Method for detecting hybrid embryo dysplasia and application thereof

Abstract

The invention relates to a method for detecting hybrid embryo dysplasia and application thereof, and the method comprises the following steps: obtaining a hybrid embryo, preparing a single-cell suspension, obtaining transcriptome data of a single cell through a single-cell multi-omics sequencing technology, distributing the obtained sequencing data to respective parent genomes to obtain allele specific data; and based on the allele specificity data, evaluating whether hybrid embryo development is abnormal or not by adopting an XCI activity scoring method or a dose compensation state method. In the pig industry, the development state of the hybrid embryos is detected in time, so that a subsequent parent hybridization scheme is guided, abortion caused by unknown reasons due to parent genome problems is reduced, and the economic benefits of the pig industry are improved.

Description

Technical Field

[0001] This invention relates to the field of animal breeding and genetics, specifically to a method for detecting developmental abnormalities in hybrid embryos and its application. Background Technology

[0002] In modern livestock breeding, hybridization is a key means of achieving genetic improvement. However, when there is significant genetic differentiation between parent breeds, the embryo failure rate can reach 20-30%, as seen in the hybridization of European and Asian pig breeds, which has resulted in considerable economic losses. Currently, embryo selection mainly relies on morphological assessment, but this method cannot detect genomic incompatibility at the molecular level, making a preimplantation molecular screening technique urgently needed.

[0003] At the molecular level, the key to mammalian embryonic development lies in proper X chromosome inactivation (XCI) and dose compensation mechanisms to balance gene expression on sex chromosomes and autosomes. XCI failure leads to severe dose imbalance and embryonic lethality, and dysregulation of dose compensation effect genes plays a crucial role in speciation and hybridization failure. However, current research lacks methods to quantify parental genome expression contributions at the single-cell level, identify dose compensation failure mechanisms, and link them to embryonic developmental defects. In practice, even after embryos selected through morphological evaluation are successfully transferred, there is still a 10-15% early miscarriage rate and delayed embryonic development, a significant proportion of which are miscarried embryos without obvious morphological abnormalities. Traditional miscarriage etiology analysis mainly relies on chromosome karyotype analysis, which cannot reveal molecular-level genomic incompatibility and epigenetic regulatory defects, leading to a large number of "unexplained" miscarriage cases. This not only causes economic losses and maternal burden but also fails to provide effective genetic guidance for subsequent mating. Therefore, there is an urgent need to establish a complete molecular testing system from pre-implantation screening to post-implantation etiological diagnosis.

[0004] In existing technologies, Chen et al. (2016) provided a methodological basis for classifying mouse embryonic stem cell (XCI) status using single-cell RNA sequencing, assessing XCI status by calculating allele-specific expression ratios. However, this method has several key drawbacks when applied to actual breeding: its research subjects are laboratory mouse hybrids with minimal genetic differentiation, which are not representative; the method lacks means to quantify genome-wide allele expression bias, making it unable to detect the dominant expression of one parent's genome; it does not include the assessment of evolutionary differentiation between parents, making it unable to predict the compatibility of high-risk hybrid combinations; most importantly, this method is essentially descriptive, providing no prediction of embryonic developmental outcomes, risk scores, or clinical decision thresholds, and it also fails to perform parent-specific dose-compensation failure analysis.

[0005] Therefore, there is an urgent need in the art for a comprehensive diagnostic method that can simultaneously quantify allelic expression bias between parental genomes, integrate evolutionary genomic differentiation metrics, separately analyze the dosage compensation status of the X chromosomes of parental genomic origin, and integrate these molecular parameters into a unified risk assessment system to provide a validated prediction threshold, thereby enabling rapid pre-implantation screening of high-risk hybrid embryos, significantly improving breeding efficiency, and reducing economic losses. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method for detecting abnormal development of hybrid embryos and its application.

