Kit for predicting development state of bovine embryos and CNVR combination used by kit

Abstract

The invention discloses a kit for predicting the development state of a bovine embryo and a CNVR combination used by the kit. The kit comprises a product for detecting the expression quantity and / or copy number of the CNVR combination in the bovine embryo genome; the CNVR combination comprises CNVR-Vst5, CNVR-Vst11, CNVR-Vst7 and / or CNVR-Vst4, and the CNVR-Vst5, the CNVR-Vst11, the CNVR-Vst7 and the CNVR-Vst4 If the expression quantities and / or copy numbers of CNVR-Vst5, CNVR-Vst11, CNVR-Vst7 and / or CNVR-Vst4 in a bovine embryo genome subjected to in-vitro fertilization culture are obviously different from those of a normal bovine embryo, the development of the bovine embryo is predicted to be stagnated, and embryo implantation is not suggested. The method can be used for scientifically judging the transplantation strategy and decision of the bovine embryo, and has important application value.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetic biology, and in particular to a kit for predicting the developmental state of bovine embryos and a CNVR combination used therein. Background Art

[0002] Livestock breeding technologies such as in vitro production (IVP) have been widely adopted in animal husbandry. They are a key means of expanding high-quality breed resources, further shortening generation intervals, increasing selection intensity, and accelerating population genetic progress. Currently, in vitro fertilization (IVP) in cattle is gradually increasing and has become an industry trend. Embryonic development is also affected by various factors, such as genetic mutations and chromosomal abnormalities in the embryo itself, which can lead to failure to successfully transfer the embryo or to a successful pregnancy after transfer. Chromosomal abnormalities can occur, for example, during meiosis in the oocyte or during mitosis during preimplantation embryonic development. Studies have found that 25-40% of bovine embryos produced by IVP contain chromosomal abnormalities, which can lead to early embryonic development failure, pregnancy failure, or the birth of infertile animals, a significant loss for the dairy industry. Therefore, in-depth research is needed to identify the factors that cause embryonic developmental arrest and key genomic structural variants that can determine embryonic developmental status. Ultimately, by detecting relevant genetic variants or molecular markers, the rate of embryonic developmental arrest can be effectively reduced, pregnancy rates after transfer can be improved, and the efficiency of IVF production can be further enhanced.

[0003] Common structural variations in the genome can be divided into three categories based on their size: single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs) ranging from 2 to 50 bp, and copy number variations (CNVs) larger than 50 bp. CNVs are typically caused by genomic rearrangements and generally refer to multi-site variations such as deletions, duplications, and insertions ranging from 50 bp to several megabases compared to a reference genome. CNVs are widely distributed across the genome, and common methods for detecting CNVs include microarrays and sequencing. With the rapid development and rapid reduction in cost of high-throughput sequencing technology, whole-genome sequencing has become the preferred method for detecting CNVs. Studies in humans have already used CNV detection to diagnose the health of early embryos. Therefore, identifying key CNVs in bovine early embryonic development is crucial for screening and diagnosis of early embryos. This not only improves pregnancy rates after embryo transfer and reduces economic losses, but also provides a key theoretical basis for efficient bovine in vitro embryo production and embryo breeding. Summary of the Invention

[0004] The purpose of the present invention is to predict the developmental state of bovine embryos cultured by in vitro fertilization, and then to guide the implantation of bovine embryos, that is, to diagnose the bovine embryos before implantation.

[0005] The present invention first protects a kit for predicting the developmental status of bovine embryos and / or preimplantation diagnosis of bovine embryos, which may include a product for detecting the expression level and / or copy number of a CNVR combination in the genome of a bovine embryo; the CNVR combination may include CNVR-Vst5, CNVR-Vst11, CNVR-Vst7 and / or CNVR-Vst4;

[0006] The nucleotide sequence of CNVR-Vst5 can be as shown in the nucleotide sequence of chromosome 5 on the bovine genome.

[0007] 60638401-60642400 as shown;

[0008] The nucleotide sequence of CNVR-Vst11 can be as shown in the nucleotide sequence of chromosome 11 on the bovine genome.

[0009] 71203201-71205200 are shown;

[0010] The nucleotide sequence of CNVR-Vst7 can be as shown in the nucleotide sequence of chromosome 7 on the bovine genome.

