A method for identifying bovine red coat color phenotype using the 8403bp sequence of the ASIP gene

Abstract

This invention discloses a method utilizing Breast Milk A method for identifying the red coat color phenotype in cattle using an 8403 bp gene sequence. Analysis of third-generation sequencing data from domestic cattle samples with different coat color phenotypes identified, for the first time, a gene significantly associated with the red coat color phenotype. Breast Milk An 8403bp structural variation in the gene sequence overlaps with a LINE-1 transposon, leading to... Breast Milk Gene transcripts produce different mRNA isoforms. This invention achieves the detection of bovine mRNA using two pairs of primers. Breast Milk Gene detection and accurate genotyping can be performed, enabling marker-assisted selection of the red coat color trait in cattle at the DNA level.

Description

Technical Field

[0001] This invention belongs to the field of animal breeding and biotechnology, and relates to marker-assisted selection in bovine genetic breeding, specifically involving the detection of... ASIP Methods for identifying genetic structural variations, and identifying genetic markers (specifically, structural variation molecular markers) associated with the red coat color trait in cattle at the DNA level. Background Technology

[0002] Structural variation (SV) refers to variations occurring at the chromosomal structural level of the genome, including insertions, deletions, duplications, and inversions. Structural variations can affect not only molecular and cellular processes, gene function, and transcriptional and translational levels, but also alter the linear and three-dimensional structure of the genome. Retrotransposable long nuclear element-1 (LINE-1) is a jumping gene, considered a family of still-active mobile elements that can form new insertions in the genome through transposition, thereby affecting transcript expression or splicing.

[0003] Previous studies have shown that the Agouti gene (i.e. ASIP ) is one of the important genes that determine the coat color of mammals, and it has multiple promoters and alternative non-coding first exons. ASIP Genes can be transcribed into transcripts with different mRNA isoforms, thereby regulating the temporal and spatial expression of genes and producing different pigmentation patterns.

[0004] The coat color of cattle is one of the important traits of breed, mainly determined by the relative content and distribution of eumelanin and phenotypic pigments in the skin and hair. Yellow cattle is a general term for various local cattle breeds or populations, with yellow being the most common coat color. Black, red, brown, and blue coat colors are also relatively common. This rich variety of coat color phenotypes provides natural material for studying the formation mechanism of cattle coat color. Research on the gene mapping and functional mechanism of cattle coat color traits not only helps to reveal the molecular mechanism of coat coloring (i.e., pigmentation), but can also be used to develop molecular markers related to cattle coat color traits for marker-assisted selection. This provides scientific means for the protection and breeding of local cattle populations. For example, the Qinchuan cattle, one of the five major improved breeds, is characterized by red coat color and is highly favored in its main distribution areas. It is a distinctive breed in the livestock resource treasure trove and has significant value for introduction and crossbreeding. However, to date, there are no reports, either domestically or internationally, on the molecular mechanism of red coat color formation in cattle or methods for accurate DNA typing, which is detrimental to the comprehensive development of selected breeds.

[0005] Additionally, CN112210607A (publication date 2021-01-12) proves... ASIPThe SNP sites g.19953331 and g.19970628 in the intron region of the gene serve as molecular markers associated with the rare white coat phenotype in buffalo and suggest potential applications in assisted breeding, as further pointed out in CN114075566A (publication date 2022-02-22). ASIP The presence of a LINE-1 transposon (2825bp insertion sequence) upstream of the gene results in the white coat phenotype of buffalo; while the latest study, "Pangenome Construction and Adaptive Introgression in Chinese Zebu cattle," suggests... ASIP The haplotype containing the 6.3kb insertion variant downstream of the gene is associated with the brown coat phenotype of zebu cattle. Summary of the Invention

[0006] The purpose of this invention is to provide a method for utilizing ASIP A method for identifying the red coat color phenotype in cattle using an 8403 bp gene sequence. This method involves... ASIP The detection of structural variation sites with a length of 8403 bp on the gene can enable marker-assisted selection for the red coat color trait in cattle.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Firstly, it provides a method for detecting cattle. ASIP Methods for studying gene structural variations include the following steps:

[0009] Genomic DNA was extracted from the bovine sample and then located at positions 63639802 to 63648204 on chromosome 13 of the bovine reference genome. ASIP The carriage status of gene structural variations in the genome of the cattle to be tested was identified.