[0007] The present invention provides a method for detecting abnormal development of hybrid embryos for non-disease diagnosis purposes, comprising the following steps:

[0008] Obtain a hybrid embryo, prepare a single-cell suspension, and obtain the transcriptome data of a single cell through single-cell multi-omics sequencing technology. The data contains allelic-specific information that can distinguish parental origins, and allocate the obtained sequencing data to their respective parental genomes to obtain allelic-specific data;

[0009] Based on the allelic-specific data, evaluate whether the development of the hybrid embryo is abnormal using the XCI activity score method or the dosage compensation status method. Among them,

[0010] The XCI activity score method is as follows:

[0011] For female hybrid embryos, separately count the gene expression levels of the X chromosomes of paternal and maternal origins in each cell, and calculate the X chromosome activity:

[0012] X chromosome activity = 1 + minimum value of gene expression levels of two X chromosomes / maximum value of gene expression levels of two X chromosomes; <00​​​​​​​​​​​​​​

[0018] The dose compensation state method is as follows:

[0019] For female or male hybrid embryos, the average gene expression ratio between the X chromosome and autosomes is calculated for each cell, i.e.:

[0020] X:A ratio = gene expression level on X chromosome / gene expression level on autosomes;

[0021] When the mean X:A ratio of all cells in an embryo is higher or lower than the mean of a normal embryo population, and the difference exceeds 10%, it indicates that dose compensation has failed, and the embryo is evaluated as "developmental abnormality".

[0022] Furthermore, the hybrid embryo is a hybrid embryo in which the X chromosome inactivation process has theoretically been completed.

[0023] Furthermore, when the hybrid embryo comes from a pig, the hybrid embryo is an embryo that is more than 27 days after fertilization.

[0024] Furthermore, when obtaining allele-specific data, the sequencing data is masked to eliminate alignment bias. The masking method involves replacing all differential sites in the parental genome with the ambiguous base 'N' in the reference genome, and then reconstructing the alignment index for the masked reference genome.

[0025] The method for detecting developmental abnormalities in hybrid embryos for non-disease diagnostic purposes provided by this invention is applied in guiding hybridization breeding. For hybrid embryos with developmental abnormalities, the dose ratio of the parent genome is analyzed to locate the source of dose imbalance and to screen for parents that do not produce developmental abnormalities for hybridization.

[0026] The method for analyzing the dose ratio of the parental genome is as follows:

[0027] Intragenomic dose ratio of parent 1 = gene expression level on the X chromosome of parent 1 / gene expression level on the autosome of parent 1;

[0028] Intragenomic dose ratio of parent 2 = gene expression level on the X chromosome of parent 2 / gene expression level on the autosome of parent 2;

[0029] Transgenomic dosage ratio 1 = gene expression level on the X chromosome of parent 1 / gene expression level on the autosome of parent 2;

[0030] Transgenomic dosage ratio 2 = gene expression level on the X chromosome of parent 2 / gene expression level on the autosome of parent 1;

[0031] When the intragenomic dose ratio of a parent is normal but the transgenomic dose ratio is abnormal, it is determined that there is a dose imbalance in the parent's genome, which will produce a hybrid embryo with abnormal development.

[0032] The method for detecting developmental abnormalities in hybrid embryos for non-disease diagnostic purposes provided by this invention can be applied to guide pig breeding.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. This invention elevates the assessment of hybrid embryonic development from morphological description to molecular detection, significantly increasing sensitivity and specificity compared to existing methods. For the first time, it reveals the complete molecular mechanism chain of hybridization incompatibility at the single-cell multi-omics level. Retrospective analysis of aborted tissue or developmentally abnormal embryos can clearly identify molecular defects such as allelic effect imbalance, XCI failure, or dose compensation abnormalities, transforming unexplained abortions into detectable mechanistic abnormalities. Based on accumulated data, a risk warning database is established, providing genetic guidance for subsequent breeding, avoiding the repeated use of high-risk parental combinations, and fundamentally reducing ineffective pregnancy cycles and feeding costs. In pig breeding, screening and excluding high-risk embryos can guide the selection of parents less prone to embryonic developmental abnormalities, significantly improving breeding efficiency.

