Molecular marker in bovine KCNIP1 gene and application thereof

Abstract

The invention discloses a molecular marker in a bovine KCNIP1 gene and application of the molecular marker. The molecular marker is an SNP (Single Nucleotide Polymorphism) site g.2487025Ggt in a bovine genome; a. The molecular marker or the substance for detecting the single nucleotide polymorphism of the cattle molecular marker provided by the invention can be applied to cattle genetic breeding, so that the number of red blood cells and the hemoglobin concentration of improved cattle offspring in Tibetan are reduced, and the problem of hypoxia faced by cattle breeding in a plateau environment is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to molecular markers in cattle KCNIP1 genes and applications 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.

[0004] KCNIP1 is a member of the cytosolic voltage-gated potassium (Kv) channel interacting protein family. It binds to the Kv4α subunit to form a native Kv4 channel complex. The encoded protein can regulate the fast inactivating current and thus the excitability of the membrane of neurons in response to changes in intracellular calcium concentration. Diseases associated with KCNIP1 include epilepsy and hypertension, and their related pathways include cardiac conduction. K channel interacting protein (KCNIP1) potassium channels are a major class of fast inactivating K channels in the brain and heart, and may also help cytoplasmic messengers regulate Kv4 channel gating. There is evidence that KCNIP1 may affect the trafficking or stability of native Kv4 proteins, because co-expression of KCNIP1 with Kv4 subunits leads to enhanced current amplitude in oocytes or mammalian cells. There are currently no reports on the association of the KCNIP1 gene with red blood cell number and hemoglobin concentration. Therefore, it is urgent to screen new molecular markers associated with the traits of red blood cell number and hemoglobin concentration in cattle, provide new molecular marker resources for molecular marker-assisted selection of plateau dairy cattle, and promote the process of plateau dairy cattle breeding improvement. Summary of the invention

[0005] The purpose of the present invention is to discover the molecular markers in the cattle KCNIP1 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 KCNIP1 gene, wherein the site of the molecular marker is g.2487025G>A in the bovine genome.

[0007] Furthermore, the molecular site g.2487025G>A means that there is a G>, A base mutation at the 61 bp in the sequence shown in SEQ ID NO.1.

[0008] The second aspect of the present application also provides a method for detecting the number of red blood cells and hemoglobin concentration of cattle, the method comprising the following steps:

[0009] 1) Detect the base type at the 61st bp in the SEQ ID NO:1 sequence;

[0010] 2) When the base type at the 61st bp in the sequence of 1) SEQ ID NO: 1 is A, the number of red blood cells and the hemoglobin concentration of the cattle are low, and the cattle are GG type individuals at the 61st bp;

[0011] When 1) the base type at the 61st bp in the SEQ ID NO: 1 sequence is G, the number of red blood cells and the hemoglobin concentration of the cattle are relatively high, and the cattle are AA type individuals at the 61st bp or GA type individuals at the 61st bp.

[0012] The third aspect of the present application also provides an application of molecular markers in the successive breeding of Tibetan improved cattle, and the specific steps include:

[0013] (1) Determine the gene type at the locus of the above molecular markers in Tibetan improved cattle, and conduct successive breeding based on the gene type at the locus;

[0014] (2) Select cattle for successive breeding with individuals whose base at the 61 bp in the sequence SEQ ID NO.1 is G type, and eliminate individuals whose base at the 61 bp is A type.

[0015] Beneficial effects of the present invention:

[0016] 1. The present invention provides molecular markers associated with the number of red blood cells and hemoglobin concentration in the bovine KCNIP1 gene, wherein the molecular markers include the g.2487025G>A molecular marker; the above molecular markers can be used as molecular markers for the number of red blood cells and hemoglobin concentration of the bovine, respectively.

[0017] 2. The present invention verifies the effect of the above molecular markers on the number of red blood cells and hemoglobin concentration in 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 G of the molecular locus g.2487025G>A can be selected. Individuals with this gene type have lower red blood cell numbers and hemoglobin concentrations. This improves the hypoxia adaptability of the offspring of Tibetan improved cattle.

