A method for identifying spotted neapolitan cow breeds using bovine chromosome 6 copy number variation

Abstract

The application discloses a method for identifying spotted neapolitan cow breeds by using bovine chromosome 6 copy number variation. Genome candidate region copy number variation analysis is carried out on selected neapolitan cow breeds with different spotted hair color traits; according to the found copy number variation segment of the genomic region 6:70230801-70291600 located on bovine chromosome 6, the copy number variation types of individuals are identified, low-cost and accurate identification of spotted neapolitan cow and solid color neapolitan cow breeds at the DNA level is realized, and the genetic breeding efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to detecting copy number variations related to the spotted coat color trait of zebu in introduction and breeding, and in particular to identifying the pure color zebu (i.e. zebu with pure color coat color trait) and the spotted zebu breed by using copy number variation types. BACKGROUND

[0002] The most distinctive feature of zebu is the presence of hump. The existing zebu genetic resources are widely distributed, mainly in tropical and subtropical regions, such as the South Asian subcontinent, Southeast Asia, southern East Asia, Africa and the Americas. There are many zebu breeds, among which the South Asian zebu can be specifically divided into Nellore, Tharparkar, Bhagnari, Dajal, Hariana, Cholistani, Dhanni, Sahiwal, Guzerat and other breeds. Due to the different geographical conditions and ecological characteristics of zebu producing areas, each breed has obvious regional differences and regional adaptation ability. For example, Cholistani is a zebu breed adapted to the hot regions of Pakistan, and Dhanni is a dairy and light drought-tolerant zebu breed. At the same time, zebu has rich coat color traits, including white, yellow, black (black brown), red-brown and other pure color coat colors, as well as white black spot, white brown spot and other spotted coat colors. The spotted coat color of zebu is obviously different from the pure color coat color, which not only attracts attention in scientific research on animal behavior and physiology, but also the zebu population with spotted coat color trait itself can be used as high-quality germplasm resources and used in introduction, breeding and other related breeding work. For cattle genetic breeding, it is usually not possible to only refer to the limited reliability of phenotypic markers such as coat color, especially for zebu. It is necessary to explore the genetic variation preserved at the genomic or DNA level for a specific zebu population.

[0003] Copy number variation (CNV) is a variation in the copy number of a DNA segment of 1 Kb or more on the genome. In mammalian populations, most autosomal genes exist in each of the parents from one copy, and the change in the normal gene copy dose will significantly affect the quantitative or qualitative phenotypic characteristics.

[0004] KIT The gene is a member of the receptor tyrosine kinase protein family, KIT Different genotypes of the gene can affect the overall coat color trait of mammals by regulating the amount and distribution of melanin, as well as maintaining the color of the eyes and skin. It is pointed out in "Copy Number Variation Research of Bos Taurus Genome" that the gene containing KITThe CNV of the gene (located at chr6: 69975194-70467027) is related to the piebald color variation of the tumor cattle, and the result of the coverage depth detection on the CNV segment shows that the copy number of the piebald individual is 2, the copy number of the white and yellow individual is 1-1.5, and the copy number of the piebald tumor cattle breed is twice that of the solid color tumor cattle breed (for details, see CN114717334A disclosed on July 8, 2022). However, it is actually difficult to use coverage depth to genotype the CNV segment, which is affected by factors such as the size of the detection range and the composition of the test population, and is not conducive to popularization in breeding; in addition, a larger genetic variation region (the above-mentioned CNV segment is about 490 Kb) will increase the calculation cost and reagent cost of detection in practical application due to the long segment. SUMMARY

[0005] The purpose of the present application is to provide a method for identifying piebald tumor cattle breed by using the copy number variation of bovine chromosome 6, which can improve the efficiency of genetic breeding related to high-quality tumor cattle germplasm resources.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, a method for identifying tumor cattle breed by using copy number variation is provided, which comprises the following steps:

[0008] Genomic DNA of the test tumor cattle is extracted, and then the copy number variation segment located at the 6th chromosome of the bovine reference genome (70230801-70291600, i.e. 6: 70230801-70291600) is detected and analyzed. If it is found by detection and analysis that the genotype (i.e. copy number variation type) of the test tumor cattle in the copy number variation segment is a multiple copy type, the test tumor cattle is determined to belong to the piebald tumor cattle breed.