[0035] 2. This invention employs unbiased reference genome preprocessing: To overcome the reference genome bias problem commonly encountered in hybridization analysis, this invention uses masking technology. By identifying all SNP sites with fixed differences between the parents and replacing these sites with ambiguous nucleotides (N) in the reference genome, it ensures that reads from both parents have a fair chance of alignment, reducing the alignment rate difference from over 5% to less than 0.5%, thus laying an unbiased foundation for subsequent accurate allele separation.

[0036] 3. This invention achieves, for the first time, the precise localization of genomic differences, enabling the diagnosis of dose balance abnormalities between parental genomes in hybrid embryos. By calculating the dose balance between different parents within the same individual: X chromosome and autosomes, X chromosomes from different parents, and autosomes from different parents, the dose balance status of the parental genomes is systematically assessed. These ratios include: intragenomic ratios (X chromosome of one parent / autosome of that parent) and transgenomic ratios (X chromosome of one parent / autosome of another parent). Abnormal transgenomic ratios combined with normal intragenomic ratios can clearly diagnose dose imbalances caused by insufficient expression of the weaker genome, thereby linking dose compensation failure with abnormal embryonic development.

[0037] 4. This invention constructs an XCI activity scoring method to evaluate the developmental status of hybrid embryos. Based on the overall expression level of X-linked genes, the X chromosome activity score is calculated, and cells are quantitatively classified into XCI initiation, in progress, or completed states. Cells that have failed XCI can be detected, which provides an important detection method for the study of abnormal embryonic development.

[0038] 5. The XCI activity scoring method or dose compensation status method of this invention can effectively evaluate whether the development of hybrid embryos is abnormal and locate the source of dose imbalance. In the modern large-scale breeding system of pigs, timely detection of the development status of hybrid embryos can guide the subsequent parent hybridization mating program, reduce unexplained abortions caused by parent genome problems, and improve the economic benefits of pig farming. Attached Figure Description

[0039] Figure 1 The flowchart illustrates the validation of the embryonic developmental abnormality detection method in a crossbred Eurasian domestic pig population, as shown in the example.

[0040] Figure 2 The XCI activity score of S7 in the example;

[0041] Figure 3 The peak verification result of ATAC for S8 in the embodiment;

[0042] Figure 4 This represents the percentage of reads expressed by heterozygotes in S8 of the embodiment;

[0043] Figure 5 This is the dose compensation anomaly detection result of S9 in the example;

[0044] Figure 6 The results of the parent-specific dose compensation effect detection for the abnormal embryos in S10 in the example are shown. Detailed Implementation

[0045] The present invention will be further described below with reference to the embodiments.

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Example: Validation of the embryonic developmental abnormality detection method in a Eurasian domestic pig hybrid population:

[0048] This embodiment uses hybrid embryos of European Large White pigs and Yunnan Small-eared pigs from China to verify the ability of the method of the present invention to detect developmental abnormalities in hybrid embryos between genetically differentiated parents. The verification process is as follows: Figure 1 As shown. The method includes the following steps:

[0049] S1. Establish reciprocal hybridization combinations and collect embryo samples:

[0050] Two domestic pig breeds with significant genetic differentiation were selected: the European Large White (LW, adult weight approximately 263 kg) and the Yunnan Small-eared Pig (SE, adult weight approximately 64 kg). Five Large White and five Yunnan Small-eared Pig parents were selected to establish reciprocal crosses, and embryos were collected on day 27 post-fertilization. In the LSC group, three mating combinations of LW boars and SE sows yielded seven embryos (four females, labeled FLS1, FLS2, FLS3, and FLS4; and three males, labeled MLS1, MLS2, and MLS3). In the SLC group, two mating combinations of SE boars and LW sows yielded six embryos (three females, labeled FSL1, FSL2, and FSL3; and three males, labeled MSL1, MSL2, and MSL3).

