Molecular marker in bovine PSMC2 gene and application thereof

Abstract

The invention discloses a molecular marker in a bovine PSMC2 gene and application of the molecular marker. The molecular marker is an SNP (Single Nucleotide Polymorphism) site g.44569796Tgt in a bovine genome; the method comprises the following steps: A, an SNP (Single Nucleotide Polymorphism) site g.44570274 C, an SNP (Single Nucleotide Polymorphism) site is g.44570306Tgt; c, an SNP (Single Nucleotide Polymorphism) site is g.44570978Ggt; c and an SNP (Single Nucleotide Polymorphism) site g.44573224Cgt; t is one or more. The molecular marker or the substance for detecting the single nucleotide polymorphism of the cattle molecular marker provided by the invention can be used in cattle genetic breeding so as to reduce the number of red blood cells and the hemoglobin concentration of improved cattle offspring in Tibetan and solve the bottleneck problem of hypoxia faced by plateau cattle breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a molecular marker in a bovine PSMC2 gene and an application thereof. Background Art

[0002] When cattle from low altitude areas migrate to the low oxygen environment of high altitude areas, the hemoglobin concentration will increase to increase the oxygen carrying capacity and meet the body's oxygen supply needs. However, excessive red blood cell count and hemoglobin concentration may increase blood viscosity, increase cardiovascular risk, hinder microcirculation, and cause diseases such as high altitude polycythemia. The Tibetan improved cattle (Holstein cattle ♂×Tibetan cattle ♀) began in the 1980s, and the progressive hybridization has been carried out to the F3 generation or above. Therefore, in the selection and breeding process of Tibetan improved cattle on the plateau, the appropriate red blood cell count and hemoglobin concentration are more conducive to the adaptation of Tibetan improved cattle to the plateau environment.

[0003] The number of red blood cells and hemoglobin concentration are complex and comprehensive traits that are regulated by multiple genes. However, the molecular markers currently reported to be related to the number of red blood cells and hemoglobin concentration in cattle lack more reliable sites for exploring gene changes under hypoxic environments. In addition, gene-gene or gene-environment interactions can also affect the physiological characteristics of plateau cattle.

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

[0005] The purpose of the present invention is to discover the molecular marker in the cattle PSMC2 gene, and to provide a new molecular marker for the detection of cattle red blood cell number and hemoglobin concentration or auxiliary molecular marker breeding in Tibetan improved cattle breeding.

[0006] In a first aspect, the present application provides a molecular marker in the bovine PSMC2 gene, wherein 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 the bovine genome.

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

[0008] Site g.44570274T>C means that there is a T>, C base mutation at the 71 bp in the sequence shown in SEQ ID NO.2;

[0009] Site g.44570306T>C means there is a T>, C base mutation at the 81 bp in the sequence shown in SEQ ID NO.3;

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

[0011] The site g.44573224C>T means that there is a C>, T base mutation at the 101bp position 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 hemoglobin concentration of a bovine, the method comprising the steps of:

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

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

[0015] When 1) the base type at the 71 bp in the SEQ ID NO:2 sequence is C, the cattle individual is a CC type individual at the 71 bp; when 1) the base type at the 71 bp in the SEQ ID NO:2 sequence is T, the cattle individual is a TT type individual at the 71 bp or a TC type individual at the 71 bp; the red blood cell number and hemoglobin concentration of the CC type individual at the 71 bp are lower than those of the TT type individual at the 71 bp and the TC type individual at the 71 bp;

[0016] When 1) the base type at the 81st bp in the SEQ ID NO:3 sequence is C, the cattle individual is a CC type individual at the 81st bp; when 1) the base type at the 81st bp in the SEQ ID NO:3 sequence is T, the cattle individual is a TT type individual at the 81st bp or a TC type individual at the 81st bp; the red blood cell number and hemoglobin concentration of the CC type individual at the 81st bp are lower than those of the TT type individual at the 81st bp and the TC type individual at the 81st bp;

[0017] When 1) the base type at the 91st bp in the SEQ ID NO:4 sequence is C, the cattle individual is a CC type individual at the 91st bp; when 1) the base type at the 91st bp in the SEQ ID NO:4 sequence is G, the cattle individual is a GG type individual at the 91st bp or a CG type individual at the 91st bp; the red blood cell number and hemoglobin concentration of the CC type individual at the 91st bp are lower than those of the GG type individual at the 91st bp and the CG type individual at the 91st bp;

