

The invention also aims to provide application of the SNP molecular marker, the primer pair or the kit in screening of the body weight traits of the Hu sheep or assisted breeding of the Hu sheep molecular marker.

The invention also aims to provide a method for screening the weight traits of Hu sheep, which comprises the following steps: extracting Hu sheep genome DNA, carrying out PCR amplification by using the primer pair, and detecting the SNP3, the SNP5, the SNP7-1, the SNP7-2, the SNP10-1, the SNP10-2 and the SNP10-3 in an amplification product so as to screen the weight traits of the Hu sheep. In one embodiment, the nucleotide sequences of the primer pairs are set forth in Table 3-1. In one embodiment, the primer pair is 3F +3R in Table 3-1, SEQ ID NO.5 and SEQ ID NO. 6. In another embodiment, the primer pair is 5F +5R in Table 3-1, SEQ ID NO.7 and SEQ ID NO. 8. In another embodiment, the primer pair is 7F +7R in Table 3-1, SEQ ID NO.9 and SEQ ID NO. 10. In another embodiment, the primer pair is 10F +10R in Table 3-1, SEQ ID NO.11 and SEQ ID NO. 12.

In a specific embodiment of any one of the above methods for screening the weight traits of Hu sheep, the reaction procedure of PCR amplification is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; the reaction system for PCR amplification is shown in Table 1:

TABLE 1

The invention also aims to provide the application of the method for screening the weight traits of the Hu sheep in molecular marker-assisted breeding of the Hu sheep.

The invention has the beneficial effects that: the molecular marker, the primer pair and the related kit can be used for screening the weight characters of the Hu sheep or the Hu sheep molecular marker assisted breeding and can be used for early breeding of Hu sheep meat use line core group individuals.

Drawings

FIG. 1 is a technical route for obtaining SNP sites that can be used for molecular breeding.

FIG. 2-1 shows the detection result of Hu sheep genomic DNA by agarose gel electrophoresis (Marker: DL2000 plus).

FIGS. 2-2 are Manhattan plots (Manhattan plots) of GLM body height whole genome association analysis.

FIGS. 2-3 are Manhattan plots (Manhattan plots) of GLM chest circumference genome-wide association analysis.

FIGS. 2-4 are Manhattan plots (Manhattan plots) of MLM somatic height whole genome association analysis.

FIGS. 2-5 are Manhattan plots (Manhattan plots) from MLM chest circumference genome-wide association analysis.

FIGS. 2-6 are the Hu sheep body high Q-Q Plot (A: GLM; B: MLM).

FIGS. 2-7 are Q-Q Plot of chest circumference of Hu sheep (A: GLM; B: MLM).

FIG. 3 shows the PCR amplification results of primers for detecting high-trait related SNPs in Hu sheep. M: DL2000 plus; lanes 1-8 correspond to amplification products designed for OAR6_95218086.1, OAR15_18440393.1, s10476.1, OARX _120998827.1, OAR1_164254640.1, s10347.1, s11279.1, and OAR6_90337552.1, respectively.

Detailed Description

The invention is further illustrated by the following examples.

Example 1 Whole genome Association analysis of Hu sheep size traits

The ovine SNP50 Genotyping BeadChip chip is developed by cooperation of Illumina's iSelect project and International sheep genome Association, integrates gene differences of multiple sheep varieties, has 1 marker per 46kb on average, provides enough SNP density to cover the whole genome, and can be applied to whole genome association research. From 2006, the inventor respectively constructs new cluster core groups for Hu mutton in Huzhou and Xiaoshan, and through breeding, the parent and the offspring clusters of the core groups show obvious differences on body size indexes such as body length, body height, chest circumference, tail length, tail width and the like. The parent population and the offspring population of the core population of the new cluster for the Hu sheep meat are genotyped by using an ovine SNP50 Genotyping BeadChip chip, and the body size traits of the core population for the meat, such as length, body height, chest circumference, tail length, tail width, and the like, constructed by the professional cooperation of the Hu Tai lake Hu sheep breeding are subjected to whole Genome Association analysis (GWAS) by using a General Linear Model (GLM) and a Mixed Linear Model (MLM), and the identification research of the body size traits and the major genes of the meat traits is carried out by using the latest sheep Genome Ovis _ aries _ v3.1 sequence information and an AS method. The research result can locate sheep body size character candidate genes and also provide important theoretical basis and reference for exploring sheep body size character functional genes.

2.1 test materials

2.1.1 test animals

240 Hu sheep are selected from Hu sheep meat use system core group of Hu sheep cooperative of Hu Tai lake Breeding specialty in Hu, and are bred in a breeding management mode according to a standard breeding management method of mutton sheep.

2.1.2 sample Collection and processing

10mL of jugular venous blood is collected from each test sheep, temporarily stored in a blood collection tube containing EDTA anticoagulant, and stored at the temperature of minus 20 ℃ for a long time.

2.1.3 Primary reagents

And (3) protease K: amresco Inc. of USA

NaCl, absolute ethanol, agarose, glacial acetic acid, boric acid, NaOH: beijing chemical Agents Ltd

DNA Marker 15000: hangzhou Zhike catalpi xi organism Limited

DNA chip hybridization kit: illumina Corp Ltd

2.1.4 Main solution

Taking the third edition of molecular cloning Experimental Manual of Sam Brooks as reference, preparing the required solution with ultrapure water as solvent, and sterilizing with steam at high temperature and high pressure for 40 min.

(1)1mol/L Tris-HCl (pH 8.0, 1L): after 121.1g of Tris-base was dissolved in 800mL of ultrapure water, the pH of the solution was adjusted to 8.0 with concentrated hydrochloric acid, the volume was adjusted to 1L, and autoclaving was performed.

(2)5 × TBE (Tris borate buffer): 54g Tris-base, 27.5g boric acid, 3.72g Na were weighed₂EDTA· 2H₂And O, adding ultrapure water to a constant volume of 1L, and uniformly mixing.

(3)0.5M EDTA (pH 8.0): 186.1g of Na₂EDTA·2H₂Dissolving O in 800mL double distilled water, adjusting pH to 8.0, diluting to 1000mL, and sterilizing at high temperature and high pressure.

(4)0.5M NaCl: 5.844g NaCI was dissolved in ultrapure water, and the volume was adjusted to 200mL, and autoclaved.

(5) 10% SDS: 10g of SDS was dissolved in ultrapure water at 65 ℃ to a constant volume of 100mL, and the solution was filtered through a 0.2nm filter and stored.

(6) TE buffer (pH 8.0) contained 20mM Tris/HCl (pH 8.0), 1mM edta (pH 8.0): 2mL of 1M Tris/HCl (pH 8.0), 0.2mL of 0.5M EDTA (pH 8.0), a volume of 100mL, and autoclaving.

(7) Proteinase K (20 mg/mL): 100mg of proteinase K was dissolved in 5mL of ultrapure water, and 400. mu.L of proteinase K was dispensed into each tube and frozen at-20 ℃.

(8)3M NaAc (pH 5.2): 12.305g of anhydrous NaAc is dissolved in double distilled water, the volume is determined to be 50mL, glacial acetic acid is added to adjust the pH value to 5.2, and autoclaving is carried out.

(9)PBS：8.0g NaCl，0.2g KCl，3.48g Na₂HPO₄·12H₂O，0.2g KH₂PO₄And (5) fixing the volume to 200mL, and autoclaving.

(10) STE: 5mL of 1M Tris HCl (pH 8.0), 20mL of 0.5M EDTA (pH 8.0), 20mL of 0.5M NaCl, 10mL of 10% SDS, and ddH was added thereto₂And O, double-distilled water is added until the volume is 100mL, and the mixture is uniformly mixed.

2.1.5 Main instrumentation

A constant-temperature water bath kettle: jiangsu Taicang laboratory Equipment Co Ltd

Thermo Fisher-80 ℃ ultra-low temperature refrigerator: united states of America

Haier 4 ℃/— 20 ℃ refrigerator: shandong (mountain east)

Centrifuge 58108 high speed cryogenic refrigerated Centrifuge: eppendorf Co, Germany

F100 icemaker: italy (chemical vapor deposition)

Bio-Rad CHEMI DOC gel imaging analysis System: united states of America

TOMY ES-315 model autoclave: japanese

Sartorius electronic balance: germany

Model DYY-7C electrophoresis apparatus: beijing six one

DYY-III32 type electrophoresis tank: beijing six one

QL-901 vortex oscillator: jiangsu

DSHZ-300 multipurpose water bath constant temperature oscillator, Jiangsu Taicang laboratory plant

AstraGene AstraNet uv spectrophotometer: great Britain

10 μ L, 100 μ L, 200 μ L, 1000 μ L pipette: eppendorf Co, Germany

Infinium genome-wide SNP analysis system: illumina Corp Ltd

2.1.6 data analysis software and on-line software

2.1.6.1 data analysis processing software

1.Tassel 3.0

The main function of Tassel is to study the relationship between phenotype and genotype, using Java software for assessing genotype and trait associations using oral and quantitative genetics tools, with the latest and statistically most robust complex population association mapping methods, including General Linear Model (GLM) and Mixed Linear Model (MLM). In the study, GLM and MLM in a Tassel software package are used for carrying out whole genome association analysis on 5 individual size traits such as height, length (oblique), chest circumference, tail length and tail width of Hu sheep.

2.Plink 1.09

GWAS analysis software can perform quality management control on genotype or phenotype data and samples, analyze and process SNPs and estimate genotype and phenotype values.

R language

And drawing a Manhattan graph and a Q-Qplot graph in the whole genome correlation analysis result.

4.Mutation Surveyor version 5.02

Sequencing peak plot analysis and sequence assembly software.

2.1.6.2 Online Web site and database

(1) UCSC online website: http:// genome. ucsc. edu/.

(2) NCBI online website: http:// www.ncbi.nlm.nih.gov.

(3) DAVID online website: http:// david.abcc.ncifcrf.gov.

