Molecular marker in bovine psmc2 gene and application thereof

Abstract

The application discloses a molecular marker in a bovine PSMC2 gene and an application thereof. The molecular marker is one or more of the following: a SNP site g.44569796T>A, a SNP site g.44570274T>C, a SNP site g.44570306T>C, a SNP site g.44570978G>C and a SNP site g.44573224C>T in a bovine genome. The molecular marker or a substance for detecting bovine molecular marker single nucleotide polymorphism provided in the application can be used in bovine genetic breeding, so as to reduce the red blood cell number and hemoglobin concentration of offspring of a Tibet improved bovine, and solve a low-oxygen bottleneck problem faced by plateau bovine selection and breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a molecular marker in bovine PSMC2 gene and application thereof. BACKGROUND

[0002] When the cattle in low altitude area are moved to the low oxygen environment in high altitude area, the hemoglobin concentration will increase to increase the oxygen carrying capacity to meet the oxygen supply demand of the body. However, the high red blood cell count and hemoglobin concentration may cause the increase of blood viscosity, increase the cardiovascular risk, hinder the microcirculation and cause the high altitude polycythemia and other diseases. The Tibet improved cattle (Holstein male x Tibet female) originated in the 1980s, and the advanced cross has been carried out to F3 generation or more. Therefore, in the breeding process of the Tibet improved cattle in the plateau, the appropriate red blood cell count and hemoglobin concentration are more conducive to the adaptation of the Tibet improved cattle to the plateau environment.

[0003] The red blood cell count and hemoglobin concentration are a complex comprehensive trait, which is regulated by multiple genes. However, the reported molecular markers related to the red blood cell count and hemoglobin concentration of cattle lack more reliable sites for exploring the gene changes in the low oxygen environment. In addition, the gene-gene or gene-environment interaction also affects the physiological characteristics of the plateau cattle.

[0004] PSMC2 (26S proteasome complex) is involved in the ATP-dependent protein degradation process. In the long-term adaptive evolution mechanism research of Tibet quail in high altitude environment, it is found that PSMC2 is related to biological processes such as cell growth and apoptosis regulation, oxidative stress and immune response. At present, there is no research report on the correlation between PSMC2 gene and red blood cell count and hemoglobin concentration. Therefore, it is urgent to screen new molecular markers related to the red blood cell count and hemoglobin concentration of cattle, to provide new molecular marker resources for the molecular marker assisted selection of plateau dairy cattle, and to promote the process of breeding improvement of plateau dairy cattle. SUMMARY

[0005] The purpose of the present application is to find a molecular marker in bovine PSMC2 gene, which provides a new molecular marker for the detection of red blood cell count and hemoglobin concentration of cattle or the auxiliary molecular marker breeding in the breeding of Tibet improved cattle.

[0006] The first aspect of the present application provides a molecular marker in bovine PSMC2 gene, and the site of the molecular marker is one or more of g.44569796T>A, g.44570274T>C, g.44570306T>C, g.44570978G>C and g.44573224C>T in bovine genome.

[0007] Further, the site g.44569796T>A is a T>A base mutation at the 61st bp in the sequence shown in SEQ ID NO. 1;

[0008] The site g.44570274T>C is a T>C base mutation at the 71st bp in the sequence shown in SEQ ID NO. 2;

[0009] The site g.44570306T>C is a T>C base mutation at the 81st bp in the sequence shown in SEQ ID NO. 3;

[0010] The site g.44570978G>C is a G>C base mutation at the 91st bp in the sequence shown in SEQ ID NO. 4;

[0011] The site g.44573224C>T is a C>T base mutation at the 101st bp in the sequence shown in SEQ ID NO. 5.

[0012] The second aspect of the present application provides a method for detecting the number of red blood cells and the concentration of hemoglobin of a bovine, the steps of the method comprising:

[0013] 1) detecting the base type of at least one of the 61st bp in the sequence shown in SEQ ID NO: 1, the 71st bp in the sequence shown in SEQ ID NO: 2, the 81st bp in the sequence shown in SEQ ID NO: 3, the 91st bp in the sequence shown in SEQ ID NO: 4, and the 101st bp in the sequence shown in SEQ ID NO: 5;

[0014] 2) when the base type of the 61st bp in the sequence shown in SEQ ID NO: 1 is A, the individual bovine is a CC type individual at the 61st bp; when the base type of the 61st bp in the sequence shown in SEQ ID NO: 1 is T, the individual bovine is a TT type individual at the 61st bp or a TA type individual at the 61st bp; the number of red blood cells and the concentration of hemoglobin of an AA type individual at the 61st bp are lower than those of a TT type individual at the 61st bp and a TA type individual at the 61st bp;