[0018] 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

[0019] Figure 1 Manhattan plot and QQ plot for GWAS of red blood cell number index.

[0020] Figure 2 Manhattan plot and QQ plot for GWAS of hemoglobin concentration index. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1 Screening of hypoxia-resistant sites in Tibetan cattle

[0023] 1. Blood sample collection of cattle at different altitude gradients

[0024] 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-altitude 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.

[0025] 2. Genomic DNA Extraction and Whole Genome Resequencing

[0026] 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.

[0027] 3.SNP calling and sequence alignment

[0028] 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.

[0029] Then download the cattle genome gff file from Ensemble and use ANNOVAR software to perform SNP annotation.

[0030] 4. Screening of hypoxia-tolerant sites in Tibetan cattle

[0031] (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;

[0032] (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;

[0033] (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;

[0034] (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.

[0035] Example 2: Application of low-density liquid phase chip

[0036] 1. Synthesis of Probes

[0037] 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.

[0038] 2. Detection of Tibetan Improved Cattle Plateau Adaptation Breeding Chip

[0039] 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.

[0040] 3. GWAS analysis

[0041] (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;

[0042] (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;

[0043] (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;

[0044] (4) Functional annotation of molecular markers using ANNOVAR;

[0045] (5) GWAS analysis was performed using the general linear model (GLM) in Tassel software. The model is as follows:

[0046] y=Wα+xβ+ε

[0047] In the formula:

[0048] y represents the phenotypic traits to be studied, i.e., RBC and HGB indices in this study;

[0049] Wα is the fixed effect (age, sex and feeding level), other factors affecting y, mainly referring to the group structure;

[0050] xβ is the labeling effect;

[0051] ε represents the residual.

[0052] GWAS results showed that the red blood cell number and hemoglobin concentration indicators were annotated to potassium voltage-gated channel interacting protein 1 (KCNIP1) at the selected signal site on chromosome 20 ( Figure 1 and Figure 2 ), the selected signal is strong. KCNIP1 is a member of the cytosolic voltage-gated potassium (Kv) channel interacting protein family, which binds to the Kv4α subunit to form a native Kv4 channel complex. The encoded protein can regulate the fast inactivating current, thereby regulating the excitability of the membrane of neurons in response to changes in intracellular calcium concentration. Diseases associated with KCNIP1 include epilepsy and hypertension, and their related pathways include cardiac conduction. K channel interacting protein (KCNIP1) potassium channels are a major class of fast inactivating K channels in the brain and heart, and may also help cytoplasmic messengers regulate Kv4 channel gating. There is evidence that KCNIP1 may affect the trafficking or stability of native Kv4 proteins, because co-expression of KCNIP1 with Kv4 subunits leads to enhanced current amplitude in oocytes or mammalian cells (Boland LM et al., 2003).

[0053] Example 3: Correlation analysis and application of molecular markers with red blood cell count and hemoglobin concentration

[0054] In order to further determine whether the KCNIP1 gene SNP site g.2487025G>A is related to the differences in the number of red blood cells and hemoglobin concentration of 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. The model used was:

[0055] Y ijk =μ+G i +H j +S k +e ijkl

[0056] Among them, Y ijk is the observed value of physiological index, μ is the population mean, G i is the genotype effect of KCNIP1 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.

[0057] 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.

[0058] The allele frequency and genotype frequency of SNP site g.2487025G>A are shown in Table 1.

[0059] The results of statistical analysis are shown in Table 1: The red blood cell number and hemoglobin concentration of the GG genotype in the SNP site g.2487025G>A were significantly lower than those of the AA genotype and GA genotype.

[0060] Table 1 Allele frequency and genotype frequency of SNP loci

[0061]

[0062] Table 2 Association analysis between KCNIP1 gene polymorphism and blood physiology of Tibetan improved cattle

[0063]

[0064] Note: The values ​​in the table are expressed as mean ± standard error. Different capital letters in the same row of data indicate extremely significant differences (P<0.01), and the same letters indicate insignificant differences (P>0.01).