[0009] Preferably, the method for identifying tumor cattle breed further comprises the following steps: if it is found by detection and analysis that the genotype of the test tumor cattle in the copy number variation segment is a deletion type or a normal type, the test tumor cattle is determined to belong to the solid color tumor cattle breed.

[0010] Preferably, the detection and analysis specifically comprises the following steps: genomic sequencing of the test tumor cattle is performed, then the genomic data of the test tumor cattle is aligned to the bovine reference genome, then the copy number variation segment is extracted and copy number calculation is performed by using software, and then the genotype of the test tumor cattle in the copy number variation segment is identified according to the copy number of the copy number variation segment.

[0011] Preferably, the identifying specifically comprises the following steps: the copy number ≤ 1.5, determining that the genotype of the test Bos indicus at the copy number variation segment is deletion type; the copy number > 1.5 and ≤ 2.9, determining that the genotype of the test Bos indicus at the copy number variation segment is normal type; the copy number > 2.9, determining that the genotype of the test Bos indicus at the copy number variation segment is multiple copy type.

[0012] Preferably, the Bos indicus reference genome is ARS-UCD1.2.

[0013] Preferably, the coat color trait of the spotted Bos indicus breed is having white ground color and the spot color is brown (e.g. Cholistani) or black (e.g. Dhanni); the coat color trait of the solid colored Bos indicus breed is having white, yellow or black (black-brown) ground color and no spot.

[0014] In a second aspect, a system for identifying Bos indicus breed by copy number variation is provided, which comprises a sequencing module, a data segmentation module, a genotyping module and a Bos indicus breed identification module.

[0015] The sequencing module is used to read or detect the genomic data of Bos indicus;

[0016] The data segmentation module is used to extract the corresponding part of the copy number variation segment located at the 70230801-70291600 site of the 6th chromosome of the above Bos indicus reference genome (i.e. 6: 70230801-70291600) in the genomic data;

[0017] The genotyping module is used to identify the genotype of Bos indicus at the copy number variation segment according to the copy number of the copy number variation segment (specifically identify the above deletion type, normal type or multiple copy type);

[0018] The Bos indicus breed identification module is used to determine that the Bos indicus with the genotype of multiple copy type belongs to the spotted Bos indicus breed (i.e. classified as the spotted Bos indicus breed), and the Bos indicus with the genotype of deletion type or normal type belongs to the solid colored Bos indicus breed (i.e. classified as the solid colored Bos indicus breed) according to the genotype of Bos indicus at the copy number variation segment.

[0019] In a third aspect, the application provides an application of Bos taurus chromosome 6 copy number variation or a detection method thereof in Bos taurus molecular marker assisted selection breeding, and the detection method of the copy number variation comprises the following steps: extracting the genomic DNA of the test Bos taurus (e.g. Bos indicus), and then genotyping the test Bos taurus according to the copy number of the copy number variation segment located at the 70230801-70291600 site of the 6th chromosome of the above Bos indicus reference genome (i.e. 6: 70230801-70291600) (the genotyping result is specifically identified as the above deletion type, normal type or multiple copy type).

[0020] Preferably, the multiple copy genotype of the copy number variation segment is significantly associated with the piebald color trait of the zebu cattle.

[0021] In a fourth aspect, the application provides a use of a copy number variation marker of bovine chromosome 6 or a detection method thereof in the introduction of piebald zebu cattle, wherein the copy number variation marker is a multiple copy genotype of a copy number variation segment located at 70230801-70291600 of the bovine reference genome (i.e. 6: 70230801-70291600), and the multiple copy genotype of the copy number variation segment is significantly associated with the piebald color trait of the zebu cattle.

