Method for identifying cattle red hair color phenotype by using 8403bp sequence of ASIP gene

Abstract

The invention discloses a method for identifying cattle red hair color phenotypes by using an 8403bp sequence of an ASIP gene. Three-generation sequencing data of genomes of domestic cattle samples with different hair color phenotypes are analyzed, the 8403bp structural variation sequence of the ASIP gene remarkably related to the red hair color phenotype is identified for the first time, and the sequence is overlapped with an LINE-1 transposon and causes an ASIP gene transcript to generate different mRNA subtypes. By adopting two pairs of primers, detection of the cattle ASIP gene can be realized, and genotyping can be accurately carried out, so that marker-assisted selection can be carried out on cattle red hair color characters on the DNA (Deoxyribose Nucleic Acid) level.

Description

Technical Field

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

[0002] Structural variation (SV) refers to variations that occur at the chromosomal structural level of the genome, including insertions, deletions, duplications, inversions, etc. Structural variation can not only affect molecular and cellular processes, gene functions, and transcription and translation levels, but can also change the linear and three-dimensional structure of the genome. The retrotransposon long interspersed nuclear element-1 (LINE-1) is a jumping gene that is considered to be a family of mobile elements that are still active. They can form new insertions in the genome through transposition, thereby affecting the expression or splicing of transcripts.

[0003] Studies have shown that the Agouti gene (i.e. ASIP ) is one of the important genes that determine mammalian coat color, with 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 the breed, which is mainly determined by the relative content and distribution of eumelanin and pheomelanin in the skin and coat. Yellow cattle is a general term for local cattle breeds or groups. Most of their coats are yellow, and black, red, brown and cyan are also common. The rich coat color phenotypes provide natural materials for studying the formation mechanism of cattle coat color. The location of cattle coat color trait genes and their functional mechanism research not only helps to reveal the molecular mechanism of coat coloring (i.e. pigmentation), but also can be used to develop molecular markers related to cattle coat color traits to achieve marker-assisted selection, thereby providing scientific means for the protection and breeding of local cattle populations. For example, Qinchuan cattle, as one of the five fine breeds, has red coat as the main coat color trait. It is deeply loved in its main distribution area and is a special breed in the treasure house of livestock and poultry resources. It has great value for introduction and hybrid breeding. However, so far, there have been no reports at home and abroad on the molecular mechanism of the formation of cattle red coat color phenotype and the method of accurate typing at the DNA level, which is not conducive to the comprehensive development of breeding varieties.

[0005] In addition, CN112210607A (published on 2021-1-12) proves ASIPThe SNP sites g.19953331 and g.19970628 in the intron region of the gene have the potential to be used as molecular markers associated with the rare white coat phenotype of buffalo and suggest that they can be used for assisted breeding. CN114075566A (published on 2022-2-22) further states ASIP The LINE-1 transposon (2825 bp insertion sequence) contained upstream of the gene causes the white coat phenotype of buffalo; and the latest "Pan-genome construction and adaptive introgression research of Chinese zebu cattle" suggests ASIP The haplotype where the 6.3kb insertion mutation is located downstream of the gene is associated with the brown coat phenotype of zebu cattle. Summary of the invention

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

[0007] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, a method for detecting a cow is provided. ASIP The method for gene structural variation comprises the following steps: Extract the bovine genomic DNA to be tested, and then locate the 63639802nd to 63648204th position of chromosome 13 of the bovine reference genome. ASIP The presence of gene structural variations in the genome of the tested cattle was identified.

[0008] Preferably, the identification is specifically carried out by sequencing analysis, for example, using the third generation sequencing technology to obtain the long read sequencing data of the cattle genome to be tested, and using the obtained sequencing data and software to identify the cattle to be tested. ASIP Genotyping.

[0009] Preferably, the identification is specifically carried out by PCR and electrophoresis detection analysis, comprising the following steps: using the bovine genomic DNA to be tested as a template, ASIP Primer pair P1 designed for the corresponding structural variation on the gene and primer pair P2 designed for the corresponding structural variation on the gene ASIP The primer pair P2 designed on the gene that does not carry the corresponding structural variation is used as the primer for PCR amplification, and the amplified products are subjected to agarose gel electrophoresis. The electrophoresis results of the corresponding amplified products of these two pairs of primers (i.e., the primer pair P1 and P2) are used to judge the cattle to be tested. ASIP The genotype of a gene.

