Application of GYPB gene in cattle molecular breeding

Abstract

The invention discloses application of a GYPB gene in cattle molecular breeding, and belongs to the technical field of molecular genetics research. The method comprises the following steps: amplifying a copy number variation region of a GYPB gene through a qPCR (Quantitative Polymerase Chain Reaction) technology by taking bovine genome DNAs of different varieties as templates and a BTF3 gene as a contrast; and analyzing the copy number and judging the copy number variation type of the individual by adopting a 2 * 2-delta delta Ct statistical method. The method provided by the invention is beneficial to correlation analysis between the cattle GYPB gene CNV and growth traits, and the application of molecular marker-assisted selection in cattle genetic breeding is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics research, and in particular relates to the application of the GYPB gene in cattle molecular breeding. Background Art

[0002] Chinese yellow cattle possess unique meat quality traits, which are used to improve beef quality. To accelerate genetic improvement, molecular marker-assisted selection (MAM) has become increasingly popular. The evolution from traditional phenotype-to-gene approach to current gene-to-phenotype approach has undoubtedly improved the precision and effectiveness of breeding. Therefore, research on animal genomes is particularly important. There are many factors contributing to genomic variability, including single nucleotide polymorphisms (SNPs), variable numbers of tandem repeats, transposons, and structural alterations (deletions, duplications, inversions, and translocations). Copy number variation (CNV), which encompasses changes in the copy number of DNA fragments ranging from 0 to 1 kilobase (kb) or larger, has been found to be common in normal individuals and can also be considered a form of genomic variation. In animal husbandry research, CNV, as a molecular breeding method, has been applied to genetic improvement efforts in pigs, chickens, cattle, and sheep.

[0003] The GYPB gene, located on chromosome 17, encodes a major glycoprotein of the red blood cell membrane that has antigenic determinants for the MN and Ss blood groups. Diseases associated with GYPB include malaria, atrial fibrillation, and stroke. However, to date, no studies have reported an association between the GYPB gene and growth traits in cattle. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an application of the GYPB gene in cattle molecular breeding.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention discloses the application of the GYPB gene in cattle molecular breeding.

[0007] The second aspect of the present invention discloses the application of a method for detecting copy number variation of the cattle GYPB gene in molecular breeding of yellow cattle.

[0008] Preferably, cattle gene DNA is used as a template and primers are designed to amplify the copy number variation region of the GYPB gene and a partial fragment of the BTF3 gene as a control by real-time quantitative PCR, and then the copy number variation type of the cattle GYPB gene is identified based on the quantitative results.

[0009] Further preferably, the copy number variation of the GYPB gene is located at Chr17: 14,389,601bp~14,391,200bp, a total of 1,600bp.

[0010] Preferably, the primer pair Primer1 for amplifying the copy number variation region of the GYPB gene is:

[0011] Upstream primer F1: 5′-ACAGAGTGTGAAAGACCTTGG-3′

[0012] Downstream primer R1: 5'-GCTCCTTCTTTGGATGGGAA-3'

[0013] The primer pair Primer2 for the copy number variation region of the BTF3 gene used as a control is:

[0014] Upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'

[0015] Downstream primer R2: 5′-TTCGGTGAAATGCCTCTCG-3′.

[0016] Further preferably, the amplification system used in the real-time quantitative PCR comprises: 1 μL 10 ng / μL genomic DNA, 0.5 μL 10 μmol / L upstream primer F1, 0.5 μL 10 μmol / L downstream primer R1, 0.5 μL 10 μmol / L upstream primer F2, 0.5 μL 10 μmol / L downstream primer R2, 5 μL SYBR PreMix Ex TaqTMII and 3μL ddH2O.

[0017] Preferably, the reaction procedure used in the real-time quantitative PCR is: pre-deformation at 95°C for 1 min; denaturation at 95°C for 10 s, and annealing at 60°C for 30 s, for a total of 40 cycles.

[0018] Preferably, the copy number variation types of the GYPB gene are divided into three categories according to -ΔΔCt: deletion type, i.e., -ΔΔCt<-0.5; normal type, i.e., -0.5≤-ΔΔCt≤0.5; and duplication type, i.e., -ΔΔCt>0.5.

[0019] Preferably, the Pinan cattle individuals with a deletion-type copy number variation type have better growth traits than those with a duplication-type copy number variation type or a normal-type copy number variation type; the Yunling cattle individuals with a duplication-type copy number variation type have better growth traits than those with a deletion-type copy number variation type or a normal-type copy number variation type.

