Application of ZNF133 gene copy number variation in cattle molecular marker-assisted selective breeding

Abstract

The invention provides application of ZNF133 gene copy number variation in cattle molecular marker-assisted selection breeding, ZNF133 gene copy number variation types closely related to Yunling cattle growth traits are detected on the DNA level, and normal individuals can serve as important candidate molecular markers for marker-assisted selection of the Yunling cattle growth traits. The method is used for accelerating the Yunling cattle molecular marker-assisted selection breeding work.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and in particular relates to an application of ZNF133 gene copy number variation in cattle molecular marker-assisted selection breeding. Background Art

[0002] In marker-assisted selection, either we know that certain QTLs (major effect genes) exist for the traits to be evaluated and their genotypes can be directly determined (such as QTLs discovered through candidate gene analysis), or we cannot determine their genotypes but know their linkage relationship with certain markers (such as QTLs discovered through marker-QTL linkage analysis) and the genotypes of these markers can be determined. In this case, this information can be used in the genetic assessment of individuals to improve accuracy.

[0003] Copy number variation (CNV) is a common genetic polymorphism, primarily manifested as submicroscopic deletions and duplications. It is caused by genomic rearrangements and generally refers to an increase or decrease in the copy number of large genomic segments larger than 1 kb in length. Commonly used CNV detection methods fall into two categories: one is primarily used to detect unknown CNVs genome-wide, including genomic microarrays and high-throughput sequencing technologies; the other is primarily used for targeted detection or verification of known CNVs.

[0004] Microarray methods primarily include comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays. Oligonucleotide probe arrays are widely used in CGH due to their high precision, high sensitivity, and low sample volume requirements. SNP arrays do not require a control sample for detection; instead, they analyze the SNP signal intensity within the sample being tested. Their primary advantage is that they simultaneously provide copy number and genotype information, and can also reveal loss of heterozygosity. However, the probes on SNP arrays are unevenly distributed across the genome, making probe design difficult in many complex regions. Therefore, SNP arrays have certain limitations for CNV detection. With the maturity of next-generation sequencing technology, direct resequencing has become the most effective method for detecting genomic structural variations. Compared with hybridization techniques, sequencing-based CNV detection offers numerous advantages: improved CNV resolution; the ability to define CNV boundaries; the ability to determine the absolute copy number of individual CNVs; and high efficacy for detecting CNVs with complex structural variations. However, this method is relatively expensive.

[0005] Among the various methods for detecting known CNVs, qPCR is a widely used quantitative technique. This method is simple to operate, highly sensitive, and fast. In PCR, a single copy of a gene is selected as an internal reference gene, and then 2 -ΔΔCt The method was used to determine the copy number variation type and relative copy number of an individual.

[0006] Zinc finger protein 133 (ZNF133) consists of a KRAB (krüppel-associated box, KRAB) domain and 14 consecutive zinc finger motifs. ZNF133 is considered a transcriptional repressor because the KRAB domain has potent inhibitory activity and the zinc finger motifs typically bind to DNA. Existing research has identified ZNF133 as overexpressed in osteosarcoma (Li et al. 2010). However, little research has been conducted on ZNF133 in cattle. Exploring the association between ZNF133 gene copy number variation and growth traits at the individual level offers a new approach for animal breeding. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides an application of ZNF133 gene copy number variation in cattle molecular marker-assisted selection breeding, which detects the type of ZNF133 gene copy number variation closely related to the growth traits of Yunling cattle at the DNA level. Normal individuals can be used as important candidate molecular markers for marker-assisted selection of Yunling cattle growth traits, and are used to accelerate the use of molecular marker-assisted selection breeding of Yunling cattle.

[0008] To achieve the above objectives, the present invention provides an application of a ZNF133 gene in assisting cattle breeding.

[0009] Furthermore, the invention specifically provides an application of bovine ZNF133 gene CNV markers in assisting cattle breeding.

[0010] Furthermore, the copy number variation region in the bovine ZNF133 gene is Chr13:37972164-37986962.

[0011] Furthermore, the normal copy number variation site of the bovine ZNF133 gene was used as a molecular marker for bovine growth traits.

[0012] Furthermore, the growth traits include body height and head length.

[0013] Furthermore, the cattle are Yunling cattle.

[0014] Furthermore, using the Yunling cattle genomic DNA to be tested as a template, the copy number variation region of the ZNF133 gene and a partial fragment of the BTF3 gene as an internal reference were amplified by real-time fluorescence quantitative PCR, and then the copy number variation type of the Yunling cattle ZNF133 gene was identified based on the quantitative results.

