Method for identifying spotted tumor cattle variety by using copy number variation of cattle chromosome 6

Abstract

The invention discloses a method for identifying a spotted tumor cattle variety by using cattle chromosome 6 copy number variation. Respectively carrying out genome candidate region copy number variation analysis on the selected tumor cattle varieties with different spot hair color characters; the copy number variation type of an individual is identified according to the found copy number variation section of the genomic region 6: 70230801-70291600 positioned on the cattle chromosome 6, so that low-cost and accurate identification of spotted tumor cattle and pure-colored tumor cattle varieties on the DNA level is realized, and the genetic breeding efficiency is improved.

Description

Technical Field

[0001] The present invention relates to detecting copy number variation related to the spotted coat color trait of zebu cattle during introduction and breeding, and specifically to distinguishing pure-color zebu cattle (i.e. zebu cattle with pure-color coat color trait) from spotted zebu cattle breeds by using copy number variation types. Background Art

[0002] The most notable physical feature of zebu cattle is that they generally have a shoulder hump. The existing zebu cattle 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 varieties of zebu cattle, among which the South Asian zebu cattle can be specifically divided into Nellore, Tharparkar, Bhagnari, Dajal, Hariana, Cholistani, Dhanni, Sahiwal, Guzerat and other varieties. The geographical conditions and ecological characteristics of zebu cattle producing areas are different, and each variety has obvious regional differences and regional adaptability. For example, Cholistani is a zebu cattle breed adapted to the hot areas of Pakistan, and Dhanni is a zebu cattle breed adapted to dairy and mild arid environments. At the same time, zebu cattle have rich coat color traits, mainly including pure coat colors such as white, yellow, black (black-brown), reddish brown, and spotted coat colors such as white with black spots and white with brown spots. The spotted coat of zebu cattle is obviously different from the pure coat color, which not only attracts attention in scientific research on animal behavior and physiology, but also the zebu cattle population with spotted coat color can be used as high-quality germplasm resources and used in related breeding work such as introduction and selection. For cattle genetic breeding, it is usually not enough to refer to only phenotypic markers with limited reliability such as coat color. Especially for zebu cattle, it is very necessary to discover the genetic variation retained by a specific zebu cattle population at the genome or DNA level.

[0003] Copy number variation (CNV) is a change in the number of copies of DNA fragments of 1 Kb or larger in the genome. In mammalian populations, most autosomal genes exist in one copy each from their parents, and this normal change in gene copy dosage can significantly affect quantitative or qualitative phenotypic characteristics.

[0004] KIT The gene is a member of the receptor tyrosine kinase protein family. KIT Different genotypes of genes can affect the overall coat color of mammals by regulating the amount and distribution of melanin, as well as maintaining the color of eyes and skin. KITThe CNV of the gene (located at chr6: 69975194-70467027) is related to the spotted coat color variation of zebu cattle, and the results of coverage depth detection on this CNV segment show that the copy number of spotted coat individuals is 2, the copy number of white and yellow individuals is 1~1.5, and the copy number of spotted zebu breeds is twice that of pure-color zebu breeds (see also CN114717334A published on 2022-7-8). However, it is actually difficult to type CNV segments using coverage depth, which will be affected by factors such as the size of the detection range and the composition of the population to be tested, which is not conducive to promotion in breeding; in addition, the larger genetic variation region (the above CNV segment is about 490 Kb) will also increase the computational cost and reagent cost of the detection in practical applications due to the longer segment. Summary of the invention

[0005] The purpose of the present invention is to provide a method for identifying spotted zebu breeds using the copy number variation of bovine chromosome 6, which can improve the efficiency of genetic breeding related to high-quality zebu germplasm resources.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, a method for identifying a zebu breed using copy number variation is provided, the method for identifying a zebu breed comprising the following steps: The genomic DNA of the zebu cattle to be tested is extracted, and then the copy number variation segment located at position 70230801-70291600 (i.e. 6: 70230801-70291600) of chromosome 6 of the bovine reference genome is detected and analyzed. If the detection and analysis finds that the genotype (i.e. copy number variation type) of the zebu cattle to be tested in the copy number variation segment is a multi-copy type, the zebu cattle to be tested is determined to belong to the spotted zebu breed.

[0007] Preferably, the method for identifying zebu breeds further comprises the following steps: if the genotype of the zebu to be tested in the copy number variation segment is found to be a deletion type or a normal type through detection and analysis, the zebu to be tested is determined to belong to a pure color zebu breed.

[0008] Preferably, 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 in the copy number variation segment based on the copy number of the copy number variation segment.