[0020] Further preferably, the Pinan cattle individuals with the deletion-type copy number variation type are superior to the individuals with the duplication-type copy number variation type or the normal-type copy number variation type in the growth trait cross height.

[0021] Preferably, the Pinan cattle individuals with a deletion-type copy number variation type are superior to those with a duplication-type copy number variation type or a normal-type copy number variation type in terms of the growth trait height.

[0022] Preferably, the Pinan cattle individuals with the deletion-type copy number variation type are superior to the individuals with the duplication-type copy number variation type or the normal-type copy number variation type in the growth trait of body length.

[0023] Preferably, the Yunling cattle individuals with a repeat-type copy number variation type are superior to those with a deletion-type copy number variation type or a normal-type copy number variation type in the growth trait hip circumference.

[0024] The third aspect of the present invention discloses a real-time fluorescence quantitative PCR kit for detecting copy number variation of the bovine GYPB gene, which includes a primer pair for amplifying the copy number variation region of the bovine GYPB gene; the copy number variation of the GYPB gene is located at Chr17: 14,389,601bp to 14,391,200bp, a total of 1,600bp.

[0025] Preferably, the primer pair Primer1 for amplifying the copy number variation region of the GYPB gene is:

[0026] Upstream primer F1: 5′-ACAGAGTGTGAAAGACCTTGG-3′

[0027] Downstream primer R1: 5′-GCTCCTTCTTTGGATGGGAA-3′.

[0028] A fourth aspect of the present invention discloses the use of a real-time fluorescence quantitative PCR kit for detecting copy number variation of the cattle GYPB gene in molecular breeding of cattle.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The application of the GYPB gene provided by the present invention in cattle molecular breeding is to detect the copy number variation of the GYPB gene of yellow cattle, and to perform statistics on the types of different CNV types. Finally, the CNV types of the GYPB gene of different cattle breeds and their corresponding growth traits are analyzed for correlation. The results show that the repetitive copy number variation type has a significant advantage in Pinan cattle and Yunling cattle, and the copy number difference is relatively obvious. In Pinan cattle, the GYPB gene copy number variation is significantly correlated with the cross height, body height, and body oblique length; the individuals with the deletion copy number variation type are superior to the individuals with the normal and repetitive copy number variation types in terms of growth traits. In Yunling cattle, the GYPB gene copy number variation is extremely significantly correlated with hip circumference; the individuals with the repetitive copy number variation type are superior to the individuals with the deletion copy number variation type in terms of growth traits. Therefore, the CNV of the GYPB gene can be used as a molecular marker for early selection and breeding of Chinese cattle.

[0031] Furthermore, the copy number variation region of the GYPB gene was amplified by qPCR using genomic DNA of different cattle breeds as templates and the BTF3 gene as a control; -ΔΔCt Statistical methods are used to analyze copy number and determine the copy number variation type of an individual. This method is convenient and rapid to operate, and can accurately identify the copy number type of an individual, allowing for rapid promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is an amplification curve diagram drawn by qPCR (GYPB gene) in an embodiment of the present invention;

[0033] Figure 2 This is a melting curve diagram drawn by qPCR (GYPB gene) in an embodiment of the present invention;

[0034] Figure 3 This is a distribution diagram of the GYPB gene copy number variation detected in the examples of the present invention in the cattle population. DETAILED DESCRIPTION

[0035] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0036] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0037] In this article, 2 -△△Ct The gene expression abundance was indexed (Log22 -△△Ct ), and variance homogeneity test was performed to statistically analyze the differences among the groups.

[0038] In the cattle genome resequencing study, the CNV region of the GYPB gene in the Chinese cattle genome was detected: Chr17: 14,389,601bp~14,391,200bp, and Primer v5.0 software was used to design a CNV in this region. Qinchuan cattle (QC), Pinan cattle (PN), Xianan cattle (XN), Yunling cattle (YL), Guyuan cattle (GY) and Jiaxian red cattle (JX) were used as experimental subjects. Their genomic DNA was used as template and BTF3 was used as the internal reference gene. The GYPB gene copy number variation was amplified by qPCR technology. 2*2 -ΔΔCt Methods The copy number variation type of the GYPB gene was determined. Based on the physiological role of the GYPB gene and the regulatory mechanism of CNV, the association between GYPB gene CNV and growth traits was studied, providing a scientific basis for the application of GYPB gene CNV as a molecular marker for assisted selection in molecular breeding of cattle.