[0010] Preferably, the identification specifically employs sequencing analysis, for example, using third-generation sequencing technology to obtain long-read sequencing data of the bovine genome to be tested, and using the obtained sequencing data and software to analyze the bovine genome... ASIP Gene typing.

[0011] Preferably, the identification specifically employs PCR and electrophoresis detection and analysis, including the following steps: using the bovine genomic DNA to be tested as a template, and targeting... ASIP Primer pair P1 designed to carry corresponding structural variations on the gene and targeting ASIP Primers designed to not carry the corresponding structural variation in the gene, using primer pair P1 and P2 as primers, were used for PCR amplification. The amplification products were then subjected to agarose gel electrophoresis. The results of the electrophoresis of the amplification products corresponding to these two primer pairs (i.e., primer pairs P1 and P2) were used to interpret the genetic information of the tested cattle. ASIP The genotype of a gene.

[0012] Preferably, the primer pair P1 is:

[0013] F1:5`-GGCAACACTAAGTCACAACC-3`;

[0014] R1: 5`-TATCGTAGGGCTCGCAATCT-3`.

[0015] Preferably, the primer pair P2 is:

[0016] F2:5`-GGCAACACTAAGTCACAACC-3`;

[0017] R2: 5`-TAAAGAACCCACCTACCAAT-3`.

[0018] Preferably, the interpretation is based on the following criteria: if the amplification product of primer pair P1 shows a 751bp band after electrophoresis and the amplification product of primer pair P2 shows a band of approximately 500bp (from non-specific amplification of primer pair P2), then the genotype is homozygous carrying the structural variation (hereinafter referred to as genotype SS); if the amplification product of primer pair P1 shows a 751bp band after electrophoresis and the amplification product of primer pair P2 shows two bands of approximately 500bp and 739bp after electrophoresis, then the genotype is heterozygous carrying the structural variation (hereinafter referred to as genotype SW); if the amplification product of primer pair P1 shows no band after electrophoresis and the amplification product of primer pair P2 shows two bands of approximately 500bp and 739bp after electrophoresis, then the genotype is homozygous not carrying the structural variation (hereinafter referred to as genotype WW).

[0019] Preferably, the bovine reference genome is ARS-UCD 1.2_Btau5.0.1Y.

[0020] Preferably, the nucleotide sequence of the structural variation is shown in SEQ.ID.NO.1.

[0021] Preferably, the cattle to be tested are domestic cattle.

[0022] Secondly, it provides a method for detecting cattle. ASIP A kit for gene structural variation, comprising amplification of gene structures located at positions 63639802 to 63648204 on chromosome 13 of the bovine reference genome. ASIP Primer sets for gene structural variations, specifically including primer pairs P1 and P2 mentioned above.

[0023] Thirdly, providing a type of cow ASIP The application of gene structural variation in marker-assisted selection breeding of cattle, wherein the structural variation is located at positions 63639802 to 63648204 on chromosome 13 of the bovine reference genome.

[0024] Preferably, the sequence of the structural variation overlaps with the LINE-1 transposon with a length of 8403 bp, and the specific sequence of the overlapping part is shown in SEQ.ID.NO.1 above.

[0025] Preferably, the structural variation is a molecular marker that is significantly associated with the red coat color trait, specifically involving the above genotypes SS and SW.

[0026] Preferably, in the application, the coat color phenotype (i.e., red or non-red) of unborn calves can be predicted based on the genotype of the parents, and the red coat color phenotype and genotype can be fixed by selecting homozygous individuals carrying the structural variation, thereby shortening the breeding time and improving the breeding efficiency.

[0027] Fourthly, the aforementioned testing cattle are also provided. ASIP The application of gene structure variation methods in marker-assisted selection breeding of domestic cattle.

[0028] The beneficial effects of this invention are reflected in:

[0029] This invention utilizes long-read sequencing data combined with differentiation and fixation indices to detect and identify an 8403bp structural variation that causes stratification between red-haired and non-red-haired cattle populations. ASIP (The gene produces different transcripts), and this localization in cattle was verified through association analysis. ASIP An 8403bp structural variation in the gene can serve as a molecular marker significantly associated with the red coat color trait in domestic cattle, and PCR typing can accurately identify the corresponding marker. ASIP The genotype of a gene can help accelerate the breeding process. Attached Figure Description

[0030] Figure 1 The example uses long-read data of red and non-red cattle to analyze the differentiation index, which yields the differentiation signal.