[0015] Furthermore, the primer pair for amplifying the copy number variation region of the ZNF133 gene is:

[0016] Upstream primer F1: 5′-GACCCAAACGGTGGAATTT-3′

[0017] Downstream primer R1: 5′-AGGAAGGAGCCGTGATCTTAAC-3′;

[0018] Furthermore, the copy number variation type is divided into three categories based on the quantitative results of -ΔΔCt: multi-copy type, -ΔΔCt>0.5; deletion type, -ΔΔCt<-0.5; normal type, -0.5≤-ΔΔCt≤0.5.

[0019] Furthermore, the amplification system used for real-time fluorescence quantitative PCR includes: 1 μL of 50 ng / L template DNA, 3.6 μL of sterile double-distilled water, 0.2 μL of the upstream and downstream primers corresponding to the amplification primer pair of the copy number variation region of the ZNF133 gene at 10 μmol / L, and Premix Ex TaqTMII 5 μL;

[0020] The reaction procedure used in the real-time fluorescence quantitative PCR was as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 30 s, for a total of 40 cycles;

[0021] The PCR product fragment size amplified by the primer pair based on the copy number variation region of the ZNF133 gene is 190 bp.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] The present invention introduces ZNF133 gene copy number variation (CNV) as a molecular marker in the breeding work of Yunling cattle, which accelerates the molecular marker-assisted selection breeding work of Yunling cattle. The traditional Yunling cattle breeding method mainly relies on phenotypic selection, which requires waiting for the cattle to grow to a certain stage and evaluating the genetic value by observing the phenotypic characteristics such as growth and reproduction. This process often takes several years. By using ZNF133 gene copy number variation as a molecular marker, in the early stage of cattle or even in the embryonic stage, it can be accurately judged whether it carries a gene type related to excellent growth traits by detecting the copy number variation of its genome. For example, individuals with normal gene copy number variation can be quickly identified, and these individuals are more likely to show ideal body height and head length traits in the subsequent growth process. By screening out individuals with excellent genotypes in advance for key cultivation, the breeding cycle can be significantly shortened and the establishment of excellent populations can be accelerated.

[0024] The growth traits of Yunling cattle, such as body height and head length, have an important impact on their production performance and economic benefits. By detecting the copy number variation region of the Yunling cattle gene (Chr13:37972164-37986962), it is possible to accurately distinguish individuals with different copy number variation types (deletion type, multiple copy type and normal type), and clarify the relationship between each type and growth traits. Studies have shown that the Deletion type has a negative effect on the body height trait of Yunling cattle, and the Duplication type has a negative effect on the head length trait of Yunling cattle, while normal-type individuals have the greatest advantage in body height and head length growth traits. Based on this discovery, breeders can selectively select individuals with normal gene copy number variation for breeding, thereby directionally improving the growth traits of Yunling cattle and cultivating superior breeds whose body height and head length are more in line with production needs.

[0025] Using ZNF133 gene copy number variation as a molecular marker has high genetic stability and is unaffected by environmental factors. Selecting for superior traits by detecting gene copy number variation patterns can more accurately reflect an individual's genetic potential, avoid misjudgments due to environmental factors, and improve the accuracy of trait selection. Molecular marker-assisted selection breeding methods based on ZNF133 gene copy number variation can more accurately screen individuals with excellent genetic potential for focused breeding, concentrating limited resources on these individuals and improving resource utilization efficiency.

[0026] In summary, applying the ZNF133 gene copy number variation of Yunling cattle to molecular marker-assisted selection breeding will provide strong support for the genetic improvement and population optimization of Yunling cattle, promote the breeding of Yunling cattle in a more efficient, more precise and more economical direction, and make important contributions to the development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the electrophoresis diagram of the PCR amplification products of the ZNF133 gene CNV detection primers; the first lane on the right is DNA marker I, and the remaining six lanes are the PCR amplification products of the ZNF133 gene. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. The embodiments are merely intended to explain the present invention rather than to limit the scope of protection of the present invention.

[0029] In the previous study of cattle genome resequencing, a copy number variation was found in the non-coding region of the cattle ZNF133 gene, Chr13: 37972164-37986962. Therefore, the present invention designed specific primers based on the region where the copy number variation occurred in the resequencing of the cattle ZNF133 gene sequence, and then used cattle (Qinchuan cattle, Xianan cattle, Jiaxian cattle, Yunling cattle) genomic DNA as a template for qPCR amplification, and used the BTF3 gene as an internal reference gene, using 2 -△△Ct Method, determine the copy number variation type of the individual. Based on the physiological role of the ZNF133 gene and the regulatory mechanism of CNV, study the correlation between the copy number variation of the ZNF133 gene and growth traits such as waist width, and provide data for molecular breeding of cattle. -△△Ct The gene expression abundance was indexed (Log22 -△△Ct ), perform a variance homogeneity test, and count the differences between groups. The specific operations of the above steps are as follows:

[0030] 1. Sample Collection and Genomic DNA Extraction

[0031] (1) Blood sample collection

[0032] Blood samples from 79 Qinchuan cattle were collected in the present invention from the Qinchuan Beef Cattle Breeding Center in Baoji, Shaanxi Province; blood samples from 324 Xianan cattle were collected from Xianan Cattle Technology Co., Ltd. in Biyang County, Zhumadian City, Henan Province; blood samples from 147 Jiaxian Red Cattle were collected from villages and towns in the Jiaxian Red Cattle Conservation Area in Pingdingshan City, Henan Province; and blood samples from 108 Yunling cattle were collected from Xiaoshao Township, Guandu District, Kunming City, Yunnan Province. Blood from a total of 658 cattle of these four breeds was collected through the jugular vein. Growth trait data, such as height, length, chest circumference, rump length, ischial width, and cruciate height, were recorded for subsequent correlation analysis.

[0033] (2) Extraction of DNA from blood samples (phenol-chloroform method)

[0034] ① Thaw the frozen blood sample in a room temperature water bath and transfer 2 mL of whole blood into a sterile 2 mL centrifuge tube.

[0035] ② Centrifuge at 4°C and 12,000 rpm for 10 min, discard the liquid, retain the precipitate, add 1.5 mL of PBS buffer, vortex to suspend the precipitate, and gently shake on ice for 15 min.

[0036] ③ Centrifuge at 4°C, 12,000 rpm for 10 min, discard the liquid, and retain the precipitate. Repeat step 3 once.

[0037] ④ Crush the precipitate until it becomes flocculent, add 500 μL of DNA extraction solution and 6 μL of proteinase K to the centrifuge tube, and incubate in a constant temperature water bath at 37°C overnight (about 16 hours) until the cell precipitate is digested and the solution is clear.

[0038] ⑤ Add 1 mL of Trips saturated phenol, shake gently on ice for 20 min, centrifuge at 4°C and 12,000 rpm for 10 min, and transfer the upper aqueous phase to another 2 mL centrifuge tube.

[0039] ⑥ Add 1 mL of chloroform, shake gently on ice for 20 min, centrifuge at 4°C and 12,000 rpm for 10 min, and transfer the upper aqueous phase to a new 1.5 mL centrifuge tube.

[0040] ⑦ Add 1 mL of pre-cooled anhydrous ethanol (-20°C), shake gently until DNA precipitates, place at -20°C for 30 min, centrifuge at 4°C, 12,000 rpm for 10 min, and discard the ethanol.

[0041] ⑧Add 1 mL of 70% ethanol, shake gently for 10 minutes, centrifuge at 4°C and 12,000 rpm for 10 minutes, discard the ethanol, and repeat the wash.

[0042] ⑨ Place at room temperature for 30 minutes, and then bake in a 60℃ oven for 30 seconds to evaporate the ethanol.

[0043] ⑩ Add 50 μL of ultrapure water and store at 4°C until the DNA is completely dissolved. Measure the concentration with a spectrophotometer and store at -80°C.

[0044] 2. Design of specific primers for amplification of target genes and internal reference genes

[0045] The bovine ZNF133 gene published by NCBI was used as the reference sequence to find the sequence of the copy number variation region screened out in the resequencing, namely the ZNF133 gene reference sequence (Chr 13:37972164-37986962), and primers containing this region were designed using Prime 5.0 software.

[0046] The primer sequence (P1) is as follows:

[0047] Sequence 1 - Upstream primer F1: 5'-GCCACCCTGACTCGTACTTAG-3'

[0048] Sequence 2 - downstream primer R1: 5'-CACCCCCTCCGTGAGAATAA-3'

[0049] The primer pair P1 ( Figure 1 )Specificity of the amplified product.

[0050] 3. Real-time Fluorescence Quantitative PCR

[0051] The qPCR reaction system is shown in Table 1:

[0052] Table 1 shows the reaction system for qPCR

[0053]

[0054]

[0055] The PCR reaction program is:

[0056] ① Pre-denaturation at 95℃ for 3 min;

[0057] ② Denaturation at 95°C for 15 s, annealing at 60°C for 30 s, for a total of 40 cycles.

[0058] 4. Identification of individual copy number variation CNV types

[0059] The experimental results were obtained using 2 -△△Ct The specific calculation method is:

[0060] ΔΔCt=ΔCt (实验组) -ΔCt (参照组)

[0061] ΔCt (实验组) =Ct (实验组目的基因) -Ct (实验组内参基因)

[0062] ΔCt (参照组) =Ct (参照组目的基因) -Ct (参照组内参基因)

[0063] In the formula, the experimental group is the individual sample to be tested for copy number variation. The reference group is the individual sample known to have no copy number variation, which can be the reference group cattle individual selected in the resequencing experiment. t That is, Cyclethreshold is the number of amplification cycles required for the fluorescence signal of the amplified product to reach the set threshold during the PCR amplification process.