[0039] Example 1

[0040] 1. Scalper sample collection

[0041] The collection method in the present invention is venous blood collection. The Qinchuan cattle collected are from the Qinchuan Beef Cattle Breeding Center in Shaanxi Province (collection time: January 2013), the Pinan cattle are from Xinye County, Nanyang City, Henan Province (collection time: January 2016), and the Yunling cattle are from the Demonstration Ranch of the Yunnan Grassland Animal Science Research Institute (collection time: September 2018). The Xianan cattle are from Xianan Cattle Technology Development Co., Ltd. in Biyang County, Zhumadian City, Henan Province (collection time: June 2015), the Jiaxian Red Cattle are from the Jiaxian Red Cattle Conservation Area in Pingdingshan City, Henan Province (collection time 2008), and the Guyuan Yellow Cattle are from Liming Village, Ying Town, Yuanzhou District, Guyuan (collection time: June 2019). Their growth trait data, such as body height, body length, chest circumference, rump length, ischium end width and cruciate height, are recorded for subsequent association analysis.

[0042] 2. DNA Extraction from Blood Samples

[0043] The phenol-chloroform method was used to extract DNA from blood samples. The specific steps are as follows:

[0044] (1) Thaw the frozen blood sample in a room temperature water bath;

[0045] (2) Transfer 2 mL of whole blood into a sterile 2 mL centrifuge tube;

[0046] (3) Centrifugation at 12,000 rpm for 10 min at 4°C;

[0047] (4) Discard the liquid, retain the precipitate, add 1.5 mL of phosphate buffered saline (PBS), vortex to suspend the precipitate, and gently shake on ice for 15 min;

[0048] (5) Centrifuge at 12000 rpm for 10 min at 4°C, discard the liquid, and retain the precipitate;

[0049] (6) Repeat steps (4) and (5) once;

[0050] (7) Use a blue gun tip to crush the precipitate into flocculent pieces (critical step, the more broken the better);

[0051] (8) Add 500 μL of DNA extraction solution and 6 μL of proteinase K to the centrifuge tube;

[0052] (9) Incubate in a constant temperature water bath at 37°C overnight (about 16 hours) until the cell pellet is completely digested and the solution is clear;

[0053] (10) Add 1 mL of Tris-saturated phenol and place on ice with gentle shaking for 20 min;

[0054] (11) Centrifuge at 12,000 rpm for 10 min at 4°C;

[0055] (12) Use a pipette to transfer the upper aqueous phase to another 2.0 mL sterile centrifuge tube;

[0056] (13) Add 0.5 mL of saturated phenol and 0.5 mL of chloroform and place on ice with gentle shaking for 20 min;

[0057] (14) Centrifuge at 12,000 rpm for 10 min at 4°C;

[0058] (15) Use a pipette to transfer the upper aqueous phase to another 2.0 mL sterile centrifuge tube;

[0059] (16) Add 1 mL of chloroform and place on ice with gentle shaking for 20 min;

[0060] (17) Centrifuge at 12,000 rpm for 10 min at 4°C;

[0061] (18) Use a pipette to transfer the upper aqueous phase to a 1.5 mL centrifuge tube;

[0062] (19) Add 1 mL of pre-cooled anhydrous ethanol (-20°C), shake gently several times until the DNA precipitates, and then place at -20°C for 30 min;

[0063] (20) Centrifuge at 12000 rpm for 10 min at 4°C and discard the ethanol;

[0064] (21) Add 1 mL of 70% ethanol and shake gently for 10 min;

[0065] (22) Centrifuge at 12,000 rpm for 10 min at 4°C, discard the ethanol, and repeat the rinse (use a pipette to remove the remaining alcohol at the bottom of the tube);

[0066] (23) Place at room temperature for 30 min and then dry in a 60°C oven for 30 s to evaporate the ethanol.

[0067] (24) Add 50 μL of ultrapure water and store at 4°C until the DNA is completely dissolved. After measuring the concentration using a spectrophotometer, store at -80°C.