[0018] When 1) the base type at the 101bp in the SEQ ID NO:5 sequence is T, the cattle individual is a TT type individual at the 101bp; when 1) the base type at the 101bp in the SEQ ID NO:5 sequence is C, the cattle individual is a CC type individual at the 101bp or a CT type individual at the 101bp; the red blood cell number and hemoglobin concentration of the TT type individual at the 101bp are lower than those of the CC type individual at the 101bp and the CT type individual at the 101bp.

[0019] The third aspect of the present application provides an application of molecular markers in the successive breeding of Tibetan improved cattle, and the specific steps include:

[0020] Determine the gene type at the site of the molecular marker in the Tibetan improved cattle, and carry out successive breeding according to the gene type at the site:

[0021] Select the individuals whose base at the 61 bp in the above SEQ ID NO.1 sequence is A type, and eliminate the individuals whose base at the 61 bp is T type; and / or,

[0022] Select the individuals whose base at the 71 bp in the above SEQ ID NO.2 sequence is C type, and eliminate the individuals whose base at the 71 bp is T type; and / or,

[0023] Select the individuals whose base at the 81 bp in the above SEQ ID NO.3 is C type, and eliminate the individuals whose base at the 81 bp is T type; and / or,

[0024] Select the individuals whose base at the 91 bp in the above SEQ ID NO.4 is of type C, and eliminate the individuals whose base at the 91 bp is of type G; and / or,

[0025] The individuals whose base at the 101 bp in the above SEQ ID NO.5 sequence is T-type were selected, and the individuals whose base at the 101 bp is C-type were eliminated.

[0026] Furthermore, individuals whose gene type of the molecular marker is AA-CC-CC-CC-TT have the lowest red blood cell number and hemoglobin concentration, and individuals whose gene type of the molecular marker is AA-CC-CC-CC-TT are selected during the successive breeding of Tibetan improved cattle.

[0027] Beneficial effects of the present invention:

[0028] 1. The present invention has discovered molecular markers associated with the number of red blood cells and hemoglobin concentration in the bovine PSMC2 gene, and the molecular markers include five molecular markers, namely, 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 five molecular markers can be used as molecular markers of the number of red blood cells and hemoglobin concentration of cattle, respectively, or as molecular markers after gene-gene combination. Moreover, the haplotype individual composed of the above five molecular markers, i.e., the individual AA-CC-CC-CC-TT, has the lowest hemoglobin concentration and number of red blood cells.

[0029] 2. The present invention verifies the effects of the above five molecular markers on the number of red blood cells and hemoglobin concentration of cattle, and can be applied to the successive breeding of Tibetan improved cattle to reduce the number of red blood cells and hemoglobin concentration. Individuals with the gene type of SNP site g.44569796T>A of A, the gene type of SNP site g.44570274T>C of C, the gene type of SNP site g.44570306T>C of C, the gene type of SNP site g.44570978G>C of C or the gene type of SNP site g.44573224C>T of T can be selected. Individuals with these five gene types have lower red blood cell numbers and hemoglobin concentrations. Haplotype individuals of AA-CC-CC-CC-TT can also be selected, and the red blood cell number and hemoglobin concentration of AA-CC-CC-CC-TT individuals are the lowest. Thereby improving the hypoxia adaptability of the offspring of Tibetan improved cattle.

[0030] 3. The present invention provides a new molecular marker for molecular marker-assisted breeding of red blood cell number and hemoglobin concentration traits of Tibetan improved cattle, which realizes the early detection of red blood cell number and hemoglobin concentration traits of Tibetan improved cattle. The detection method is fast and accurate, and is not affected by breeding environment conditions. The selection and breeding of Tibetan improved cattle through the molecular marker can greatly shorten the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Manhattan plot and QQ plot for GWAS of red blood cell number index.