[0065] The results of genetic association analysis showed that the molecular marker of SNP site g.2487025G>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 G at the 61bp in the sequence shown in SEQ ID NO.1 were significantly lower than those of individuals with genotype A at the 61bp in the sequence shown in SEQ ID NO.1, indicating that genotype G 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.

[0066] Example 4 Application of molecular markers in the above KCNIP1 gene in successive breeding

[0067] 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.

[0068] The base at the 61 bp position in the SEQ ID NO.1 sequence was determined to be G type, and the number of red blood cells and hemoglobin concentration of this base type were significantly lower than those of other base types.

[0069] Therefore, the individuals whose base at the 61bp in the SEQ ID NO.1 sequence is G type (the cattle individuals are GG type individuals at the 61bp) are selected for the successive breeding of the Tibetan improved cattle, and the individuals whose base at the 61bp in the SEQ ID NO.1 sequence is A type (the cattle individuals are AA type individuals at the 61bp or GA type individuals at the 61bp) are eliminated.

[0070] The present invention screened out genes and sites associated with altitude by whole-genome resequencing of cattle genomic DNA distributed at different altitude gradients, and created a low-cost and high-throughput genotyping liquid phase chip (10K) based on these altitude-associated sites. According to the existing high-generation hybrid separation populations of backcross and crosscross of Tibetan improved cattle, the hypoxia-related blood physiological indicators were systematically measured. The 10K SNP chip was used to implement a genome-wide association study (GWAS) of hypoxia physiology, and the molecular marker SNP site g.2487025G>A on the KCNIP1 gene associated with the number of red blood cells and hemoglobin concentration was obtained.

[0071] 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, and finally screened out 11,637 molecular markers related to the hypoxia tolerance trait of cattle. Then the target site information was screened and checked, and then the probes were designed and synthesized, tested and adjusted, and the final probes and capture sites were determined to create 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 10K SNP chip was used to implement a genome-wide association analysis (GWAS) of hypoxia physiology, and 5 molecular markers related to the number of red blood cells and hemoglobin concentration on the cattle KCNIP1 gene were screened: SNP site g.2487025G>A. (Bold and underlined in the sequence)

[0072] Molecular marker of SNP site g.2487025G>A: The nucleotide sequence of the fragment is shown in SEQ ID NO: 1, and there is a G>A base mutation at the 61 bp in the sequence (bold and underlined indicate the mutation site);

[0073] SEQ ID NO.1:

[0074]

[0075] 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 KCNIP1 gene, characterized in that: The site of the molecular marker is g.2487025G>A in the cattle genome.

2. The molecular marker according to claim 1, characterized in that The molecular marker g.2487025G>A means that there is a G>, A base mutation at the 61 bp position in the sequence shown in SEQ ID NO.

1.

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) Detect the base type at the 61st bp in the SEQ ID NO:1 sequence; 2) When the base type at the 61st bp in the sequence of 1) SEQ ID NO: 1 is A, the number of red blood cells and the hemoglobin concentration of the cattle are low, and the cattle are GG type individuals at the 61st bp; When the base type at the 61st bp in the 1) SEQ ID NO: 1 sequence is G, the number of red blood cells and the hemoglobin concentration of the cattle are relatively high, and the cattle are AA type individuals at the 61st bp or GA type individuals at the 61st bp.

4. An application of molecular markers in the successive breeding of improved Tibetan cattle, characterized in that: The specific steps include: (1) determining the gene type at the site of the molecular marker as claimed in claim 1 in the Tibetan improved cattle, and performing successive breeding according to the gene type at the site; (2) Select cattle for successive breeding with individuals whose base at the 61 bp in the sequence SEQ ID NO.1 is G type, and eliminate individuals whose base at the 61 bp is A type.

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