[0015] when the base type of the 71st bp in the sequence shown in SEQ ID NO: 2 is C, the individual bovine is a CC type individual at the 71st bp; when the base type of the 71st bp in the sequence shown in SEQ ID NO: 2 is T, the individual bovine is a TT type individual at the 71st bp or a TC type individual at the 71st bp; the number of red blood cells and the concentration of hemoglobin of a CC type individual at the 71st bp are lower than those of a TT type individual at the 71st bp and a TC type individual at the 71st bp;

[0016] When the base type at position 81 in the sequence of SEQ ID NO: 3 is C, the individual of the cattle is a CC type individual at position 81; when the base type at position 81 in the sequence of SEQ ID NO: 3 is T, the individual of the cattle is a TT type individual at position 81 or a TC type individual at position 81; the red blood cell count and hemoglobin concentration of the CC type individual at position 81 are lower than those of the TT type individual at position 81 and the TC type individual at position 81;

[0017] When the base type at position 91 in the sequence of SEQ ID NO: 4 is C, the individual of the cattle is a CC type individual at position 91; when the base type at position 91 in the sequence of SEQ ID NO: 4 is G, the individual of the cattle is a GG type individual at position 91 or a CG type individual at position 91; the red blood cell count and hemoglobin concentration of the CC type individual at position 91 are lower than those of the GG type individual at position 91 and the CG type individual at position 91;

[0018] When the base type at position 101 in the sequence of SEQ ID NO: 5 is T, the individual of the cattle is a TT type individual at position 101; when the base type at position 101 in the sequence of SEQ ID NO: 5 is C, the individual of the cattle is a CC type individual at position 101 or a CT type individual at position 101; the red blood cell count and hemoglobin concentration of the TT type individual at position 101 are lower than those of the CC type individual at position 101 and the CT type individual at position 101.

[0019] The third aspect of the present application provides an application of a molecular marker in the subculture selection of the Tibet improved cattle, and the specific steps include:

[0020] Determining the gene type at the site of the above-mentioned molecular marker in the Tibet improved cattle, and performing subculture selection according to the gene type at the site:

[0021] Selecting an individual with A type base at position 61 in the above-mentioned SEQ ID NO. 1 sequence, and eliminating an individual with T type base at position 61; and / or,

[0022] Selecting an individual with C type base at position 71 in the above-mentioned SEQ ID NO. 2 sequence, and eliminating an individual with T type base at position 71; and / or,

[0023] Selecting an individual with C type base at position 81 in the above-mentioned SEQ ID NO. 3 sequence, and eliminating an individual with T type base at position 81; and / or,

[0024] Selecting an individual with C type base at position 91 in the above-mentioned SEQ ID NO. 4 sequence, and eliminating an individual with G type base at position 91; and / or,

[0025] Select the individual of which the base at 101bp in the above-mentioned SEQ ID NO. 5 sequence is T type, and eliminate the individual of which the base at 101bp is C type.

[0026] Further, the red blood cell number and hemoglobin concentration of the individual of which the molecular marker gene type is AA-CC-CC-CC-TT are the lowest, and the individual of which the molecular marker gene type is AA-CC-CC-CC-TT is selected in the subculture breeding of the Tibet improved cattle.

[0027] The beneficial effects of the present application are:

[0028] 1. The present application discovers the molecular markers in the PSMC2 gene of cattle associated with the red blood cell number and hemoglobin concentration, and the molecular markers include five molecular markers of SNP site g.44569796T>A, SNP site g.44570274T>C, SNP site g.44570306T>C, SNP site g.44570978G>C and SNP site g.44573224C>T; the above-mentioned five molecular markers can be used as the molecular markers of the red blood cell number and hemoglobin concentration of cattle respectively, and can be used as the molecular markers after gene-gene combination. Moreover, the hemoglobin concentration and red blood cell number of the individual of which the haplotype composed of the above-mentioned five molecular markers is AA-CC-CC-CC-TT are the lowest.

[0029] 2. The present application verifies the influence effect of the above-mentioned five molecular markers on the red blood cell number and hemoglobin concentration of cattle respectively, and can be applied to the subculture breeding of the Tibet improved cattle for reducing the red blood cell number and hemoglobin concentration, and the individual of which the gene type of SNP site g.44569796T>A is A, the gene type of SNP site g.44570274T>C is C, the gene type of SNP site g.44570306T>C is C, the gene type of SNP site g.44570978G>C is C or the gene type of SNP site g.44573224C>T is T, and the red blood cell number and hemoglobin concentration of the individual of the five kinds of gene types are lower. The individual of which the haplotype is AA-CC-CC-CC-TT can also be selected, and the red blood cell number and hemoglobin concentration of the individual of which the haplotype is AA-CC-CC-CC-TT are the lowest. Thus, the hypoxia adaptation ability of the offspring of the Tibet improved cattle is improved.

