Molecular marker related to cattle carcass character and application thereof

Abstract

The invention relates to the technical field of molecular biology. The invention provides a molecular marker related to cattle carcass traits. The molecular marker related to the cattle carcass traits is related to cattle carcass length, carcass depth, carcass chest depth, net meat weight, dressing percentage and net meat percentage. The invention also provides an application of the molecular marker related to the cattle carcass character in cattle genetic breeding, and an application of a reagent for detecting the molecular marker related to the cattle carcass character in preparation of a reagent for assisting cattle genetic breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a molecular marker related to cattle carcass traits and an application thereof. Background Art

[0002] Single nucleotide polymorphism (SNP) is a site polymorphism caused by the replacement of a single base. It is the most common type of variation in the genome of an organism. SNPs may be within the gene sequence or in non-coding sequences outside the gene. SNP markers have been widely used due to their high genetic stability, rich and wide distribution of sites, and fast and convenient detection.

[0003] In recent years, molecular breeding has gradually involved most areas of breeding, including the protection and development of genetic resources, seed selection, selection, hybrid vigor prediction, and the detection and utilization of quantitative trait loci (QTL). Specifically in the process of livestock and poultry breeding, such as cattle breeds, obtaining high-quality breeds with long and deep carcasses, high net meat rate, and fast growth rate is of great significance to the development of animal husbandry.

[0004] However, there are no reports on the research of genes and variant loci associated with carcass trait advantages in the Huaxi cattle breed. Summary of the invention

[0005] The purpose of the present invention is to provide a molecular marker related to cattle carcass traits and its application, and to determine the molecular sites related to cattle carcass length, carcass depth, carcass chest depth, net meat weight, slaughter rate and net meat rate by detecting and screening SNP molecular sites related to cattle carcass traits and associated genes.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a molecular marker related to cattle carcass traits. The carcass length molecular marker comprises a MUC4 gene SNP site, a VLDLR gene SNP site, a SPTLC2 gene SNP site and a KCNG2 gene SNP site.

[0008] Preferably, the carcass depth molecular markers include the PHF24 gene SNP site and the HSPB3 gene SNP site; the carcass breast depth molecular markers include the CPPED1 gene SNP site; the net meat weight molecular markers include the H2AJ gene SNP site and the CPPED1 gene SNP site; the slaughter rate molecular markers include the ERI2 gene SNP site; the net meat rate molecular markers include the AK4 gene SNP site, the PKHD1L1 gene SNP site and the CCDC85C gene SNP site.

[0009] Preferably, the MUC4 gene SNP site is chr1:70384274, the VLDLR gene SNP site is chr8:41889516, the SPTLC2 gene SNP site is chr10:88811874, the KCNG2 gene SNP site is chr24:648475, the PHF24 gene SNP site is chr8:59239108, the HSPB3 gene SNP site is chr20:24634792, and the CPPED 1 gene SNP site includes chr25:11376242 or chr25:11376552, the H2AJ gene SNP site is chr5:95091626, the ERI2 gene SNP site is chr25:18521054, the AK4 gene SNP site is chr3:80286547, the PKHD1L1 gene SNP site is chr14:55142895, and the CCDC85C gene SNP site is chr21:64466894.

[0010] The invention also provides application of the molecular markers related to cattle carcass traits in cattle genetic breeding.

[0011] The present invention also provides the use of the reagent for detecting the molecular markers related to cattle carcass traits in the preparation of reagents for assisting cattle genetic breeding.

[0012] By adopting the above technical solution, the present invention has the following beneficial effects:

[0013] The present invention analyzes and identifies variant sites associated with cattle carcass traits by combining GWAS and eQTL, and detects 12 genes (MUC4, VLDLR, SPTLC2, KCNG2, PHF24, HSPB3, AK4, PKHD1L1, CCDC85C, H2AJ, CPPED1, ERI2) that are co-regulated by six traits, namely, carcass length, carcass depth, carcass chest depth, net meat weight, slaughter rate and net meat rate, by GWAS and cis-eQTL methods; and further obtains 13 variant sites that regulate the 12 genes in both GWAS and cis-eQTL analysis, wherein the chr8:41889516 variant site regulates the VLDLR gene in GWAS, cis-eQTL and fine mapping. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of genome-wide association analysis for the traits of dressing rate;

[0015] Figure 2 The results of genome-wide association analysis for net meat weight trait;

[0016] Figure 3 This is the genome-wide association analysis of net meat percentage traits;

[0017] Figure 4 The results of genome-wide association analysis for carcass depth traits are shown;

[0018] Figure 5 This is the genome-wide association analysis of carcass length traits;

[0019] Figure 6 This is the genome-wide association analysis of the carcass breast depth trait. DETAILED DESCRIPTION

[0020] The invention provides a molecular marker related to cattle carcass traits, including a carcass length molecular marker, a carcass depth molecular marker, a carcass chest depth molecular marker, a net meat weight molecular marker, a slaughter rate molecular marker and a net meat rate molecular marker.