2.2 test methods

2.2.1 measurement of body size Properties

The body size measurement of the Hu mutton family core group individual includes body (oblique) length, body height, tail length, tail width and chest circumference. Tools such as a tape measure are used for measurement, so that the sheep is kept on a horizontal ground, quiet and relaxed to stand. The measuring staff are ensured to be the same person as much as possible so as to reduce the measuring error caused by human factors, each sheep is measured at least 2 times, and the average value is taken as the final measuring result. The specific determination method is as follows:

body length: the linear distance from the front end of the shoulder and foot bones to the back end of the ischial tuberosity;

body height: the vertical distance from the highest point of the chignon to the ground;

chest circumference: the length of one circle around the chest along the posterior edge of the scapula;

tail length: distance from tail root to tail end;

tail width: the distance at the widest position of the tail width.

2.2.2 technical route

The technical route is shown in figure 1.

2.2.3 extraction of genomic DNA from peripheral blood

After thawing frozen peripheral blood at-20 ℃, 0.5mL of the thawed peripheral blood was pipetted into a new 1.5mL LEP tube and subjected to extraction of genomic DNA from the blood by the Tris saturated phenol method.

The integrity of the genomic DNA was checked by agarose gel electrophoresis.

Taking 2 mu L of genome DNA solution, measuring the concentration and the purification effect of the DNA, and ensuring that the concentration of the extracted and purified DNA is more than 100 ng/mu L and the OD value 260/280 is 1.8-2.0.

2.2.4 genotyping of individual SNPs of Hu mutton lineage core group G1 and G2 generations

The individual SNPs were genotyped using the Ovine SNP50 BeadChip chip developed by Illumina and International sheep genome Association experts.

2.2.4.1 genotyping procedure

(1) Sample normalization: the DNA concentration was diluted to 50 ng/. mu.L.

(2) Amplification of DNA: a NaOH solution with a concentration of 1N was added to the sample, and then the reagents required for genome amplification were added, followed by standing at room temperature for 12 hours.

(3) Fragmentation of DNA: the DNA is fragmented using an enzyme preparation.

(4) And (3) precipitating DNA: the DNA was deposited on the tube wall using anhydrous isopropanol.

(5) Resuspending the DNA: after drying at room temperature, the corresponding buffer solvent is added to completely dissolve the compound.

(6) Hybridization of DNA to chip: and (5) hybridizing the DNA sample obtained in the step (5) with the chip, and then placing the hybridized DNA sample in a hybridization furnace for full reaction.

(7) Extension and staining of the chip: unbound and non-specifically bound DNA is washed away with a wash solution.

(8) Coating a chip: the chip was placed in XC4 reagent, coated with a coating solution, and then placed under vacuum for 1 hour.

(9) Chip scanning: and (4) placing the chip processed in the step (8) into a scanner for chip scanning.

2.2.4.2 genotype data quality control management

After the chip is processed, the data is input into Beadstudios software for relevant analysis. After the genotype output by the software is sorted and corrected, statistical analysis is carried out, and inaccurate SNP sites are eliminated or corrected by using the software, so that the characteristics of all data of the whole genome are obtained.

2.2.4.3 SNP locus typing and quality management

And carrying out typing research on the whole genome nucleic acid of all samples by using a whole genome genotyping system, then using genome studio software to convert the data into visual genotyping results, storing the visualized genotyping results into a txt format, and outputting the visualized genotyping results.

2.2.5 data processing

2.2.5.1 statistical analysis of phenotypic data

Statistical analysis was performed on the measured meat-based core population ruler indices using SPSS20 statistical software, and the average value, standard deviation, and the like of each individual ruler index were calculated.

2.2.5.2 GWAS handling

After the genotype data are subjected to quality control treatment, General Linear Model (GLM) and Mixed Linear Model (MLM) of TASSEL3.0 software are used for carrying out GWAS analysis on SNP, and SNPs related to core population size trait phenotypes of Hu sheep meat line are mined.

The GLM model corrects for gender, population structure 2 confounders. Since all individuals analyzed were sheep from the same feedlot under the same feeding environment and management conditions, field effects were not included in the data modeling.

The concrete model is as follows: y ═ X β + e

Wherein the content of the first and second substances,

y: the core population ruler character and the body weight character phenotype value vector of the Hu mutton line;

beta: fixed effect vectors such as phenotype mean, SNP, population structure, sex, etc.;

e: a residual effect vector;

and X is a correlation matrix of beta.

The MLM model corrected for 3 confounders of gender, population structure and affinity.

The concrete model is as follows: y ═ X β + S α + Qv + Zu + e

Wherein:

y: the core population ruler character phenotype value vector of the Hu mutton lineage;

beta: fixed effect vectors other than SNPs and population structures;

α: an SNP effect vector;

v: a population structure effect vector;

u: a multi-gene background effect vector;

e: a residual effect vector;

x, S, Q, Z are the incidence matrixes of beta, alpha, v, u, respectively.

2.2.5.3 multiple hypothesis test

When the core population ruler character correlation analysis of the Hu mutton line is carried out, if a plurality of hypothesis tests generate errors, the P value needs to be processed, analyzed and corrected. Respectively adopting GLM and MLM to analyze and calculate an F value and a P value, and then carrying out detection, wherein the specific formula is as follows:

if the P value of the site is less than alpha, the SNP site is considered to have a significant association with the size trait.

2.2.5.4 population stratification

When the Hu mutton system core population ruler character correlation analysis is carried out, the population stratification and false positive influence are large. And (3) drawing a Q-Q plot diagram of the properties of the height, body length, chest circumference, tail length and tail width of the core group of the Hu mutton system to judge whether deviation and the layering phenomenon of the sample group occur.

2.2.5.5 annotation and mining of candidate genes

After the significant SNPs sites are obtained by whole genome association analysis, 500bp base sequences at the upstream and downstream of the significant associated SNP sites are downloaded, and BLAST of the sequences is carried out with databases such as NCBI and Ovis aries _ v4.0(UCSC) and the like to determine the positioning information and adjacent gene information of the SNP.

2.3 data analysis results

2.3.1 t-test of individual size traits of Hu mutton family core group G1 and G2 generations

Before the GWAS analysis of data, first, the body size traits of the hu-sheep meat family core group of G1 (n-161) and G2 (n-79) individuals were subjected to t-test.

TABLE 2-1 t test of individual size traits of Hu sheep core group G1 and G2 generations

Note: different lower case letters in the same row indicate significant difference (P <0.05), different upper case letters indicate significant difference (P <0.01), and the same letter indicates insignificant difference.

The results show that the individuals of the generation G1 and the generation G2 have very significant differences in 5 individual size characters of body height, body length, chest circumference, tail length and tail width, and the body height, body length, chest circumference, tail length and tail width of the individuals of the generation G2 of the Hu mutton lineage core group after breeding are all very significantly higher than those of the individuals of the generation G1 (P <0.01) (Table 2-1).

2.3.2 analysis of correlation between weight of 6 months old and body size traits in Hu mutton systems core group

The production records of the hu-sheep meat-based core group G1 and G2 included 6-month-old weight, and correlation analysis was performed on 5-body-size traits, namely body height, body length, chest circumference, tail length, and tail width, and 6-month-old weight of 240 individuals in total of the hu-sheep meat-based core group G1 (n: 161) and G2 (n: 79), and as a result, as shown in table 2-2, the 5-body-size traits and 6-month-old weight of the hu-sheep core group individuals showed very significant positive correlation, the 6-month-old weight-related trait having the lowest number of 6-month-old weight was tail width (r: 0.640, P <0.01), and the 6-month-old weight-related trait having the highest number of 6-month-old weight was chest circumference (r: 0.893, P < 0.01). In addition, there is a very significant positive correlation between the body size traits, with the highest correlation coefficient between body height and chest circumference (r ═ 0.896, P <0.01), and the lowest correlation coefficient between body length and tail width (r ═ 0.589, P < 0.01).

TABLE 2-2 analysis of the relationship between the size traits and 6-month-old weight of individuals (Huzhou) from the G1 and G2 generation core clusters of Hu sheep meat

Note: indicates significant correlation (P < 0.05); indicates very significant correlation (P < 0.01).

2.3.3 genomic DNA detection

All individual extracted and purified blood genomic DNA was tested for fragment length, purity and concentration. The detection result of the 1% agarose gel electrophoresis of the genome DNA is shown in figure 2-1, the genome achieves the standard of single band, brightness and no tailing, and the genome DNAOD value 260/280 is 1.8-2.0, and can be used for SNP typing.

The detection result is shown in FIG. 2-1.

2.3.4 SNP typing and quality management

The qualified genomic DNA of the sample is detected by a complete genome typing platform, and the collected 240 individual samples and 54241 SNPs sites are subjected to the following quality control management by adopting Plink1.09 software. For SNP sites, we eliminated: (1)3577 chromosome loci with a typing success rate of less than 90%; (2)4435 chromosomal loci with an allele frequency equal to 0.05; (3)20 chromosomal loci not eligible for HWE testing. For an individual. We eliminated 12 individuals with a typing success rate of less than 90%.

According to the quality management principle, 228 individual samples and 46209 effective sites are finally screened for GWAS analysis.

2.3.5 determination of level of significance between chromosomes

To reduce the false positive rate from multiple assays, the whole genome association analysis results P values were corrected with linkage disequilibrium corrected Bonferroni corrections. The final estimated LD block and single independent SNP number was 35161, so the Bonferroni corrected significant P-value threshold at 5% genome level was 1.42203 × 10^-6(0.05/35161), i.e., SNPs with a P-value below this threshold are considered to be significantly associated with a phenotype; to achieveThe threshold of the extremely significant P value to the genome level is 2.844 x10^-7(0.01/35161)。

2.3.6 size trait GWAS results

GWAS analysis was performed on hu mutton family of core group individuals of G1 and G2 generations according to the software and model provided in "materials and methods" 2.2.5.