[0009] Preferably, the identification specifically includes the following steps: when the copy number is ≤1.5, the genotype of the zebu cattle to be tested in the copy number variation segment is determined to be a deletion type; when the copy number is >1.5 and ≤2.9, the genotype of the zebu cattle to be tested in the copy number variation segment is determined to be a normal type; when the copy number is >2.9, the genotype of the zebu cattle to be tested in the copy number variation segment is determined to be a multi-copy type.

[0010] Preferably, the cattle reference genome is ARS-UCD1.2.

[0011] Preferably, the spotted zebu breed has a coat color trait of having a white base color and brown (such as Cholistani) or black (such as Dhanni) spots; the solid-colored zebu breed has a coat color trait of having a white, yellow or black (dark brown) base color and no spots.

[0012] In a second aspect, a system for identifying zebu breeds using copy number variation is provided, the system comprising 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 (i.e., 6: 70230801-70291600) of chromosome 6 of the above-mentioned cattle reference genome in the genomic data; The genotyping module is used to identify the genotype of the zebu cattle in the copy number variation segment according to the copy number of the copy number variation segment (specifically identifying it as the above-mentioned deletion type, normal type or multi-copy type); The zebu breed identification module is used to determine the zebu cattle with a multi-copy genotype as belonging to the spotted zebu breed (i.e., classified as a spotted zebu breed) based on the genotype of the zebu cattle in the copy number variation segment, and to determine the zebu cattle with a deletion type or normal genotype as belonging to the pure-color zebu breed (i.e., classified as a pure-color zebu breed).

[0013] In a third aspect, there is provided an application of a copy number variation of bovine chromosome 6 or a detection method thereof in molecular marker-assisted selection breeding of cattle, wherein the copy number variation detection method comprises the following steps: extracting genomic DNA of the cattle to be tested (such as zebu cattle, etc.), and then performing genotyping on the cattle to be tested based on the copy number of the copy number variation segment located at positions 70230801-70291600 (i.e., 6:70230801-70291600) of chromosome 6 of the above-mentioned bovine reference genome (the typing result is specifically identified as the above-mentioned deletion type, normal type or multiple copy type).

[0014] Preferably, the multi-copy genotype of the copy number variation segment is a molecular marker (specifically a copy number variation marker) that is significantly associated with the spotted coat color trait of zebu cattle.

[0015] In a fourth aspect, a copy number variation marker for bovine chromosome 6 or a detection method thereof for use in the introduction of spotted zebu cattle is provided, wherein the copy number variation marker is a multi-copy genotype of a copy number variation segment located at positions 70230801-70291600 (i.e., 6: 70230801-70291600) of chromosome 6 of the above-mentioned bovine reference genome, and the multi-copy genotype of the copy number variation segment is significantly correlated with the spotted coat color trait of zebu cattle.

[0016] In the fifth aspect, a copy number variation marker of bovine chromosome 6 or a detection method thereof is provided for use in identifying spotted zebu breeds, wherein the copy number variation marker is a multi-copy genotype of the copy number variation segment located at positions 70230801-70291600 (i.e., 6: 70230801-70291600) of chromosome 6 of the above-mentioned bovine reference genome, and the multi-copy genotype of the copy number variation segment is significantly correlated with the spotted coat color trait of zebu cattle.

[0017] The beneficial effects of the present invention are embodied in: The present invention performs copy number variation analysis of candidate regions of the genome on selected target zebu breeds with different spotted coat color traits (and pure-color zebu breeds as controls), and relocates the variation segments related to the spotted coat color traits of zebu cattle. The multi-copy genotype of the obtained new copy number variation segments (for example, 70230801bp-70291600bp of chromosome 6 of the cattle reference genome ARS-UCD1.2) is significantly correlated with the spotted coat color traits of zebu cattle. By identifying the genotypes, spotted zebu cattle and pure-color zebu cattle breeds (the latter has a deletion type or a normal type) can be accurately identified at the DNA level, and a spotted zebu cattle population with excellent genetic resources can be quickly established. The present invention not only solves the problem that coverage depth typing was difficult to apply in actual sample populations in the past (for example, false positives occur when identifying the spotted coat color trait of zebu samples at a higher copy number coverage depth than normal, i.e., repeated copy number types, resulting in a decrease in identification accuracy after changes in sample size and composition), but also provides an important basis and a more convenient approach for improving the efficiency of molecular marker-assisted selection of spotted coat color traits, introduction of spotted zebu and variety identification (for example, the new copy number variation segment is smaller in length, which is conducive to reducing detection costs). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Results of genome-wide association analysis of spotted zebu breeds (samples from Cholistani) and pure-colored zebu breeds.