[0068] 3. Primer Design

[0069] The CNV region of the GYPB gene in the Chinese cattle genome was detected to be located at Chr17: 14,389,601 bp to 14,391,93 bp. Primers were designed for this region using Primer v5.0 software (PREMIER Bio Soft International, California, UFA). In primer pair Primer1, the sequence of upstream primer F1 is shown as SEQ ID NO. 1 in Table 1, and the sequence of downstream primer R1 is shown as SEQ ID NO. 2 in Table 1. Simultaneously, primers were designed to amplify a specific fragment (166 bp) of the BTF3 gene (internal reference gene) using the bovine BTF3 gene (AC000177.1) published by NCBI as a reference sequence. In primer pair Primer2, the sequence of upstream primer F2 is shown as SEQ ID NO. 3 in Table 1, and the sequence of downstream primer R2 is shown as SEQ ID NO. 4 in Table 1.

[0070] Table 1 Sequence Listing

[0071]

[0072]

[0073] The primers were determined to be suitable for real-time fluorescence quantitative PCR analysis by drawing amplification curves and melting peaks. A smooth amplification curve indicated that the qPCR reagents were of good quality and the amplification system and conditions were appropriate ( Figure 1 ); The melting curves of the samples were drawn in a consistent manner, with smooth curves, high and sharp peaks, and no miscellaneous peaks caused by primer dimers or non-specific amplification, indicating that the primers were of good quality ( Figure 2 In summary, the GYPB primer pair and the BTF3 primer pair have good specificity and can be used in subsequent experiments.

[0074] 4. Real-time Quantitative PCR

[0075] The qPCR reaction system is shown in Table 2.

[0076] Table 2 qPCR reaction system

[0077]

[0078] The qPCR reaction procedure is:

[0079] (1) Pre-deformation at 95°C for 1 min;

[0080] (2) Denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 40 cycles.

[0081] 5. Analysis of GYPB gene CNV types in cattle

[0082] The cycle threshold (Ct) is used as a parameter to draw a standard curve to detect the initial copy number of the template in the sample to be tested. Each sample is amplified with primers (Primer1 and Primer2) of the target sequence and the internal reference sequence, and three parallel replicates are set up at the same time. -ΔΔCt The copy number was analyzed by statistical methods, where ΔCt = Ct target gene - Ct internal reference gene.

[0083] According to the judgment criteria (as follows), the copy number variation types of the GYPB gene are divided into three categories: deletion type, normal type and duplication type.

[0084] Judgment criteria: when -ΔΔCt<-0.5, it is a loss type (Loss); when -0.5≤-ΔΔCt≤0.5, it is a normal type (Median); -ΔΔCt>0.5, it is a duplication type (Gain).

[0085] Test results such as Figure 3 As shown, copy number variation in Pinan cattle (PN), Xianan cattle (XN), Yunling cattle (YL), Guyuan yellow cattle (GY), and Jiaxian red cattle (JX) is concentrated in deletion types, while Qinchuan cattle (QC) have a relatively high number of duplication types. The CNV distribution of PN, YL, XN, GY, and JX is relatively concentrated, while that of QC is relatively dispersed. Furthermore, PN and GY have the most duplication types, each far exceeding half of the population. XN, YL, and JX have relatively more duplication types, slightly exceeding half of the population. The CNV distribution of PN, YL, GY, and JX is similar: duplication types > normal types > deletion types. The GY population has no deletion CNVs. As summarized, duplication types account for a relatively high proportion of CNVs in the population.

[0086] 5. Data Processing

[0087] The number of individuals with various copy number variation types in different cattle breeds was counted.

[0088] SPSS software was used to conduct a univariate analysis of the correlation between the GYPB gene copy number variation type and growth traits. Based on the different factors affecting body shape traits, age and genetic effects were considered, and a fixed model was used for analysis. The simplified model was then simplified according to the actual situation.

[0089] Yijk=μ+Ai+Gj+Eijk

[0090] where Yijk is the phenotypic record of individual growth traits; μ is the population mean; Ai is the age effect; Gj is the copy number effect of each sample; and Eijk is the random error.

[0091] The results of data processing are shown in Table 3.

[0092] Table 3. Association analysis between GYPB gene copy number variation and growth traits in cattle

[0093]

[0094]

[0095] Note: Mean values ​​with the same letters on their shoulders indicate no significant difference (P>0.05); mean values ​​with different letters on their shoulders indicate significant difference (P<0.05), *P<0.05.

