Use of molecular markers for beef cattle meat quality traits detection, and chips, methods and applications

Abstract

The application relates to the technical field of molecular detection, and particularly discloses the use, chip, method and application of a molecular marker for beef cattle meat trait detection, wherein the molecular marker comprises 62 SNP sites; the physical positions of the 62 SNP sites are determined based on whole genome sequence alignment of a Hereford cattle reference genome, the version number of the whole genome sequence of the Hereford cattle reference genome is GCF_002263795.3; and the 62 SNP sites are shown in Table 1. The molecular marker of the application can be applied to detection of growth traits and meat quality traits of different breeds of beef cattle, wherein the growth traits include body weight, body height, cross section height, body slant length, chest circumference and pipe circumference; and the meat quality traits include backfat thickness, eye muscle area, protein, intramuscular fat content, amino acid, myristic acid, palmitoleic acid, linoleic acid and arachidonic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular detection, in particular to the use of molecular markers for beef cattle meat trait detection, a chip, a method and application. BACKGROUND

[0002] The important economic traits of beef cattle mainly include growth traits and meat quality traits, wherein the growth traits include body weight, body height, crossbreds height, body slant length, chest circumference and pipe circumference; the meat quality traits include backfat thickness, eye muscle area, protein, intramuscular fat content, amino acid, myristic acid, palmitoleic acid, linoleic acid and arachidonic acid.

[0003] The prior art only discloses SNP markers related to part of growth traits and meat quality traits of specific breeds of beef cattle, and cannot well detect the growth traits and meat quality traits of different breeds of beef cattle. For example, CN202110034261.5 discloses a SNP marker related to beef cattle meat traits and its application, which discloses that A / G mutation exists at OLR1 gene exon 4 at Chr5:99812807bp of grassland red cattle, and the SNP marker is related to water loss rate and eye muscle area; for example, CN201910026712.3 discloses a ARID5B gene mutation site affecting intramuscular fat content of beef cattle and its application, which discloses that the ARID5B gene mutation site affecting the intramuscular fat content of Yanhuang cattle is located at the 10th exon of ARID5B gene 261bp.

[0004] Therefore, in order to adapt to the detection of growth traits and meat quality traits of different breeds of beef cattle, it is necessary to provide a more comprehensive and effective combination of molecular markers. SUMMARY

[0005] The purpose of the present application is to provide the use of molecular markers for beef cattle meat trait detection, a chip, a method and application. The molecular markers of the present application can be used to detect the growth traits and meat quality traits of different breeds of beef cattle, including body weight, body height, crossbreds height, body slant length, chest circumference and pipe circumference; the meat quality traits include backfat thickness, eye muscle area, protein, intramuscular fat content, amino acid, myristic acid, palmitoleic acid, linoleic acid and arachidonic acid.

[0006] The present application is realized by the following technical solutions:

[0007] A molecular marker for beef cattle meat trait detection, the molecular marker comprising 62 SNP sites; the physical positions of the 62 SNP sites are determined based on whole genome sequence alignment of a Hereford cattle reference genome, and the version number of the whole genome sequence of the Hereford cattle reference genome is GCF_002263795.3; wherein the 62 SNP sites are shown in Table 1:

[0008] Table 1

[0009]

[0010] Wherein, the number before the colon in the genomic position represents the chromosome, and the number after the colon represents the physical position of the locus on the corresponding chromosome.

[0011] The 62 SNP sites of the application are obtained based on DNA samples of two beef cattle breeds, i.e., Yanhuang cattle and grassland red cattle, through chip detection and GWAS analysis, and the 62 SNP sites can be used for detecting growth traits and meat quality traits of different breeds of beef cattle, the growth traits including body weight, body height, cross section height, body slant length, chest circumference and pipe circumference, and the meat quality traits including backfat thickness, eye muscle area, protein, intramuscular fat content, amino acid, myristic acid, palmitoleic acid, linoleic acid and arachidonic acid, and the application has the advantages of comprehensive detection of growth traits and meat quality traits.

[0012] The application of the molecular marker for detecting beef cattle meat traits in a gene breeding chip, and the gene breeding chip includes a liquid chip.

