Detection of structural variations on bovine chromosome 6 and its application

Abstract

The application discloses detection of a structural variation of a bovine chromosome 6 and application thereof, the structural variation is a heterozygous inversion at a region 6:69772181-71772202, can be used as an inversion marker for identifying blue cattle, and provides a basis for further exploring genetic mechanisms of influencing blue cattle hair color and adaptability.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and molecular breeding, and relates to the identification of structural variation of chromosome 6 of the cattle genome, and specifically to the screening and verification of inversion markers for identifying the coat color of Qinghai-Tibet Plateau blue cattle. Background Art

[0002] The Qinghai-Tibet Plateau is known for its low oxygen levels, low temperatures, and strong ultraviolet radiation. Cattle migrated to the plateau approximately 3,700 years ago, making them one of the earliest domesticated animals to arrive there. Tibetan Plateau cattle are the oldest and most primitive breed of cattle. Due to their high altitude and cold habitat, they possess robust cardiopulmonary function, are able to efficiently utilize the forage resources of alpine pastures, and provide fuel and labor for local herders. These cattle are naturally adaptable to the plateau environment and are a vital germplasm resource for developing new breeds of cattle suitable for this plateau environment.

[0003] Coat color, or the color of the coat, is one of the most important characteristics of cattle. It is also an important genetic marker for identifying individuals, bloodlines, and breeds (see: Coat Color Inheritance of BMY and Brahman Cattle. Yellow Cattle Magazine, 2005, (04): 19-21, etc.). Some populations of Yellow Cattle on the Qinghai-Tibet Plateau include individuals with a completely blue coat, which are collectively referred to locally as blue cattle.

[0004] Genetic genes play a role in the formation of animal coat color, including by influencing the development, migration, and survival of melanocytes, which in turn influence coat color. For example, the ASIP gene, where the SNP associated with white and tile-gray coat color in buffaloes, mentioned in CN112210607A, is located. Currently, no research has been reported on candidate genes that determine coat color in blue cattle, and molecular markers for identifying blue cattle coat color are also lacking.

[0005] In addition, current research on the main effect genes of cattle coat color traits often only involves the exploration of markers that affect coat color differences between individuals, such as the copy number variation of the candidate genomic region containing the KIT gene mentioned in CN114717334A. This to a certain extent limits the practical application value of related markers in cattle breed selection. Summary of the Invention

[0006] The purpose of the present invention is to provide a detection method for the structural variation of chromosome 6 of the cattle genome and its application, so as to quickly establish a cattle population with excellent genetic resources by utilizing inversion markers related to the coat color of blue cattle.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, a method for detecting an inversion marker for identifying the hair color of a Tibetan Plateau blue cattle is provided, the method comprising the following steps:

[0009] Structural variations located in a candidate genomic region of cattle individuals were detected. The structural variations were inversions, and the inversion region was located at bp 69772181–71772202 of chromosome 6 of the cattle reference genome.

[0010] Preferably, the method specifically includes the following steps: performing PCR using individual cattle genomic DNA as a template, and then identifying the target fragment obtained by PCR amplification (for example, if an amplified band is found after electrophoresis of the amplified product, or an amplified band of the correct size is found, it is determined that the target fragment is amplified).

[0011] Preferably, when the target fragment is amplified, the coat color of the cattle (such as Langka cattle, Bailang cattle, Dingjie cattle) is blue, that is, the inversion marker is located in the cattle reference genome 6:69772181–71772202 (usually the corresponding genotype of the inversion marker on the genome is a heterozygous inversion).

[0012] Preferably, the amplification primers used in the PCR are:

[0013] Upstream primer: 5'-ATCTTAGCTGTAGCATGT -3'

[0014] Downstream primer: 5'-ACTTGTAGGTTACTGGTT -3'.

[0015] Preferably, the target fragment is 584 bp (using the above-mentioned amplification primers).

[0016] Preferably, the candidate genomic region is located at bp 69750001–72725001 of chromosome 6 of the bovine reference genome.

[0017] Preferably, the cattle reference genome is ARS-UCD1.2.

[0018] In a second aspect, a kit is provided for detecting inversion markers for identifying the hair color of Qinghai-Tibet Plateau blue cattle. The detection object of the kit is a structural variation located in the candidate genome region of a cattle individual, and the structural variation is the above-mentioned inversion.