GWAS results for the 2.3.6.1 size trait GLM

GLM analysis results showed that 4 SNPs were significantly associated with body height at the genomic level, OAR6_90337552.1 located on chromosome 6, s11279.1 on chromosome 8, s44173.1 on chromosome 10 and s55179.1 on chromosome 17, respectively; the 7 SNPs were extremely significantly associated with body height at the genomic level, located at OAR1_164254640.1 on chromosome 1, s10476.11 on chromosome 2, OAR6_95218086.1 on chromosome 6, s10347.1 on chromosome 9, OAR15_18440393.1 on chromosome 15, OARX _76354330.1 and OARX _120998827.1 on chromosome 27, respectively (tables 2-3); at the same time, OARX _76354330.1 located on chromosome 27 also significantly correlated with bust size at the genomic level (tables 2-4). No SNP was significantly associated with the 3 individual size traits body length, tail width and tail length at the genomic level.

TABLE 2-3 Hu sheep SNPs (GLM analysis results) significantly related to body height at the genomic level

Note: SNPs with gray shading are very significantly related SNPs at the genomic level. The same applies below.

Tables 2-4 Hu sheep SNPs (GLM analysis results) significantly related to breast circumference at the genomic level

Manhattan plots (Manhattan plots) of GLM body height and chest circumference genome-wide association analysis are shown in FIGS. 2-2 and 2-3, wherein the X axis of the Manhattan plot is the chromosome position of the SNP locus, the Y axis is the P value (-log10) of the SNP, and the higher the Y value is, the more significant the P value is.

GWAS results for MLM in 2.3.6.2 size traits

MLM analysis results showed that 4 SNPs were significantly associated with body height at the genomic level, OAR1_164254640.1 located on chromosome 1, s10347.1 located on chromosome 9, OAR15_18440393.1 located on chromosome 15, and OARX _120998827.1 located on chromosome 27; 2 SNPs were associated with body height to be extremely significant at the genomic level, OAR6_90337552.1 located on chromosome 6 and OARX _76354330.1 located on chromosome 27 (tables 2-5), respectively; at the same time, OARX _76354330.1 located on chromosome 27 was also significantly correlated with bust size at the genomic level (tables 2-6). No SNP achieved significant association at the genomic level with the 3 individual size traits body length, tail width and tail length.

TABLE 2-5 Hu sheep SNPs (MLM analysis results) significantly related to body height at the genomic level

TABLE 2-6 Hu sheep SNPs (MLM analysis results) significantly associated with chest circumference at the genomic level

The MLM analysis results are different from the GLM analysis results in that s10476.11 located on chromosome 2, OAR6_90337552.1 on chromosome 6, s11279.1 on chromosome 8, s44173.1 on chromosome 10, and s55179.1 on chromosome 17 did not reach a significant level at the whole genome level nor at the height.

As with the GLM analysis results, MLM analysis also yielded "chromosome 27 OARX _76354330.1 showed significant or very significant correlation at the whole genome level with both body height and chest circumference"; the conclusion that OAR6_95218086.1 located on chromosome 6 and OARX _120998827.1 located on chromosome 27 were extremely significantly correlated with body height at the genomic level.

Manhattan plots (Manhattan plots) for MLM body height and bust genome-wide association analysis are shown in FIGS. 2-4, 2-5.

2.3.7 population stratification assessment

Population stratification refers to the presence of other sub-populations within a population that result in the appearance of false positive results when performing association analysis, thereby affecting the results.

GLM and MLM were used in this study to plot Q-Q plot for the body height (FIGS. 2-6) and chest circumference (FIGS. 2-7) traits detected to have significant associations, respectively.

The Q-Q plot is used primarily to measure the difference between observed and predicted values. The abscissa of fig. 2-6 and fig. 2-7 represents the quantile result corresponding to the actual observed value, the ordinate represents the quantile result corresponding to the theoretical predicted value of the established model, the two values should be approximately equal, the slash in the graph represents the prediction line, if the deviation occurs, the deviation between the actual value and the predicted value is shown, and if the deviation is larger, the deviation is shown to be caused by the genetic action generated by the mutation of the SNP site.

Actual values of GLM (figures 2-6A, figures 2-7A) and MLM (figures 2-6B, figures 2-7B) Q-Q plot in body height and chest circumference basically fall on a prediction line, and the graphs are basically consistent, which shows that correlation analysis results of GLM and MLM are reliable, and a test population is corrected without population stratification.

2.3.8 Gene annotation of significantly related SNPs at the Whole genome level

According to the method described in '2.2.5.5 candidate gene annotation and mining', BLAST is carried out on SNP loci which are significantly related to Hu sheep body height and chest circumference GWAS whole genome level, the relation between the SNP loci and information such as efficacy and the like of walking places in the sheep whole genome is confirmed, and then annotation is carried out.

At the genome-wide level, 11 SNPs loci annotation information that are significantly associated with the height or bust of Hu sheep are shown in tables 2-7. 5 of these SNPs are located intragenously, e.g., OARX _76354330.1 is located in intron 2 of the CAPN6 gene; OAR1_164254640.1 is located in intron 7 of the CADM2 gene; s11279.1 is located in intron 3 of the RNF217 gene; s10347.1 is located in intron 3 of the SAMD12 gene; OAR15_18440393.1 is located in intron 8 of the DDX10 gene. In addition, there are 6 significantly related SNP sites at the genome-wide level located in intergenic regions, e.g., s55179.1 located between ACADS and SPPL3, 16kb downstream of ACADS; s10476.1 is located between the SLC38A11 and COBALL 1 genes, 93kb downstream of SLC38A 11; OAR6 — 95218086.1 is located between GC and NPFFR2, 209kb downstream of GC; OAR6_90337552.1 is located between EPHA5 and LOC101120496, 1446kb downstream of EPHA 5; s44173.1 is located between LOC101106088 and FAM124A, 25kb downstream of LOC 101106088; OARX _120998827.1 is located between the LOC101103048 and PRR32 genes, 270kb downstream of LOC 101103048;

on the distributed chromosomes, 11 SNPs sites which are obviously related to the height of the Hu sheep or the chest circumference at the whole genome level are distributed on 9 chromosomes, and are respectively 1 site of the No.1 chromosome, 1 site of the No.2 chromosome, 2 sites of the No.6 chromosome, 1 site of the No.8 chromosome, 1 site of the No.9 chromosome, 1 site of the No.10 chromosome, 1 site of the No. 15 chromosome, 1 site of the No. 17 chromosome and 2 sites of the No. 27 chromosome.

Tables 2-7 alignment results (GLM) of 500bp sequences at the upstream and downstream of 11 SNPs significantly associated with the height or bust of Hu sheep

2.4 discussion

2.4.1 selection of phenotypic traits

The results of the correlation analysis of the height, length, chest circumference, tail length and tail width of the Hu mutton ethnic core group individuals and the 6-month-old body weight show that the body size and the body weight have obvious positive correlation, and the results are consistent with the research results and the conditions of production practice. For example, in the case of high shijin, the height, chest circumference and the like of the sheep with wave are significantly related to the body weight^[35]Zhao Zi Gui discovers that there is an extremely significant linear regression relationship between body weight and body size traits such as body height, chest circumference, and body length^[36]. In the process of sheep raising, the subsequent production performance can be indirectly predicted by measuring the body size and the body weight of the animals, and the aim of early breeding is fulfilled.

At present, the GWAS research of sheep is mostly focused on reproductive traits^[37,38]Meat, meat and foodQualitative character^[39]And milk production traits^[40]Disease resistance^[41]Quality of wool^[42]Hair color, hair color^[19,28,43]Angle type^[44,45]And tail type^[46,47]Etc. the GWAS analysis related to body size character is not much studied, but Al-Mamun et Al also found that NCAPG and LCORL genes on sheep chromosome 6 can influence body size index of Australian merino sheep^[6]. In the research, GWAS analysis is carried out on 5 individual size indexes of height, body length, chest circumference, tail length and tail width of the Hu sheep, the indexes are closely related to the growth traits and variety characteristics of the Hu sheep, the research result is helpful for obtaining candidate genes related to the Hu sheep body size traits, related SNP can also be used for early breeding of core group individuals of the Hu sheep meat line, and the method has important significance for improving the Hu sheep meat performance.

2.4.2 population stratification

Population stratification causes a false positive phenomenon generated by GWAS analysis, and the phenomenon is not related to a significantly related trait but appears as a false association although the phenomenon appears to be significantly related^[48]. Population stratification is therefore also considered to be one of the most important reasons for influencing GWAS results^[49]. Population stratification interweaves with false positives, making many correlations indistinguishable, and also affecting inter-population validation^[50]。

In previous studies, the influence of GWAS on the results of the analysis was studied in both bovine and porcine GWAS studies^[51]. From the sampling population, since the test sheep are from a semi-open core group constructed in the same test field, theoretically, no population stratification should be present. The actual values of the Q-Q plot of the body height and chest circumference GLM and MLM are substantially coincident with the prediction line, and the Q-Q plot of GLM and MLM are substantially coincident. According to the conditions of the significant association sites obtained by combining the GLM and MLM analysis results, the chest circumference GLM and MLM analysis results are completely consistent, the difference between the body height GLM and MLM is small, the GLM has only 5 significant sites more than the MLM, and the results of the association analysis of the GLM and the MLM are reliable, and the test population is corrected without population stratification.

2.4.3 Gene annotation

Since there are 11 sites of significant SNPs we analyzed, spread over 9 chromosomes. After gene annotation is carried out on the 11 SNPs (the gene annotation result is mainly based on the sequencing and splicing result of an Oar _ v4.0 whole genome shotgun method in an NCBI database at 11/5/2018), we obtain some candidate genes possibly related to the height and chest circumference of the Hu sheep.