[0019] Figure 2 VST results for CNVs of spotted zebu breed (sample from Cholistani) and pure-colored zebu breed.

[0020] Figure 3 Results of genome-wide association analysis of spotted zebu breed (sample from Dhanni) and pure-colored zebu breed.

[0021] Figure 4 The VST results for CNV of spotted Zebu breed (sample from Dhanni) and pure-colored Zebu breed.

[0022] Figure 5 The coverage depth calculation results of the CNV segments of spotted zebu breed (sample from Dhanni) and pure zebu breed.

[0023] Figure 6 Calculation results for coverage depth of CNV segments for spotted zebu breeds (samples from Cholistani) and pure-colored zebu breeds. DETAILED DESCRIPTION

[0024] 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.

[0025] (I) Relocalization of genomic variants associated with spotted coat color in zebu cattle using whole-genome resequencing, whole-genome association analysis, and whole-genome selective elimination (1.1) Sample collection, genomic DNA extraction and resequencing Ear tissue samples of 19 spotted zebu cattle were collected from Pakistan (9 from Cholistani and 10 from Dhanni, the ear tissue samples were collected in November 2020 by Quratulain Hanif, a member of this team), and 20 pure-colored zebu cattle (10 from Bhagnari and 10 from Dajal, ear tissue samples were also collected in Pakistan) were used as controls.

[0026] The coat color traits of the 19 spotted zebu collected were as follows: Cholistani's spots were brown and the base was white; Dhanni's spots were black and the base was white.

[0027] The coat color traits of the 20 pure-colored zebu cattle collected were: Bhagnari was pure white, Dajal was pure white, and there were 10 heads of each breed.

[0028] The collected ear tissue samples were stored in a -80° freezer and transported on dry ice to Shenzhen BGI Co., Ltd. for genomic DNA extraction and whole genome resequencing.

[0029] (1.2) Genome alignment and SNP detection The resequencing results of 39 cattle were aligned to the cattle reference genome ARS-UCD1.2 (NCBI version number GCF_000003205.7) using BWA software, and the whole-genome single nucleotide polymorphism (SNP) detection was performed using GATK software to construct the whole-genome SNP set of 39 cattle.

[0030] (1.3) Genome-wide association analysis of a single spotted zebu breed The spotted zebu breed Cholistani was used as the target cattle population for the experiment. Whole-genome association analysis was performed using whole-genome SNPs from Cholistani and two control cattle populations from unspotted zebu breeds Bhagnari and Dajal. The association analysis results located the candidate region associated with the spotted coat color trait of zebu cattle (specifically Cholistani, Bhagnari and Dajal) on chromosome 6, and the region with the strongest signal was located between 70321916 bp and 70345225 bp (the lowest signal value was 2.652758×10 -10 , Figure 1 ).

[0031] The spotted zebu breed Dhanni was used as the target cattle population for the experiment. The genome-wide SNPs of three zebu breeds, Dhanni and two control cattle populations from unspotted zebu breeds Bhagnari and Dajal, were used for genome-wide association analysis. The results of the association analysis located the candidate region associated with the spotted coat color trait of zebu cattle (specifically Dhanni, Bhagnari and Dajal) on chromosome 6, and the region with the strongest signal was located between 70210464 bp and 70403106 bp (the lowest signal value was 1.289627×10 -09 , Figure 3 ).

[0032] (1.4) Screening for regions of differentiation in the genomes of spotted and pure-colored zebu For the resequencing data of all experimental zebu cattle, CNVcaller software was used to detect all CNV segments on the zebu cattle genome; then the whole genome selective elimination signal calculation (Vst) method was used to compare the obviously differentiated regions 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 whole genome association analysis) was obviously differentiated in pure-colored zebu cattle and spotted zebu cattle (specifically Cholistani), and the lowest Vst signal value was 0.7300 ( Figure 2), where the Vst signal value is between 0 and 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 whole genome association analysis) is obviously differentiated in pure-colored zebu and spotted zebu (specifically Dhanni), with the lowest Vst signal value of 0.7051 ( Figure 4 ).

[0033] (1.5) Screening for CNV segments unique to spotted zebu The CNVs of chromosome 6 of all zebu cattle detected by CNVcaller software were checked. The results showed that spotted zebu cattle (specifically Dhanni) had three CNV segments in the corresponding genomic differentiation regions (70157601 bp-70228400 bp, 70230801 bp-70291600 bp, and 70341601 bp-70407200 bp, respectively, see Figure 5 ), while the spotted zebu (specifically Cholistani) has 5 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 figure).