[0013] A liquid chip for detecting beef cattle meat traits, and the genotyping of the liquid chip includes the 62 SNP sites described above.

[0014] The liquid chip further includes probes designed according to the gene sequences covering the 62 SNP sites.

[0015] Specifically, the GC content ratio of the probe sequence is 20-80%, the probe length is 120 bp, the maximum upper limit of the number of specific similar fragments on the reference genome is less than 5, and the maximum distance from the designed region is less than 10 bp.

[0016] A design method of a liquid chip, including the following steps:

[0017] S1, sample collection: obtaining a DNA sample of beef cattle;

[0018] S2, data acquisition: performing chip detection based on the DNA sample, acquiring genotype data, and performing performance determination on the growth traits of the sample and detecting the meat quality traits, so as to obtain phenotype data related to the growth traits and the meat quality traits of the sample;

[0019] S3, quality control: performing genotype quality control and phenotype quality control on the genotype data and the phenotype data, respectively;

[0020] S4, filling: filling the missing genotypes after quality control;

[0021] S5, GWAS analysis: performing GWAS analysis on the genotype data and the phenotype data after quality control and filling; obtaining significant sites and positioning candidate genes;

[0022] S6, probe design: design the probe sequence for detecting 62 SNP sites.

[0023] The liquid chip is applied to detecting beef cattle growth traits and meat quality traits, the growth traits include body weight, body height, cross section height, body slant length, chest circumference and pipe circumference; the meat quality traits include back fat thickness, eye muscle area, protein, intramuscular fat content, amino acid, myristic acid, palmitoleic acid, linoleic acid and arachidonic acid.

[0024] Specifically, the liquid chip is applied to detecting beef cattle growth traits and meat quality traits, and specifically includes the following steps:

[0025] Step A, obtaining the DNA sample of the beef cattle individual to be detected;

[0026] Step B, obtaining the genotype data of the beef cattle individual to be detected based on the liquid chip;

[0027] Step C, analyzing the detection result to determine whether the beef cattle individual to be detected contains the genes related to the growth traits and the meat quality traits.

[0028] The liquid chip is applied to beef cattle molecular breeding.

[0029] The liquid chip is applied to beef cattle whole genome association analysis.

[0030] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0031] The 62 SNP sites of the present application can be used for detecting the growth traits and the meat quality traits of different breeds of beef cattle, the growth traits include body weight, body height, cross section height, body slant length, chest circumference and pipe circumference; the meat quality traits include back fat thickness, eye muscle area, protein, intramuscular fat content, amino acid, myristic acid, palmitoleic acid, linoleic acid and arachidonic acid, and the present application has the comprehensive advantage of detecting the growth traits and the meat quality traits. When the liquid chip designed based on the 62 SNP sites is used for detecting the growth traits and the meat quality traits of beef cattle, the detection efficiency is high, the gene detection of the target traits of different breeds of beef cattle can be realized quickly and at low cost, and the present application can be used for screening the breeding of high-quality beef cattle. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below, the illustrative embodiments of the present application and the description thereof are only used for explaining the present application, and are not used as the limitation of the present application, the following described embodiments are part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without making creative efforts belong to the protection scope of the present application.

[0033] In the following description, numerous specific details are set forth to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details. In other instances, well-known structures, materials, or processes have not been described in detail in order to avoid obscuring the present application. Materials, instruments, and reagents used in the following examples, and throughout the specification, are those that are conventionally used and are available to those skilled in the art unless otherwise specified. Techniques used in the examples are those conventionally used and are well known and available to those skilled in the art unless otherwise specified.

[0034] Example 1

[0035] Screening of molecular markers for detecting beef cattle growth traits and meat quality traits

[0036] This example is based on the raw sites (raw sequencing data) obtained by chip sequencing of 346 beef cattle samples, GWAS analysis is carried out based on the raw data, significant sites and candidate genes are obtained, and finally 62 SNP sites as shown in Table 1 or Table 2 are obtained.

[0037] Among them, the 346 beef cattle samples include 202 Yanhuang cattle and 144 grassland red cattle, 96 Yanhuang cattle and 144 grassland red cattle are used for beef cattle growth trait analysis, and 106 Yanhuang cattle are used for beef cattle meat quality trait analysis.