[0022] In a fifth aspect, the application provides a use of a copy number variation marker of bovine chromosome 6 or a detection method thereof in the identification of a piebald zebu cattle breed, wherein the copy number variation marker is a multiple copy genotype of a copy number variation segment located at 70230801-70291600 of the bovine reference genome (i.e. 6: 70230801-70291600), and the multiple copy genotype of the copy number variation segment is significantly associated with the piebald color trait of the zebu cattle.

[0023] The application has the following advantages:

[0024] The application analyzes the copy number variation of the candidate genomic region of the target zebu cattle with different piebald color traits (and uses the solid-color zebu cattle as a control), repositions the variation segment related to the piebald color trait of the zebu cattle, and finds that the multiple copy genotype of the new copy number variation segment (e.g. 70230801bp-70291600 of the bovine reference genome ARS-UCD1.2 chromosome 6) is significantly associated with the piebald color trait of the zebu cattle. The genotype can be used to accurately identify the piebald zebu cattle and the solid-color zebu cattle (the latter has a deletion genotype or a normal genotype) at the DNA level, and quickly establish a piebald zebu cattle population with excellent genetic resources. The application not only solves the problem that the previous coverage depth typing is difficult to apply in an actual sample population (e.g. false positives occur when identifying the piebald color trait of the zebu sample at a coverage depth higher than the normal copy number, i.e. a repeated copy number type, resulting in a decrease in the identification accuracy when the sample size and composition change), but also provides an important basis and a more convenient way to improve the efficiency of molecular marker-assisted selection of the piebald color trait, the introduction of piebald zebu cattle, and the identification of zebu cattle breeds (e.g. the new copy number variation segment is shorter, which is beneficial to reduce the detection cost). BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Results of whole genome association analysis for spotted zebu breed (samples from Cholistani) and solid zebu breed.

[0026] Figure 2 VST results for CNVs of spotted zebu breed (samples from Cholistani) and solid zebu breed.

[0027] Figure 3 Results of whole genome association analysis for spotted zebu breed (samples from Dhanni) and solid zebu breed.

[0028] Figure 4 VST results for CNVs of spotted zebu breed (samples from Dhanni) and solid zebu breed.

[0029] Figure 5 Coverage depth calculation results for CNV segments of spotted zebu breed (samples from Dhanni) and solid zebu breed.

[0030] Figure 6 Coverage depth calculation results for CNV segments of spotted zebu breed (samples from Cholistani) and solid zebu breed. DETAILED DESCRIPTION

[0031] The application will be further described below in conjunction with the accompanying drawings and examples. The examples are only used to explain the application, and are not a limitation on the scope of protection of the application.

[0032] (I) Genome-wide repositioning of genomic variants associated with the spotted coat color trait in zebu using genome-wide resequencing, genome-wide association analysis, and genome-wide selective sweep

[0033] (1.1) Sample collection, genomic DNA extraction, and resequencing

[0034] Ear tissue samples were collected from 19 spotted zebu (9 Cholistani, 10 Dhanni, collected in November 2020 by Quratulain Hanif, a member of the team) and 20 solid zebu (10 Bhagnari, 10 Dajal, also collected in Pakistan as controls.

[0035] The coat color traits of the 19 spotted zebu collected were as follows: the spots of Cholistani were brown, and the base color was white; the spots of Dhanni were black, and the base color was white.

[0036] The coat color traits of the 20 solid zebu collected were as follows: Bhagnari was pure white, and Dajal was pure white, with 10 heads of each breed.

[0037] The collected ear tissue samples were stored in a -80 ° refrigerator and transported with dry ice to Shenzhen Huada Gene Company Limited for extraction of genomic DNA and whole genome resequencing.