[0011] 18346401-18350000 is shown;

[0012] The nucleotide sequence of CNVR-Vst4 can be as shown in the nucleotide sequence of chromosome 4 on the bovine genome.

[0013] 44117201-44118800 are shown.

[0014] In the above kit, the expression level and / or copy number of the CNVR combination can be the expression level and / or copy number obtained by detecting bovine embryo genomic DNA using real-time fluorescence quantitative PCR.

[0015] In the above-mentioned kit, the product for detecting the expression level and / or copy number of the CNVR combination in the bovine embryo genome may include a primer pair Vst5 for detecting CNVR-Vst5, a primer pair Vst11 for detecting CNVR-Vst11, a primer pair Vst7 for detecting CNVR-Vst7 and / or a primer pair Vst4 for detecting CNVR-Vst4.

[0016] The primer pair Vst5 can specifically consist of a primer CNVR-Vst 5F having a nucleotide sequence as shown in SEQ ID NO: 3 and a primer CNVR-Vst 5R having a nucleotide sequence as shown in SEQ ID NO: 4.

[0017] The primer pair Vst11 may specifically consist of a primer CNVR-Vst 11F having a nucleotide sequence as shown in SEQ ID NO: 5 and a primer CNVR-Vst 11R having a nucleotide sequence as shown in SEQ ID NO: 6.

[0018] The primer pair Vst7 can specifically consist of a primer CNVR-Vst 7F having a nucleotide sequence as shown in SEQ ID NO:7 and a primer CNVR-Vst 7R having a nucleotide sequence as shown in SEQ ID NO:8.

[0019] The primer pair Vst4 may specifically consist of a primer CNVR-Vst 4F having a nucleotide sequence as shown in SEQ ID NO: 1 and a primer CNVR-Vst 4R having a nucleotide sequence as shown in SEQ ID NO: 2.

[0020] Any of the above-mentioned products for detecting the expression level and / or copy number of the CNVR combination in the bovine embryo genome can specifically be composed of a primer pair Vst5 for detecting CNVR-Vst5, a primer pair Vst11 for detecting CNVR-Vst11, a primer pair Vst7 for detecting CNVR-Vst7 and / or a primer pair Vst4 for detecting CNVR-Vst4.

[0021] In the above kit, the expression level and / or copy number of the CNVR combination may be the expression level and / or copy number of the CNVR combination relative to the internal reference gene.

[0022] The internal reference gene may be the BTF3 gene.

[0023] Any of the above-mentioned products for detecting the expression level and / or copy number of a CNVR combination in a bovine embryo genome may further include a primer pair for detecting an internal reference gene.

[0024] Any of the aforementioned primer pairs for detecting an internal reference gene may be a primer pair BTF3 for detecting a BTF3 gene. Specifically, the primer pair BTF3 may be composed of a primer BTF3F having a nucleotide sequence as shown in SEQ ID NO: 9 and a primer BTF3R having a nucleotide sequence as shown in SEQ ID NO: 2.

[0025] In the above kit, the bovine embryo may be a bovine embryo cultured by in vitro fertilization.

[0026] In the above kit, the bovine embryo may be an embryo that has developed to the blastocyst or expanded blastocyst stage.

[0027] The above-mentioned kit can specifically be composed of any of the above-mentioned products for detecting the expression level and / or copy number of the CNVR combination in the bovine embryo genome.

[0028] Any of the above-mentioned kits may also include a carrier having the following contents recorded thereon: using a bovine embryo that has developed to the blastocyst or expanded blastocyst stage as a sample to be tested, and using a normal bovine embryo that has developed to the blastocyst or expanded blastocyst stage as a reference sample, detecting the expression level and / or copy number of the CNVR combination in the sample to be tested and the reference sample; and then predicting the embryonic developmental status of the sample to be tested or performing pre-implantation diagnosis based on the difference in the expression level and / or copy number of the CNVR combination in the sample to be tested and the reference sample. Specifically, if the expression level and / or copy number of the CNVR combination in the sample to be tested is significantly higher than that in the reference sample, it indicates that a large number of CNV fragments in the corresponding CNVR region in the CNVR combination are repeated, which is not conducive to embryonic health, and the sample to be tested is prone to developmental arrest or has a high risk of developmental arrest.