[0038] The specific screening process includes the following steps:

[0039] S1, sample collection: obtain DNA samples of 202 Yanhuang cattle and 144 grassland red cattle;

[0040] S2, data acquisition: 202 Yanhuang cattle and 144 grassland red cattle DNA samples are detected by 150k chip (BeadChip Array 24X1 Infinium HTS iSelect GGP-HDv3) and 20k chip (12 Reactions, Panel Name:062023035-cattle20K) respectively, genotype data is obtained, and performance determination is performed on the growth traits (such as body weight, body height, cross section height, body oblique length, etc.) of the above samples, and the meat quality traits (such as different amino acid and fatty acid content, etc.) are detected, thereby obtaining the phenotype data related to the growth traits and meat quality traits of the samples;

[0041] S3, quality control: genotype data and phenotype data are subjected to genotype quality control and phenotype quality control respectively. The genotype quality control process will delete sites with missing typing number >10%, minimum allele frequency lower than 0.01, and Hardy-Weinberg test P value <0.000001, and for phenotype quality control, the individual with missing phenotype should be deleted;

[0042] S4, filling: for the SNP not detected on individual sites, using Beagle5.0 can maximize the use of known site information to infer unknown sites, and the filling accuracy is more than 95%;

[0043] S5, GWAS analysis: the GWAS analysis adopts a mixed linear model, wherein y is a phenotype vector, is a population structure effect and a fixed effect of field, year, season, etc., is a marker effect to be tested, is a polygenic effect, is a residual effect. K in the polygenic effect is a kinship matrix inferred by markers. Finally, GEMMA is used to quickly and accurately solve, and ANNOVAR software is used to annotate significant SNPs to the corresponding genes, to obtain significant sites and candidate genes of different traits of beef cattle.

[0044] Table 2

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] Note: The red cattle in Table 2 are grassland red cattle.

[0053] Example 2

[0054] The liquid chip for detecting beef cattle meat traits includes a probe combination for identifying the genotypes of 62 SNP sites.

[0055] The purpose of this embodiment is to apply 62 SNP sites in Table 1 or Table 2 to probe design. Using the GenoBaits Probe Designer software, the probe is designed according to the evaluation results of the upstream and downstream sequences of the target site, mainly evaluating the complexity of the upstream and downstream sequences of the target site, GC content, etc., and the target site is preferentially placed in the middle position of the probe. The designed probe is 120 bp in length. Generally, the performance of the probe is comprehensively evaluated from the aspects of GC content (20%-80%), Tm value (60-80℃), number of alignments on the reference genome (≤5), etc. The probe is 120 bp in length, and the 5' end is modified with a biotin group DNA nucleotide sequence.

[0056] Example 3:

[0057] The method for detecting the genotypes of 62 SNP sites in Table 1 using the liquid chip developed in Example 2 includes the following steps:

[0058] 1. Genomic DNA extraction

[0059] The magnetic bead method DNA extraction kit (Kangwei Century CW2361S) was used to extract the sample DNA.

[0060] 2. Genomic DNA fragmentation

[0061] Take 300 ng of DNA sample and add Smearase Buffer 4 μL, Smearase Enzymes 2 μL, total volume 24 μL, then place the reaction plate in the PCR instrument to execute the program: 4℃ 1 min→30℃ 10 min→72℃ 20 min→4℃ storage.

[0062] 3. Fragment selection and end repair plus A

[0063] First, the range of broken fragments is selected by magnetic beads to remove fragments that are too large and too small, so that the DNA fragments are concentrated in 200-300 bp; secondly, take 40 μL of fragmented DNA / cfDNA, End Repair&A-Tailing Buffer 6 μl, End Repair&A-Tailing Enzyme 4 μl, total reaction system is 50 ul. Mix well, take 10 μL in 96-well PCR plate, 20℃ incubate for 30 min, 65℃ incubate for 30 min, then 4℃ storage.

