Detection of bovine genome 6 # chromosome structure variation and application of bovine genome 6 # chromosome structure variation

Abstract

The invention discloses detection of bovine genome No.6 chromosome structure variation and application thereof, the structure variation is a heterozygous inversion at a region 6: 69772181-71772202, can be used as an inversion marker for identifying green cattle, and provides a basis for deeply discussing a genetic mechanism influencing the hair color and adaptability of the green cattle.

Description

Technical Field

[0001] The 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 hair color of Qinghai-Tibet Plateau blue cattle. Background Art

[0002] The Qinghai-Tibet Plateau is famous for its low oxygen, low temperature and strong ultraviolet radiation. Cattle migrated to the Qinghai-Tibet Plateau about 3,700 years ago and were one of the earliest domesticated animals to enter the Qinghai-Tibet Plateau. The Qinghai-Tibet Plateau cattle are currently the oldest and most primitive cattle. Because they are distributed in high-altitude and high-altitude areas, they have strong cardiopulmonary functions, can efficiently utilize the forage resources of high-altitude pastures, and provide fuel and labor for local herdsmen. They have natural adaptability to the plateau environment and are an important germplasm resource for breeding new varieties of cattle in plateau environments. Coat color is one of the important characteristics of cattle. Coat color is also an important genetic marker for identifying individuals, bloodlines, and breeds (reference: Coat color inheritance of BMY cattle and Brahman cattle. Yellow Cattle Magazine, 2005, (04): 19-21, etc.). Some populations of yellow cattle on the Qinghai-Tibet Plateau include individuals with blue coat color, which are collectively called blue cattle in the local area.

[0003] The role of genetic genes in the formation of animal coat color includes: affecting the development, migration and survival of melanocytes, thereby affecting coat color, such as the ASIP gene where the SNPs related to the white coat color and tile-gray coat color of buffalo mentioned in CN112210607A are located. At present, there are no reports on the candidate genes that determine the coat color of blue cattle, and there is also a lack of molecular markers for identifying the coat color of blue cattle.

[0004] In addition, current research on the major 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

[0005] The purpose of the present invention is to provide a detection method for structural variation of chromosome 6 of the cattle genome and its application, so as to rapidly establish a cattle population with excellent genetic resources by utilizing inversion markers related to the hair color of blue cattle.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: 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: The structural variation located in the candidate genomic region of the cattle individual was detected. The structural variation was an inversion, and the location of the inversion region was at 69772181-71772202 bp of chromosome 6 of the cattle reference genome.

[0007] 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 amplification band is found after electrophoresis of the amplified product, or an amplification band of the correct size is found, it is determined that the target fragment is amplified).

[0008] 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).

[0009] Preferably, the amplification primers used in the PCR are: Upstream primer: 5'-ATCTTTAGCTGTAGCATGT -3' Downstream primer: 5'-ACTTGTAGGTTACTGGTT -3'.

[0010] Preferably, the target fragment is 584 bp (using the above-mentioned amplification primers).

[0011] Preferably, the candidate genomic region is located at bp 69750001–72725001 of chromosome 6 of the bovine reference genome.

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

[0013] In a second aspect, a kit for detecting inversion markers for identifying the hair color of Qinghai-Tibet Plateau blue cattle is provided, wherein the detection object of the kit is a structural variation located in a candidate genome region of a cattle individual, and the structural variation is the above-mentioned inversion.

[0014] Preferably, the kit specifically includes the above-mentioned amplification primers.

[0015] In a third aspect, there is provided the use of the inversion marker for identifying the hair color of Qinghai-Tibet Plateau blue cattle in cattle marker-assisted selection breeding.

[0016] Preferably, cattle individuals having the inversion marker (i.e., the inversion is detected in the candidate genomic region of the cattle individual) are selected to establish a cattle population adapted to plateau environments (e.g., altitude ≥ 4000 m), thereby accelerating the breeding process of improved breeds.

[0017] In a fourth aspect, there is provided the use of the inversion marker for identifying the hair color of blue cattle on the Qinghai-Tibet Plateau in identifying the hair color of pure-color cattle.

[0018] Preferably, the cattle individual having the inversion marker has blue coat color.

[0019] A fifth aspect provides a method for identifying pure color cattle breeds, comprising the following steps: The structural variation located in the candidate genome region of the cattle individual is detected, and the structural variation is the above-mentioned inversion; for the cattle individual in which the inversion is detected, it is determined that the cattle individual belongs to Qing cattle.

[0020] Preferably, the blue cattle are from the population of blue cattle in the Qinghai-Tibet Plateau (such as Langka cattle, Bailang cattle, Dingjie cattle).