[0032] Figure 2 Manhattan plot and QQ plot for GWAS of hemoglobin concentration index. DETAILED DESCRIPTION

[0033] The molecular markers screened by the present invention can be applied to the genotype association analysis of genes related to the number of red blood cells and hemoglobin concentration in cattle, providing a new molecular marker resource for the molecular marker-assisted selection of the number of red blood cells and hemoglobin concentration traits in cattle. In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the materials used in the following embodiments are purchased from the market.

[0034] Example 1 Screening of hypoxia-resistant sites in Tibetan cattle

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

[0036] The present invention selects Tibetan cattle (3500m) and Yunnan local cattle (altitudes are: 3000m, 2500m, 1500m and 500m respectively) distributed at different altitude gradients, 20 heads at each altitude, a total of 100 heads, and the specific sample information is that 20 heads were collected in Gongbu Gyamda County (GB) of Tibet Autonomous Region at an altitude of 3500m; 20 heads were collected in Diqing Tibetan Autonomous Prefecture (DQ) of Yunnan Province at an altitude of 3000m; 10 heads were collected in Laojun Mountain area (JS) of Wenshan Prefecture of Yunnan Province and Lijiang City (LJ) of Yunnan Province at an altitude of 2500m; 10 heads were collected in Tengchong City (TC) of Yunnan Province and Wuding County (WD) of Yunnan Province at an altitude of 1500m; 10 heads were collected in Damaidi Town (SB) of Shuangbai County of Yunnan Province and Yingjiang County (YJ) of Yunnan Province at an altitude of 500m. Use a high-pressure vacuum blood collection tube containing EDTA anticoagulant to collect 5 ml of blood from the bovine jugular vein. Place the anticoagulant tube in an ice box with a large number of ice packs and bring it back to the laboratory, where it will be stored in a -80°C refrigerator for subsequent DNA extraction for whole genome resequencing.

[0037] 2. Genomic DNA Extraction and Whole Genome Resequencing

[0038] Bovine genomic DNA was extracted using a blood genomic DNA extraction kit (Tiangen Biochemical Technology, DP348) according to the instructions. The specific method is described in the instructions. The genomic DNA that passed the quality inspection was sent to Beijing Berry Genomics Biotech Co., Ltd. (Beijing, China) for secondary quality inspection and library construction, and paired-end whole genome resequencing was performed using the Illumina HiSeq2500 platform. The sequencing coverage of 100 individual genomes was about 5X, and the sequencing data volume was not less than 15G. The entire sequencing process was controlled by Illumina HiSeq Control Software.

[0039] 3.SNP calling and sequence alignment

[0040] First, the original data from the sequencing machine was quality controlled, and the bases with sequencing quality less than 20 (i.e., sequencing accuracy less than 99%) were removed from the reads, and the reads with a length of less than 35bp after trimming were discarded. For the quality-controlled data, the 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. The samtools software was used to sort and mark the repeated sequences. Next, the Genome Analysis Toolkit (GATK) v3.7 standard process was used for SNP calling to obtain a high-quality gene variation information set. First, the HaplotypeCaller in GATK was used to perform variants calling to generate an individual mutation information set g.vcf file. The g.vcf files of all individuals were merged, and GenotypeGVCFs was used for joint calling to obtain the variation information of this group and the accurate genotype of each individual. Next, the variant information was quality controlled. Due to the lack of high-quality known variant sets as references, hard filtering was used. The SelectVariants command was used to split SNP (single nucleotide polymorphism) and InDel (Insertion-Deletion). For the initial SNP file, the VariantFiltration command was used to filter out the SNP information that did not meet the quality conditions and generate the final VCF file. 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 VCF files, PLINK v1.90b was used for further SNP quality control, and SNPs with multiple alleles, missing rates greater than 10%, and a minimum allele frequency of 0 were eliminated. Individuals with a SNP missing rate greater than 20% were eliminated, and the data after quality control were used for subsequent analysis.

[0041] Then download the cattle genome gff file from Ensemble and use ANNOVAR software to perform SNP annotation.