2.4.3.1 CADM2(cell adhesion molecule 2)

OAR1_164254640.1 is located in intron 7 of the CADM2(Gene ID:101120371) Gene. Cell adhesion molecule (CADM) consists of a family of proteins whose functions include maintaining cell polarity and tumor suppression, and low expression of CADM2 gene expression can be observed in several cancers including hepatocellular carcinoma (HCC)^[52]. The CADM2 gene is repressed in human renal cell carcinoma by DNA promoter methylation and/or loss of heterozygosity. Acts as a novel tumor inhibitor and may be a potential therapeutic target for human renal cell carcinoma^[53]. A genome-wide association study using microsatellites (561 cases and 561 controls) in the Japanese population has also identified CADM2, a candidate gene for psoriasis^[54]. The learners identified CADM2 as a potent regulator of systemic energy homeostasis and decreased CADM2 expression reversed a number of metabolic syndrome-associated symptoms including obesity, impaired insulin resistance and glucose homeostasis^[55]。

2.4.3.2 RNF217(ring finger protein 217)

s11279.1 is located in intron 3 of the RNF217(Gene ID: 101119141) Gene. Human RNF217 encodes a highly conserved RING finger protein, expressed predominantly in testis and skeletal muscle with different splice variants. One of the members of the RBR ubiquitin ligase subfamily of RNF217 containing the TM domain, all 5 Transmembrane (TM) -containing RBR E3 ligases comprising RNF144A and RNF144B, RNF19A/Dorfin, RNF19B and RNF217 (also known as IBRDC1) have an upper layer structure of RBR-TM (GXXKG). High expression of certain human leukemic RNF217, indicating that dysregulation of this gene may be associated with leukemic development^[56]. Furthermore, mutations in the motif GXXXG of RNF217 protein have also been found in human gastric cancer, gastric adenocarcinoma and liver cancer^[57]。

2.4.3.3 SAMD12(sterile alpha motif domain containing 12)

s10347.1 is located in intron 3 of SAMD12(Gene ID: 101114621). SAMD12 is one of SAM (SAM) family members, SAMD12 may exert functions and actions affecting male sterility mainly through SAM domain^[58]. Studies have shown that abnormal amplification of TTTCA and TTTTA repeats in intron 4 of SAMD12 is associated with myoclonic epilepsy in adults, with TTTCA and TTTTA repeats estimated in SAMD12 in the range of 2.2-18.4 kb, corresponding to 440-3680 repeat units^[59]。

2.4.3.4 DDX10(DEAD-box helicase 10)

OAR15_18440393.1 is located in intron 8 of DDX10(Gene ID: 101106358). DDX10 coding RNA helicase, relating to ovarian cancer, liver cancer and acute myeloid leukemia^[60]And the like.

2.4.3.5 CAPN6(calpain-6)

OARX _76354330.1 is located in intron 2 of CAPN6(Gene ID: 101110122). Calpain 6(CAPN6) is one of the calcium-dependent intracellular non-lysosomal proteases. CAPN6 is a non-proteolytic protease with microtubule-binding and stabilizing activity, and can promote cytoskeletal structure and microtubule stability in osteoclasts^[61]. CAPN6 acts as a potential regulator of RAC1 activity by interacting with the Rho guanine nucleotide exchange factor GEF-H1 to control lipid membrane formation and cellular motility^[62]. CAPN6 expressed in embryonic tissue can be used as microtubule stabilizing protein and participate in microtubule dynamics and cytoskeletal tissue regulation^[63]. During embryogenesis, CAPN6 mRNA expression was observed during skeletal and myocardial development, and the gene was also expressed in specific cells of the lung, kidney and placenta, as well as in various epithelial cell types^[64]。

Through gene annotation, 11 SNPs (single nucleotide polymorphism) sites which are significantly related to the height or chest circumference of the Hu sheep at the whole genome level are distributed on 9 chromosomes, wherein 1 site of each of chromosomes 1, 2, 8, 9, 10, 15 and 17 is significantly related to the core population size traits for the Hu sheep meat, and 2 sites of each of chromosomes 6 and 27 are significantly related to the core population size traits for the Hu sheep meatThe body size traits are significantly related. Researchers have found multiple body weights with sheep on this chromosome^[6]Multiple tyre property^[65]And yield of wool^[66]And the like, and candidate genes related to the production traits. Sheep chromosome 27 is a sex chromosome, and SNPs on the chromosome are mostly related to fat deposition, tail fat fullness and tail type of sheep^[67-69]. The existing research results show that chromosomes 6 and 27 have important influence on the production traits and germplasm characteristics of sheep, so that 4 SNPs discovered on the chromosomes possibly have important significance on breeding of Hu mutton line varieties.

In addition, the results of gene annotation showed that 6 SNPs among 11 SNPs sites significantly associated with the height of the body or chest circumference of the hu sheep at the genome-wide level were located in intergenic regions, and 5 SNPs already located in the gene were also distributed in introns of the gene, so that the way these SNPs affect the body size traits of the hu sheep still needs further research and analysis.

Example 2 group validation of Hu sheep body high trait related SNPs

By using millions of SNPs in a genome, GWAS can perform molecular genetic marker control analysis or correlation analysis on the whole genome level, and find out genetic variation affecting complex traits through comparison. GWAS studies are currently mainly conducted using a two-stage or multi-stage method. In the first stage, control analysis of different groups is carried out on the basis of the SNP chip covering the whole genome range, and a small amount of positive SNP at the genome level is obtained after statistical analysis of different mathematical models. The results of genotyping and data model analysis are validated in the second or subsequent stages using a larger sample. Such a design is required to ensure the sensitivity and specificity of screening for SNPs associated with a target trait in the first stage, minimize false positives or false negatives of the analysis, and use a sufficiently large sample group for genotyping validation in the second stage.

With the development of genomics research and gene chip technology, a large number of genetic variations associated with complex traits are discovered and identified by a GWAS method, and the method is widely applied to screening and identifying major genes of important economic traits of agricultural animals. By utilizing an ovine SNP50 Genotyping foundry chip and by comparatively analyzing G1 generation and G2 generation groups of the core group of the new group for the Hu sheep meat, which is constructed by the inventor, 11 SNPs which obviously influence the height and the chest circumference of the Hu sheep at the genome level are discovered. Whether these SNPs can be used for early breeding of Hu mutton family core group individuals still needs repeated verification of large groups.

At present, no reports of economic traits GWAS of Hu sheep exist, 11 base sequences of 500bp at the upper and lower reaches of SNPs which significantly affect the height and chest circumference of the Hu sheep at the genome level are downloaded, PCR amplification primers are designed, 11 SNPs for the Hu sheep meat line core group G3 generation individuals constructed by Hangzhou huge agriculture development Limited company are taken as research objects, genotyping and population genetics analysis of 11 SNPs for the Hu sheep meat line core group G3 generation 203 individuals and correlation analysis of SNPs and Hu sheep meat line core group ruler traits (height and chest circumference) and weight traits are carried out, and the GWAS verification result of 11 sites can be used for subsequent functional gene verification, explanation of molecular genetic mechanisms of trait variation and establishment of important bases for Hu sheep meat trait molecular marker assisted breeding.

3.1 Collection of materials and samples

3.1.1 selection of animal populations

The experimental animals are from Hu mutton system core group G3 individuals constructed by Hangzhou huge agriculture development limited company. And carrying out SNP genotype analysis on different sites of the individual of the 203 Hu sheep, and carrying out population genetics analysis on the corresponding sites.

3.1.2 blood sample Collection and preservation

See example 1 for details.

3.2 configuration of Primary reagents, solutions and test instrumentation

See example 1 for details.

3.3 test methods

3.3.1 descriptive statistical analysis of phenotypes

The SPSS20 software was used to perform preliminary statistical analyses including minimum, maximum, mean and standard deviation on the height, bust, birth weight, weaning weight, 6 month old weight and adult weight phenotype values of the hu mutton panel core group G3 individuals.

3.3.2 genomic DNA extraction and quality control

See example 1 for details.

3.3.3 PCR amplification and detection

(1) Primer design and Synthesis

By taking 11 SNPs sites which are screened out in the example 1 and are obviously related to the Hu sheep body height and chest circumference traits as the center, downloading sequences of 500bp respectively at the upstream and downstream of 11 SNPs in a UCSC database, and designing amplification primers at the upstream and downstream of the SNPs. 2F and 2R designed for OARX _76354330.1 site, 11F and 11R designed for s44173.1 site and 12F and 12R designed for s55179.1 site have obvious non-specific amplification in a pre-test, and the genotype conditions of 8 sites comprising OAR6_95218086.1, OAR15_18440393.1, s10476.1, OARX _120998827.1, OAR1_164254640.1, s10347.1, s11279.1, OAR6_90337552.1, s44173.1 and s55179.1 can only be verified due to the fact that the sequence of primers at each site cannot be revised, and the sequences of primers at each site are detailed in tables 3-1

TABLE 3-1 primer design results

(2) PCR amplification system for Hu sheep body high-character related SNP detection

Amplification was performed using a 30. mu.L PCR reaction program: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃ after the reaction is finished. The amounts of the specific reactants used are shown in Table 3-2.

TABLE 3-2 PCR amplification system for Hu sheep body high trait related SNP detection

(3) Detecting the fragment of interest and sequencing

And uniformly mixing 5 mu L of PCR product with 1 mu L of 6 XOrangeLoading Buffer, then spotting the mixture on 1.5% agarose gel, marking the mixture by using DL2000plus as a Marker, performing electrophoresis at 200V for about 15min, and after EB (Electron beam) dyeing for 10min, observing whether the PCR product has an amplification band, recording, photographing and storing. Each SNP site of each sample was subjected to direct sequencing of the upstream and downstream primers of the PCR amplification product.

3.3.4 sequence analysis

And (3) analyzing the positive and negative phase sequencing peak images of each individual by using the Mutation Surveyor 5.02 software, and determining the Mutation sites and Mutation modes of the amplification product sequencing results at different sites of each sample. And (4) performing secondary sequencing on the abnormal sequencing result or the peak-free map or the abnormal peak map until a stable and consistent sequencing result is obtained.