[0029] The above-mentioned kit can specifically be composed of any of the above-mentioned products for detecting the expression level and / or copy number of the CNVR combination in the bovine embryo genome and any of the above-mentioned vectors.

[0030] The use of any of the above-mentioned products for detecting the expression level and / or copy number of CNVR combinations in the bovine embryo genome in predicting the developmental status of bovine embryos and / or pre-implantation diagnosis of bovine embryos also falls within the scope of protection of the present invention.

[0031] The use of any of the above-mentioned products for detecting the expression level and / or copy number of CNVR combinations in the bovine embryo genome and any of the above-mentioned vectors in predicting the developmental status of bovine embryos and / or pre-implantation diagnosis of bovine embryos also falls within the scope of protection of the present invention.

[0032] In the above application, the bovine embryo may be a bovine embryo cultured by in vitro fertilization.

[0033] In the above application, the bovine embryo may be an embryo that has developed to the blastocyst or expanded blastocyst stage.

[0034] Any of the above-mentioned cattle can specifically be Chinese Holstein cows.

[0035] Any of the above developmental states may be normal development or developmental arrest.

[0036] The present invention clarifies the effects of in vitro fertilization on bovine embryo development and discovers differences in the expression levels and / or copy numbers of four CNVRs (i.e., CNVR-Vst5, CNVR-Vst11, CNVR-Vst7, and / or CNVR-Vst4). If the expression levels and / or copy numbers of CNVR-Vst5, CNVR-Vst11, CNVR-Vst7, and / or CNVR-Vst4 in the genome of a bovine embryo cultured through in vitro fertilization and developing to the blastocyst or expanded blastocyst stage do not differ significantly from those of a normal bovine embryo that has developed to the blastocyst or expanded blastocyst stage, the bovine embryo can develop normally and embryo transfer can be performed. If the expression level and / or copy number of CNVR-Vst5, CNVR-Vst11, CNVR-Vst7, and / or CNVR-Vst4 in the genome of a bovine embryo cultured through in vitro fertilization and developing to the blastocyst or expanded blastocyst stage is significantly different from that of a normal bovine embryo developing to the blastocyst or expanded blastocyst stage (e.g., the expression level and / or copy number in the former is significantly increased), then the bovine embryo is developmentally arrested and embryo implantation is not recommended. This invention can scientifically determine the transplantation strategy and decision-making for bovine embryos and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the number of CNVR intersections between the embryonic groups of the normal development group and the developmental arrest group.

[0038] Figure 2 Distribution of all CNVR populations subjected to selection analysis (Vst).

[0039] Figure 3 These are the specific detection results of the detection primers for the four CNVRs and the internal reference gene in Example 2.

[0040] Figure 4 This is the detection of differential CNVR copy numbers in embryos at different developmental states in Example 2. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0042] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0043] The CNV region (CNVR) in the following embodiments refers to the CNVs detected by different individuals in the detection group. If there are overlapping regions, the CNNs are integrated and merged into one CNVR.

[0044] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0045] Example 1. Copy Number Variation Detection and Identification of Different CNVRs in Embryos at Different Developmental States

[0046] 1. Acquisition and grouping of bovine embryo samples

[0047] The bovine embryo samples used in this example were all Chinese Holstein cow embryos produced by in vitro fertilization and developed to the 6-7 day blastocyst or expanded blastocyst stage after in vitro culture. They were provided by Shandong Aux Animal Husbandry and Seed Co., Ltd. The names of the 46 bovine embryo samples are shown in column 1 of Table 2. Based on the embryonic developmental status, the 46 embryos were divided into a normal development group (hereinafter referred to as the normal group) and a developmental arrest group (hereinafter referred to as the arrest group), with 23 embryos in each group.

[0048] 2. Acquisition and Amplification of Genomic DNA from Bovine Embryo Samples

[0049] Using REPLI- Single Cell Kit (Qiagen) was used to amplify genomic DNA from bovine embryo samples. The details are as follows:

[0050] 1. Add 500 μL of HO (sc) to one tube of Buffer DLB, mix thoroughly to dissolve, and centrifuge briefly to obtain Buffer DLB. Next, mix 3 μL of DTT (1 M) and 33 μL of Buffer DLB, vortex thoroughly, and centrifuge to obtain 36 μL of Buffer D2. 36 μL of Buffer D2 is sufficient for 12 reactions.

[0051] A total of 46 reaction volumes were prepared.