[0034] Combining CNV segments associated with spotted coat color traits in two different zebu breeds (Cholistani and Dhanni), we identified KIT The 70230801 bp-70291600 bp interval on chromosome 6 at the 3' end of the gene and partially overlapping with the gene (specifically, a certain length extending from the gene to the downstream of the gene) is a CNV segment unique to spotted zebu cattle (i.e., 6:70230801-70291600). Figure 5 and Figure 6 The coverage depth detection results shown indicate that the above CNV segments (including the CNV segments unique to spotted zebu) actually exist in the experimental zebu population.

[0035] 2. Genotyping of spotted and pure-colored zebu cattle (2.1) Copy number detection and typing of the CNV segment between 70230801 bp and 70291600 bp on chromosome 6 The above 39 zebu cattle and other pure-colored zebu cattle collected (specific sampling information: 22 Burma cattle, including 11 males white and without spots, and 11 females yellow and without spots, and the ear tissue samples were collected in December 2019; 10 Sahiwal cattle, with black-brown fur and no spots, and the ear tissue samples were also collected from Pakistan;) were typed and the copy number of the genome target region 6: 70230801-70291600 was calculated. The specific results are shown in Table 1.

[0036] The copy number typing was performed with reference to the method proposed by Zhang et al. (Fengwei Zhang, ChongWang, Haiyue Xu, Xiaoting Xia, Xiaoyu Luo, Kaihui Li, Jianlin Han, ChuzhaoLei, 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.), and the specific typing criteria are given as follows: When the copy number of an individual at the interval of 70230801 bp-70291600 bp on chromosome 6 is less than or equal to 1.5, the genotype of the individual (i.e., the copy number variation type) is determined to be deletion type; when the copy number of an 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, the genotype of the individual (i.e., the copy number variation type) is determined to be normal type; when the copy number of an individual at the interval of 70230801 bp-70291600 bp on chromosome 6 is greater than 2.9, the genotype of the individual (i.e., the copy number variation type) is determined to be multiple copy type.

[0037] (2.2) The coverage depth detection and typing were performed using the 69975000 bp-70467000 bp interval of chromosome 6 as the CNV segment (i.e. 6: 69975000-70467000) According to the typing method used in the patent CN114717334A filed by the applicant earlier, the above 71 cattle were genotyped. According to the analysis results of the individual genome coverage depth, when the coverage depth of the interval 69975000 bp-70467000bp of chromosome 6 is higher than the coverage depth of the normal copy number 1, the individual genotype is determined to be a repeated copy number type; when the coverage depth of the interval 69975000 bp-70467000 bp of chromosome 6 is consistent with the coverage depth of the normal copy number 1 (for example, equal to or less than the coverage depth of the normal copy number 1), the individual genotype is determined to be a 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 of 8.67906 corresponding to the Dajal individual with sample number SRR12632078 and absolute copy number 1.00095 in Table 1. The specific results are shown in Table 1.

[0038] Table 1. Sample information

[0039] Table 1 (continued). Sample information

[0040] Table 1 (continued). Sample information

[0041] Table 1 (continued). Sample information

[0042] 3. Molecular marker identification of CNV in spotted zebu (3.1) Sample population and its genotype data The genotypes of all individuals in the above Table 1 in the specific CNV segment (i.e., 6: 70230801-70291600) were used (the three genotypes were: deletion type, normal type, and multi-copy type).

[0043] (3.2) Sample population phenotypic data The coat color traits of all individuals recorded in Table 1 above are used (coat color is divided into: spots, solid color).

[0044] (3.3) Large group association analysis Statistical tests were performed by constructing a cross-tabulation (see Table 2 for details) and using three different calculation methods.

[0045] Table 2. Cross-tabulation of coat color × CNV segment (6: 70230801-70291600) genotype type

[0046] The analysis results showed that the multi-copy type of the CNV segment (6: 70230801-70291600) was a molecular marker significantly associated with the spotted coat color of zebu cattle (see Table 3 for details).

[0047] Table 3. Chi-square test of coat color and genotype type (CNV segment position 6: 70230801-70291600)

[0048] Note: In Table 3, superscript a means that the expected counts of 2 cells (33.3%) are less than 5; the minimum expected count is 54.

[0049] In addition, the genotypes of all individuals in Table 1 above in the range of 69975000 bp-70467000 bp on chromosome 6 (i.e. 6: 69975000-70467000) were counted (two genotypes: wild homozygous type and repeated copy number type). The results are as follows: all spotted zebu were of repeated copy number type (a total of 19), while 33 of the pure-colored zebu were of wild homozygous type (and 19 pure-colored zebu were of repeated copy number type).