[0021] In the present invention, the carcass length molecular marker includes a MUC4 gene SNP site, a VLDLR gene SNP site, a SPTLC2 gene SNP site, and a KCNG2 gene SNP site. In the present invention, the MUC4 gene SNP site is chr1:70384274, the VLDLR gene SNP site is chr8:41889516, the SPTLC2 gene SNP site is chr10:88811874, and the KCNG2 gene SNP site is chr24:648475.

[0022] In the present invention, the carcass deep molecular markers include the PHF24 gene SNP site and the HSPB3 gene SNP site. In the present invention, the PHF24 gene SNP site is chr8:59239108, and the HSPB3 gene SNP site is chr20:24634792.

[0023] In the present invention, the carcass breast depth molecular marker includes a CPPED1 gene SNP site. In the present invention, the CPPED1 gene SNP site includes chr25:11376242 or chr25:11376552.

[0024] In the present invention, the net meat weight molecular marker includes the H2AJ gene SNP site and the CPPED1 gene SNP site. In the present invention, the H2AJ gene SNP site is chr5:95091626, and the CPPED1 gene SNP site includes chr25:11376242 or chr25:11376552.

[0025] In the present invention, the slaughter rate molecular marker includes the ERI2 gene SNP site. The ERI2 gene SNP site in the present invention is chr25:18521054.

[0026] In the present invention, the net meat rate molecular marker includes AK4 gene SNP site, PKHD1L1 gene SNP site and CCDC85C gene SNP site. In the present invention, the AK4 gene SNP site is chr3:80286547, the PKHD1L1 gene SNP site is chr14:55142895, and the CCDC85C gene SNP site is chr21:64466894.

[0027] The invention also provides application of the molecular markers related to cattle carcass traits in cattle genetic breeding.

[0028] The present invention also provides the use of the reagent for detecting the molecular markers related to the cattle carcass traits in preparing a kit.

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] 1. Experimental Materials

[0032] 1. Carcass phenotypic data collection

[0033] This study used a resource group of 1566 West China cattle for meat, which was constructed by the cattle genetic breeding innovation team of Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The average age of the group was 24 months. The carcass weight (CW), carcass length (CL), carcass depth (CD), carcass chest depth (CCD) and net meat weight (LMW), live weight before slaughter (LW), slaughter percentage (DP) and net meat percentage (LMP) were collected and measured. The measurement and calculation of the above traits were strictly in accordance with the relevant industry standards (NY / T2660-2014).

[0034]

[0035]

[0036] 2. Genotype Data Collection

[0037] In this study, blood samples from 1566 cattle were collected, and the TIANamp Blood DNA kit was used to extract genomic DNA from the blood. The Illumina BovineHD SNP chip was used to perform genotyping on high-standard DNA samples (DNA concentration>50ng / μL, A260 / A280 between 1.8-2.0). For detailed information on this chip, please refer to (https: / / www.illumina.com / documents / products / datasheets / datasheet_bovineHD.pdf). After genotyping, the offline data was quality controlled using PLINK (v1.90) software to eliminate unqualified SNP sites and individuals. The elimination criteria are as follows:

[0038] (1) The SNP detection rate is less than 95%;

[0039] (2) The frequency of the smallest allele of the SNP locus is less than 5%;

[0040] (3) individuals with a SNP deletion greater than 10%;

[0041] (4) The SNP locus does not meet the Hardy-Weinberg equilibrium test P value 1×10 -5 ;

[0042] (5) The SNP site is located on the sex chromosome.

[0043] Download the Qianniu Project public data (https: / / www.ebi.ac.uk / ena / browser / view / ERZ1738264), use Beagle software to fill in the genotype data, and obtain the filled genotype data.

[0044] 3. Transcriptome Sample Collection

[0045] Transcriptome data were collected from 227 Huaxi cattle from the resource population, which were born at similar times and from the same cattle farm. After weaning, the calves were transported to the same fattening farm for fattening, and the feeding conditions were kept consistent during the fattening period. They were slaughtered when they were 24 months old. The longissimus dorsi muscle samples on the left side of the cattle were placed in 5 mL cryovials and stored in liquid nitrogen tanks.