[0019] Preferably, the kit specifically includes the above-mentioned amplification primers.

[0020] In a third aspect, the present invention provides the use of the above-mentioned inversion marker for identifying the hair color of Qinghai-Tibet Plateau blue cattle in cattle marker-assisted selection breeding.

[0021] Preferably, cattle individuals with the inversion marker are selected (i.e., the inversion is detected in the candidate genomic region of the cattle individual) to establish a cattle population adapted to the plateau environment (e.g., altitude ≥ 4000 m), thereby accelerating the breeding process of improved breeds.

[0022] In a fourth aspect, the invention provides the use of the inversion marker for identifying the hair color of Tibetan Plateau blue cattle in identifying the hair color of pure-color cattle.

[0023] Preferably, the cattle individual having the inversion marker has blue coat color.

[0024] A fifth aspect provides a method for identifying pure color cattle breeds, comprising the steps of:

[0025] Structural variations located in the candidate genome region of the cattle individual are detected, wherein the structural variations are the above-mentioned inversions; and for the cattle individual in which the inversions are detected, it is determined that the cattle individual belongs to the blue cattle.

[0026] Preferably, the blue cattle are from the population of blue cattle in the Qinghai-Tibet Plateau (such as Langka cattle, Bailang cattle, Dingjie cattle).

[0027] Preferably, for cattle individuals in which the inversion is not detected, it is determined that the cattle individual does not belong to blue cattle (i.e., belongs to non-blue cattle, such as black, white, yellow and other coat colors).

[0028] The beneficial effects of the present invention are embodied in:

[0029] By analyzing the genomic structural variation of blue cattle and non-blue cattle, this study identified an approximately 2Mb inversion region 6:69772181–71772202, which is associated with the coat color of blue cattle on the Qinghai-Tibet Plateau. The corresponding inversion markers obtained through screening were then validated. The results show that the inversion marker disclosed in this study, located at bp 69772181–71772202 on bovine chromosome 6, not only provides a basis for distinguishing blue cattle from non-blue cattle, but also significantly correlates with the cattle's adaptability to the plateau environment, facilitating the rapid breeding of improved breeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The association signals were obtained by genome-wide association analysis using whole-genome resequencing data of Qingniu and non-Qingniu.

[0031] Figure 2 The differentiation signals were obtained by analyzing the population differentiation index using the three-generation ONT data of Qingniu and non-Qingniu.

[0032] Figure 3 The association signals are obtained from genome-wide association analysis using the variation scanning results of three-generation ONT data.

[0033] Figure 4These are the electrophoresis results of PCR products of blue cattle and non-blue cattle: "Gray" indicates cyan, representing the corresponding lanes as blue cattle samples (DJ16, DJ17, DJ18, DJ19, and DJ20); "Non-gray" indicates the corresponding lanes as non-blue cattle samples (ALT2, ALT4, ALT8, ALT9, and ALT10). DETAILED DESCRIPTION

[0034] The present invention will be described in further detail below with reference to the accompanying drawings and examples. Obviously, the described examples are only used to explain the present invention, rather than to limit the scope of protection of the present invention.

[0035] (1) Experimental animals and sample collection

[0036] (1) Collect cattle resequencing data; collect blood samples from some cattle, extract DNA, and perform resequencing (see Table 1 for sample information).

[0037] (2) Blood samples were collected from samples of different breeds (including five blue cattle), and third-generation ONT (Oxford Nanopore Technologies) data from several non-blue cattle were collected. All collected samples were sent to Xi'an Haorui Gene Technology Co., Ltd. for ONT sequencing. A total of 85 third-generation ONT data were obtained (see Table 2 for sample information).

[0038] (3) 23 blue cattle and 10 non-blue cattle under the same feeding and management conditions were selected, and their blood samples were collected, DNA was extracted, and resequencing was performed (sample information is shown in Table 3).

[0039] Table 1. Information of resequencing data used for genome-wide association analysis

[0040]

[0041] Table 1. Information on resequencing data used for genome-wide association studies (continued)

[0042]

[0043] Table 1. Information on resequencing data used for genome-wide association studies (continued)

[0044]

[0045] Table 2. Basic information of 85 third-generation cattle ONT data

[0046]

[0047] Note: BL18, LK04, LK08, LK09, and LK14 in Table 2 are samples of Tibetan Plateau blue cattle, and the rest are non-blue cattle.