[0021] Preferably, for cattle individuals in which the inversion is not detected, it is judged that the cattle individual is not a blue cattle (i.e., a non-blue cattle, such as a black, white, yellow or other coat color).

[0022] The beneficial effects of the present invention are embodied in: The present invention analyzes the genomic structural variation of blue cattle and non-blue cattle, identifies an inversion region of about 2Mb 6:69772181-71772202 related to the coat color of blue cattle on the Qinghai-Tibet Plateau, and verifies the corresponding inversion markers obtained by screening. The results show that the inversion marker located at 69772181-71772202bp of chromosome 6 of cattle disclosed by the present invention not only provides a basis for distinguishing blue cattle from non-blue cattle, but also is significantly related to the adaptability of cattle to the plateau environment, which is conducive to accelerating the process of breeding of fine breeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The association signals obtained from genome-wide association analysis using whole-genome resequencing data of Qingniu and non-Qingniu.

[0024] Figure 2 The differentiation signals were obtained by analyzing the population differentiation index using the three-generation ONT data of Qingniu and non-Qingniu.

[0025] Figure 3 The association signals are obtained by genome-wide association analysis using the variation scanning results of three-generation ONT data.

[0026] Figure 4 These are the electrophoresis results of PCR products of blue cattle and non-blue cattle: “Gray” means cyan, representing the corresponding lanes as blue cattle samples (DJ16, DJ17, DJ18, DJ19 and DJ20); “Non-gray” means the corresponding lanes as non-blue cattle samples (ALT2, ALT4, ALT8, ALT9 and ALT10). DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only used to explain the present invention, rather than to limit the protection scope of the present invention.

[0028] 1. Experimental animals and sample collection (1) Collect cattle resequencing data; collect blood samples from some cattle, extract DNA, and perform resequencing (sample information see Table 1).

[0029] (2) Blood samples were collected from samples of different breeds (including five blue cattle), and the third-generation ONT (Oxford Nanopore Technologies) data of several non-blue cattle were collected. All the collected samples were sent to Xi'an Haorui Gene Technology Co., Ltd. for ONT sequencing. A total of 85 cattle third-generation ONT data were obtained (sample information is shown in Table 2).

[0030] (3) A total of 23 blue cattle and 10 non-blue cattle under the same feeding and management conditions were selected, and blood samples were collected. DNA was extracted and resequencing was performed (sample information is shown in Table 3).

[0031] Table 1. Information of resequencing data used for genome-wide association analysis

[0032] Table 1. Information on resequencing data used for genome-wide association studies (continued)

[0033] Table 1. Information on resequencing data used for genome-wide association studies (continued)

[0034] Table 2. Basic information of 85 third-generation cattle ONT data

[0035] Note: BL18, LK04, LK08, LK09, and LK14 in Table 2 are samples of Tibetan Plateau blue cattle, and the rest are non-blue cattle.

[0036] Table 3. Basic information of Tibetan Plateau blue cattle samples and non-blue cattle samples used for PCR

[0037] 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, tan, or black-brown).

[0038] 2. Main reagents and instruments Whole blood genomic DNA extraction kit (centrifugal column type, China Tiangen Biochemical Technology Beijing Co., Ltd.); agarose (Haijin Biological Company, USA); DNA Marker (molecular weight 2000, China Tiangen Biochemical Technology Beijing Co., Ltd.); electrophoresis instrument (model JY200C, Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.); gel imager supporting analysis software (named 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 (10000× aqueous solution).

[0039] 3. Screening of candidate genes and functional variation regions for cyan coat color traits In order to explore the causes of blue coat color at the molecular level, the whole genome resequencing data of blue cattle and non-blue cattle were used to perform genome-wide association analysis based on single nucleotide polymorphisms (SNPs), and the strongest association signal was determined to be at 69765842–72752916 bp 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), which intersected with the strongest association signal obtained from the above whole-genome association analysis. After scanning for variation in the region with significant population differentiation (i.e., 69750001–72725001 bp on chromosome 6), a heterozygous inversion of about 2 Mb was detected, the region is 6: 69772181–71772202 (Table 4), that is, the structural variation (SV) in the genomic region of the KIT gene and its vicinity on chromosome 6. The above whole-genome association analysis, population differentiation index analysis, and scanning variation were all analyzed based on the cattle reference genome ARS-UCD1.2.

[0040] In addition, other genome-wide association analyses showed that this inversion region was the strongest association signal for 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 (6: inversion at 69772181–71772202).

[0041] Table 4. Frequency table of inversion in three generations of ONT data

[0042] Note: In Table 4, the 0 / 0 genotype is homozygous without inversion, and the corresponding samples are all non-black cattle; the 0 / 1 genotype is heterozygous for inversion, and the corresponding samples are all black cattle; the 1 / 1 genotype is homozygous for inversion, which is lethal, so it was not detected; the percentage in brackets is in black cattle.