[0038] (1.2) Genomic alignment and SNP detection

[0039] The resequencing results of 39 cows were aligned to the bovine reference genome ARS-UCD1.2 (NCBI version number GCF_000003205.7) using BWA software, and whole genome single nucleotide polymorphism (SNP) detection was performed using GATK software to construct a whole genome SNP set of 39 cows.

[0040] (1.3) Whole genome association analysis of single-spotted tumor cow breeds

[0041] The target cow population of the experiment was established with the Cholistani spotted tumor cow breed, and whole genome SNPs of three spotted tumor cow breeds of Cholistani, Bhagnari and Dajal from two control cow populations of non-spotted tumor cow breeds were used for whole genome association analysis. The results of the association analysis will locate the candidate region related to the spotted coat color trait of the tumor cow (specifically Cholistani, Bhagnari and Dajal) on chromosome 6, and the region with the strongest signal is located between 70321916 bp-70345225 bp (the lowest signal value is 2.652758 x 10 -10 , Figure 1 ).

[0042] The target cow population of the experiment was established with the Dhanni spotted tumor cow breed, and whole genome SNPs of three spotted tumor cow breeds of Dhanni, Bhagnari and Dajal from two control cow populations of non-spotted tumor cow breeds were used for whole genome association analysis. The results of the association analysis will locate the candidate region related to the spotted coat color trait of the tumor cow (specifically Dhanni, Bhagnari and Dajal) on chromosome 6, and the region with the strongest signal is located between 70210464 bp-70403106 bp (the lowest signal value is 1.289627 x 10 -09 , Figure 3 ).

[0043] (1.4) Screening of differentiated regions in the genomes of spotted tumor cows and solid color tumor cows

[0044] The resequencing data of all experimental zebu cattle were used to detect all CNV segments on the zebu cattle genome using CNVcaller software. Then, the genome-wide selective elimination signal calculation (Vst) method was used to compare the regions with obvious differentiation in the genomes of pure-colored zebu cattle and spotted zebu cattle. The results showed that the 70167601 bp-70407200 bp interval of chromosome 6 (including the strongest signal interval located by genome-wide association analysis) was clearly differentiated in pure-colored zebu cattle and spotted zebu cattle (specifically Cholistani), with the lowest Vst signal value of 0.7300 ( Figure 2 ), where the Vst signal value ranges from 0 to 1, representing the degree of differentiation of the population. The larger the Vst signal value, the higher the degree of differentiation of the population and the higher the degree of selection. The 70157601 bp-70407200 bp interval of chromosome 6 (including the strongest signal interval located by genome-wide association analysis) is clearly differentiated in pure-colored zebu and spotted zebu (specifically Dhanni), with the lowest Vst signal value of 0.7051 ( Figure 4 ).

[0045] (1.5) Screening for CNV segments unique to spotted zebu

[0046] The CNVs of all zebu chromosome 6 detected by CNVcaller software were examined. The results showed that the spotted zebu (specifically Dhanni) had three CNV segments within the corresponding genomic differentiation regions (70157601 bp-70228400 bp, 70230801 bp-70291600 bp, and 70341601 bp-70407200 bp, respectively; see also Figure 5 ), while spotted zebu (specifically Cholistani) has five CNV segments in the corresponding genomic differentiation regions (70167601 bp-70228400 bp, 70230801 bp-70291600 bp, 70346001 bp-70355600 bp, 70356801 bp-70361200 bp, and 70362001 bp-70407200 bp, see also Figure 6 The interval marked above the gray scale column in the .

[0047] Combining the CNV segments associated with spotted coat color traits in two different zebu breeds (Cholistani and Dhanni), we identified the CNV segments close to KITThe interval of 70230801 bp-70291600 bp on chromosome 6, which is partially overlapped with the 3' end of the gene and partially overlaps with the gene (specifically, a certain length of interval extending from within the gene to downstream of the gene), is a CNV segment specific to the spotted cattle (i.e., 6:70230801-70291600). Meanwhile, according to the CNV segments shown in Table 1, the CNV segment specific to the spotted cattle is also present in the experimental cattle population. Figure 5 and Figure 6 The coverage depth detection results shown can draw a conclusion that the CNV segments (including the CNV segment specific to the spotted cattle) are actually present in the experimental cattle population.