[0046] (II) Experimental methods

[0047] 1. Genome-wide association study (GWAS)

[0048] Based on the mixed linear model, GWAS analysis was conducted on carcass weight (CW), carcass length (CL), carcass depth (CD), carcass chest depth (CCD), net meat weight (LMW), dressing percentage (DP) and net meat percentage (LMP). The factors affecting the relevant carcass traits were comprehensively considered, and the farm, year and sex were used as fixed effects. The analysis was performed using GCTA (v1.93.0). The specific model is as follows:

[0049] y=u+bx+g+e

[0050] Where y is the phenotypic value vector; b is the coefficient of the fixed effect covariate; u is the mean; x is the genotype indicator variable, and the three different genotypes BB / Bb / bb are coded with 0 / 1 / 2 numbers respectively; e is the random residual and follows the normal distribution N(0,σ e 2 ); g represents the vector of small-effect polygenic effects and follows the normal distribution N(0,σ a 2 G) SNP significance test The Bonferroni multiple test method was used to set the SNP genome significance level threshold to 1 / SNP number.

[0051] 2. RNA library construction, quality control, sequencing and analysis

[0052] (1) Cut a soybean-sized muscle sample (100 mg), add it to a sterile centrifuge tube (2 mL) containing steel beads and RNA extraction reagent (1 mL TRIzol), and mix it using a homogenizer (40 Hz, 20 s / time, repeated 3 times);

[0053] (2) Centrifuge at 12,000 g for 15 min at 4°C, extract the supernatant, transfer it into a centrifuge tube (1.5 mL) containing 200 μL of chloroform (CHCl3), and shake for 15 s;

[0054] (3) Centrifuge at 12,000 g for 15 min at 4 °C, extract the supernatant, transfer it into a centrifuge tube (1.5 mL) containing 500 μL of isopropanol (C3H8O), shake gently to mix, and let stand for 20 min;

[0055] (4) Centrifuge at 12,000 g for 15 min at 4°C, discard the supernatant, add 1 mL of 75% ethanol, and repeat the washing process;

[0056] (5) 4°C, 8000 g, 10 min, discard the supernatant, dry for 5 min, add 30 μL of RNase-free H2O2 to dissolve the RNA precipitate;

[0057] (6) Gel electrophoresis imager, Nanodrop spectrophotometer, and Agilent2100 were used to detect the purity, concentration, and integrity of RNA, and high-quality RNA (OD260 / OD280>1.8, integrity value>7) was selected for library construction;

[0058] (7) Enrich mRNA from all samples using magnetic beads attached with Oligo(dT), obtain mRNA fragments using Fragmentation buffer, and use the fragments as templates to synthesize the first strand of cDNA;

[0059] (8) Using the first strand of cDNA as a template, synthesizing the second strand of cDNA, and purifying the double-stranded cDNA using a nucleic acid purification kit;

[0060] (9) Filling the sticky ends of the purified double-stranded cDNA, adding A-tails and connecting sequencing adapters to amplify the library;

[0061] (10) Qubit 2.0 and Agilent 2100 were used to detect library concentration and fragment size, respectively. The libraries that passed the quality inspection were sequenced using Illumina Nova Seq 6000 according to the PE150 strategy.

[0062] (11) After obtaining the original sequencing data, the transcriptome data was filtered using Fastp (v0.23.0) software to obtain high-quality Cleandata;

[0063] (12) Cleandata was aligned to the latest reference genome of cattle using Hisat2 (v2.2.1) software;

[0064] (13) The expression abundance (TPM) of all genes in the genome was quantified using StringTie (v2.1.1) software.

[0065] 3. Expression quantitative trait loci (eQTL)

[0066] (1) Genes with TPM ≥ 0.1 in 20% of the samples were selected for subsequent analysis.

[0067] (2) To reduce the impact of batch effect and population structure on genes, the first two population structures and 15 batch effects were selected to correct gene expression.

[0068] (3) PLINK was used to extract the filling genotypes of 227 heads in the resource population, and the corrected gene expression levels were used as phenotypes. FastQTL was used for cis-eQTLs analysis, and the Benjamini-Hochberg method was used to screen significant cis-regulatory sites (cis-variants).

[0069] 4. eQTL finemapping

[0070] The DAP-g algorithm was used to perform fine mapping analysis (eQTLfinemapping) of cis-eQTLs, and sites with posterior probability (PIP)>0.8 were selected.