[0030] 3. The present application provides a new molecular marker for the molecular marker assisted breeding of the red blood cell number and hemoglobin concentration traits of the Tibet improved cattle, realizes the early detection of the red blood cell number and hemoglobin concentration traits of the Tibet improved cattle, and the detection method is rapid, accurate and not affected by the breeding environmental conditions. The breeding of the Tibet improved cattle through the molecular marker can greatly shorten the breeding cycle. Attached Figure Description

[0031] Figure 1 The GWAS Manhattan plot and QQ plot are indicators of red blood cell count.

[0032] Figure 2 The GWAS Manhattan plot and QQ plot are indicators of hemoglobin concentration. Detailed Implementation

[0033] The molecular markers screened in this invention can be applied to genotype association analysis of genes related to bovine erythrocyte number and hemoglobin concentration, providing a new molecular marker resource for marker-assisted selection of bovine erythrocyte number and hemoglobin concentration traits. To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of this invention. Unless otherwise specified, all materials used in the following embodiments were purchased commercially.

[0034] Example 1: Screening of hypoxia-tolerant loci in Tibetan cattle

[0035] 1. Blood sample collection from cattle at different altitude gradients

[0036] This invention selected Tibetan cattle (3500m) and Yunnan native yellow cattle (at altitudes of 3000m, 2500m, 1500m and 500m respectively) distributed at different altitude gradients, with 20 cattle at each altitude, for a total of 100 cattle. Specifically, 20 cattle were collected from Gongbo'gyamda County, Tibet Autonomous Region (GB) at an altitude of 3500m; 20 cattle were collected from Diqing Tibetan Autonomous Prefecture, Yunnan Province (DQ) at an altitude of 3000m; 10 cattle were collected from Laojunshan area, Wenshan Prefecture, Yunnan Province (JS) and Lijiang City, Yunnan Province (LJ) at an altitude of 2500m; 10 cattle were collected from Tengchong City, Yunnan Province (TC) and Wuding County, Yunnan Province (WD) at an altitude of 1500m; and 10 cattle were collected from Damai Town, Shuangbai County, Yunnan Province (SB) and Yingjiang County, Yunnan Province (YJ) at an altitude of 500m. Using high-pressure vacuum blood collection tubes containing EDTA anticoagulant, 5 ml of blood was collected from the bovine jugular vein. The anticoagulant tube was placed in an ice box with plenty of ice packs and brought back to the laboratory, where it was stored at -80°C for subsequent DNA extraction and whole-genome resequencing.

[0037] 2. Genomic DNA extraction and whole-genome resequencing

[0038] The bovine genomic DNA was extracted using a blood genomic DNA extraction kit (Tiangen Biotech, DP348) according to the instructions, and the specific method was shown in the instructions. The qualified genomic DNA was sent to Beijing Berry & GENE Biotech Co., Ltd. (Beijing, China) for secondary inspection and library construction, and the Illumina HiSeq2500 platform was used for pair-end whole genome resequencing. The coverage of 100 individual genomes was about 5X, and the sequencing data was not less than 15G. The whole sequencing process was controlled by the Illumina HiSeq Control Software.

[0039] 3. SNP calling and sequence alignment

[0040] Firstly, the raw sequencing data was quality controlled, and bases with sequencing quality less than 20 (i.e. sequencing accuracy less than 99%) were removed from reads, and reads with length less than 35 bp after trimming were discarded. For the quality controlled data, Burrows-Wheeler-Alignment (BWA) software (Li & Durbin, 2010) was used to align to the corresponding reference genome (Bos taurus UMD 3.1), and the alignment result bam file was obtained, and samtools software was used for sorting and marking duplicate sequences. Next, Genome Analysis Toolkit (GATK) v3.7 standard process was used for SNP calling to obtain a high-quality gene variation information set. Firstly, HaplotypeCaller in GATK was used for variants calling to generate individual mutation information set g.vcf file. The g.vcf files of all individuals were merged, and GenotypeGVCFs was used for joint calling, thereby obtaining the variation information of the population and the accurate genotype of each individual. Next, the variation information was quality controlled, and hard filtering was used to complete due to the lack of a high-quality known variation set as a reference. SelectVariants command was used to split SNP (single nucleotide polymorphism) and InDel (Insertion-Deletion), and for the initial SNP file split out, VariantFiltration command was used to filter out SNP information not meeting the quality condition, and the final VCF file was generated. The filtering parameters were "-cluster 3-window 20; QD<2.0; QUAL<30; MQ<40.0; FS>60.0; SOR>3.0; MQRankSum<-12.5; ReadPosRankSum<-8.0; DP<10; --missing-values-evaluate-as-failing true". For the VCF file, PLINK v1.90b was used for further SNP quality control, and SNPs with multiple alleles, deletion rate greater than 10%, and minimum allele frequency of 0 were removed, and individuals with SNP deletion rate greater than 20% were removed, and the quality controlled data would be used for subsequent analysis.