[0042] 4. Screening of hypoxia-tolerant sites in Tibetan cattle

[0043] (1) Based on population genetics methods, the MEMEA method was established using likelihood estimation, and the multivariate normal distribution was used to identify molecular markers whose allele frequencies were selected by environmental factors. By introducing the random effect u between sites, adjacent sites were allowed to be subject to different selection intensities. At the same time, this method fully considers the genetic piggyback effect and uses a sliding window model to detect sites subject to environmental selection pressure. In the present invention, the altitude gradient of cattle is used as an environmental variable to calculate the P value of each molecular marker in the model related to the molecular mechanism of adaptation to the plateau environment. Then, the -log(P) values ​​are sorted from large to small, and the top 1% of molecular markers are selected as candidate signals;

[0044] (2) Based on population genomic data, BayPass was used for regression analysis to screen molecular markers that were highly correlated with the changing trends of environmental factors;

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

[0046] (4) Combining MEMEA and BayPass analysis methods to detect genetic polymorphic loci that are subject to environmental adaptability selection among populations at continuous altitude gradients, and then combining single locus F ST The analysis results verified and supplemented each other, and the candidate sites of the three analysis methods were intersected. The F ST Sliding window results (20 kb window, 5 kb step size), merge the top 1% Fst windows into regions (overlapping or distance < 20 kb), and combine BayPass, MEMEA, and single-site F ST The intersection sites falling within the regional range were selected as SNPs related to hypoxia adaptation, and finally 11,637 molecular markers related to the hypoxia tolerance trait of cattle were screened.

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

[0048] 1. Synthesis of Probes

[0049] As described in Example 1, 11,637 molecular markers related to the hypoxia tolerance trait of cattle were analyzed and screened from the whole genome sequencing data of cattle distributed at continuous altitude gradients. The target site information was checked, and then probes were designed and synthesized, tested and adjusted, and the final probes and capture sites were determined. 10KSNP liquid phase chips were prepared at Shijiazhuang Boredy Biotechnology Co., Ltd.

[0050] 2. Detection of Tibetan Improved Cattle Plateau Adaptation Breeding Chip

[0051] A total of 1547 Tibetan improved cattle were selected for testing. Blood physiological indicators were measured and whole genome DNA was extracted. After passing the quality inspection, genotyping was performed using a 10K SNP liquid phase chip. The results were used for subsequent association analysis between genotypes and physiological indicators.

[0052] 3. GWAS analysis

[0053] (1) Use fastp software (version 0.20.0, parameters: -n 10-q 20-u 40) to filter the raw reads obtained by sequencing, and finally obtain clean reads, which are 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 BWA software; the UnifiedGenotyper module of the GATK software (version v3.5-0-g36282e4) was used for variation detection;

[0055] (3) The VariantFiltration module was used for filtering, and the alignment results and variant results were used to extract the genotyping information of molecular markers using a self-written Perl script;

[0056] (4) Functional annotation of molecular markers using ANNOVAR;

[0057] (5) GWAS analysis was performed using the general linear model (GLM) in Tassel software. The model is as follows:

[0058] y=Wα+xβ+ε

[0059] In the formula:

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

[0061] Wα is the fixed effect (age, sex and feeding level), other factors affecting y, mainly referring to the group structure;

[0062] xβ is the labeling effect;

[0063] ε represents the residual.

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

[0065] Example 3: Correlation 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 the PSMC2 gene are related to the differences in the number of red blood cells and hemoglobin concentration in Tibetan improved cattle, the GLM program in the SAS statistical analysis software was used to perform association analysis between the traits of individuals with different genotypes of molecular markers, and the model used was:

[0067] Y ijk =μ+G i +H j +S k +e ijkl

[0068] Among them, Y ijk is the observed value of physiological index, μ is the population mean, G i is the genotype effect of PSMC2 gene, H j is the jth age effect, S k is the kth gender effect, e ijkl is a random error and follows a normal distribution.

[0069] According to the above model, the least square means of the red blood cell number and hemoglobin concentration indexes corresponding to each genotype of the molecular marker were calculated using the GLM process of SAS (Ver.9.4), and the difference significance test was performed.

[0070] The allele frequencies and genotype frequencies 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 results of statistical analysis are shown in Table 2: The red blood cell number 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 number 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 number 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 number 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 number 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 frequencies and genotype frequencies of the five SNP loci

[0073]

[0074]

[0075] Table 2 Association analysis between PSMC2 gene polymorphisms and red blood cell count and hemoglobin concentration in Tibetan improved cattle

[0076]

[0077] Note: The values ​​in the table are expressed as mean ± standard error; different capital letters in the same column and the same SNP locus data indicate extremely significant differences (P<0.01), and the same letters indicate insignificant differences (P>0.01). The same below.