[0031] Figure 2 In this example, long read length data is used to... ASIP Visualization of IGV in the 8403bp structural variant sequence of the gene.

[0032] Figure 3 In this example, long-read data is used to analyze cattle with different coat color phenotypes. ASIP The gene was genotyped based on the 8403bp structural variation sequence.

[0033] Figure 4 For the example of cattle ASIP The design principle of PCR genotyping primers used in gene typing (refer to...) ASIP(LINE-1 transposon site in the gene that forms an 8403bp structural variation sequence).

[0034] Figure 5 The bovine markers used in the examples for large-scale molecular marker analysis validation are shown. ASIP Typical electrophoretic patterns of different genotypes obtained from gene PCR typing; where: SS represents a homozygous individual carrying an 8403bp structural variation sequence, SW represents a heterozygous individual carrying an 8403bp structural variation sequence, WW represents a homozygous individual without carrying an 8403bp structural variation sequence (wild type), and M represents DNA marker D2000. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0036] 1. Domestic cattle sample collection

[0037] Ear tissue samples were collected from a total of 83 cattle from 10 local breeds / groups in this experiment. The collected ear tissue samples were quickly brought back after being soaked in 75% alcohol and stored at -80℃. Phenotypic data of coat color of the samples were also collected (Table 1).

[0038] Table 1. Sampling Information Table for Domestic Cattle

[0039]

[0040] 2. Genome sequencing and variant detection analysis

[0041] DNA was extracted from the above-mentioned bovine ear tissues using the Tiangen Genomic DNA Extraction Kit. All extracted genomic DNA samples were subjected to whole-genome long-read sequencing on the commercial third-generation sequencing platform Oxford Nanopore, and sequencing data were collected from each sample. For each sample, the sequencing reads were aligned to the bovine reference genome ARS-UCD 1.2_Btau5.0.1Y using the "-ont" parameter in NGMLR software. Structural variations in the sample sequences were then detected using Sniffle, CuteSV, and SVIM software. Simultaneously, VCFtools software was used to perform inter-population divergence tests on 29 red-coat phenotype bovine samples (specifically Qinchuan cattle, Jiaxian red cattle, and Guyuan cattle as shown in Table 1) and 54 samples of other coat color phenotypes (i.e., non-red-coat phenotypes). F ST -SV). Then, ANNOVER software was used for annotation. Finally, in the cow... ASIPA structural variation of 8403 bp was found in the genes, which showed the highest fixation index between red-coated and non-red-coated cattle populations. F ST = 0.81, see Figure 1 The sequence details of this structural variation are shown in Table 2-1, SEQ.ID.NO.1.

[0042] Table 2-1. Cattle ASIP Gene structural variation sequence information

[0043]

[0044] The following is SEQ.ID.NO.1:

[0045]

[0046] The 8403bp structural variation sequence was visualized using IGV software (results are shown below). Figure 2 As shown), the "-freq" parameter of the VCFtools software was used to perform genotyping on individuals with different coat color phenotypes (see Table 1) in a domestic cattle sample population (83 individuals) based on long read data (results are shown in Table 1). Figure 3 (As shown). The results indicate that the 8403bp structural variant sequence is carried in all red-coated cattle, while its frequency is lower in non-red-coated cattle, and this structural variant is actually present in cattle with a specific phenotype (e.g., red-coated). ASIP In genes.

[0047] Furthermore, the 8403 bp structural variation sequence was aligned to a ruminant family repetitive sequence library using RepeatMasker software, and known mobile elements were searched. The results showed that the 8403 bp structural variation sequence was annotated to completely overlap with a LINE-1 transposon.

[0048] 3. Cow ASIP Detection of structural variations in LINE-1 transposons

[0049] 3.1 Sample collection and genomic DNA extraction

[0050] To verify the findings of the above experiments regarding cattle using large-group association analysis... ASIP To investigate the correlation between gene LINE-1 transposon structural variations (13: 63639802-63648204) and the red coat phenotype, ear tissues from 74 Qinchuan cattle samples were randomly collected at the Qinchuan Cattle Breeding Farm in Baoji City, Shaanxi Province (June 2024), and ear tissues from 97 Wenshan cattle samples were randomly collected at the Wenshan Cattle Farm in Wenshan City, Yunnan Province (March 2024). Coat color phenotype data from these 171 samples were collected, and then whole-genome DNA was extracted from these samples using the Tiangen Genomic DNA Extraction Kit.