[0041] Then the bovine genome gff file was downloaded from Ensemble, and ANNOVAR software was used for SNP annotation.

[0042] 4. Screening of low-oxygen-tolerant sites of Tibetan cattle

[0043] (1) Based on population genetics method, MEMEA method is established using likelihood estimation, using multivariate normal distribution to identify the molecular marker of allelic frequency selected by environmental factors, by introducing the random effect u between sites, allowing adjacent sites to be subjected to different selection intensity. At the same time, the method fully considers the genetic hitchhiking effect, and uses the sliding window mode to detect sites subjected to environmental selection pressure. In this invention, the altitude gradient of yellow cattle is used as the environmental variable, and the P value of each molecular marker in the model related to the molecular mechanism of highland environment adaptation is calculated. Then the -log(P) value is sorted from large to small, and the top 1% of molecular markers are selected as candidate signals;

[0044] (2) Based on population genomic data, regression analysis is performed using BayPass to screen molecular markers with high correlation with the trend of environmental factors;

[0045] (3) Select 3500m Tibetan cattle and 500m altitude distributed cattle, calculate the F ST of each molecular marker by vcftools software, and select the top 1% of molecular markers as candidate signals;

[0046] (4) Comprehensive analysis of MEMEA and BayPass two analysis methods, detect genetic polymorphic sites subjected to environmental adaptability selection between populations under continuous altitude gradient, combined with unit point F ST , the analysis results are verified and supplemented with each other, and the candidate sites of the three analysis methods are intersected. Calculate F ST of each molecular marker by vcftools software, and select the top 1% of molecular markers as candidate signals; ST of the intersection sites of BayPass, MEMEA and unit point F ST within the region range as hypoxia adaptation related SNPs, and finally 11637 molecular markers related to cattle hypoxia tolerance traits are screened.

[0047] Example 2: Application of low-density liquid chip

[0048] 1. Synthesis of probe

[0049] As described in Example 1, 11637 molecular markers related to cattle hypoxia tolerance traits were screened from continuous altitude gradient distributed cattle whole genome sequencing data. The target site information was checked, and then the probe was designed and synthesized, tested and adjusted, and the final probe and capture site were determined. 10K SNP liquid chip was prepared in Shijiazhuang Boruidi Biotechnology Co., Ltd.

[0050] 2. Detection of Tibetan improved cattle plateau adaptation breeding chip

[0051] 1547 heads of Tibet improved cattle were used as the detection cattle, blood physiological indexes were determined, whole genome DNA was extracted, and after quality inspection, 10K SNP liquid chip was used for genotyping, and the results were used for subsequent genotype and physiological index association analysis.

[0052] 3.GWAS analysis

[0053] (1) The raw sequencing sequences (raw reads) obtained by sequencing were filtered using the software fastp (version 0.20.0, parameters: -n 10-q 20-u 40), and finally clean reads were obtained, which were used for subsequent analysis;

[0054] (2) The clean reads after quality control were aligned with the reference genome (Bos taurus UMD3.1) sequence using the software BWA; the UnifiedGenotyper module of the software GATK (version v3.5-0-g36282e4) was used for variation detection;

[0055] (3) The VariantFiltration module was used for filtering, and the genotype information of the molecular markers was extracted using the self-compiled Perl script for the alignment results and variation results;

[0056] (4) ANNOVAR was used to annotate the function of the molecular markers;

[0057] (5) The linear regression model (GLM) general linear model in the Tassel software was used for GWAS analysis, and the model was as follows:

[0058] y = Wα + xβ + ε

[0059] In the formula:

[0060] y represents the phenotype traits to be studied, i.e. RBC and HGB indexes in this study;

[0061] Wα is the fixed effect (age, sex and feeding level), which affects y, and mainly refers to the population structure;

[0062] xβ is the marker effect;

[0063] ε represents the residual error.