[0064] 4. Linker ligation and enrichment

[0065] In the 96-well PCR plate of step 3, 5 μL CAGT Universal Adapters, 20 μL Ligation Master Mix were added, vortexed to mix, centrifuged briefly to collect the reaction solution to the bottom of the tube, and incubated at 20°C for 15 min in a PCR instrument to complete the ligation of the sequencing adapters. After purification of the ligation product, PCR amplification enrichment was performed, 15 μL of the purified ligation product was taken, 10 μL CAGT UDI Primer was added, 25 μL Equinox Library Amp Mix (2x) was added, mixed, 35 μL of the reaction solution was taken to the PCR instrument for library amplification. After the construction of the whole genome library, the library was quantified using the dsDNA HS Assay Kit for Qubit kit; at the same time, electrophoresis was used to detect whether the main peak of the library was in the range of 300-500 bp.

[0066] 5. Liquid chip hybrid capture

[0067] The whole genome library of each sample was pooled, and the total amount of the final hybrid capture library was 4 μg. The pooled library was concentrated and hybridized with probes to capture the fragments of the target region from the whole genome library. Excess probes, hybridization reagents and other reagent components were removed by elution. Finally, the target region was enriched by PCR amplification after hybridization to obtain the library for sequencing.

[0068] 6. Library quality control and sequencing

[0069] After the construction of the hybrid capture library, the library was quantified using the dsDNA HS Assay Kit for Qubit kit; at the same time, electrophoresis was used to detect whether the main peak of the library was in the range of 300-500 bp. The constructed library was sequenced on a DNBSEQ-T7 sequencer.

[0070] The 346 samples in Example 1 were used for liquid chip detection of 62 sites, and the average detection rate reached 100%.

[0071] Example 4:

[0072] Application of the liquid chip developed in Example 2 in beef cattle growth traits and meat quality traits

[0073] The whole genome resequencing data of 40 samples of 4 beef cattle breeds (grassland red cattle, Yan cattle, Yanbian cattle and Simmental cattle) were used to detect 62 SNP sites of the above samples, and the detection rate was above 99.58%, which was sufficient to prove that the screened sites supported the detection of growth and meat quality traits of beef cattle samples of other breeds. The specific embodiments are shown in Table 3:

[0074] Table 3

[0075] .

[0076] Example 5

[0077] Application of the liquid phase chip developed in Example 2 in whole genome association analysis of beef cattle

[0078] Using the liquid phase chip developed in Example 2, the genotype data of 202 Yanhuang cattle and 144 Caoyuanhong cattle in Example 1 were obtained and filled by genotype detection in Example 3, and based on the mixed linear model, the growth traits and meat quality traits of 202 Yanhuang cattle and 144 Caoyuanhong cattle were analyzed.

[0079] The phenotypic variance explained (PVE) of each SNP site was calculated based on the PVE calculation formula, and the PVE of 62 SNP sites was greater than 14%. Combined with the P value (derived from TASSEL software) and the set threshold, 62 SNP sites related to the growth traits and meat quality traits of beef cattle were screened out.

[0080] Example 6

[0081] Application of the liquid phase chip developed in Example 2 in beef cattle breeding

[0082] The whole genome resequencing data of the beef cattle to be tested can be obtained by using the liquid phase chip developed in Example 2, and 62 SNP sites in Table 1 are detected. Whether the growth traits and meat quality traits of the detected beef cattle meet the requirements can be judged according to the detection rate of 62 SNP sites:

[0083] For example: when the detection rate of 62 SNP sites is 100%, i.e. the sample contains 62 SNP sites on the chromosome, i.e. the sample meets the requirements of the growth traits and meat quality traits of beef cattle; when the detection rate of 62 SNP sites is less than 100%, i.e. the sample contains only part of the 62 SNP sites on the chromosome, further analysis is needed to determine which of the 62 SNP sites and the corresponding traits are missing, and whether the sample meets the requirements according to the missing traits and the feeding target requirements: if the missing traits do not affect the feeding target, it meets the requirements.

[0084] In a specific case:

[0085] Suppose the feeding target is to obtain Yanhuang cattle containing high protein beef, a Yanhuang cattle can be selected to obtain the chip data developed by the liquid chip in Example 2, and 62 SNP sites in Table 1 are detected. If all 62 SNP sites are detected, the Yanhuang cattle meets the requirements. If only part of the 62 SNP sites is detected, it is necessary to determine whether the site is indeed a site related to the protein content of beef. If it is related, it still meets the requirements, otherwise it does not meet the requirements.