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

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

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

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

[0082] The molecular marker of SNP site g.44573224C>T (101bp in the sequence shown in SEQ ID NO.5) is significantly correlated with the number of red blood cells and hemoglobin concentration of cattle. The number of red blood cells and hemoglobin concentration of individuals with genotype T at the 101bp in the sequence shown in SEQ ID NO.5 are significantly lower than those of individuals with genotype C at the 101bp in the sequence shown in SEQ ID NO.5, indicating that genotype T at the 101bp in the sequence shown in SEQ ID NO.5 is an allele that is beneficial to reducing the number of red blood cells 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 of cattle, and when the genotype at the 61bp position in the sequence shown in SEQ ID NO.1 is A, the genotype at the 71bp position in the sequence shown in SEQ ID NO.2 is C, the genotype at the 81bp position in the sequence shown in SEQ ID NO.3 is C, the genotype at the 91bp position in the sequence shown in SEQ ID NO.4 is C, and the genotype at the 101bp position 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 Tibetan improved cattle. It can solve the low oxygen bottleneck problem faced by plateau cattle breeding, provide a rapid and accurate molecular means for breeding plateau tolerant individuals, and provide a reliable technical platform for carrying out genetic breeding work such as breeding of cattle stress resistance traits, promote the development of my country's plateau cattle industry, and also provide theoretical reference and method guidance for the breeding of other plateau animals.

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

[0085] Haploview software was used to analyze linkage disequilibrium of five molecular markers of PSMC2 gene. The five loci, SNP g.44569796T>A, SNP g.44570274T>C, SNP g.44570306T>C, SNP g.44570978G>C and SNP g.44573224C>T, were closely linked (r 2 =0.98). A total of five genotype combinations were detected in the Tibetan 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 61bp in the sequence shown in SEQ ID NO.1 is T, the genotype at the 71bp in the sequence shown in SEQ ID NO.2 is T, the genotype at the 81bp in the sequence shown in SEQ ID NO.3 is T, the genotype at the 91bp in the sequence shown in SEQ ID NO.4 is G, and the genotype at the 101bp in the sequence shown in SEQ ID NO.5 is C).

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

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

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

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

[0091] Table 3 Association analysis between PSMC2 gene haplotype and red blood cell count and hemoglobin concentration in Tibetan improved cattle

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

[0093] The above-mentioned PSMC2 gene 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 can be used as new molecular markers for genetic improvement of red blood cell number and hemoglobin concentration traits in Tibetan improved cattle.

[0094] Example 5 Application of the molecular markers in the PSMC2 gene in subgeneration breeding

[0095] First, the gene type of the Tibetan improved cattle at the molecular marker site in the above embodiment is determined, and then successive breeding is carried out according to the gene type of the site.

[0096] It was determined that the base at the 61st bp in the SEQ ID NO.1 sequence was A type, the base at the 71st bp in the SEQ ID NO.2 sequence was C type, the base at the 81st bp in the SEQ ID NO.3 sequence was C type, the base at the 91st bp in the SEQ ID NO.4 sequence was C type, and the base at the 101st bp in the SEQ ID NO.5 sequence was T type. The red blood cell number and hemoglobin concentration of these five base types were significantly lower than those of other base types.