[0096] Results indicate that CNVs in the GYPB gene do affect growth traits in Chinese Yellow cattle. Repeated copy number variants significantly predominated in Pinan and Yunling cattle, with significant differences in copy number. Furthermore, in Pinan cattle, GYPB gene copy number variants were significantly associated with cross height, body height, and body length; individuals with deletion-type copy number variants exhibited superior growth traits compared to those with normal and repeat-type copy number variants. In Yunling cattle, GYPB gene copy number variants were highly significantly associated with hip circumference; individuals with duplication-type copy number variants exhibited superior growth traits compared to those with deletion-type and normal-type copy number variants. The GYPB locus was not significantly associated with growth traits in Xianan cattle, but the deletion-type variant exhibited an advantage in body height and weight, making it a candidate molecular marker for marker-assisted selection in Xianan cattle. Therefore, CNVs in the GYPB gene could serve as a molecular marker for early breeding of Chinese Yellow cattle.

[0097] Example 2

[0098] Real-time fluorescence quantitative PCR kit for detecting bovine GYPB gene copy number variation, including:

[0099] Primer pair Primer 1 for amplifying the copy number variation region of the bovine GYPB gene, the primer pair Primer 1 comprising an upstream primer F1 (sequence shown as SEQ ID NO. 1 in Table 1) and a downstream primer R1 (sequence shown as SEQ ID NO. 2 in Table 1);

[0100] Primer pair Primer2 for amplifying a partial fragment of the cattle BTF3 gene as an internal reference, wherein the primer pair Primer2 includes an upstream primer F2 sequence as shown in SEQ ID NO.3 and a downstream primer R2 sequence as shown in SEQ ID NO.4;

[0101] Other reagents include dNTPs, PCR reaction buffer, and DNA polymerase.

[0102] The kit can be used for molecular breeding of cattle.

[0103] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of GYPB gene in cattle molecular breeding.

2. Application of the method for detecting copy number variation of the cattle GYPB gene in molecular breeding of cattle.

3. The use according to claim 2, characterized in that Using cattle gene DNA as template and primers designed, the copy number variation region of the GYPB gene and a partial fragment of the BTF3 gene as a control were amplified by real-time quantitative PCR, and the copy number variation type of the cattle GYPB gene was identified based on the quantitative results.

4. The use according to claim 3, characterized in that The copy number variation of the GYPB gene is located at Chr17: 14,389,601bp~14,391,200bp, a total of 1,600bp.

5. The use according to claim 3, characterized in that The primer pair Primer1 for amplifying the copy number variation region of the GYPB gene is: Upstream primer F1: 5′-ACAGAGTGTGAAAGACCTTGG-3′ Downstream primer R1: 5'-GCTCCTTCTTTGGATGGGAA-3' The primer pair Primer2 for the copy number variation region of the BTF3 gene used as a control is: Upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3' Downstream primer R2: 5′-TTCGGTGAAATGCCTCTCG-3′.

6. The use according to claim 3, characterized in that The copy number variation types of the GYPB gene are divided into three categories according to -ΔΔCt: deletion type, i.e., -ΔΔCt<-0.5; normal type, i.e., -0.5≤-ΔΔCt≤0.5; and duplication type, i.e., -ΔΔCt>0.

5.

7. The use according to claim 3, characterized in that Pinan cattle individuals with deletion-type copy number variation have better growth traits than those with duplication-type copy number variation or normal-type copy number variation; Yunling cattle individuals with duplication-type copy number variation have better growth traits than those with deletion-type copy number variation or normal-type copy number variation.

8. A real-time fluorescence quantitative PCR kit for detecting bovine GYPB gene copy number variation, characterized in that: The kit comprises a primer pair for amplifying a copy number variation region of a bovine GYPB gene; the copy number variation of the GYPB gene is located at Chr17: 14,389,601 bp to 14,391,200 bp, a total of 1,600 bp.

9. A real-time fluorescence quantitative PCR kit for detecting bovine GYPB gene copy number variation according to claim 8, characterized in that: The amplification primer pair Primer1 for the copy number variation region of the GYPB gene is: Upstream primer F1: 5′-ACAGAGTGTGAAAGACCTTGG-3′ Downstream primer R1: 5′-GCTCCTTCTTTGGATGGGAA-3′.

10. Use of the real-time fluorescence quantitative PCR kit for detecting copy number variation of the cattle GYPB gene according to claim 8 or 9 in molecular breeding of cattle.

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