[0071] (III) Experimental results

[0072] 1. Genome-wide association analysis

[0073] 239 variants were screened by the significant threshold and 184 significant genes were annotated (1 / N = 1.13 × 10 -7 ), a total of 2205 candidate variants and 822 genes were screened by the optional threshold (P = 10 -5 ), among which 122 genes including MUC4, VLDLR, SPTLC2, KCNG2 were identified for carcass length, 95 genes including PHF24, HSPB3 were identified for carcass depth, 71 genes including AK4, PKHD1L1, CCDC85C were identified for net meat percentage, 174 genes including H2AJ, CPPED1 were identified for net meat weight, 114 genes including ERI2 were identified for dressing percentage, 122 genes including CPPED1 were identified for carcass breast depth, 124 genes were identified for carcass weight (such as Figure 1-6 ).

[0074] 2. eQTL Analysis of Dorsal Muscle Tissue

[0075] Through eQTL analysis of dorsal muscle tissue, a total of 1,370,681 cis-eQTLs regulated 5,860 cis-regulated genes (cis-eGenes), including MUC4, VLDLR, SPTLC2, KCNG2, PHF24, HSPB3, AK4, PKHD1L1, CCDC85C, H2AJ, CPPED1, and ERI2.

[0076] 3. eQTL finemapping

[0077] eQTLfinemapping analysis of 1,370,681 cis-eQTLs revealed 410 eQTLs, among which the chr8:41889516 variant site regulated the VLDLR gene.

[0078] 4. The intersection of GWAS and cis-eQTL

[0079] By comparing the genes identified by GWAS and cis-eQTL (Table 1), it was found that 13 variant sites regulated 12 genes (MUC4, VLDLR, SPTLC2, KCNG2, PHF24, HSPB3, AK4, PKHD1L1, CCDC85C, H2AJ, CPPED1, ERI2) in both GWAS and cis-eQTL analysis. It was further found that the chr8:41889516 variant site regulated VLDLR in GWAS, cis-eQTL and finemapping.

[0080] Table 113 variant sites regulate 12 common genes

[0081]

[0082]

[0083] Note: CL stands for carcass length, CD stands for carcass depth, CCD stands for carcasschest depth, LMW stands for leanmeat weight, and LMP stands for leanmeat percentage.

[0084] It can be seen from the above embodiments that the present invention screened out 12 common genes related to cattle carcass traits through GWAS and cis-eQTL methods, namely MUC4, VLDLR, SPTLC2, KCNG2, PHF24, HSPB3, AK4, PKHD1L1, CCDC85C, H2AJ, CPPED1 and ERI2; it was also found that the above 13 sites have a regulatory effect on these 12 genes in GWAS or cis-eQTL analysis.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A molecular marker associated with a bovine carcass trait, characterized in that: The carcass length molecular markers include the MUC4 gene SNP site, the VLDLR gene SNP site, the SPTLC2 gene SNP site, and the KCNG2 gene SNP site; Carcass deep molecular markers include the PHF24 gene SNP site and the HSPB3 gene SNP site; Carcass breast depth molecular markers include the CPPED1 gene SNP locus; The net meat weight molecular markers include the H2AJ gene SNP site and the CPPED1 gene SNP site; Molecular markers for slaughter rate include the ERI2 gene SNP locus; The molecular markers of net meat rate include AK4 gene SNP site, PKHD1L1 gene SNP site and CCDC85C gene SNP site; The MUC4 gene SNP site is chr1:70384274, the VLDLR gene SNP site is chr8:41889516, the SPTLC2 gene SNP site is chr10:88811874, the KCNG2 gene SNP site is chr24:648475, the PHF24 gene SNP site is chr8:59239108, the HSPB3 gene SNP site is chr20:24634792, the CPPED1 gene SNP site is chr14:88811874, the KCNG2 gene SNP site is chr24:648475, the PHF24 gene SNP site is chr8:59239108, the HSPB3 gene SNP site is chr20:24634792, the The SNP sites include chr25:11376242 or chr25:11376552, the H2AJ gene SNP site is chr5:95091626, the ERI2 gene SNP site is chr25:18521054, the AK4 gene SNP site is chr3:80286547, the PKHD1L1 gene SNP site is chr14:55142895, and the CCDC85C gene SNP site is chr21:64466894.

2. Application of the molecular markers related to cattle carcass traits as described in claim 1 in cattle genetic breeding.

3. Use of a reagent for detecting molecular markers associated with cattle carcass traits as claimed in claim 1 in the preparation of reagents to assist cattle genetic breeding.

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