[0097] Therefore, the individuals whose base at the 61st bp in the sequence of SEQ ID NO.1 is A-type (the cattle individuals are AA-type individuals at the 61st bp), and the individuals whose base at the 61st bp in the sequence of SEQ ID NO.1 is T-type (the cattle individuals are TT-type individuals at the 61st bp or AT-type individuals at the 61st bp); and / or, the individuals whose base at the 71st bp in the sequence of SEQ ID NO.2 is C-type (the cattle individuals are CC-type individuals at the 71st bp), and the individuals whose base at the 71st bp in the sequence of SEQ ID NO.2 is T-type (the cattle individuals are TT-type individuals at the 71st bp or TC-type individuals at the 71st bp); and / or, the individuals whose base at the 81st bp in the sequence of SEQ ID NO.3 is C-type (the cattle individuals are CC-type individuals at the 81st bp), and the individuals whose base at the 81st bp in the sequence of SEQ ID NO. The individuals whose base at the 81st bp in the NO.3 sequence is T-type (the cattle individuals are TT-type individuals at the 81st bp or TC-type individuals at the 80th bp); and / or, the individuals whose base at the 91st bp in the SEQ ID NO.4 sequence is C-type (the cattle individuals are CC-type individuals at the 91st bp), and the individuals whose base at the 91st bp in the SEQ ID NO.3 sequence is G-type (the cattle individuals are GG-type individuals at the 91st bp or CG-type individuals at the 91st bp); and / or, the individuals whose base at the 101st bp in the SEQ ID NO.5 sequence is T-type (the cattle individuals are TT-type individuals at the 101bp), and the individuals whose base at the 101st bp in the SEQ ID NO.5 sequence is C-type (the cattle individuals are CC-type individuals at the 101bp or CT-type individuals at the 101bp).

[0098] The gene type of the molecular marker of the Tibetan improved cattle is AA-CC-CC-CC-TT, and the red blood cell number and hemoglobin concentration of the genotype individual are the lowest. During the successive breeding of the Tibetan improved cattle, the genotype individuals with the molecular marker AA-CC-CC-CC-TT are selected.

[0099] The present invention screens out genes and sites associated with altitude by whole-genome resequencing of cattle genomic DNA distributed at different altitude gradients, creates a low-cost and high-throughput genotyping liquid phase chip (10K) based on these altitude-associated sites, systematically measures hypoxia-related blood physiological indicators based on existing high-generation hybridization segregation populations of backcross and crosscross of Tibetan improved cattle, and uses the 10K SNP chip to perform genome-wide association analysis (GWAS) of hypoxia physiology, and obtains molecular markers 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 on the PSMC2 gene that are associated with the number of red blood cells and hemoglobin concentration.

[0100] In the embodiment of the present invention, Tibetan cattle (3500 m) and Yunnan local cattle (3000 m, 2500 m, 1500 m and 500 m above sea level) with different altitude gradients were selected for whole genome resequencing, and the resequencing data were compared with the cattle reference genome (Bos taurus UMD 3.1). ST , MEMEA and BayPass analysis methods were used to screen 11,637 molecular markers related to the hypoxia tolerance trait in cattle. Then, the target site information was screened and verified, and then the probes were designed and synthesized, tested and adjusted, and the final probes and capture sites were determined, creating a low-cost and high-throughput genotyping liquid phase chip (10K). Finally, based on the existing high-generation hybrid segregation populations of backcross and cross-cross of Tibetan improved cattle, the hypoxia-related blood physiological indicators were systematically determined, and the genome-wide association study (GWAS) of hypoxia physiology was carried out using the 10K SNP chip, and five molecular markers related to the number of red blood cells and hemoglobin concentration on the bovine PSMC2 gene were screened: 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. (Bold and underlined in the sequence)

[0101] Molecular marker of 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 61 bp in the sequence;

[0102] SEQ ID NO.1:

[0103]

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

[0105] SEQ ID NO.2:

[0106]

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

[0108] SEQ ID NO.3:

[0109]

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

[0111] SEQ ID NO.4:

[0112]

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

[0114] SEQ ID NO.5:

[0115]

[0116]

[0117] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A molecular marker in the bovine PSMC2 gene, characterized in that The sites of the molecular markers are one or more of g.44569796T>A, g.44570274T>C, g.44570306T>C, g.44570978G>C and g.44573224C>T in the cattle genome.

2. The molecular marker according to claim 1, characterized in that The site g.44569796T>A means that there is a T>, A base mutation at the 61 bp of the sequence shown in SEQ ID NO.1; The site g.44570274T>C means that there is a T>, C base mutation at the 71 bp in the sequence shown in SEQ ID NO.2; The site g.44570306T>C means that there is a T>, C base mutation at the 81 bp in the sequence shown in SEQ ID NO.3; The site g.44570978G>C means that there is a G>, C base mutation at the 91 bp in the sequence shown in SEQ ID NO.4; The site g.44573224C>T means that there is a C>, T base mutation at the 101 bp in the sequence shown in SEQ ID NO.

5.