[0051] 3.2 Primer Design and Synthesis for PCR Typing

[0052] Due to the findings of the above experiments on cattle ASIP The LINE-1 transposon structural variant sequence is long and consists of a transposon sequence, posing a certain amplification challenge. Therefore, two primer pairs, P1 and P2, were designed for the two alleles carrying and not carrying the aforementioned 8403 bp structural variant sequence at this site (see...). Figure 4 The specific primer sequences are shown in Table 2-2.

[0053] Table 2-2. PCR typing primer sequence information

[0054]

[0055] 3.3 PCR typing

[0056] Using the genomic DNA of each sample as a template, two independent PCRs were performed using primer pairs P1 and P2. The PCR products were electrophoresed on 2% agarose gels, and the genotyping results (genotype) were interpreted.

[0057] The PCR reaction system (total volume 25 μL) consisted of: 9.7 μL ddH2O, 0.4 μL forward primer F1 or F2 in P1 or P2, 0.4 μL reverse primer R1 or R2 in P1 or P2, 2 μL template DNA, and 12.5 μL dNTP Mixture.

[0058] The PCR amplification program for primer pair P1 was as follows: 94℃ for 5 min; 94℃ for 30 s, 53℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 7 min; store at 4℃.

[0059] The PCR amplification program for primer pair P2 was as follows: 94℃ for 5 min; 94℃ for 30 s, 53℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 7 min; store at 4℃.

[0060] The PCR products of primer pairs P1 and P2 showed the following results after electrophoresis detection: Figure 5 In contrast, homozygous or heterozygous individuals carrying the 8403bp structural variant sequence showed a 751bp band after PCR amplification of P1 using primers (recovered via gel extraction and sequencing). Homozygous individuals without the 8403bp structural variant sequence showed no band after PCR amplification and electrophoresis of P1 using primers, indicating that the 751bp band was a specific PCR product targeting the 8403bp structural variant sequence. In contrast, homozygous, heterozygous, and homozygous individuals without the 8403bp structural variant sequence all showed a band of approximately 500bp after PCR amplification of P2 using primers (recovered via gel extraction and sequencing). The latter two groups also showed an additional 739bp band (recovered via gel extraction and sequencing). This indicates that primers for P2 only produce specific amplification results if the individual does not carry the 8403bp structural variant sequence in at least one allele.

[0061] The final PCR typing results are as follows: Among the 171 samples tested, the Qinchuan cattle population consisted of 67 homozygous individuals carrying the 8403bp structural variation sequence (i.e., genotype SS) and 7 heterozygous individuals carrying the 8403bp structural variation sequence (i.e., genotype SW); the Wenshan cattle population consisted of 1 homozygous individual carrying the 8403bp structural variation sequence (i.e., genotype SS), 3 heterozygous individuals carrying the 8403bp structural variation sequence (i.e., genotype SW), and the remaining 93 homozygous individuals without the 8403bp structural variation sequence (i.e., genotype WW).

[0062] 3.4 Correlation Analysis

[0063] Based on the PCR typing data and corresponding coat color phenotype data of the above 171 samples, contingency tables for chi-square test were compiled, and the chi-square test was performed using SPSS software. The results are shown in Tables 3, 4, 5, and 6.

[0064] Table 3. Coat color × genotype cross table

[0065]

[0066] Note: In Table 3, the red-coated cattle are all from Qinchuan cattle, and the yellow-coated cattle are all from Wenshan cattle.

[0067] Table 4. Chi-square test of coat color and genotype

[0068]

[0069] Note: In Table 4, a. the expected count for a single cell (16.7%) is less than 5; the minimum expected count is 4.33.

[0070] Table 5. Cross-tabulation of coat color × allele type

[0071]

[0072] Note: In Table 5, the red-haired cattle are all from Qinchuan cattle, and the yellow-haired cattle are all from Wenshan cattle.

[0073] Table 6. Chi-square test of coat color and allele type

[0074]

[0075] Note: In Table 6, a. The expected count for 0 cells (.0%) is less than 5; the minimum expected count is 63.18.