[0064] The quantitative results were divided into three categories according to -ΔΔCt: Duplication, -ΔΔCt>0.5; Deltion, -ΔΔCt<-0.5; Normal, -0.5≤-ΔΔCt≤0.5.

[0065] 5. Data Processing

[0066] Production data: body height, cross height, body length, chest circumference, chest width, chest depth, rump length, ischial width, waist angle width, weight.

[0067] The number of individuals of three types (Deltion, Normal, Duplication) in the detection group was counted.

[0068] SPSS was used for correlation analysis. In data processing, a fixed model was used for analysis based on the different indices affecting body size traits, taking into account environmental effects, age, breed, genetic effects, and their interaction effects. At the same time, it was simplified according to actual conditions. The complete model is as follows:

[0069] Y ijk =μ+A+B+G j +E ijk

[0070] Where Y ijk is the individual phenotypic record; μ is the population mean; G j is the copy number variation type of each site; E ijk is a random error.

[0071] 6. After completing basic research on multiple varieties of yellow cattle, the present invention focused on the correlation analysis between gene copy number variation and growth traits in Yunling cattle. The results of the correlation analysis between ZNF133 gene copy number variation and growth traits in Yunling cattle are shown in Table 2:

[0072] Table 2 shows the association analysis between ZNF133 gene copy number variation and growth traits in Yunling cattle (108 heads)

[0073]

[0074] Note: Different letters on the shoulders of the mean values ​​indicate significant differences (P < 0.05), *P < 0.05; the numbers in brackets indicate the frequency of individuals with different copy number variation types.

[0075] Results showed that the copy number variation of the ZNF133 gene in Yunling cattle was significantly associated with body height and head length. The growth trait (body height) of individuals with the Deltion type was significantly inferior to that of individuals with the Duplication and Normal types; the growth trait (head length) of individuals with the Duplication type was significantly inferior to that of individuals with the Deltion and Normal types. Overall, the Normal type was the most advantageous. Therefore, the Normal type of the ZNF133 gene could serve as a molecular marker for early selection of growth traits in Yunling cattle, thereby accelerating molecular marker-assisted selection breeding in Yunling cattle.

[0076] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of ZNF133 gene in assisted cattle breeding.

2. The use according to claim 1, characterized in that Specifically, it is the application of bovine ZNF133 gene CNV markers in assisting cattle breeding.

3. The use according to claim 2, characterized in that The copy number variation region in the bovine ZNF133 gene is Chr13:37972164-37986962.

4. The use according to claim 2, characterized in that The normal copy number variation sites of the bovine ZNF133 gene were used as molecular markers for bovine growth traits.

5. The use according to claim 4, characterized in that The growth traits include body height and head length.

6. The use according to any one of claims 1 to 5, characterized in that The cattle are Yunling cattle.

7. The use according to claim 6, characterized in that Using the Yunling cattle genomic DNA as the template, the copy number variation region of the ZNF133 gene and a partial fragment of the BTF3 gene as an internal reference were amplified by real-time fluorescence quantitative PCR, and then the copy number variation type of the Yunling cattle ZNF133 gene was identified based on the quantitative results.

8. The use according to claim 7, characterized in that The primer pair for amplifying the copy number variation region of the ZNF133 gene is: Upstream primer F1: 5′-GACCCAAACGGTGGAATTT-3′ Downstream primer R1: 5′-AGGAAGGAGCCGTGATCTTAAC-3′.

9. The use according to claim 7, characterized in that The copy number variation types are divided into three categories based on the quantitative results of -ΔΔCt: multi-copy type, -ΔΔCt>0.5; deletion type, -ΔΔCt<-0.5; normal type, -0.5≤-ΔΔCt≤0.

5.

10. The use according to claim 1, characterized in that The amplification system used for real-time fluorescence quantitative PCR includes: 1 μL of 50 ng / L template DNA, 3.6 μL of sterile double-distilled water, 0.2 μL of the upstream and downstream primers corresponding to the amplification primer pair of the copy number variation region of the ZNF133 gene at 10 μmol / L, and Premix Ex TaqTMII 5 μL; The reaction procedure used in the real-time fluorescence quantitative PCR was as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 30 s, for a total of 40 cycles; The PCR product fragment size amplified by the primer pair based on the copy number variation region of the ZNF133 gene is 190 bp.

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