3.3.5 statistical analysis

3.3.5.1 Gene frequency and genotype frequency

The gene frequency and genotype frequency of each SNPs were calculated using PopGen32 software.

First, the frequency of a certain genotype in a population is equal to the number of genotype individuals/the number of individuals in the population x 100%;

② the frequency of a certain gene in the body is equal to the homozygous genotype frequency of the character +1/2 multiplied by the heterozygous genotype frequency.

3.3.5.2 Hardy-Weinberg equilibrium assay

Exercise chi²And identifying whether the genotype frequency and the gene frequency of each variety meet Hardy-Weinberg balance.

Wherein: m represents the number of genotypes; fi represents the number of individuals of the ith genotype observed; n represents the total number of samples; p is a radical of_iIndicates the theoretical genotype frequency for the ith genotype.

3.3.5.3 Polymorphic Information Content (PIC)

Wherein: pi and Pj are the ith and jth allele frequencies, respectively; n is the allelic factor.

3.3.5.4 locus heterozygosity (H)

Wherein: pi is the gene frequency; m is the allelic factor; r is the number of sites; h is the average heterozygosity.

3.3.5.5 Shannon Information Content (SIC)

SIC＝-ClogP_i

Wherein: p_iC is a constant, is the frequency of the ith allele in the population.

3.3.5.6 Association analysis

SNPs associated with the core population size trait phenotype of the hu mutton line were mined based on the General Linear Model (GLM) using SPSS20 software.

Since all individuals analyzed were sheep from the same feedlot under the same feeding environment and management conditions, field effects were not included in the data modeling.

The concrete model is as follows: y ═ X β + e

Wherein the content of the first and second substances,

y: the core population ruler character and the body weight character phenotype value vector of the Hu mutton line;

beta: fixed effect vectors such as phenotypic mean, SNP, etc.;

e: a residual effect vector;

and X is a correlation matrix of beta.

When Y is the vector of the birth weight phenotype value of the Hu mutton family core group, the analysis is carried out according to the following model:

Y＝Xβ+Sα+e；

wherein:

y: the core population ruler character phenotype value vector of the Hu mutton lineage;

α: fixed effect vector of sibling number;

beta: an SNP effect vector;

e: a residual effect vector;

x, S are the incidence matrixes of beta and alpha respectively.

3.4 results and analysis

3.4.1 descriptive statistical analysis of phenotypes

The description statistics of the body size and weight trait related indexes of the Hu mutton family core group G3 generation individuals (Xiaoshan) are shown in tables 3-3, and the related analysis results are shown in tables 3-4.

The correlation analysis result shows that the sibling number of the Hu mutton family core group G3 generation individual is extremely obviously and negatively correlated with the birth weight (r is 0.640, P is less than 0.01) and the weaning weight (r is-0.164, P is less than 0.05); the body height and the body weight of the whole year are in extremely obvious positive correlation (r is 0.281, P is less than 0.01), and the chest circumference and the body weight of the whole year are in extremely obvious positive correlation (r is 0.721, P is less than 0.01). In addition, body height and chest circumference are also in extremely obvious positive correlation (r is 0.186, and P is less than 0.01).

TABLE 3-3 descriptive statistical analysis of the somatic size and weight traits of Hu mutton family core group G3 individuals (Xiaoshan)

TABLE 3-4 correlation analysis of body size traits and body weights of Hu mutton family core group G3 generation individuals (Xiaoshan)

3.4.2 PCR amplification of Hu sheep body high-character related SNPs detection primers

As can be seen from FIG. 3, the PCR products of the primers have high brightness, single band and no non-specific amplification, and the actual PCR products are consistent with the estimated PCR amplification products in size, so that the subsequent direct sequencing test of the PCR-products can be performed.

3.4.3 Hu sheep body high character related SNPs site amplification product mutation analysis

The results of analysis of mutation of the amplification products of 8 Hu sheep body size trait-related SNPs loci are shown in tables 3-5.

TABLE 3-5 Hu sheep body high character related SNPs locus, mutation type and mode

In addition to the mutation detected in the amplification products of the primer pairs 9F and 9R corresponding only to the SNP site of interest, the amplification products of the remaining 7 primer pairs detected other mutations near the SNP site of interest.

Wherein, 2 mutation sites are detected by 3 pairs of primers of 3F and 3R (the length of an amplification product is 385bp), 4F and 4R (the length of the amplification product is 418bp), 6F and 6R (the length of the amplification product is 500 bp); the amplification products of 5F and 5R are 3 mutation sites (the length of the amplification product is 269 bp); 4 mutation sites were detected in the amplification products of 7F and 7R (the length of the amplification product is 439bp), and 8F and 8R (the length of the amplification product is 307 bp); the amplification products of 10F and 10R detected most mutations, and the amplification products with the length of 262bp found up to 9 mutation sites, and the 27 mutations and the mutation modes found in the 8 pairs of primers Hu mutton lineage core group G3 individuals are shown in tables 3-4.

The results indicate that there may be many other types of mutations to be deeply explored near the SNP used by the ovine SNP50 Genotyping beacon chip in the group of the Hu sheep.

In addition, as a more interesting result, although the mutation sites directly found by sequencing the 8 pairs of primer PCR products are 27, the mutation modes of 100bp downstream of s10476.1 in the amplified products of 5F and 5R are 2, so that the alleles of the sites are not common 2 and are changed into 3. Accordingly, 27 mutation sites, with a turnover of 82.1% (23/28) and a turnover of 17.9% (5/28) in 28 mutation patterns.

From the results of gene annotation of 27 SNPs, 22 of them were located in the intergenic region, and the other 5 were located in the gene intron, respectively OAR15_18440393.1 was located in intron 8 of DDX10 gene, s10347.1 was located in intron 3 of SAMD12 gene, s11279.1 was located in intron 3 of RNF217 gene, OAR1_164254640.1 was located in intron 7 of CADM2 gene, and OARX _76354330.1 was located in intron 2 of CAPN6 gene.

3.4.4 genetic parameters of Hu sheep body high trait related SNPs

The effective allelic factors, Shannon information content and average heterozygosity calculation results of 27 SNP sites are shown in tables 3-6.

The A → G and A → C mutation genetic parameters corresponding to 100bp downstream of s10476.1 in the PCR amplification products of the 5F and 5R primer pairs are the highest, the effective allelic factors, Shannon information content and average heterozygosity are 2.1584, 0.8353 and 0.5367 respectively, and the PCR amplification products have abundant genetic polymorphisms. While the genetic parameters of OAR6_95218086.1(C → T) in the PCR amplification products of the 3F and 3R primer pairs and the C → A mutation at 149bp downstream of OAR1_164254640.1 in the PCR amplification products of the 7F and 7R primer pairs are the lowest, the effective allele factor, Shannon information content and average heterozygosity are 1.0713, 0.1500 and 0.0666 respectively, and the genetic variation range is relatively narrow.

3.4.5 group genetics analysis of Hu sheep body high trait related SNPs

Polymorphic Information Content (PIC) is used to determine and analyze the information content expressed by a genetic marker, with high polymorphic sites when PIC >0.5, moderate polymorphic sites when 0.25< PIC <0.5, and low polymorphic sites when PIC < 0.25.

Genetic parameters of SNPs sites related to body height traits of 3-6 Hu sheep

TABLE 3-7 group genetics analysis of SNPs loci related to Hu sheep body high trait

TABLE 3-8 Hardy-Weinberg equilibrium test of SNPs sites related to Hu sheep body height traits

TABLE 3-9 Association analysis of Hu sheep body high trait related SNPs loci and body weight traits

Note: the same column of data followed by no letters, or the same letters, indicates that the difference is not significant (P > 0.05). A small letter indicates significant difference (P < 0.05). The different capitalized letters showed significant differences (P < 0.01).

In this experiment, the PIC values of 14 sites were less than 0.25, and were respectively the OAR6_95218086.1 upstream 44bpG → a mutation, OAR6_95218086.1C → T mutation, s10476.1a → C mutation, s10476.1 downstream 16bpT → C mutation, OARX _120998827.1 upstream 126bpA → C mutation, OARX _120998827.1C → T mutation, OAR1_164254640.1G → a mutation, OAR1_164254640.1 downstream 149bp C → a mutation, OAR1_164254640.1 downstream 192bp and 235bp T → C mutation, s10347.1 downstream 72bp T → C mutation, s10347.1 downstream 83bp T → C mutation, OAR6_90337552.1 upstream 41bp G → a mutation, and OAR6_90337552.1 downstream 83bp C → a mutation, and these sites were shown to be low-polymorphic. The PIC values of the remaining 13 SNPs were all between 0.25 and 0.5, and were moderately polymorphic (tables 3-7).

3.4.6 Hard-Weinberg equilibrium test of Hu sheep body height trait related SNPs

The results of the χ 2 test showed that 25 SNPs, except for the 50bpT → C mutation downstream of OAR6_90337552.1 and the 79bp C → T downstream of OAR6_90337552.1, did not reach Hard-Weinberg equilibrium (P <0.05), all were in Hard-Weinberg equilibrium (tables 3-8).

3.4.7 Association analysis of Hu sheep body high trait related SNPs and body size trait and body weight trait

The high-trait related SNPs of Hu sheep were subjected to correlation analysis with body size traits and body weight traits using the GLM model listed in "3.3.5.6 correlation analysis" (tables 3 to 9).

3.4.7.1 Association analysis of Hu sheep body high trait related SNPs and Hu sheep meat use system core group G3 generation individual body size trait

The analysis result shows that the influence of the 27 mutation sites of the 8 pairs of primers on the individual size traits of the Hu mutton lineage core group G3 generation does not reach the significance level (P is more than 0.05).