[0048] (II) Genotype identification of spotted cattle and solid-colored cattle

[0049] (2.1) Copy number detection and typing of the CNV segment with the interval of 70230801 bp-70291600 bp on chromosome 6

[0050] The genomic target region 6: 70230801-70291600 of the above 39 cattle and other solid-colored cattle (specific sampling information: Burma 22, of which the male is white and spotless, and the female is yellow and spotless, 11 males and 11 females, ear tissue samples were collected in December 2019; Sahiwal 10, with black-brown color and no spots, ear tissue samples were also collected from Pakistan) were typed and the copy number was calculated, and the specific results are shown in Table 1.

[0051] In which, when typing the copy number, the method proposed by Zhang et al. (Fengwei Zhang, Chong Wang, Haiyue Xu, Xiaoting Xia, Xiaoyu Luo, Kaihui Li, Jianlin Han, Chuzhao Lei, Ningbo Chen, Xiangpeng Yue. Genomic analysis reveals a KIT-related chromosomal translocation associated with the white coat phenotype in yak. Journal of Animal Breeding and Genetics. 140(3):330-342.) was used, and the specific typing criteria are as follows:

[0052] When the copy number of the individual at the interval of 70230801 bp-70291600 bp on chromosome 6 is less than or equal to 1.5, then the genotype (i.e. the copy number variation type) of the individual is determined to be the deletion type; when the copy number of the individual at the interval of 70230801 bp-70291600 bp on chromosome 6 is greater than 1.5 and less than or equal to 2.9, then the genotype (i.e. the copy number variation type) of the individual is determined to be the normal type; when the copy number of the individual at the interval of 70230801 bp-70291600 bp on chromosome 6 is greater than 2.9, then the genotype (i.e. the copy number variation type) of the individual is determined to be the multiple copy type.

[0053] (2.2) Perform coverage depth detection and typing with the interval of 69975000 bp-70467000 bp on chromosome 6 as the CNV segment (i.e. 6: 69975000-70467000)

[0054] According to the typing method adopted in the patent CN114717334A early declared by the applicant, the above 71 cattle were genotyped. According to the coverage depth analysis results of the individual genome, when the coverage depth of the interval of 69975000 bp-70467000 bp on chromosome 6 is higher than the coverage depth of the normal copy number 1, the genotype of the individual is determined to be the repeated copy number type; when the coverage depth of the interval of 69975000 bp-70467000 bp on chromosome 6 is consistent with (for example, equal to or less than) the coverage depth of the normal copy number 1, the genotype of the individual is determined to be the wild homozygous type. And the coverage depth of the normal copy number 1 in the typing is determined according to the coverage depth detection result 8.67906 of the Dajal individual corresponding to the sample number SRR12632078 in Table 1, the absolute copy number of which is 1.00095. The specific results are shown in Table 1.

[0055] Table 1. Sample information

[0056]

[0057] Table 1 (continued). Sample information

[0058]

[0059] Table 1 (continued). Sample information

[0060]

[0061] Table 1 (continued). Sample information

[0062]

[0063] (III) Identification of molecular marker of CNV in spotted cattle

[0064] (3.1) Sample population and its genotype data

[0065] The genotype of all individuals in Table 1 above at the specific CNV segment (i.e. 6: 70230801-70291600) was determined (three genotypes: deletion, normal, and multiple copy).

[0066] (3.2) Phenotype data of sample population

[0067] The coat color trait (coat color is divided into: spotted, solid) of all individuals recorded in Table 1 above was determined.

[0068] (3.3) Large population association analysis

[0069] Statistical tests were performed by constructing a cross table (see Table 2) and using three different calculation methods.