[0064] The GWAS results show that the red blood cell number and hemoglobin concentration indexes at the 4th chromosome selection signal site are annotated to the 26S proteasome complex (Proteasome 26S subunit, ATPase 2, PSMC2) Figure 1 and Figure 2), the selection signal is strong. PSMC2 is involved in ATP-dependent protein degradation process. In the long-term adaptive evolution mechanism research of Tibetan partridge at high altitude environment, PSMC2 is found to be related to biological processes such as cell growth and apoptosis regulation, oxidative stress and immune response.

[0065] Example 3: Association analysis and application of molecular markers with red blood cell count and hemoglobin concentration

[0066] In order to further determine whether the molecular marker sites (SNP sites) g.44569796T>A, g.44570274T>C, g.44570306T>C, g.44570978G>C and g.44573224C>T in PSMC2 gene are related to the differences in red blood cell count and hemoglobin concentration traits of Tibetan improved cattle, the GLM program in SAS statistical analysis software was used for association analysis of traits between individuals with different genotypes of molecular markers, and the model was as follows:

[0067] Y ijk = μ + G i + H j + S k + e ijkl

[0068] Wherein, Y ijk is the observed value of physiological index, μ is the population mean, G i is the i th genotype effect of PSMC2 gene, H j is the j th age effect, S k is the k th sex effect, e ijkl is random error, which is subject to normal distribution.

[0069] According to the above model, the least square means of red blood cell count and hemoglobin concentration of each genotype of molecular markers were calculated by GLM process of SAS (Ver. 9.4) statistics, and the significance test of difference was carried out.

[0070] The allele frequency and genotype frequency of SNP site g.44569796T>A, SNP site g.44570274T>C, SNP site g.44570306T>C, SNP site g.44570978G>C and SNP site g.44573224C>T are shown in Table 1.

[0071] The statistical analysis results are shown in Table 2: the red blood cell count and hemoglobin concentration of AA genotype in SNP site g.44569796T>A were significantly lower than those of TT genotype and TA genotype (P<0.05); the red blood cell count and hemoglobin concentration of CC genotype in SNP site g.44570274T>C were significantly lower than those of TT genotype and TC genotype (P<0.05); the red blood cell count and hemoglobin concentration of CC genotype in SNP site g.44570306T>C were significantly lower than those of TT genotype and TC genotype (P<0.05); the red blood cell count and hemoglobin concentration of CC genotype in SNP site g.44570978G>C were significantly lower than those of GG genotype and CG genotype (P<0.05); the red blood cell count and hemoglobin concentration of TT genotype in SNP site g.44573224C>T were significantly lower than those of CC genotype and CT genotype (P<0.05).

[0072] Table 1 Allele frequency and genotype frequency of 5 SNP sites

[0073]

[0074]

[0075] Table 2 Association analysis of PSMC2 gene polymorphic sites with red blood cell count and hemoglobin concentration of Tibet improved cattle

[0076]

[0077] Note: the values in the table are expressed as mean ± standard error; the same column and the same SNP site data with different superscript capital letters represent extremely significant difference (P<0.01), and the same letter represents no significant difference (P>0.01). The same below.

[0078] The genetic association analysis results show that the molecular marker of SNP site g.44569796T>A (61bp in the sequence shown in SEQ ID NO.1) is significantly related to red blood cell count and bovine hemoglobin concentration, and the red blood cell count and hemoglobin concentration of individuals with genotype A at 61bp in the sequence shown in SEQ ID NO.1 are significantly lower than those of individuals with genotype T at 61bp in the sequence shown in SEQ ID NO.1, indicating that genotype A at 61bp in the sequence shown in SEQ ID NO.1 is an allele that is beneficial to reducing red blood cell count and hemoglobin concentration.

[0079] The molecular marker of SNP site g.44570274 T>C (at 71 bp in the sequence shown in SEQ ID NO. 2) is significantly associated with the red blood cell number and hemoglobin concentration of the cattle, the red blood cell number and hemoglobin concentration of the individual with genotype C at 71 bp in the sequence shown in SEQ ID NO. 2 are significantly lower than those of the individual with genotype T at 71 bp in the sequence shown in SEQ ID NO. 2, indicating that the genotype C at 71 bp in the sequence shown in SEQ ID NO. 2 is an allele beneficial to reducing the red blood cell number and hemoglobin concentration.

[0080] The molecular marker of SNP site g.44570306 T>C (at 81 bp in the sequence shown in SEQ ID NO. 3) is significantly associated with the red blood cell number and hemoglobin concentration of the cattle, the red blood cell number and hemoglobin concentration of the individual with genotype C at 81 bp in the sequence shown in SEQ ID NO. 3 are significantly lower than those of the individual with genotype T at 81 bp in the sequence shown in SEQ ID NO. 3, indicating that the genotype C at 81 bp in the sequence shown in SEQ ID NO. 3 is an allele beneficial to reducing the red blood cell number and hemoglobin concentration.