[0010] Preferably, the primer pair P1 is: F1:5`-GGCAACACTAAGTCACAACC-3`; R1: 5`-TATCGTAGGGCTCGCAATCT-3`.

[0011] Preferably, the primer pair P2 is: F2:5`-GGCAACACTAAGTCACAACC-3`; R2: 5`-TAAAGAACCCACCTACCAAT-3`.

[0012] Preferably, the basis for the interpretation is: if the corresponding amplification product of primer pair P1 shows a band of 751 bp after electrophoresis and the corresponding amplification product of primer pair P2 shows a band of nearly 500 bp after electrophoresis (from non-specific amplification of primer pair P2), the genotype is a homozygote carrying the structural variation (referred to as genotype SS); if the corresponding amplification product of primer pair P1 shows a band of 751 bp after electrophoresis and the corresponding amplification product of primer pair P2 shows two bands of nearly 500 bp and 739 bp after electrophoresis, the genotype is a heterozygote carrying the structural variation (referred to as genotype SW); if the corresponding amplification product of primer pair P1 shows no band after electrophoresis and the corresponding amplification product of primer pair P2 shows two bands of nearly 500 bp and 739 bp after electrophoresis, the genotype is a homozygote not carrying the structural variation (referred to as genotype WW).

[0013] Preferably, the cattle reference genome is ARS-UCD 1.2_Btau5.0.1Y.

[0014] Preferably, the nucleotide sequence of the structural variation is shown in SEQ.ID.NO.1.

[0015] Preferably, the source of the cattle to be tested is domestic cattle.

[0016] In the second aspect, a method for detecting cattle is provided. ASIP A kit for detecting gene structural variation, the kit comprising a kit for amplifying a gene located at position 63639802 to position 63648204 of chromosome 13 of a bovine reference genome ASIP The primer set for gene structural variation, for example, specifically includes the above-mentioned primer pair P1 and primer pair P2.

[0017] A third aspect provides a cattle ASIP The application of gene structural variation in cattle marker-assisted selection breeding, wherein the structural variation is located at position 63639802 to position 63648204 of chromosome 13 of the cattle reference genome.

[0018] Preferably, the sequence of the structural variation overlaps with the LINE-1 transposon with a length of 8403 bp, and the sequence of the overlapping part is specifically shown in the above SEQ.ID.NO.1.

[0019] Preferably, the structural variation is a molecular marker significantly associated with the red coat color trait, specifically involving the above genotypes SS and SW.

[0020] Preferably, in the application, the coat color phenotype (i.e., red or non-red) of the unborn calf can be predicted based on the genotype of the parents, and by selecting homozygous individuals carrying the structural variation, the fixation of the red coat color phenotype and genotype can be achieved, thereby shortening the breeding time and improving the breeding efficiency.

[0021] In a fourth aspect, the above-mentioned detection cattle are also provided ASIP Application of gene structure variation methods in marker-assisted selection breeding of cattle.

[0022] The beneficial effects of the present invention are embodied in: The present invention uses long-read sequencing data combined with differentiation index and fixation index to detect and discover an 8403bp structural variation (which causes red and non-red phenotype cattle populations to be stratified) ASIP The gene produces different transcripts), and this localization was verified by association analysis in cattle ASIP The 8403bp structural variation on the gene can be used as a molecular marker significantly associated with the red coat color trait of cattle, and PCR typing can accurately identify the corresponding ASIP The genotype of the gene is thus helpful to speed up the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the differentiation signal obtained by analyzing the population differentiation index using the long read data of red-haired cattle and non-red-haired cattle in the embodiment.

[0024] Figure 2 In the embodiment, long read length data is used to ASIP IGV visualization of 8403bp structural variation sequence of the gene.

[0025] Figure 3 In the embodiment, long read data is used to analyze the different coat color phenotypes of cattle. ASIP The gene was genotyped according to the 8403bp structural variation sequence.

[0026] Figure 4 In the embodiment for cattle ASIP Principles of PCR typing primer design for genes (refer to ASIP The LINE-1 transposon site formed an 8403 bp structural variation sequence in the gene).