[0052] 2. Take a sterile centrifuge tube (200 μL) containing the bovine embryo sample and add enough PBS sc 1× to bring the final volume to 4 μL. Then add 3 μL of Buffer D2, flick the tube gently, and centrifuge briefly to mix.

[0053] 3. After completing step 2, perform PCR amplification: set the lid temperature to 70°C, incubate at 65°C for 10 minutes, and maintain at 4°C.

[0054] 4. After completing step 3, remove the PCR tube and temporarily place it on ice. Add 3 mL of Stop Solution, flick the tube to mix, centrifuge briefly, and place on ice to obtain the PCR sample.

[0055] 5. Prepare the master mix according to Table 1 in an ice bath. Centrifuge briefly after preparation.

[0056] Table 1. Master mix

[0057] Element Single reaction volume <![CDATA[H2Osc]]> 9μL REPLI-gscReactionBuffer 29μL REPLI-gscDNA Polymerase 2μL Total volume 40 μL

[0058] Note: The data listed in Table 1 is for a single reaction system. The actual preparation should be scaled up to 46 reactions. Add the ingredients in the order listed in the table.

[0059] 6. After completing step 4, add 40 μL of the master mix prepared in step 5 to each tube of PCR sample, bringing the total volume to 50 μL. Mix thoroughly and centrifuge. Then perform PCR amplification: set the lid temperature to 70°C and incubate at 30°C for 8 hours to amplify; inactivate REPLI-g sc DNA Polymerase at 65°C for 3 minutes; and maintain at 4°C.

[0060] 7. After completing step 6, remove the PCR tube, shake to mix, and centrifuge briefly. Place the amplified DNA in a 4°C refrigerator for short-term use or in a -20°C refrigerator for long-term storage.

[0061] All amplified bovine embryo genomic DNA samples were subjected to agarose gel electrophoresis and concentration determination.

[0062] The concentration, volume, and total amount of genomic DNA from all amplified bovine embryo samples are shown in columns 2-4 of Table 2, respectively.

[0063] Table 2

[0064]

[0065]

[0066]

[0067] 3. Copy Number Variation Detection and Identification of Different CNVs in Embryos at Different Developmental Stages

[0068] 1. Library construction and sequencing

[0069] All amplified bovine embryo genomic DNA samples were subjected to Illumina small fragment library construction (library type: WGS) and high-depth sequencing of 60G / sample (see columns 5-6 in Table 2 for details) to obtain whole-genome resequencing data of bovine embryo samples.

[0070] 2. Genome resequencing data analysis

[0071] The whole genome resequencing data of all bovine embryo samples were analyzed. The specific steps are as follows:

[0072] (1) Sequencing data quality control.

[0073] Fastp software was used to perform quality control on the raw sequencing sequences (Raw Reads) converted from the raw image data files obtained by sequencing through base calling analysis: specifically, read pairs with adapters were removed; paired reads in which the N content in the single-end sequencing reads exceeded 10% of the read length ratio, and reads containing low-quality (Q≤5) bases exceeding 50% of the read length ratio were removed to obtain clean reads.

[0074] (2) Use BWA software to align the clean reads after quality control to the bovine reference genome (ARS-UCD 1.3) to obtain a sam file. Use the view command of Samtools software to convert the sam format file to bam format, and then use the sort command to sort the bam file.

[0075] (3) Alignment quality assessment: The flagstat module of Samtools (1.9) software was used to calculate the alignment rate of paired-end reads to the reference genome to evaluate the quality of sequencing data and alignment efficiency.

[0076] (4) Copy number variation detection. CNVcaller software was used to detect and genotype CNVs for each sample, and only two types, Deletion and Duplication, were detected. The main steps were as follows: first, the reference genome was divided into 800bp windows to construct a reference genome database; the absolute copy number of each window was calculated based on the bam file in step (2) (Individual.Process.sh). After the operation was completed, three default folders (RD_raw, RD_absolute, and RD_normalized) were generated, which respectively contained the raw read segment counts of all windows in the whole genome of each sample, the read segment counts after merging through the link file, and the absolute copy number of each sample after GC sequencing skew correction and normalization; finally, the copy number variation region was determined, and the sample files in the RD_normalized folder were merged using CNV.Discovery.sh. The boundaries of the CNVR (Copy Number Variation Region) were preliminarily determined by comprehensively considering the distribution of absolute copy number, the frequency of variation, and the significant correlation between adjacent windows, and the primary CNVR was generated. Finally, adjacent CNVRs with significantly correlated copy number distributions within the population were merged to obtain the final merged CNVR, which determined the copy number region for each sample. Finally, genotype determination was performed using Genotype.py to generate genotypeCNVR.vcf.