[0048] Table 3. Basic information of Tibetan Plateau blue cattle samples and non-blue cattle samples used for PCR

[0049]

[0050] Note: The blue cattle samples in Table 3 were collected from Dingjie cattle, and the Altai cattle were all non-blue cattle (hair color was yellow, yellow-brown, or black-brown).

[0051] (2) Main reagents and instruments

[0052] Whole blood genomic DNA extraction kit (spin column type, Tiangen Biochemical Technology Beijing Co., Ltd., China); agarose (Haijin Biotechnology, USA); DNA marker (molecular weight 2000, Tiangen Biochemical Technology Beijing Co., Ltd., China); electrophoresis instrument (model JY200C, Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.); gel imager analysis software (LaneID); PCR amplification instrument (Gene Company Limited); multi-function vortex mixer (model G560E); electronic balance (model BS210S, Beijing Sartorius Instrument System Co., Ltd.); microwave oven (model MF-2070MGZ, Haier Group); DuRed nucleic acid dye (10,000× aqueous solution).

[0053] (III) Screening of candidate genes and functional variation regions for the cyan coat color trait

[0054] To explore the molecular causes of blue coat color, we used whole-genome resequencing data from blue cattle and non-blue cattle to conduct genome-wide association analysis based on single nucleotide polymorphisms (SNPs). We determined that the strongest association signal was at bp 69765842–72752916 on chromosome 6 ( Figure 1 ; P = 1.022608e-11), about 2.99Mb, this region contains 36 protein-coding genes, including KIT (position 6: 70166681–70254049), KDR, PDGFRA and other genes. The results of the SNP-based population genetic differentiation analysis of Qing cattle and non-Qing cattle showed that the most significant differentiation signal was located at 69750001–72725001 bp on chromosome 6 ( Figure 2; P = 6.97766e-06), intersecting with the strongest association signal from the genome-wide association analysis above. Subsequently, a variant scan within a region of significant population differentiation (i.e., bp 69750001–72725001 on chromosome 6) detected a heterozygous inversion of approximately 2 Mb, located at bp 6: 69772181–71772202 (Table 4). This structural variant (SV) is located within the KIT gene and its surrounding genomic region on chromosome 6. The above genome-wide association analysis, population differentiation index analysis, and variant scanning were all performed using the bovine reference genome ARS-UCD1.2.

[0055] In addition, other genome-wide association analyses showed that this inversion region was the strongest association signal associated with blue cattle coat color ( Figure 3 ; P = 0×10 ^ -8), that is, the difference in coat color between blue cattle and non-blue cattle is most likely determined by the above structural variation (inversion at 6: 69772181–71772202).

[0056] Table 4. Frequency of inversions in three generations of ONT data

[0057]

[0058] Note: In Table 4, the 0 / 0 genotype is homozygous for no inversion, and the corresponding samples are all non-teal cattle; the 0 / 1 genotype is heterozygous for inversion, and the corresponding samples are all teal cattle; the 1 / 1 genotype is homozygous for inversion, which is lethal and therefore not detected; the percentage in brackets is in teal cattle.

[0059] In addition, the samples in Table 2 were grouped with altitudes of 1000m and 4000m as the limits, where samples above 4000m were set as the high-altitude group and samples below 1000m were set as the low-altitude group. After association analysis, it was found that the heterozygous inversion was also significantly associated with high altitude (P = 3.927587×10^-2, i.e. P < 0.05).

[0060] (IV) Polymerase chain reaction (PCR) experiments to verify the application effect of inversion tagging

[0061] 4.1 DNA extraction

[0062] Genomic DNA was extracted from the samples listed in Table 3 using a whole blood genomic DNA extraction kit. The extracted DNA was quality tested and used for future use.

[0063] 4.2 Primer design and synthesis

[0064] In order to facilitate the detection of inversion marks (i.e., heterozygous inversions) in samples, primers were designed at the breakpoint of the above-mentioned inversion region (amplification site: 69772181 bp on chromosome 6, i.e., 6: 69772181).