3.4.7.2 Association analysis of Hu sheep body height character related SNPs and Hu sheep meat use line core group G3 generation individual body weight character

The analysis result shows that 7 mutation sites of 4 primers in 8 primers can significantly influence the weight traits of individuals of the Hu mutton lineage core group G3 generation (P <0.05 or P < 0.01).

Mutation of 44bp G → A upstream of OAR6_95218086.1 in the amplification products of primer pair 3F and 3R significantly affects the weaning weight (P <0.05) and 6-month-old weight (P <0.05) of individuals in the core group G3 of the Hu mutton lineage. Wherein the weaning weight (17.83 +/-2.35 kg) of the mutation homozygous AA individuals is remarkably higher than that of the mutation heterozygous GA individuals (16.21 +/-1.76 kg) (P is less than 0.01), the 6-month-old weight (37.82 +/-2.64 kg) of the mutation homozygous AA individuals is remarkably higher than that of the mutation heterozygous GA individuals (36.19 +/-2.09 kg) (P is less than 0.05), and the site is remarkably related to the weaning weight and the 6-month-old weight.

Although the 16bp T → C mutation downstream of s10476.1 in the amplified products of the primer pairs 5F and 5R can significantly affect the weaning weight (P <0.05) and the 6-month-old weight (P <0.01) of the Hu sheep meat lineage core group G3 individuals, the site is significantly related to the weaning weight and the 6-month-old weight.

192bp T → C mutation downstream of OAR1_164254640.1 and 235bp T → C mutation downstream of OAR1_164254640.1 in the amplification products of the primer pairs 7F and 7R are completely linked, and the two sites can significantly influence the weaning weight and the 6-month-old weight of the Hu mutton ethnic group G3 individuals (P <0.05) and greatly influence the birth weight of the Hu mutton ethnic group G3 individuals (P <0.01) singly or in combination. The birth weight (3.59 +/-0.04 kg) of the mutant CC individual of 192bp or 235bp downstream of OAR1_164254640.1 is remarkably higher than that of the T T type individual (2.91 +/-0.33 kg) and the mutant TC individual (2.88 +/-0.32 kg) (P < 0.01); the weaning weight of mutant CC haplotypes (22.55 + -1.63 kg) was significantly higher than that of TT type individuals (17.66 + -2.36 kg), and mutant TC individuals (17.57 + -2.10 kg) (P < 0.01). The 6-month-old weight (45.90 + -0 kg) of the mutant CC haplotype was significantly greater than that of TT type individuals (37.62 + -2.65 kg) and TC type individuals (37.66 + -33 kg) (P < 0.01). The birth weight (3.59 +/-0.04 kg) of mutant homozygous haplotype CCCC individuals 192bp and 235bp downstream of OAR1_164254640.1 is remarkably higher than that of wild homozygous haplotype TTTT individuals (2.91 +/-0.33 kg) and mutant heterozygous haplotype TCTC individuals (2.88 +/-0.32 kg) (P is less than 0.01); the weaning weight of mutant homozygous CCCC haplotype individuals (22.55 +/-1.63 kg) is remarkably higher than that of wild homozygous TTTT haplotype individuals (17.66 +/-2.36 kg) and that of mutant heterozygous haplotype TCTC individuals (17.57 +/-2.10 kg) (P is less than 0.01). The 6-month-old weight (45.90 + -0 kg) of the mutant homozygous CCCC haplotype individuals is significantly greater than that of the TTTT haplotype individuals (37.62 + -2.65 kg) and the TCTC haplotype individuals (37.66 + -33 kg) (P < 0.01).

The 41bp G → A mutation upstream of OAR6_90337552.1 in the amplification products of the primer pair 10F and 10R can obviously influence the birth weight, the daily weight gain before weaning, the weaning weight and the 6-month-old weight of the Hu mutton ethnic group G3 generation individuals.

79bp C → T downstream of OAR6_90337552.1 in the amplification products of the primer pair 10F and 10R significantly affect the weaning weight and the 6-month age weight (P <0.01) of the Hu mutton germline core group G3 generation individuals, and the weaning weight (18.10 +/-2.28 kg) and the 6-month age weight (38.13 +/-2.44 kg) of the CT genotype individuals are significantly larger than the weaning weight (16.11 +/-2.31 kg) and the 6-month age weight (35.92 +/-2.79 kg) (P <0.01) of the TT individuals. Although there was no significant difference in birth weight, daily weight gain before weaning, and daily weight gain from weaning to 6 months of age among individuals of different genotypes, a trend of CT > CC > TT was presented. The 83bp C → A mutation at the downstream of OAR6_90337552.1 in the primer amplification product can also significantly influence the weight of 6 months old and the daily gain from 6 months to the week old of the Hu sheep meat use line core group G3 generation individuals (P <0.05), the weight of 6 months old of the mutation heterozygous CA individuals (38.09 +/-2.81 kg), the daily gain from 6 months old to the week old (0.23 +/-0.06 kg) are significantly larger than the weight of 6 months old of the mutation homozygous AA individuals (33.80 +/-3.91 kg) and the daily gain from 6 months old to the week old (0.31 +/-0.10 kg) (P <0.05), and the weight of 6 months old of the wild type individual CC (37.64 +/-2.51 kg, P <0.01), the daily gain from 6 months old to the week old (0.24 +/-0.06 kg, P <0.05) are significantly larger than the mutation homozygous AA.

3.5 discussion

3.5.1 study of SNP sites and mutation types

3.5.1.1 mutant forms of SNP

SNP is a DNA sequence polymorphism caused by a mutation of a single base for transversion, transition, deletion or insertion^[72-74]Usually, the frequency of variation is greater than 1%^[73]. Since SNP sites involve a single base variation, this variation may be a transition (mutations between pyrimidines and pyrimidines, purines and purines are generally referred to as transitions, i.e.

) Or else transversion (mutations between pyrimidines and purines are known as transversions, i.e.

). If the base mutation is a free mutation, the transversion should be twice that of the transition, whereas in fact the transition variation accounts for 70.1% of the base mutation and the transversion accounts for approximately 29.1% of the mutation^[74]. The reason for this is that cytosine is methylated in the C → T mutation, and spontaneously loses amino groups and is converted into thymine, which becomes a mutation hot spot.

In the core group of the Hu mutton lineage studied, we found 28 types of mutations at 27 sites in total, with a proportion of conversion mutations of 82.1% (23/28) and a transversion proportion of 17.9% (5/28), with conversion higher than that generally found in the genomeThe ratio of occurrence is lower than that of the transversion which generally occurs in the genome. In addition, theoretically, a SNP site should have 4 alleles, i.e., A, T, C, G, but generally only 2 alleles occur, and thus is called a biallelic gene. Among the 27 mutation sites, we found an interesting result that the mutation pattern 100bp downstream of s10476.1 in the amplification products of 5F and 5R was 2, namely A → G, A → C, wherein A → G accounted for 91.6% (186/203) and A → C accounted for 8.4% (17/203), and the allele at this site corresponded to 3 of A, G, C. It is not uncommon to report SNP triallelic and tetraallelic genes^[75]However, the specific mechanism of generation is not clear. The mutation of 100bp downstream of s10476.1 is located in the intergenic region, and compared with other sites, because the site presents a three-allele, the polymorphism information content is higher than that of other sites.

The neutral theory proposed by japanese scientist m.kimura (1968) states that at the molecular level, most evolutionary evolvements and most variations within species are not caused by natural selection, but by genetic drift of those mutant alleles which are neutral or near neutral to selection. I.e., mutated forms of these sites are neither detrimental nor beneficial to the survival and growth of the individual organism. Although the A → G, A → C mutation of 100bp downstream of s10476.1 is the SNP site screened by GWAS and related to the size traits of individuals of the Hu mutton generation core group G1 and G2 generation, in the group verification of individuals of the Hu mutton generation core group G3 generation, the GLM analysis result of the site shows that the SNP does not affect the size and weight traits of individuals of the Hu mutton generation core group G3 generation, so that the SNP site of the three alleles is considered to be more prone to neutral mutation.

3.5.1.2 SNP mutation position distribution

SNPs are often artificially divided into two forms: one is a functional mutation, occurring mainly in the coding region of a gene. The other is single base mutation, mainly in non-coding regions. Due to selection pressure, the distribution of SNPs in the genome is often heterogeneous, with mutations at non-coding regions being much more frequent than at coding regions. Taking human genome SNP as an example, in 100 ten thousand SNPs, about 24-40 ten thousand SNPs exist in a coding region, about 50 ten thousand SNPs exist in a non-coding region, but about 20% -30% of coding region mutations can cause non-synonymous coding SNPs, thereby causing functional changes of proteins.

The verification result of SNPs sites obtained by GWAS analysis results in the test shows that only 4 pairs of primers and 7 sites in the designed primers can obviously influence the weight index of Hu mutton use system core group G3 generation individuals, wherein 5 SNPs are located in a gene spacer region, and 2 SNPs are located in an intron of a gene.

3.5.2 genetic diversity analysis of SNPs

It is widely accepted by modern geneticists that genetic variation is a prerequisite for an organism to adapt to environmental changes^[76]. The existence of genetic diversity greatly improves the viability of species. The index for measuring the genetic variation size in the population not only comprises the genetic heterozygosity, but also comprises the polymorphic information content. Heterozygosity can reflect the degree of genetic variation of a population at a plurality of gene loci, and the larger the value of the average heterozygosity is, the larger the degree of genetic variation occurring in the population is, and the smaller the degree is vice versa. Polymorphic information content is used to describe the information content expressed by a genetic marker.

Through the analysis of genetic variation and population heterozygosity of the test population, the PIC of the SNP locus of the Hu sheep is found to be moderate polymorphism except 14 shown as low polymorphism. This indicates that the population has a higher genetic diversity.