[0077] 3. Analysis of selection on CNVR among populations

[0078] Across the genome, 234,579 CNVRs were detected in the embryos of the normal development group, and 99,891 CNVRs were detected in the embryos of the developmental arrest group, of which 517 CNVRs were shared by the two groups. Figure 1 (CW is the normal development group, SW is the developmental arrest group). st ) to screen for differential CNVR between the two groups. st It is a statistical indicator used to quantify the degree of differentiation of copy number variation between populations. By comparing the variance of copy number variation within and between populations, it can identify CNV regions that are significantly differentiated under the influence of natural selection or genetic drift. st The value of is between 0 and 1. The larger the value, the greater the difference in copy number variation in the region between populations, and vice versa. The calculation formula is:

[0079]

[0080] Where V1 refers to the variance of the copy number of group 1; N1 refers to the number of individuals in group 1; V2 refers to the variance of the copy number of group 2; N2 refers to the number of individuals in group 2; V total is the variance of the copy number of all individuals; N total Refers to the total number of individuals.

[0081] The experimental group was divided into two groups: normal development group and developmental arrest group. The absolute copy number of CNVR was obtained by CNVcaller software, and the V difference between the two groups was calculated. st Value, all V on autosomes 1 to 29 and chromosome X st The distribution of CNVR values ​​between 0 and 1 is shown in Figure 2 , where V st There were 29 CNVRs with values ​​greater than 0.25, indicating that these CNVRs were significantly differentiated between the two groups.

[0082] Keep V st CNVRs with values ​​greater than 0.3 were considered as differential CNVRs between the normal development group and the developmental arrest group to be verified. The copy number changes between the normal development group and the developmental arrest group are shown in Table 3.

[0083] Table 3. Differences in CNVR between the normal development group and the developmental arrest group

[0084]

[0085] Note: In the CNVR position, the number before the colon is the chromosome on which it is located, and the number after the colon is the physical position of the CNVR on the chromosome.

[0086] It can be seen from this that the large-scale duplication of CNV fragments in the CNVR-Vst4, CNVR-Vst5, CNVR-Vst7 and CNVR-Vst11 regions may be detrimental to the healthy development of the embryo and are potential sites that cause embryonic developmental arrest.

[0087] Example 2: Quantitative PCR detection of CNVR differences in embryos at different developmental states

[0088] 1. Recollect 6 samples of normal and developmentally arrested bovine embryos for repeated validation. The bovine embryo samples used in this example were all Chinese Holstein cow embryos produced by in vitro fertilization, cultured in vitro to the 6-7 day blastocyst or expanded blastocyst stage, and provided by Shandong Aokes Animal Husbandry and Seed Co., Ltd.

[0089] Six bovine embryo samples with normal development were used as the normal embryo development group.

[0090] Six developmentally arrested bovine embryo samples were used as the embryo developmentally arrested group.

[0091] 2. Amplify the genomic DNA of the bovine embryo samples collected in step 1 according to the method of step 2 in Example 1, and dilute the amplified genomic DNA of all bovine embryo samples to the same concentration.

[0092] 3. According to TB Premix Ex Taq TM According to the instructions of the II (Tli RNaseH Plus) (RR820, Takara) kit, real-time fluorescence quantitative PCR was used to detect the relative expression levels of CNVR-Vst4, CNVR-Vst5, CNVR-Vst11, and CNVR-Vst7 in the genomic DNA of bovine embryo samples (with the bovine BTF3 gene as an internal reference), that is, the relative copy number.

[0093] The nucleotide sequences of the detection primers for each CNVR and internal reference gene are shown in Table 4. The primer specificity detection results are shown in Figure 3 ,The results showed that the primers had good specificity.