[0081] The molecular marker of SNP site g.44570978 G>C (at 91 bp in the sequence shown in SEQ ID NO. 4) is significantly associated with the red blood cell number and hemoglobin concentration of the cattle, the red blood cell number and hemoglobin concentration of the individual with genotype C at 91 bp in the sequence shown in SEQ ID NO. 4 are significantly lower than those of the individual with genotype G at 91 bp in the sequence shown in SEQ ID NO. 4, indicating that the genotype C at 91 bp in the sequence shown in SEQ ID NO. 4 is an allele beneficial to reducing the red blood cell number and hemoglobin concentration.

[0082] The molecular marker of SNP site g.44573224 C>T (at 101 bp in the sequence shown in SEQ ID NO. 5) is significantly associated with the red blood cell number and hemoglobin concentration of the cattle, the red blood cell number and hemoglobin concentration of the individual with genotype T at 101 bp in the sequence shown in SEQ ID NO. 5 are significantly lower than those of the individual with genotype C at 101 bp in the sequence shown in SEQ ID NO. 5, indicating that the genotype T at 101 bp in the sequence shown in SEQ ID NO. 5 is an allele beneficial to reducing the red blood cell number and hemoglobin concentration.

[0083] The molecular marker can be used as a molecular marker for detecting the number of red blood cells and hemoglobin concentration associated with the number of red blood cells and hemoglobin concentration, and when the genotype at the 61st bp in the sequence shown in SEQ ID NO. 1 is A, the genotype at the 71st bp in the sequence shown in SEQ ID NO. 2 is C, the genotype at the 81st bp in the sequence shown in SEQ ID NO. 3 is C, the genotype at the 91st bp in the sequence shown in SEQ ID NO. 4 is C, and the genotype at the 101st bp in the sequence shown in SEQ ID NO. 5 is T (AA-CC-CC-CC-TT), it is more conducive to reducing the number of red blood cells and hemoglobin concentration of the Tibet improved cattle. The low oxygen bottleneck problem faced by the breeding of plateau cattle can be solved, a fast and accurate molecular method is provided for breeding plateau tolerant individuals, and a reliable technical platform is provided for carrying out cattle stress tolerance trait breeding and other related genetic breeding work, promoting the development of China's plateau cattle industry, and also providing a theoretical reference and method guidance for the breeding of other plateau animals.

[0084] Example 4: Linkage disequilibrium analysis of PSMC2 gene polymorphism sites

[0085] The five molecular markers of the PSMC2 gene were analyzed for linkage disequilibrium using Haploview software, and the five sites of SNP site g.44569796 T>A, SNP site g.44570274 T>C, SNP site g.44570306 T>C, SNP site g.44570978 G>C and SNP site g.44573224 C>T were tightly linked (r 2 =0.98). Five combined genotypes were detected in the Tibet improved cattle population: AA-CC-CC-CC-TT (the genotype at the 61st bp in the sequence shown in SEQ ID NO. 1 is A, the genotype at the 71st bp in the sequence shown in SEQ ID NO. 2 is C, the genotype at the 81st bp in the sequence shown in SEQ ID NO. 3 is C, the genotype at the 91st bp in the sequence shown in SEQ ID NO. 4 is C, and the genotype at the 101st bp in the sequence shown in SEQ ID NO. 5 is T).

[0086] TT-TT-TT-GG-CC (the genotype at the 61st bp in the sequence shown in SEQ ID NO. 1 is T, the genotype at the 71st bp in the sequence shown in SEQ ID NO. 2 is T, the genotype at the 81st bp in the sequence shown in SEQ ID NO. 3 is T, the genotype at the 91st bp in the sequence shown in SEQ ID NO. 4 is G, and the genotype at the 101st bp in the sequence shown in SEQ ID NO. 5 is C).

[0087] TA-TC-TC-CG-CT (genotype T at 61bp in the sequence shown in SEQ ID NO. 1, genotype T at 71bp in the sequence shown in SEQ ID NO. 2, genotype T at 81bp in the sequence shown in SEQ ID NO. 3, genotype G at 91bp in the sequence shown in SEQ ID NO. 4, and genotype C at 101bp in the sequence shown in SEQ ID NO. 5).

[0088] TA-TT-TC-CG-CT (genotype T at 61bp in the sequence shown in SEQ ID NO. 1, genotype T at 71bp in the sequence shown in SEQ ID NO. 2, genotype T at 81bp in the sequence shown in SEQ ID NO. 3, genotype G at 91bp in the sequence shown in SEQ ID NO. 4, and genotype C at 101bp in the sequence shown in SEQ ID NO. 5).