[0027] Figure 5 The cattle used for molecular marker large group analysis and verification in the embodiment ASIPTypical electrophoretic patterns of different genotypes obtained by gene PCR typing; where SS represents a homozygous individual carrying a 8403bp structural variation sequence, SW represents a heterozygous individual carrying a 8403bp structural variation sequence, WW represents a homozygous individual not carrying a 8403bp structural variation sequence (wild type), and M represents DNA marker D2000. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0029] 1. Cattle Sample Collection In this experiment, ear tissues of 83 cattle samples from 10 local breeds / groups were collected. The collected ear tissues were soaked in 75% alcohol and quickly brought back and stored at -80°C. At the same time, the phenotypic data of the coat color of the samples were collected (Table 1).

[0030] Table 1. Cattle sampling information

[0031] 2. Genome Sequencing and Variant Detection Analysis The Tiangen genomic DNA extraction kit was used to extract DNA from the ear tissues of the above-mentioned cattle. The extracted genomic DNA of all samples was sent to the commercial third-generation sequencing Oxford Nanopore platform for whole-genome long-read sequencing, and the sequencing data of each sample was collected. For each sample, the "-ont" parameter of the NGMLR software was used to align the sequenced reads to the cattle reference genome ARS-UCD 1.2_Btau5.0.1Y, and then the sample sequences were tested for structural variations using Sniffle, CuteSV, and SVIM software. At the same time, the VCFtools software was used to perform inter-group divergence tests on 29 red-hair phenotype cattle samples (specifically Qinchuan cattle, Jiaxian red cattle, and Guyuan cattle in Table 1) and 54 cattle samples with other coat color phenotypes (i.e., non-red coat color phenotypes) ( F ST -SV). ANNOVER software was then used for annotation. ASIP An 8403bp structural variation was found in the gene, which showed the highest fixation index between the red coat phenotype cattle population and the non-red coat phenotype cattle population ( F ST = 0.81, see Figure 1 ), the sequence of this structural variation is detailed in SEQ.ID.NO.1 in Table 2-1.

[0032] Table 2-1. Cattle ASIPGene structure variation sequence information

[0033] The following is SEQ.ID.NO.1: The IGV software was used to visualize the above 8403bp structural variation sequence (the results are shown in Figure 2 As shown in the figure), combined with the "-freq" parameter of VCFtools software, genotyping of individuals with different coat color phenotypes (see Table 1) in the cattle sample population (83 individuals) was performed based on long read data (the results are shown in the figure). Figure 3 The results showed that the 8403bp structural variation sequence was carried in all cattle with red coat phenotypes, but was carried at a lower frequency in cattle with non-red coat phenotypes, and that the structural variation actually existed in cattle with specific phenotypes (e.g., red coat phenotypes). ASIP In genes.

[0034] In addition, RepeatMasker software was used to align the 8403 bp structural variant sequence to the ruminant family repeat sequence library and search for known mobile elements. The results showed that the 8403 bp structural variant sequence was annotated to completely overlap with a LINE-1 transposon.

[0035] 3. Cow ASIP Detection of structural variation of LINE-1 transposon 3.1 Sample collection and genomic DNA extraction In order to verify the above experimental findings through large group association analysis ASIP The correlation between the structural variation of the gene LINE-1 transposon (13: 63639802-63648204) and the red coat color phenotype. In this experiment, ear tissues of Qinchuan cattle samples (74 heads) were randomly collected at the Qinchuan Cattle Conservation Farm in Baoji City, Shaanxi Province (June 2024), and ear tissues of Wenshan cattle samples (97 heads) were randomly collected in Wenshan City, Yunnan Province (Wenshan Cattle Farm) (March 2024). The coat color phenotype data of these 171 samples were collected at the same time, and then the whole genome DNA of these samples was extracted using the Tiangen Genomic DNA Extraction Kit.

[0036] 3.2 Primer design and synthesis for PCR typing method Due to the above experimental findings ASIP The sequence fragment of the LINE-1 transposon structural variation is long and is a transposon sequence, which is difficult to amplify. Therefore, two pairs of primers, P1 and P2, were designed for the two alleles carrying and not carrying the above 8403bp structural variation sequence at the structural variation site (see Figure 4 ), the specific sequences of the primers are shown in Table 2-2.