In Hu mutton germline core group G3 generation individual primer pair 10F and 10R, 50bp T → C (P) at downstream of OAR6_90337552.1<0.05) and 79bp C → T (P) downstream of OAR6_90337552.1<0.01) deviate significantly or very significantly from the Hardy-Weinberg equilibrium, the observed numbers of both site-mutant heterozygotes exceed their theoretical numbers, while the observed numbers of wild-type individuals and mutant homozygotes are smaller than the corresponding theoretical numbers. The lack of Hardy-Weinberg balance in the population may be due to changes in gene frequency caused by the presence of other factors such as artificial selection or close relatives^[77]。

The GWAS analysis result of 8 pairs of primer SNP sites shows that the site 79bp C → T at the downstream of OAR6_90337552.1 can greatly influence the weaning weight and the 6-month-old weight of the Hu mutton ethnic group core group G3 individuals. The weaning weight (18.10 +/-2.28 kg) and the 6-month-old weight (38.13 +/-2.44 kg) of the mutant heterozygous CT individuals are remarkably greater than those of the mutant homozygous TT individuals (16.11 +/-2.31 kg) and 6-month-old weight (35.92 +/-2.79 kg) (P <0.01), and in addition, the weaning weight and the 6-month-old weight of the CT individuals are also higher than those of the wild CC individuals (17.41 +/-2.29 kg) and 6-month-old weight (37.40 +/-2.71 kg), but the weaning weight and the 6-month-old weight are not remarkably different (P > 0.05). As the site is very obviously related to the weaning weight and the 6-month-old weight of the Hu sheep core group G3 generation individuals, the mutant heterozygous individuals are selected due to high weaning weight and 6-month-old weight in the artificial breeding process of the Hu sheep meat strain, and finally the mutant heterozygous genotype frequency is obviously increased. The result of Hardy-Weinberg equilibrium detection at this site can also be used to confirm this conclusion. The result not only shows that the breeding work of the Hu mutton line in the sheep farm is very successful, but also shows the wide prospect of the molecular marker-assisted breeding work of the Hu mutton line breeding for the early breeding of the subsequent individuals.

3.5.3 population genetic variation and differentiation

Heterozygosity (H), i.e., the frequency of heterozygotes at the site to be detected in the population, indicates that Ne and H are greater if PIC is greater, and that a greater value of these parameters indicates a high variability in the population at the point of change. The research result shows that the genetic parameters of the downstream 100bp A → G, A → C of s10476.1 are the highest, and the genetic diversity is higher.

3.5.4 SNPs related to individual body weight traits of Hu mutton family core group G3 generation

3.5.4.1 weight-related

The analysis result of 44bp G → A mutation GLM upstream of OAR6_95218086.1 in the 3F +3R amplification product shows that the SNP is remarkably related to the weaning weight and the 6-month-old weight of the Hu mutton ethnic group core group G3 individuals (P<0.05). This site is located in the GC and NPFFR2 intergenic region, 209kb downstream of the GC gene. NPFFR2 belongs to RF aminopeptide family members, NPFF plays an important role in fluid balance, pain, food intake and cardiovascular function^[76]. Therefore, mutation at this site may be related to the NPFF groupThe mode of action of these compounds, due to their functional relevance in the regulation of food intake, is yet to be confirmed by further studies.

(ii) weaning weight of individuals of 16bp T → C mutation downstream of 5F +5R amplification product s10476.1 and Hu mutton germline core group G3 (P)<0.05) and 6 months of age are significantly related (P)<0.01). This site is located in the SLC38A11 and COBALL 1 intergenic region, 93kb downstream of SLC38A 11. The mutant heterozygote individuals and the mutant homozygote individuals have no significant difference between the weaning weight and the 6-month-old weight (P)>0.05) but all are numerically higher than the wild type individuals. Studies have shown that COBALL 1 is involved in chronic lymphocytic leukemia and B cell development^[77]. Therefore, whether the individuals of the mutation heterozygote type and the mutation homozygote type show higher B cell development level and more complete body immunity, so that the disease resistance is stronger, the phenotype of diarrhea prevention and the like is shown, and finally the higher weaning weight and the 6-month-old weight are reached, of course, the inference also needs the verification of a subsequent large population.

7F +7R amplification product OAR1_164254640.1 downstream 192bp T → C and downstream 235bp T → C, alone or in combination, and the primary weight of Hu mutton germline core group G3 individual (P)<0.01), weaning weight (P)<0.05) and 6 months old body weight (P)<0.05) significantly correlated. This site is located at intron 7 of the CADM2 gene. CADM2 is cell adhesion molecule 2, is one of cell adhesion molecule family members, and can maintain cell polarity and inhibit tumor^[77]And potent modulators of systemic energy homeostasis. It has been found that reduction of CADM2 expression can reverse a variety of symptoms associated with metabolic syndrome including obesity, impaired insulin resistance and glucose homeostasis^[55]. Our study results show that the birth weight of mutant CC individuals (3.59 + -0.04 kg) is significantly higher than that of T T type individuals (2.91 + -0.33 kg) and mutant TC individuals (2.88 + -0.32 kg) (P)<0.01); the weaning weight (22.55 +/-1.63 kg) of the mutant CC haploid individual is remarkably higher than that of the TT type individual (17.66 +/-2.36 kg) and that of the mutant TC individual (17.57 +/-2.10 kg) (P)<0.01). The 6-month-old weight (45.90 +/-0 kg) of the mutant CC haploid individual is remarkably greater than that of TT type individual (37.62 +/-2.65 kg) and TC type individual (37.66 +/-33 kg) (P<0.01). The birth weight (3.59 plus or minus 0.04kg) of the mutant homozygous haplotype CCCC individual is remarkably higher than that of the wild homozygous singlePloidy TTTT individuals (2.91 + -0.33 kg) and mutant heterozygous haplotype TCTC individuals (2.88 + -0.32 kg) (P)<0.01); the weaning weight (22.55 +/-1.63 kg) of the mutant homozygous CCCC haplotype individual is remarkably higher than that of the wild homozygous TTTT haplotype individual (17.66 +/-2.36 kg) and that of the mutant heterozygous haplotype TCTC individual (17.57 +/-2.10 kg) (P)<0.01). The 6-month-old weight (45.90 +/-0 kg) of the mutant homozygous CCCC haplotype individual is remarkably larger than that of the TTTT haplotype individual (37.62 +/-2.65 kg) and the TCTC haplotype individual (37.66 +/-2.33 kg) (P)<0.01). But the genotype frequency of the site mutation homozygous haplotype individual is still low, and the fact that the targeted breeding of the site mutation homozygous haplotype individual can achieve better breeding effect in the meat character breeding aspect is shown.

The 41bp G → A mutation upstream of the amplification product OAR6_90337552.1 of 10F +10R can significantly influence the birth weight, the daily weight gain before weaning, the weaning weight and the 6-month-old body weight of the Hu mutton family core group G3 individuals. Downstream 79bp C → T of OAR6_90337552.1 is significantly related to the weaning weight and 6-month-old weight of Hu sheep meat use generation G3 individuals, and downstream 83bp C → A of OAR6_90337552.1 is significantly related to 6-month-old weight and 6-month-old to week-old daily weight of Hu sheep meat use generation G3 individuals. These 3 SNPs are all located between EPHA5 and LOC101120496 genes, 1446kb downstream of EPHA 5. EPHA5 is a receptor tyrosine kinase and a novel regulator of DNA damage repair. Research shows that the gene not only can regulate the DNA damage repair process induced by ionizing radiation, but also is a functional target spot of lung cancer^[78]Also plays a key role in the process of embryo development^[79]. Therefore, the conclusion that whether 3 sites detected by the amplification product of 10F +10R can influence the growth and development of animals and further influence the meat performance of Hu sheep through the EPHA5 gene still needs further experimental verification.

3.5.4.2 GWAS analysis result of core population size character related SNPs of Hu mutton system and significance of population verification thereof

In the production practice, the body size and the weight of the livestock and the poultry are measured, and the indexes of the later meat production performance, the milk production performance and the like of the livestock and the poultry are indirectly predicted, so that the breeding period can be shortened, and the early breeding is realized. The body size and weight of the livestock and poultry are measured by a plurality of scholarsSubject related studies. For example, Wu Zhaofu (2014) found that the body weight of a long-tail cock on the dam can be better estimated by finding that the shank is long and the chest circumference is long^[80]. The stone Biru (2010) finds that the body weight and the breast angle of the compound cocks of the black-bone chicken from the north are extremely obviously related, the breast angle is a body size index determined by the meat type chicken, and the index can be used for breeding chicken strains^[81]. The Russian koji and the like establish a linear regression model of body size and weight data of sheep in 2012, the difference between a predicted value obtained through the data model and an actual measured value is not significant, the weight change condition of ewes in a group can be estimated through body size character measurement indexes in actual breeding work, and the Russian koji and the like think that in the strain breeding and production research of black group adult ewes of Jianyang big-ear sheep, the chest circumference should be used as a first breeding index to comprehensively plan the index of body length, so that a relatively ideal breeding and propagation effect is obtained^[82]. In view of the correlation between the size trait and the weight trait, population verification is carried out on 8 SNPs obtained by GWAS analysis in example 1, and SNP which can be used for early breeding of the size trait or the weight trait of a lake sheep in a large population range is expected to be obtained.

The validation results showed that the sites detected by the chips were ubiquitous in the population, and the remaining 5 SNPs were moderately polymorphic, except for OAR1_164254640.1(G → A), s10476.1(A → C), and OAR6_95218086.1(C → T), which were low-grade polymorphic. However, the correlation analysis results of these sites with the body size and body weight data of the core group G3 generation for hu sheep showed that these sites did not affect the body size trait of the core group G3 generation for hu sheep, and that these 8 SNP sites also did not significantly affect the body weight trait of the core group G3 generation for hu sheep. Purely from the experimental results, it seems that the results obtained from the GWAS analysis are of no practical significance.

However, comparing the data of the two mathematical models and the source thereof, we have found the following problems:

(1) the stability and accuracy of the sheep body ruler index measurement in different batches of tests can not be completely guaranteed.