[0089] TT-TT-TC-GG-CC (genotype T at 61bp in the sequence shown in SEQ ID NO. 1, genotype T at 71bp in the sequence shown in SEQ ID NO. 2, genotype T at 81bp in the sequence shown in SEQ ID NO. 3, genotype G at 91bp in the sequence shown in SEQ ID NO. 4, and genotype C at 101bp in the sequence shown in SEQ ID NO. 5)

[0090] The results of the association analysis of different combination genotypes with the red blood cell count and hemoglobin concentration of the Tibet improved cattle are shown in Table 3: the hemoglobin concentration of the individuals with the AA-CC-CC-CC-TT genotype was significantly lower than that of the individuals with the TT-TT-TT-GG-CC genotype and the TT-TT-TC-GG-CC genotype (P<0.01), and lower than that of the individuals with the TA-TC-TC-CG-CT and TA-TT-TC-CG-CT genotypes (P>0.01).

[0091] Table 3 Association analysis of PSMC2 gene haplotype with red blood cell count and hemoglobin concentration of Tibet improved cattle

[0092] Haplotype Hemoglobin concentration Red blood cell number AA-CC-CC-CC-TT 125.17 ± 11.76 B ]]> 7.05 ± 1.69 B ]] TT-TT-TT-GG-CC 154.99 ± 10.23 A ]] 9.28 ± 2.21 A <!-- 8 -->]] TA-TC-TC-CG-CT 129.81 ± 14.33 B ]] 7.28 ± 1.36 B ]] TA-TT-TC-CG-CT 137.46 ± 9.56 AB ]] 7.68 ± 1.15 AB ]] TT-TT-TC-GG-CC 145.48 ± 7.98 A ]] 8.46 ± 1.64 A ]]

[0093] The above-mentioned SNP site g.44569796 T>A, SNP site g.44570274 T>C, SNP site g.44570306 T>C, SNP site g.44570978 G>C, and SNP site g.44573224 C>T of the PSMC2 gene can be used as a new molecular marker for genetic improvement of the red blood cell count and hemoglobin concentration traits of the Tibet improved cattle.

[0094] Application of the molecular marker in the PSMC2 gene described above in the subline breeding

[0095] First, determine the gene type of the Tibet improved cattle at the molecular marker site in the above examples, and then carry out subline breeding according to the gene type of the site.

[0096] Determine that the base at position 61 bp in SEQ ID NO. 1 sequence is A type, the base at position 71 bp in SEQ ID NO. 2 sequence is C type, the base at position 81 bp in SEQ ID NO. 3 sequence is C type, the base at position 91 bp in SEQ ID NO. 4 sequence is C type, and the base at position 101 bp in SEQ ID NO. 5 sequence is T type. Because the number of red blood cells and hemoglobin concentration of these five base types are significantly lower than those of other base types.

[0097] Therefore, select the individuals of Tibet improved cattle with A type base at position 61 bp in SEQ ID NO. 1 sequence (the individual of the cattle is AA type individual at position 61 bp) for subline breeding, and eliminate the individuals with T type base at position 61 bp in SEQ ID NO. 1 sequence (the individual of the cattle is TT type individual at position 61 bp or AT type individual at position 61 bp); and / or, select the individuals of the cattle with C type base at position 71 bp in SEQ ID NO. 2 sequence (the individual of the cattle is CC type individual at position 71 bp) for subline breeding, and eliminate the individuals with T type base at position 71 bp in SEQ ID NO. 2 sequence (the individual of the cattle is TT type individual at position 71 bp or TC type individual at position 71 bp); and / or, select the individuals of the cattle with C type base at position 81 bp in SEQ ID NO. 3 sequence (the individual of the cattle is CC type individual at position 81 bp) for subline breeding, and eliminate the individuals with T type base at position 81 bp in SEQ ID NO. 3 sequence (the individual of the cattle is TT type individual at position 81 bp or TC type individual at position 80 bp); and / or, select the individuals of the cattle with C type base at position 91 bp in SEQ ID NO. 4 sequence (the individual of the cattle is CC type individual at position 91 bp) for subline breeding, and eliminate the individuals with G type base at position 91 bp in SEQ ID NO. 3 sequence (the individual of the cattle is GG type individual at position 91 bp or CG type individual at position 91 bp); and / or, select the individuals of the cattle with T type base at position 101 bp in SEQ ID NO. 5 sequence (the individual of the cattle is TT type individual at position 101 bp) for subline breeding, and eliminate the individuals with C type base at position 101 bp in SEQ ID NO. 5 sequence (the individual of the cattle is CC type individual at position 101 bp or CT type individual at position 101 bp).