[0070] Table 2. Coat color x CNV segment (6: 70230801-70291600) genotype type cross table

[0071]

[0072] The results of the analysis showed that the multiple copy type of the CNV segment (6: 70230801-70291600) was a molecular marker significantly associated with the spotted coat color of the cattle (see Table 3 for details).

[0073] Table 3. Coat color and genotype type (CNV segment position is 6: 70230801-70291600) chi-square test

[0074]

[0075] Note: In Table 3, the superscript a indicates that the expected count of 2 cells (33.3%) is less than 5; the minimum expected count is 54.

[0076] In addition, the genotype of all individuals in Table 1 above at the 6: 69975000-70467000 interval (i.e. 6: 69975000-70467000) was determined (two genotypes: wild homozygous, repeated copy number type), and the results were as follows: all 19 spotted cattle were of the repeated copy number type, while 33 of the solid color cattle were of the wild homozygous type (19 of the solid color cattle were of the repeated copy number type).

[0077] (IV) Analysis of the effectiveness of identifying the spotted cattle breed

[0078] (4.1) Polka breed identification based on unique CNV segment (i.e. 6: 70230801-70291600)

[0079] S1 Whole genome sequencing is performed on the polka individual to be tested, the sequencing data is aligned on the reference genome ARS-UCD1.2 to obtain a whole genome bam file, and then CNVcaller software is used to extract the unique CNV segment.

[0080] S2 The copy number of the unique CNV segment of the polka individual to be tested is calculated according to the Genotype.py script provided by the CNVcaller software.

[0081] S3 On the basis of step S2, the breed of the individual is determined, and the specific description is as follows: deletion type, the individual is determined to belong to solid polka; normal type, the individual is determined to belong to solid polka; multiple copy type, the individual is determined to belong to polka.

[0082] As can be seen from the above table 1, the method of using the copy number typing of the unique CNV segment (i.e. 6: 70230801-70291600) is verified, and the accuracy rate of identifying polka and solid polka reaches 100%.

[0083] (4.2) Polka breed identification based on 6: 69975000-70467000 bp interval

[0084] C1 Whole genome sequencing is performed on the polka individual to be tested, the sequencing data is aligned on the reference genome ARS-UCD1.2 to obtain a whole genome bam file, and samtools software is used to extract the bam file of the target interval 6: 69975000-70467000.

[0085] C2 The coverage depth of the target interval 6: 69975000-70467000 of the polka individual to be tested is calculated using sambamba, and the coverage depth graph is drawn.

[0086] C3 On the basis of step C2 (and referring to CN114717334A), the breed of the individual is determined, and the specific description is as follows: wild homozygous type, the individual is determined to belong to solid polka; repeated copy number type, the individual is determined to belong to polka.

[0087] As can be seen from the above table 1, the method of using the coverage depth typing of the 6: 69975000 bp-70467000 bp interval is verified, and the accuracy rate of identifying polka and solid polka is only 52 / 71 (i.e. 73.23%).

[0088] In summary, the present application discloses a unique CNV segment (i.e. 6: 70230801-70291600) of the chromosome 6 of the bovine reference genome existing in the spotted tumor cattle population, and a gene mutation (copy number variation) occurring by using the CNV segment can accurately identify the spotted color trait of the tumor cattle breed at the DNA level, so as to more effectively utilize the tumor cattle for introduction and marker-assisted selection.

Claims

1. A method for identifying zebu breeds using copy number variation, characterized by: The method for identifying the Zebu breed comprises the following steps: Extracting genomic DNA from the zebu cattle to be tested, and then detecting and analyzing the copy number variation segment located at positions 70230801-70291600 of chromosome 6 of the bovine reference genome ARS-UCD1.2; if the genotype of the zebu cattle to be tested is a multi-copy type, the zebu cattle to be tested is a spotted zebu cattle; If the copy number of the copy number variation segment is ≤1.5, the genotype is determined to be a deletion type; if the copy number of the copy number variation segment is >1.5 and ≤2.9, the genotype is determined to be a normal type; if the copy number of the copy number variation segment is >2.9, the genotype is determined to be a multi-copy type.