[0076] Association analysis results show that cattle ASIPThe structural variation of the LINE-1 transposon (13: 63639802-63648204) is significantly associated with the red coat phenotype. This means that the 8403bp structural variation sequence carried in the domestic cattle genome can be used as a molecular marker to assist in the selection of red coat traits at the DNA level. For example, in the introduction and crossbreeding of Qinchuan cattle, the red coat phenotype can be fixed in the selected domestic cattle population based on the carrying status of the above 8403bp structural variation sequence in the individual genome (especially homozygotes), thereby accelerating the breeding process.

[0077] 4. Cow ASIP Gene Transcript Analysis

[0078] Skin tissues were collected from a blue-haired Langkazi cattle (sampling in August 2024, Shannan City, Tibet Autonomous Region) without the aforementioned 8403 bp structural variant sequence and from a red-haired Qinchuan cattle (homozygous) carrying the aforementioned 8403 bp structural variant sequence (sampling in June 2024, Baoji City, Shaanxi Province). mRNA libraries were constructed, and third-generation full-length transcriptome sequencing was performed on the Oxford Nanopore platform. Subsequently, the sequencing data were aligned and transcripts were assembled using minimap2 and stringtie2 software based on the bovine reference genome. Results showed that Langkazi cattle and Qinchuan cattle... ASIP The gene (the latter contains the bovine gene found in this gene) ASIP (Gene LINE-1 transposon structural variation) is transcribed into transcripts with different mRNA isoforms.

[0079] In summary, this invention reveals for the first time that the region 63639802 bp-63648204 bp on chromosome 13 of the bovine reference genome is a location specific to red-haired cattle. ASIP Structural variation sites on genes: Homozygous and heterozygous domestic cattle (such as Qinchuan cattle, Jiaxian red cattle, and Guyuan cattle) carrying the corresponding 8403bp structural variation sequence at these sites generally exhibit a red coat phenotype. Furthermore, the 8403bp structural variation sequence can be used as a molecular marker to predict the red coat trait in domestic cattle. Combined with PCR genotyping, this lays the foundation for effectively utilizing this molecular marker for the introduction of domestic cattle and marker-assisted selection breeding.

Claims

1. A type of cattle ASIP The application of gene structural variation in marker-assisted selection breeding for the red coat color trait in domestic cattle is characterized by: The structural variation is located at positions 63639802 to 63648204 on chromosome 13 of the bovine reference genome ARS-UCD 1.2_Btau5.0.1Y. The nucleotide sequence of the structural variation is shown in SEQ.ID.NO.

1.

2. A method for detecting cattle ASIP The application of gene structural variation methods in marker-assisted selection breeding of red coat color in domestic cattle is characterized by: The detection cow ASIP The methods for gene structural variation include the following steps: Genomic DNA was extracted from the bovine sample and then analyzed at positions 63639802 to 63648204 on chromosome 13 of the bovine reference genome ARS-UCD 1.2_Btau5.0.1Y. ASIP The carriage status of gene structural variations in the tested cattle was identified; The nucleotide sequence of the structural variation is shown in SEQ.ID.NO.

1.

3. The application according to claim 2, characterized in that: The detection specifically includes the following steps: Using the bovine genomic DNA to be tested as a template, and targeting ASIP Primer pair P1 designed to carry corresponding structural variations on the gene and targeting ASIP PCR amplification was performed using primer pair P2, which was designed to prevent the genes from carrying the corresponding structural variations. The amplification products were then subjected to agarose gel electrophoresis, and the genotype of the cattle to be tested was determined based on the electrophoresis results.

4. The application according to claim 3, characterized in that: The primer pair P1 is: F1:5`-GGCAACACTAAGTCACAACC-3`; R1:5`-TATCGTAGGGCTCGCAATCT-3`; The primer pair P2 is: F2:5`-GGCAACACTAAGTCACAACC-3`; R2: 5`-TAAAGAACCCACCTACCAAT-3`.

5. The application according to claim 3, characterized in that: The interpretation criteria are as follows: if the amplification product of primer pair P1 shows a 751bp band after electrophoresis and the amplification product of primer pair P2 does not show a 739bp band after electrophoresis, then the genotype is homozygous carrying the aforementioned structural variation; if the amplification product of primer pair P1 shows a 751bp band after electrophoresis and the amplification product of primer pair P2 shows a 739bp band after electrophoresis, then the genotype is heterozygous carrying the aforementioned structural variation; if the amplification product of primer pair P1 does not show a 751bp band after electrophoresis and the amplification product of primer pair P2 shows a 739bp band after electrophoresis, then the genotype is homozygous not carrying the aforementioned structural variation.

Images