[0037] Table 2-2. PCR typing primer sequence information

[0038] 3.3 PCR typing Using the genomic DNA of each sample as a template, two independent PCRs were performed using two pairs of primers, P1 and P2. The PCR products were electrophoresed on 2% agarose gel to interpret the typing results (genotype).

[0039] The PCR reaction system (total volume 25 μL) was: ddH2O 9.7 μL, forward primer F1 or F2 in P1 or P2 0.4 μL, reverse primer R1 or R2 in P1 or P2 0.4 μL, template DNA 2 μL, and dNTP Mixture 12.5 μL; The PCR amplification program for primer pair P1 was: 94°C for 5 min; 94°C for 30 s, 53°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 7 min; storage at 4°C; The PCR amplification program for primer pair P2 was: 94°C for 5 min; 94°C for 30 s, 53°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 7 min; storage at 4°C; The PCR products of primer pair P1 and P2 were detected by electrophoresis with the following results: Figure 5 ): After PCR amplification using primer pair P1, a 751bp band can be obtained by electrophoresis detection for homozygous or heterozygous individuals carrying the 8403bp structural variation sequence (sequencing after recovery with a gel recovery kit), while no band can be found for homozygous individuals not carrying the 8403bp structural variation sequence after PCR amplification and electrophoresis detection using primer pair P1, i.e., the 751bp band is a specific PCR product for the 8403bp structural variation sequence; in contrast, after PCR amplification using primer pair P2 for homozygous individuals carrying the 8403bp structural variation sequence, heterozygous individuals and homozygous individuals not carrying the 8403bp structural variation sequence can all obtain a band of nearly 500bp by electrophoresis detection (sequencing after recovery with a gel recovery kit), and the latter two types of individuals also obtain another 739bp band (sequencing after recovery with a gel recovery kit), i.e., primer pair P2 will produce specific amplification results only when the individual does not carry the 8403bp structural variation sequence on at least one allele.

[0040] The final results of the PCR typing experiment are as follows: among the 171 samples tested, the Qinchuan cattle population consisted of 67 individuals with a genotype of homozygous carrying the 8403bp structural variation sequence (i.e., genotype SS) and 7 individuals with a genotype of heterozygous carrying the 8403bp structural variation sequence (i.e., genotype SW); the Wenshan cattle population consisted of 1 individual with a genotype of homozygous carrying the 8403bp structural variation sequence (i.e., genotype SS), 3 individuals with a genotype of heterozygous carrying the 8403bp structural variation sequence (i.e., genotype SW) and the remaining 93 individuals with a genotype of homozygous not carrying the 8403bp structural variation sequence (i.e., genotype WW).

[0041] 3.4 Correlation Analysis For the PCR typing data of the above 171 samples and the coat color phenotype data of the corresponding samples, a contingency table for chi-square test was compiled, and the chi-square test was performed using SPSS software. The results are shown in Tables 3, 4, 5 and 6.

[0042] Table 3. Cross table of coat color × genotype

[0043] Note: In Table 3, the cattle with red coat color are all from Qinchuan cattle, and the cattle with yellow coat color are all from Wenshan cattle.

[0044] Table 4. Chi-square test of coat color and genotype

[0045] Note: In Table 4, a. 1 cell (16.7%) has an expected count less than 5; the minimum expected count is 4.33.

[0046] Table 5. Cross table of coat color × allele type

[0047] Note: In Table 5, the cattle with red coat color are all from Qinchuan cattle, and the cattle with yellow coat color are all from Wenshan cattle.

[0048] Table 6. Chi-square test of coat color and allele type

[0049] Note: In Table 6, a. 0 cells (.0%) have expected counts less than 5; the minimum expected count is 63.18.

[0050] The results of association analysis showed that ASIPThe structural variation of the gene LINE-1 transposon (13: 63639802-63648204) is significantly correlated with the red coat color phenotype, that is, the 8403bp structural variation sequence carried in the cattle genome can be used as a molecular marker for marker-assisted selection of its red coat color trait at the DNA level. For example, in the introduction and hybrid breeding of Qinchuan cattle, the red coat color phenotype can be fixed in the selected cattle population based on the presence of the 8403bp structural variation sequence in the individual genome tested (especially homozygotes), thereby accelerating the breeding process.