[0043] In addition, the samples in Table 2 were grouped with 1000m and 4000m as the limits, where samples above 4000m were set as high-altitude groups and samples below 1000m were set as low-altitude groups. After association analysis, it was found that the heterozygous inversion was also significantly correlated with high altitude (P = 3.927587×10^-2, i.e. P<0.05).

[0044] 4. Polymerase chain reaction (PCR) experiments to verify the application effect of inversion markers 4.1 DNA extraction The blood genomic DNA was extracted from the samples in Table 3 using a whole blood genomic DNA extraction kit. The extracted DNA was tested for quality and used as a standby after passing the test.

[0045] 4.2 Primer design and synthesis In order to facilitate the detection of inversion marks (i.e., heterozygous inversions) in samples, primers were designed at the breakpoints of the above-mentioned inversion regions (amplification site: 69772181 bp on chromosome 6, i.e., 6: 69772181).

[0046] Specifically, Primer Premier 5.0 software was used to design primers, and the designed primers were synthesized by Shanghai Bioengineering Co., Ltd. The primer sequences are shown in Table 5.

[0047] Table 5. Primer sequence information

[0048] 4.3 PCR amplification reaction PCR was performed in 25 μL amplification reaction system (Table 6), and the reaction procedure was shown in Table 7. After the reaction, the PCR products were detected 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).

[0049] Table 6. PCR reaction system

[0050] Table 7. PCR reaction parameters

[0051] 4.4 Blood DNA extraction results 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 requirements for templates.

[0052] 4.5 PCR amplification results of inversion markers See also Figure 4 The PCR amplification results of Qingniu samples were consistent with the expected fragment size (Table 5), and there were no non-specific amplification bands, which could be directly used to determine the genotype. The results are shown in Table 8. Among them, the sequence of the amplified 584bp target fragment is as follows (i.e., SEQ.NO.ID.3): 5`--3` Table 8. Inversion frequencies of samples used for PCR

[0053] It can be concluded from Table 8 that the inversion marker located at 69772181–71772202 bp on chromosome 6 of the cattle reference genome can be used to identify Qinghai-Tibet Plateau blue cattle (with an accuracy of 100%). At the same time, the inversion marker can also be used to quickly select cattle individuals with coat color and plateau environment adaptation at an early stage.

Claims

1. A method for detecting a bovine chromosome structural variation, characterized in that: The method comprises the following steps: The structural variation located in the candidate genome region of cattle was detected, and the structural variation was an inversion, and the inversion region was located at 69772181-71772202 bp of chromosome 6 of the cattle reference genome.

2. The use of the method for detecting bovine chromosome structural variation as claimed in claim 1 in bovine marker-assisted selection breeding, characterized in that: The inversion is a molecular marker for identifying Qingniu.

3. A kit for detecting an inversion marker for identifying blue cattle, characterized in that: The kit includes primers for detecting a structural variation located in a candidate genomic region of cattle, wherein the structural variation is an inversion, and the inversion region is located at 69772181-71772202 bp of chromosome 6 of the cattle reference genome.

4. A kit for detecting an inversion marker for identifying Tingnid according to claim 3, characterized in that: The kit specifically includes the following amplification primers for PCR: Upstream primer: 5'-ATCTTTAGCTGTAGCATGT -3' Downstream primer: 5'-ACTTGTAGGTTACTGGTT -3' When the target fragment was present in the amplification result, the inversion marker located at 69772181–71772202 bp on chromosome 6 of the bovine reference genome was detected.

5. An application of an inversion marker for identifying blue cattle in cattle marker-assisted selection breeding, characterized in that: The inversion marker is located at 69772181–71772202 bp on chromosome 6 of the bovine reference genome.

6. The use according to claim 5, characterized in that: Cattle carrying the inversion marker are selected to establish a cattle population adapted to plateau environments.

7. An application of an inversion marker for identifying blue cattle in identifying the hair color of pure-color cattle, characterized in that: The inversion marker is located at 69772181–71772202 bp on chromosome 6 of the bovine reference genome.

8. The use according to claim 7, characterized in that: The hair color of cattle with the inversion marker is cyan.

9. A method for identifying pure color cattle breeds, characterized in that: The following steps are involved: A structural variation located in a candidate genome region of cattle is detected, wherein the structural variation is an inversion, and the inversion region is located at 69772181-71772202 bp of chromosome 6 of the cattle reference genome; for cattle in which the inversion is detected, it is determined that the cattle belongs to Qing cattle.

10. A method for identifying pure color cattle breeds according to claim 9, characterized in that: The Qingniu is derived from the population of Qingniu in the Qinghai-Tibet Plateau.

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