2. The method for identifying zebu breeds using copy number variation according to claim 1, characterized in that: The method for identifying the zebu breed further comprises the following steps: if the genotype of the zebu to be tested is a deletion type or a normal type, the zebu to be tested is a pure color zebu.

3. The method for identifying zebu breeds using copy number variation according to claim 1 or 2, characterized in that: The detection and analysis specifically includes the following steps: performing genome sequencing on the zebu cattle to be tested, then aligning the genome data of the zebu cattle to be tested to the bovine reference genome, then using software to extract the copy number variation segment and perform copy number calculation, and then identifying the genotype of the zebu cattle to be tested based on the copy number of the copy number variation segment.

4. A system for identifying Zebu breeds using copy number variation, characterized by: The system includes a sequencing module, a data segmentation module, a genotyping module, and a zebu breed identification module; The sequencing module is used to read or detect genomic data of zebu cattle; The data segmentation module is used to extract the corresponding part of the copy number variation segment located at position 70230801-70291600 of chromosome 6 of the bovine reference genome ARS-UCD1.2 in the genomic data; The genotyping module is used to identify the genotype of the zebu cattle according to the copy number of the copy number variation segment; The zebu breed identification module is used to classify zebu cattle with a multi-copy genotype as spotted zebu cattle according to their genotypes; If the copy number of the copy number variation segment is ≤1.5, the genotype is determined to be a deletion type; if the copy number of the copy number variation segment is >1.5 and ≤2.9, the genotype is determined to be a normal type; if the copy number of the copy number variation segment is >2.9, the genotype is determined to be a multi-copy type.

5. An application of a bovine chromosome 6 copy number variation or a detection method thereof in assisted selection breeding for spotted coat color in zebu cattle, characterized in that: The copy number variation detection method comprises the following steps: extracting genomic DNA of the zebu cattle to be tested, and then genotyping the zebu cattle to be tested according to the copy number variation segment located at positions 70230801-70291600 of chromosome 6 of the bovine reference genome ARS-UCD1.2; The multi-copy genotype of the copy number variation segment is associated with the spotted coat color trait of zebu cattle; If the copy number of the copy number variation segment is ≤1.5, the genotype is determined to be a deletion type; if the copy number of the copy number variation segment is >1.5 and ≤2.9, the genotype is determined to be a normal type; if the copy number of the copy number variation segment is >2.9, the genotype is determined to be a multi-copy type.

6. Use of a marker for copy number variation of bovine chromosome 6 or a detection method thereof in the introduction of spotted zebu cattle, characterized in that: The copy number variation marker is a copy number variation segment located at position 70230801-70291600 of chromosome 6 of the bovine reference genome ARS-UCD1.2; The multi-copy genotype of the copy number variation segment is associated with the spotted coat color trait of zebu cattle; If the copy number of the copy number variation segment is ≤1.5, the genotype is determined to be a deletion type; if the copy number of the copy number variation segment is >1.5 and ≤2.9, the genotype is determined to be a normal type; if the copy number of the copy number variation segment is >2.9, the genotype is determined to be a multi-copy type.

7. Use of a bovine chromosome 6 copy number variation marker or a detection method thereof in identifying spotted zebu breeds, characterized in that: The copy number variation marker is a copy number variation segment located at position 70230801-70291600 of chromosome 6 of the bovine reference genome ARS-UCD1.2; The multi-copy genotype of the copy number variation segment is associated with the spotted coat color trait of zebu cattle; If the copy number of the copy number variation segment is ≤1.5, the genotype is determined to be a deletion type; if the copy number of the copy number variation segment is >1.5 and ≤2.9, the genotype is determined to be a normal type; if the copy number of the copy number variation segment is >2.9, the genotype is determined to be a multi-copy type.

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