[0051] 4. Cow ASIP Gene transcript analysis The skin tissue of a blue-haired Langkazi cattle that does not carry the above-mentioned 8403bp structural variation sequence (sampled in August 2024, in Shannan City, Tibet Autonomous Region) and the skin tissue of a red-haired Qinchuan cattle (homozygous) that carries the above-mentioned 8403bp structural variation sequence (sampled in June 2024, in Baoji City, Shaanxi Province) were collected to construct an mRNA library, and third-generation full-length transcriptome sequencing was performed on the Oxford Nanopore platform. The sequencing data were then aligned and the transcripts were assembled based on the bovine reference genome using minimap2 and stringtie2 software. The results showed that the Langkazi cattle and Qinchuan cattle have the same structure. ASIP gene (the latter gene is found in cattle ASIP Genes (LINE-1 transposon structural variation) are transcribed into transcripts with different mRNA isoforms.

[0052] In summary, the present invention reveals for the first time that the 63639802 bp-63648204 bp region of chromosome 13 of the cattle reference genome is located in the red coat phenotype cattle ASIP The structural variation site on the gene, homozygous and heterozygous cattle (such as Qinchuan cattle, Jiaxian red cattle, Guyuan cattle) carrying the corresponding 8403bp structural variation sequence at this site generally have a red coat color phenotype, and the 8403bp structural variation sequence can be used as a molecular marker to predict the red coat color trait of cattle. Combined with the use of PCR typing method, it lays the foundation for the effective use of this molecular marker for the introduction of cattle and molecular marker-assisted selection breeding.

Claims

1. A method for detecting cattle ASIP A method for genetic structural variation, characterized in that: The following steps are involved: Extract the bovine genomic DNA to be tested, and then locate the 63639802nd to 63648204th position of chromosome 13 of the bovine reference genome. ASIP The presence of gene structural variation in the tested cattle is identified.

2. A method for detecting cattle according to claim 1 ASIP A method for genetic structural variation, characterized in that: The identification specifically comprises the following steps: using the bovine genomic DNA to be tested as a template, ASIP Primer pair P1 designed for the corresponding structural variation on the gene and primer pair P2 designed for the corresponding structural variation on the gene ASIP The primer pair P2 designed for genes not carrying the corresponding structural variation is used as the primer for PCR amplification, and the amplified products are subjected to agarose gel electrophoresis. The genotype of the tested cattle is determined based on the electrophoresis results.

3. A method for detecting cattle according to claim 2 ASIP A method for genetic structural variation, 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`.

4. A method for detecting cattle according to claim 2 ASIP A method for genetic structural variation, characterized in that: The basis for the interpretation is: if the corresponding amplification product of primer pair P1 shows a 751bp band after electrophoresis and the corresponding amplification product of primer pair P2 does not show a 739bp band after electrophoresis, the genotype is a homozygote carrying the structural variation; if the corresponding amplification product of primer pair P1 shows a 751bp band after electrophoresis and the corresponding amplification product of primer pair P2 shows a 739bp band after electrophoresis, the genotype is a heterozygote carrying the structural variation; if the corresponding amplification product of primer pair P1 does not show a 751bp band after electrophoresis and the corresponding amplification product of primer pair P2 shows a 739bp band after electrophoresis, the genotype is a homozygote not carrying the structural variation.

5. A method for detecting cattle according to claim 1 ASIP A method for genetic structural variation, characterized in that: The cattle reference genome is ARS-UCD 1.2_Btau5.0.1Y.

6. A method for detecting cattle according to claim 1 ASIP A method for genetic structural variation, characterized in that: The source of the cattle to be tested is domestic cattle.

7. A method for detecting cattle ASIP A kit for detecting gene structure variation, characterized in that: The kit includes amplification of the 63639802 to 63648204 position of chromosome 13 located in the bovine reference genome. ASIP Primer sets for gene structural variation.

8. A kind of cattle ASIP The application of gene structure variation in cattle marker-assisted selection breeding is characterized by: The structural variation is located at position 63639802 to position 63648204 of chromosome 13 of the bovine reference genome.

9. The detection method according to any one of claims 1 to 6 ASIP Application of gene structure variation methods in marker-assisted selection breeding of cattle.

10. The use according to claim 9, characterized in that: The structural variation is a molecular marker associated with the red coat color trait.

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