[0094] Table 4

[0095]

[0096] Some of the real-time fluorescence quantitative PCR test results are shown in Figure 4 The results showed that compared with the normal embryonic development group, the relative expression levels (i.e., copy numbers) of CNVR-Vst5, CNVR-Vst7, and CNVR-Vst11 in the embryonic genome of the developmental arrest group were significantly increased (P<0.05), indicating that there were a large number of duplications of CNV fragments in these regions, which was not conducive to the healthy development of the embryo and was a potential structural variation that led to embryonic developmental arrest.

[0097] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A kit comprising products for detecting the expression level and / or copy number of a CNVR combination in a bovine embryo genome; The CNVR combination includes CNVR-Vst5, CNVR-Vst11, CNVR-Vst7 and / or CNVR-Vst4; The nucleotide sequence of CNVR-Vst5 is as shown in the nucleotide sequence of chromosome 5 on the bovine genome. 60638401-60642400 as shown; The nucleotide sequence of CNVR-Vst11 is shown at positions 71203201-71205200 of chromosome 11 on the bovine genome; The nucleotide sequence of CNVR-Vst7 is as shown in the nucleotide sequence of chromosome 7 on the bovine genome. 18346401-18350000 is shown; The nucleotide sequence of CNVR-Vst4 is as shown in the nucleotide sequence of chromosome 4 on the bovine genome. 44117201-44118800 as shown; The kit is used for predicting the developmental status of bovine embryos and / or for pre-implantation diagnosis of bovine embryos.

2. The kit according to claim 1, wherein: The product for detecting the expression level and / or copy number of the CNVR combination in the bovine embryo genome includes a primer pair Vst5 for detecting CNVR-Vst5, a primer pair Vst11 for detecting CNVR-Vst11, a primer pair Vst7 for detecting CNVR-Vst7, and / or a primer pair Vst4 for detecting CNVR-Vst4; Primer pair Vst5 consists of primer CNVR-Vst 5F having a nucleotide sequence as shown in SEQ ID NO:3 and primer CNVR-Vst 5R having a nucleotide sequence as shown in SEQ ID NO:4; The primer pair Vst11 consists of a primer CNVR-Vst 11F having a nucleotide sequence as shown in SEQ ID NO:5 and a primer CNVR-Vst 11R having a nucleotide sequence as shown in SEQ ID NO:6; Primer pair Vst7 consists of primer CNVR-Vst 7F having a nucleotide sequence as shown in SEQ ID NO:7 and primer CNVR-Vst 7R having a nucleotide sequence as shown in SEQ ID NO:8; The primer pair Vst4 consists of a primer CNVR-Vst 4F having a nucleotide sequence as shown in SEQ ID NO: 1 and a primer CNVR-Vst 4R having a nucleotide sequence as shown in SEQ ID NO:

2.

3. The kit according to claim 1, wherein: The expression level and / or copy number of the CNVR combination is the expression level and / or copy number of the CNVR combination relative to the internal reference gene.

4. The kit according to claim 1, wherein: The bovine embryos are cultured by in vitro fertilization.

5. The kit according to claim 1 or 4, characterized in that: The bovine embryo is an embryo that has developed to the blastocyst or expanded blastocyst stage.

6. The kit according to any one of claims 1 to 5, characterized in that: The kit also includes a carrier recording the following content: using a bovine embryo that develops to the blastocyst or expanded blastocyst stage as a sample to be tested, and using a normal bovine embryo that develops to the blastocyst or expanded blastocyst stage as a reference sample, detecting the expression level and / or copy number of the CNVR combination in the sample to be tested and the reference sample; and then predicting the embryonic development status of the sample to be tested or performing pre-implantation diagnosis based on the difference in the expression level and / or copy number of the CNVR combination in the sample to be tested and the reference sample.

7. Use of the product for detecting the expression level and / or copy number of a CNVR combination in a bovine embryo genome according to any one of claims 1 to 5 in predicting the developmental status of bovine embryos and / or in bovine embryo preimplantation diagnosis.

8. Use of the product for detecting the expression level and / or copy number of CNVR combinations in the genome of bovine embryos according to any one of claims 1 to 5 and the vector according to claim 6 in predicting the developmental status of bovine embryos and / or preimplantation diagnosis of bovine embryos.

9. The use according to claim 7 or 8, characterized in that: The bovine embryos are cultured by in vitro fertilization.

10. The use according to claim 7 or 8, characterized in that: The bovine embryo is an embryo that has developed to the blastocyst or expanded blastocyst stage.

Images