[0098] The Tibetan improved cow with the molecular marker genotype of AA-CC-CC-CC-TT has the lowest red blood cell count and hemoglobin concentration, and the individual with the genotype of AA-CC-CC-CC-TT is selected in the subsequent selection of the Tibetan improved cow.

[0099] The application screens genes and sites associated with altitude by whole genome resequencing of bovine genomic DNA distributed in different altitude gradients, creates a low-cost high-throughput genotyping liquid chip (10K) based on the altitude-associated sites, determines the low-oxygen-related blood physiological indicators according to the existing high-generation crossbreeding and crossbreeding of the Tibetan improved cow, and uses the 10K SNP chip to implement whole genome association analysis (GWAS) of low-oxygen physiology, and obtains the molecular marker SNP sites g.44569796T>A, SNP site g.44570274T>C, SNP site g.44570306T>C, SNP site g.44570978G>C and SNP site g.44573224C>T associated with red blood cell count and hemoglobin concentration on the PSMC2 gene.

[0100] In the embodiments of the application, the Tibetan cow (3500m) and the Yunnan local yellow cow (altitudes are 3000m, 2500m, 1500m and 500m respectively) distributed in different altitude gradients are selected for whole genome resequencing, the resequencing data is compared with the bovine reference genome (Bos taurus UMD 3.1), F ST , MEMEA and BayPass analysis methods are used, and finally 11637 molecular markers associated with the low-oxygen-resistant traits of the bovine are screened.

[0101] The molecular marker of the SNP site g.44569796T>A: the nucleotide sequence of the fragment is shown in SEQ ID NO:1, and there is a T>A base mutation at the 61st bp in the shown sequence.

[0102] SEQ ID NO. 1:

[0103]

[0104] Molecular marker of SNP site g.44570274 T>C: the nucleotide sequence of the fragment is shown as SEQ ID NO: 2, and there is a T>C base mutation at the 71st bp in the shown sequence;

[0105] SEQ ID NO. 2:

[0106]

[0107] Molecular marker of SNP site g.44570306 T>C: the nucleotide sequence of the fragment is shown as SEQ ID NO: 3, and there is a T>C base mutation at the 81st bp in the shown sequence;

[0108] SEQ ID NO. 3:

[0109]

[0110] Molecular marker of SNP site g.44570978 G>C: the nucleotide sequence of the fragment is shown as SEQ ID NO: 4, and there is a G>C base mutation at the 91st bp in the shown sequence;

[0111] SEQ ID NO. 4:

[0112]

[0113] Molecular marker of SNP site g.44573224 C>T: the nucleotide sequence of the fragment is shown as SEQ ID NO: 5, and there is a C>T base mutation at the 101st bp in the shown sequence;

[0114] SEQ ID NO. 5:

[0115]

[0116]

[0117] Having described various embodiments of the application, it is to be understood that the above description is meant to be illustrative only, and that many modifications and variations of the embodiments described herein are possible. It is therefore to be understood that within the scope of the appended claims, and their equivalents, many alternatives to the embodiments described herein are possible. The selection of terms to be used in the description is not intended to limit the scope of the embodiments described herein, but rather to best explain the principles of the embodiments, practical application, or improvement over the technology in the art, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. The use of a molecular marker in the subcloning of Tibet improved cattle, characterized in that, The specific steps comprise: determining the gene type of the molecular marker site in the Tibet improved cattle, and selecting the individual according to the gene type of the site for the subculture breeding: selecting the individual with A type base at 61bp in the SEQ ID NO.1 sequence, eliminating the individual with T type base at 61bp; and selecting the individual with C type base at 71bp in the SEQ ID NO.2 sequence, eliminating the individual with T type base at 71bp; and selecting the individual with C type base at 81bp in the SEQ ID NO.3 sequence, eliminating the individual with T type base at 81bp; and selecting the individual with C type base at 91bp in the SEQ ID NO.4 sequence, eliminating the individual with G type base at 91bp; and selecting the individual with T type base at 101bp in the SEQ ID NO.5 sequence, eliminating the individual with C type base at 101bp; the molecular marker site is a combination of g.44569796T>A, g.44570274T>C, g.44570306T>C, g.44570978G>C and g.44573224C>T in the bovine genome.

2. Use according to claim 1, characterized in that, the gene type of the molecular marker is AA-CC-CC-CC-TT, and the red blood cell number and hemoglobin concentration of the individual are the lowest, and the individual with the gene type of the molecular marker being AA-CC-CC-CC-TT is selected for the subculture breeding of the Tibet improved cattle.

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