3. A method for detecting the number of red blood cells and hemoglobin concentration in cattle, characterized in that: The steps of the method include: 1) Detecting at least one base type at the 61 bp in the SEQ ID NO:1 sequence; the 71 bp in the SEQ ID NO:2 sequence; the 81 bp in the SEQ ID NO:3 sequence; the 91 bp in the SEQ ID NO:4 sequence; and the 101 bp in the SEQ ID NO:5 sequence; 2) When the base type at the 61 bp in the sequence 1) SEQ ID NO: 1 is A, the cattle individual is a CC type individual at the 61 bp; when the base type at the 61 bp in the sequence 1) SEQ ID NO: 1 is T, the cattle individual is a TT type individual at the 61 bp or a TA type individual at the 61 bp; the red blood cell number and hemoglobin concentration of the AA type individual at the 61 bp are lower than those of the TT type individual at the 61 bp and the TA type individual at the 61 bp; When the base type at the 71 bp in the sequence 1) SEQ ID NO: 2 is C, the cattle individual is a CC type individual at the 71 bp; when the base type at the 71 bp in the sequence 1) SEQ ID NO: 2 is T, the cattle individual is a TT type individual at the 71 bp or a TC type individual at the 71 bp; the red blood cell number and hemoglobin concentration of the CC type individual at the 71 bp are lower than those of the TT type individual at the 71 bp and the TC type individual at the 71 bp; When the base type at the 81st bp in the sequence 1) SEQ ID NO:3 is C, the cattle individual is a CC type individual at the 81st bp; when the base type at the 81st bp in the sequence 1) SEQ ID NO:3 is T, the cattle individual is a TT type individual at the 81st bp or a TC type individual at the 81st bp; the red blood cell number and hemoglobin concentration of the CC type individual at the 81st bp are lower than those of the TT type individual at the 81st bp and the TC type individual at the 81st bp; When the base type at the 91st bp in the 1) SEQ ID NO:4 sequence is C, the cattle individual is a CC type individual at the 91st bp; when the base type at the 91st bp in the 1) SEQ ID NO:4 sequence is G, the cattle individual is a GG type individual at the 91st bp or a CG type individual at the 91st bp; the red blood cell number and hemoglobin concentration of the CC type individual at the 91st bp are lower than those of the GG type individual at the 91st bp and the CG type individual at the 91st bp; When the base type at the 101bp in the 1) SEQ ID NO:5 sequence is T, the cattle individual is a TT type individual at the 101bp; when the base type at the 101bp in the 1) SEQ ID NO:5 sequence is C, the cattle individual is a CC type individual at the 101bp or a CT type individual at the 101bp; the red blood cell number and hemoglobin concentration of the TT type individual at the 101bp are lower than those of the CC type individual at the 101bp and the CT type individual at the 101bp.

4. An application of molecular markers in the successive breeding of improved Tibetan cattle, characterized in that: The specific steps include: Determine the gene type at the site of the molecular marker as claimed in claim 1 in the Tibetan improved cattle, and perform successive breeding according to the gene type at the site: Select the individuals whose base at the 61 bp in the SEQ ID NO.1 sequence of claim 1 is A type, and eliminate the individuals whose base at the 61 bp is T type; and / or, Select the individuals whose base at the 71 bp in the SEQ ID NO.2 sequence of claim 1 is C type, and eliminate the individuals whose base at the 71 bp is T type; and / or, Select the individuals whose base at the 81 bp in the SEQ ID NO.3 sequence of claim 1 is C type, and eliminate the individuals whose base at the 81 bp is T type; and / or, Select the individuals whose base at the 91 bp in the SEQ ID NO.4 sequence of claim 1 is C type, and eliminate the individuals whose base at the 91 bp is G type; and / or, The individuals whose base at the 101 bp in the SEQ ID NO.5 sequence of claim 1 is T-type are selected, and the individuals whose base at the 101 bp is C-type are eliminated.

5. The use according to claim 4, characterized in that: The individuals whose gene type of the molecular marker is AA-CC-CC-CC-TT have the lowest red blood cell number and hemoglobin concentration. During the successive breeding of Tibetan improved cattle, individuals whose gene type of the molecular marker is AA-CC-CC-CC-TT are selected.

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