[0065] Primers were designed using Primer Premier 5.0 software and synthesized by Shanghai Bioengineering Co., Ltd. The primer sequences are shown in Table 5.

[0066] Table 5. Primer sequence information

[0067]

[0068] 4.3 PCR amplification reaction

[0069] PCR was performed in a 25 μL amplification reaction system (Table 6). The reaction procedure is shown in Table 7. After the reaction, PCR products were examined by 1.5% agarose gel electrophoresis, and the genotype of the inversion was determined based on the presence and size of the amplified fragment (no amplified band, genotype 0 / 0; presence of a 584 bp band, genotype 0 / 1).

[0070] Table 6. PCR reaction system

[0071]

[0072] Table 7. PCR reaction parameters

[0073]

[0074] 4.4 Blood DNA extraction results

[0075] The genomic DNA extracted from the blood samples of blue cattle and non-blue cattle samples was tested by agarose gel electrophoresis. The results showed that the genomic DNA bands were clear and bright, without tailing, and the concentration was uniform, which fully met the PCR template requirements.

[0076] 4.5 PCR amplification results of inversion markers

[0077] See also Figure 4 The PCR amplification results of the Qingniu sample were consistent with the expected fragment size (Table 5), and there were no non-specific amplification bands, which can be directly used to determine the genotype. The results are shown in Table 8. The sequence of the amplified 584bp target fragment is as follows (i.e., SEQ.NO.ID.3):

[0078] 5`--3`

[0079] Table 8. Inversion frequencies of samples used for PCR

[0080]

[0081] Table 8 shows that the inversion marker located at bp 69772181–71772202 on chromosome 6 of the bovine reference genome can be used to identify Tibetan Plateau blue cattle (with 100% accuracy). This inversion marker can also be used for early and rapid selection of cattle with improved coat color and adaptation to the plateau environment.

Claims

1. A method for detecting bovine chromosome structural variation, characterized by: The method comprises the following steps: The structural variation in the candidate bovine genome region was detected. The structural variation was an inversion. The inversion region was located at 69772181–71772202 bp on chromosome 6 of the bovine reference genome ARS-UCD1.

2. The detection adopts PCR, and the amplification primers of PCR are as follows: Upstream primer: 5'-ATCTTAGCTGTAGCATGT -3' Downstream primer: 5′-ACTTGTAGGTTACTGGTT -3′; The cattle are Qinghai-Tibet Plateau yellow cattle.

2. A kit for detecting an inversion marker for identifying blue cattle, characterized by: The kit includes primers for detecting a structural variation located in a candidate bovine genome region, wherein the structural variation is an inversion, and the inversion region is located at 69772181–71772202 bp of chromosome 6 of the bovine reference genome ARS-UCD1.2; The kit specifically includes the following amplification primers for PCR: Upstream primer: 5'-ATCTTAGCTGTAGCATGT -3' Downstream primer: 5'-ACTTGTAGGTTACTGGTT -3' When the amplification result shows the presence of a 584 bp target fragment, the inversion marker is detected; The cattle are Qinghai-Tibet Plateau Yellow Cattle, and the population of Qinghai-Tibet Plateau Yellow Cattle with blue coat color all over its body is blue cattle.

3. Use of a reagent for detecting an inversion marker in blue cattle for identifying pure-color cattle hair color, characterized in that: The inversion marker is located at 69772181–71772202 bp on chromosome 6 of the bovine reference genome ARS-UCD1.2; The hair color of the cattle with the inversion marker is cyan; The cattle are Qinghai-Tibet Plateau Yellow Cattle, and the population of Qinghai-Tibet Plateau Yellow Cattle with blue coat color all over its body is blue cattle.

4. A method for identifying pure color cattle breeds, characterized by: The following steps are involved: Detecting a structural variation in a candidate bovine genome region, where the structural variation is an inversion, is located at bp 69772181–71772202 on chromosome 6 of the bovine reference genome ARS-UCD1.

2. Cattle in which the inversion is detected are judged to have a cyan coat color and are classified as cyan cattle. The cattle are Qinghai-Tibet Plateau Yellow Cattle, and the population of Qinghai-Tibet Plateau Yellow Cattle with blue coat color all over its body is blue cattle.

Images