[0050] 4. Analysis of the effectiveness of identifying spotted zebu breeds (4.1) Identification of spotted zebu breeds based on a unique CNV segment (i.e., 6: 70230801-70291600) S1 The whole genome of the zebu cattle to be tested was sequenced, and the sequencing data was aligned with the reference genome ARS-UCD1.2 to obtain the whole genome bam file, and then the CNVcaller software was used to extract the unique CNV segment.

[0051] S2 Calculate the copy number of the unique CNV segment of the zebu individual to be tested based on the Genotype.py script provided by the CNVcaller software.

[0052] S3 determines the breed of the individual based on step S2, specifically as follows: if the individual is a missing type, it is determined that the individual belongs to a pure-colored zebu; if the individual is a normal type, it is determined that the individual belongs to a pure-colored zebu; if the individual is a multi-copy type, it is determined that the individual belongs to a spotted zebu.

[0053] As can be seen from Table 1 above, it has been verified that the accuracy of identifying spotted zebu and pure-colored zebu reached 100% by using the copy number typing method of the unique CNV segment (i.e. 6: 70230801-70291600).

[0054] (4.2) Identification of spotted zebu breeds based on the 69975000 bp-70467000 bp region on chromosome 6 C1 The whole genome of the zebu cattle individuals to be tested was sequenced, and the sequencing data was aligned with the reference genome ARS-UCD1.2 to obtain the whole genome bam file. The bam file of the target interval 6: 69975000-70467000 was extracted using the samtools software.

[0055] C2 uses sambamba to calculate the coverage depth of the target interval 6: 69975000-70467000 of the zebu individuals to be tested, and draws a coverage depth map.

[0056] C3 Based on step C2 (and referring to CN114717334A), the breed of the individual is determined, as follows: if it is a wild homozygous type, the individual is determined to be a pure-colored zebu; if it is a repeated copy number type, the individual is determined to be a spotted zebu.

[0057] As can be seen from Table 1 above, the accuracy of identifying spotted zebu and pure-colored zebu using the coverage depth typing method of the 69975000 bp-70467000 bp interval of chromosome 6 was only 52 / 71 (i.e. 73.23%).

[0058] In summary, the present invention reveals a unique CNV segment of chromosome 6 of the bovine reference genome (i.e., 6: 70230801-70291600) that exists in the spotted zebu population. The genetic variation (copy number change) occurring in this CNV segment can be used to accurately identify the spotted coat color trait of the zebu breed at the DNA level, thereby more effectively utilizing zebu for introduction and marker-assisted selection.

Claims

1. A method for identifying zebu breeds using copy number variation, characterized in that: The method for identifying the Zebu breed comprises the following steps: The genomic DNA of the zebu cattle to be tested is extracted, and then the copy number variation segment located at position 70230801-70291600 of chromosome 6 of the bovine reference genome is detected and analyzed; 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.

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 also includes 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. A 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. The method for identifying zebu breeds using copy number variation according to claim 3, characterized in that: The identification specifically includes the following steps: if the copy number is ≤1.5, the genotype is determined to be a deletion type; if the copy number is >1.5 and ≤2.9, the genotype is determined to be a normal type; if the copy number is >2.9, the genotype is determined to be a multi-copy type.

5. A method for identifying zebu breeds using copy number variation according to claim 1 or 2, characterized in that: The cattle reference genome is ARS-UCD1.

2.

6. A system for identifying zebu breeds using copy number variation, characterized in that: 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 cattle reference genome 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 with a multi-copy genotype as spotted zebu according to the genotype of the zebu.

7. An application of a bovine chromosome 6 copy number variation or a detection method thereof in bovine molecular marker-assisted selection breeding, characterized in that: The copy number variation detection method comprises the following steps: extracting the genomic DNA of the cattle to be tested, and then performing genotyping on the cattle to be tested according to the copy number variation segment located at positions 70230801-70291600 of chromosome 6 of the cattle reference genome.

8. The use according to claim 7, characterized in that: The multi-copy genotype of the copy number variation segment is a molecular marker associated with the spotted coat color trait of zebu cattle.

9. Application of a bovine chromosome 6 copy number variation marker or a detection method thereof in the introduction of spotted zebu cattle, characterized in that: The copy number variation marker is a multi-copy genotype of the copy number variation segment located at positions 70230801-70291600 of chromosome 6 of the bovine reference genome.

10. 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 multi-copy genotype of the copy number variation segment located at positions 70230801-70291600 of chromosome 6 of the bovine reference genome.

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