[0086] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. The application of a molecular marker for detecting beef traits in the detection of growth traits and meat quality traits in beef cattle, characterized in that, The growth traits are composed of body weight, body height, cross height, body length, chest girth, and cannon bone girth; the meat quality traits are composed of backfat thickness, eye muscle area, protein content, intramuscular fat content, amino acids, myristic acid, palmitoleic acid, linoleic acid, and arachidonic acid; the molecular markers consist of 62 SNP loci; the physical locations of the 62 SNP loci were determined based on whole-genome sequence alignment of the Hereford cattle reference genome, version number GCF_002263795.3; the 62 SNP loci are shown in Table 1: Table 1 。 2. The application of the molecular markers for detecting beef traits as described in claim 1 in the gene breeding chip for growth traits and meat quality traits of beef cattle, characterized in that, The gene breeding chip includes a liquid phase chip; the growth traits consist of body weight, body height, cross height, body length, chest circumference, and canal circumference; the meat quality traits consist of backfat thickness, eye muscle area, protein, intramuscular fat content, amino acids, myristic acid, palmitoleic acid, linoleic acid, and arachidonic acid.

3. A liquid phase chip for detecting the characteristics of beef, characterized in that, This includes probes designed based on gene sequences covering the 62 SNP sites as described in claim 1.

4. The liquid phase chip according to claim 3, characterized in that, The probe sequence has a GC content of 20-80%, a probe length of 120 bp, a maximum upper limit of less than 5 specific similar fragments on the reference genome, and a maximum distance of less than 10 bp from the designed region.

5. The design method of a liquid phase chip as described in any one of claims 3-4, characterized in that, Includes the following steps: S1. Sample Collection: Obtain DNA samples from beef cattle; S2. Data Acquisition: Based on DNA samples, microarray detection is performed to obtain genotype data, and performance measurements of growth traits and meat quality traits are conducted to obtain phenotypic data related to growth and meat quality traits. The growth traits consist of body weight, body height, cross-shaped height, body length, chest circumference, and cannon bone circumference. The meat quality traits consist of backfat thickness, eye muscle area, protein content, intramuscular fat content, amino acids, myristic acid, palmitoleic acid, linoleic acid, and arachidonic acid. S3. Quality control: Perform genotype quality control and phenotypic quality control on the genotype data and the phenotypic data respectively; S4, Filling: Filling in the missing genotypes after quality control; S5. GWAS Analysis: Perform GWAS analysis on the quality-controlled and imputed genotype and phenotypic data to obtain significant loci and locate candidate genes; S6. Probe Design: Design probe sequences for detecting 62 SNP sites.

6. The application of the liquid phase chip as described in any one of claims 3-4 in detecting growth traits and meat quality traits of beef cattle, characterized in that, Growth traits consist of body weight, body height, cross-section height, body length, chest circumference, and cannon circumference; meat quality traits consist of backfat thickness, eye muscle area, protein, intramuscular fat content, amino acids, myristic acid, palmitoleic acid, linoleic acid, and arachidonic acid.

7. The application according to claim 6, characterized in that, The liquid phase chip is used to detect growth traits and meat quality traits in beef cattle, specifically including the following steps: Step A: Obtain DNA samples from the individual beef cattle to be tested; Step B: Obtain the genotype data of the individual beef cattle to be tested based on the liquid phase chip as described in any one of claims 3-4; Step C: Analyze the test results to determine whether the tested beef cattle individuals contain genes related to growth traits and meat quality traits.

8. The application of the liquid phase chip as described in any one of claims 3-4 in molecular breeding of growth traits and meat quality traits in beef cattle; characterized in that, The growth traits consist of body weight, body height, cross-section height, body length, chest circumference, and cannon circumference; the meat quality traits consist of backfat thickness, eye muscle area, protein, intramuscular fat content, amino acids, myristic acid, palmitoleic acid, linoleic acid, and arachidonic acid.

9. The application of the liquid phase chip as described in any one of claims 3-4 in the genome-wide association study of beef cattle.

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