When Hu sheep carries out body size measurement, the tested individual needs to be pulled to a flat ground to be stable, and the tester is fixed to reduce test errors. Example 1 the sheep used for GWAS analysis were all from the core group of the hu mutton department of the hu cooperative breeding specialty of tao lake tai, hu, and the measurement personnel were the same person, so the measurement error was small. However, the experimental sheep used in the third chapter of group verification comes from the core group of the hu mutton system constructed by the Hangzhou huge agricultural development Co., Ltd, and the number of measurement personnel is many, so that the artificial error of the measurement of the body size indexes of the lake sheep in the batch is large. Different from body size measurement, the body weight measurement has higher requirements on test sheep and a measurer, the requirements on environment and measurement are lower, and the test error is extremely small, so that the Hu sheep body weight data can be collected more easily in the actual production practice, and the accuracy of the data can be ensured more.

(2) The core population ruler data of the Hu mutton lineage is obviously related to the weight data.

Research shows that the growth rate character and the body size character are closely related to each other mainly through 6-month-old heaviness, chest circumference, body length and the like^[83]. The correlation coefficient of the body height and the body weight of 6 months old sheep in GWAS analysis of Hu Tai lake sheep breeding professional cooperative society in Hu State can reach 0.839 (P)<0.01), the correlation coefficient between the chest circumference and the body weight at 6 months of age is higher to 0.893 (P)<0.01). The Hu sheep used in the group verification of Hangzhou huge agricultural development limited company has the body height of the whole year and the body weight of 6 months (r is-0.147, P)<0.05), adult (r ═ 0.164, P)<0.05) but exhibits a significant negative correlation), the week old chest circumference and 6 months old body weight (r 0.281, P)<0.01), adult (r ═ 0.712, P)<0.01) all achieved a very significant positive correlation.

Because the size indexes and weight indexes of a population used in GWAS analysis have extremely obvious correlation, SNP sites which are obtained by GWAS analysis and are obviously correlated with the size characters at the whole genome level may have higher correlation with the weight characters, so that in subsequent population verification work, the population verification work shows that the sites are correlated with the birth weight, the weaning weight, the 6-month-old weight and the adult weight of the Hu sheep to different degrees.

Because the test management level and conditions of the test sheep are not completely the same among different farms, and the breeding directions are not completely the same, whether the related analysis result is caused by measurement errors or the breeding directions cause that the body size and weight data of two meat system core groups in different generations still need to be compared and analyzed. In addition, although the correlation analysis of the two fields clearly defines the extremely significant correlation between the chest circumference of the Hu sheep and the adult weight of the Hu sheep, unfortunately, when group verification is carried out, only one SNP site which is significantly correlated with the chest circumference at the whole genome level is obtained in GWAS analysis, OARX _76354330.1, and a primer of a corresponding single amplification product cannot be designed, so that the verification work of the site cannot be carried out, and therefore, whether the amplification primer and the amplification system of the corresponding site can be optimized in the subsequent work or not can be carried out, and the verification information of the site is supplemented, so that the reliability of the GWAS result is an important supplement.

(3) Role of GWAS analysis.

According to our validation results, only 1 of 8 pairs of primers used by us only detected the corresponding site to be detected in the target amplification region, and the other 7 pairs detected 2 SNPs or more. Furthermore, 27 detected SNP loci (containing 19 new SNPs loci) are found to have significant or extremely significant correlation with the weight traits of the Hu sheep in later verification, and the gene frequency and genotype frequency of the loci more or less show the correlation with the Hu sheep meat line core group breeding work performed by the inventor. The screening-out ratio of the functional SNPs sites as high as more than 22.2 percent is inseparable with the previous massive GWAS analysis work. These interesting sites could be included in our field of analysis, precisely because of their close distance to the positive sites obtained by GWAS analysis.

Therefore, we consider GWAS an important tool for candidate functional gene and candidate functional SNP screening, which can provide a landmark-like effect to direct the research direction for later validation work without inundating researchers with sequence information that is too expensive as in the sea. Of course, if SNP detection chips with higher density can be developed, the reliability of the result will be greatly improved. GWAS combined with a group verification result shows that GWAS is an important tool for screening candidate functional genes and candidate functional SNPs, SNPs which are obviously related to Hu sheep meat performance can be screened near SNP sites related to the screened size traits at a higher probability, and the SNPs can provide a road sign-like effect for later verification work, so that more valuable candidate functional SNPs are found and are used for early breeding of core group individuals of a subsequent Hu sheep meat line.

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Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> molecular marker comprising SNP7-1 and application thereof in assisted breeding of Hu sheep

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 385

<212> DNA

<213> CP011891.1

<220>

<221> mutation

<222> (（209）)..(（209）)

<223> r is g or a

<400> 1

gtcctacttg tggtctctgt ttcccctcaa tgggccttac aaatgctaat acatgtttaa 60

gggtaattaa cattacttag gttgattaac atggaaatta aaaagaaaaa aagctgaaga 120

gaaagttgag acttcttccc tacatggtag aaatttaaag ggactcattc ccaaaagaca 180

aatctataga tgtttattaa agaaatggra agagtgagac agacattagg attaaagcaa 240

caggttttga tacggtgcta cctaagatta gatgggccaa catgaaaggg gcccaaacaa 300

atccactcta catttatagt acagaactag ggtaaatatt taaaaggtta taaaaaattg 360

cactgagata ttaggccagc aacac 385

<210> 2

<211> 269

<212> DNA

<213> CP011887.1

<220>

<221> mutation

<222> (（129）)..(（129）)

<223> y is t or c

<400> 2

gtctgatgga tgtgctgtag tttctaggcc aactgagttc acagtggtca agataattat 60

aaagccattg aattgctttt cttctttgtg ctcacgtggt tgcacgttac acacaaagtg 120

gaagtggcyg agagaggagc agtctcctcc tcctttttgt caggtatccc agctccagga 180

caatcaggaa atagacaggg tccctatctt acaggacagc ctctgtgttt tgcggtctca 240

gcagctctcg ttattctcag gtgtgcatg 269

<210> 3

<211> 439

<212> DNA

<213> CP011886.1

<220>

<221> mutation

<222> (（303）)..(（303）)

<223> y is t or c

<220>

<221> mutation

<222> (（373）)..(（373）)

<223> y is t or c

<400> 3

gctgggatga aagagattaa ccattagcta gtttggcaga aagctaaaga agagatctgg 60

aaggttttaa gaaaccaaga ctaagtgatt aacagggttc atgagtggtt gataatccag 120

ggtttgcatt gtataaagcc tcgttctagc atgtttccca gttcccttat gctagtctta 180

ttttaaccac aaaattaccc tgttgtaaaa ggtttacaaa gacccttaga ctgttttcat 240

tttatcgata aggacagtgg gccttgtgga ggttatttga ccttttctag tcacacagta 300

aayaaatgat agaatgggga ttacaaaaat actttaagaa attttaacat cactgatggt 360

tacattttct taytttcaca gtattttcac ttctgtcatg ttggcttttc aggataaccc 420

agggaagttg acaaggatg 439

<210> 4

<211> 262

<212> DNA

<213> CP011891.1

<220>

<221> mutation

<222> (（87）)..(（87）)

<223> r is g or a

<220>

<221> mutation

<222> (（207）)..(（207）)

<223> y is t or c

<220>

<221> mutation

<222> (（211）)..(（211）)

<223> m is c or a

<400> 4

cagtctgaat cccaattatc actaacatgt tataaatgta ggaaaacttc agttttgctc 60

acccacaaat tgttggccat cactgtrtct gggtgttaag tgagaagatg aatcttgaat 120

gcttagcaca gtgctgagaa caatatagtg aagtgagtga agtcgcttag tcatgtctga 180

ccctttgcaa ctccatggac tgtagcytac magcctcatc tgtctgtgag attttccagg 240

caatagtact ggagtggatt tc 262

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtcctacttg tggtctctgt tt 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgttgctgg cctaatatct ca 22

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtctgatgga tgtgctgtag ttt 23

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

catgcacacc tgagaataac gaga 24

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gctgggatga aagagattaa cca 23

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

catccttgtc aacttccctg ggtt 24

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cagtctgaat cccaattatc act 23

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gaaatccact ccagtactat tgcct 25

Claims (8)

1. A primer pair for detecting an SNP molecular marker related to the body weight traits of Hu sheep is disclosed, wherein the SNP molecular marker is SNP7-1 located at the 303bp site of SEQ ID NO.3 and is T or C.

2. The primer pair according to claim 1, wherein the sequences are shown as SEQ ID NO.9 and SEQ ID NO. 10.

3. A kit comprising the primer pair of claim 1 or 2.

4. The use of the primer pair of claim 1 or 2 or the kit of claim 3 for screening the weight trait of Hu sheep or molecular marker assisted breeding of Hu sheep.

5. A method for screening the body weight traits of Hu sheep comprises the following steps: extracting Hu sheep genome DNA, carrying out PCR amplification by using the primer pair of claim 1, and detecting SNP molecular markers related to the Hu sheep weight traits in an amplification product so as to screen the Hu sheep weight traits; the SNP molecular marker is SNP7-1 located at the 303bp site of SEQ ID NO.3, and is T or C.

6. The method of claim 5, wherein the primer pair is the primer pair of claim 2.

7. The method of claim 5 or 6, wherein the PCR amplification is performed by the following reaction sequence: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; the reaction system for PCR amplification is as follows:

10 XDream Taq Green buffer solution 3.0 μ L

dNTP 1.5µL

Upstream primer 0.15 mu L

Downstream primer 0.15 mu L

Dream Taq DNA polymerase 0.20 μ L

DNA template 2.0 mu L

ddH2O 23.0µL

The total system is 30.0 muL.

8. Use of the method according to any one of claims 5 to 7 in molecular marker assisted breeding of Hu sheep.

Images