[0051] The reason for choosing to collect embryos on day 27 after fertilization is that at this time, the number of embryonic cells is moderate (100,000-150,000 cells), the X chromosome inactivation process has been basically completed in theory, and the main tissue types have differentiated, making it suitable for single-cell multi-omics analysis.

[0052] S2. Perform SHARE-seq single-cell multi-omics sequencing:

[0053] 2.1 Preparation of Single-Cell Suspension: Embryos were removed under aseptic conditions, and washed with PBS to remove blood and maternal tissue. A digestion solution containing 0.25% Trypsin-EDTA and Collagenase IV was used, and the cells were treated at 37°C for 15 minutes. The suspension was obtained by filtration through a 40μm cell sieve. Cell viability was assessed by trypan blue staining, requiring >85%. The cell concentration was adjusted to 1000 cells / μL. Single-cell suspensions were obtained from 13 embryos, with an average cell viability of 91.2% ± 3.8%.

[0054] 2.2 SHARE-seq Single-Cell Multi-Omic Sequencing: SHARE-seq is a single-cell multi-omics sequencing technology that can simultaneously acquire transcriptome and chromatin accessibility data for individual cells. The procedure first involves treating 50,000 cells with Tn5 transposase (37°C for 30 minutes) to insert sequencing adapters into open chromatin regions, thereby marking chromatin accessibility. Subsequently, mRNA is captured using oligo-dT primers with cell barcodes and reverse transcribed to synthesize cDNA, capturing RNA information. Next, ATAC libraries for chromatin accessibility and RNA libraries for transcriptome sequencing are constructed, followed by PCR amplification, purification, and quality testing. Finally, paired-end 150bp high-throughput sequencing is performed on the Illumina NovaSeq 6000 platform, with target sequencing depths of an average of 50,000 reads per cell for the RNA library and an average of 25,000 reads per cell for the ATAC library.

[0055] S3. Identification of differentially expressed alleles in the parental genome:

[0056] 3.1 For the five Large White pigs and five Yunnan Small Ear pigs used in this embodiment, the homozygous sites that differ between the parents were identified, which were used to separate the parental origins of the reads obtained by SHARE-seq.

[0057] 3.2 Sequence alignment and variant detection: BWA-MEM was used to align to the Duroc pig reference genome Sscrofa11.1, and GATK HaplotypeCaller was used to detect SNPs and InDels;

[0058] 3.3. Variance quality filtering: Set strict standards (sequencing depth DP≥10, genotype quality GQ≥30, quality value QD>2.0, etc.).

[0059] 3.4 Identification of Parental Genomic Differential Loci: SNPs with fixed differences between parents were screened, specifically loci where one parent showed a homozygous reference allele (0 / 0) and the other a homozygous alternative allele (1 / 1). An average of 16,619,053 differential loci were identified per parent pair, with an average density of 1 locus / 1.54 kb. These loci were evenly distributed across the entire genome, including the X chromosome, providing sufficient markers for allele splitting. This process generated haplotype counts for both the father and mother, enabling subsequent procedures to calculate gene expression and chromatin accessibility.

[0060] S4. Reference genome masking to eliminate alignment bias:

[0061] Since the reference genome Sscrofa11.1 is based on the Duroc breed, and Duroc pigs are more closely phylogenetically related to Large White pigs (LW) than to Yunnan Small-eared pigs (SE), direct alignment would result in higher alignment rates for LW-derived reads compared to SE-derived reads. Therefore, the sequencing data was masked to eliminate alignment bias.

[0062] The masking method involves replacing all differentially expressed sites in the parent genome with the ambiguous base N in the reference genome, and then reconstructing the SHARE-seq alignment index for the masked reference genome.

[0063] S5. Allele-specific splitting to obtain allele-specific data:

[0064] Sequencing reads are allocated according to parental origin: using differentially expressed sites in the parental genome, sequencing reads are split into their respective parental genomes; by matching the alleles covered in the reads, it is determined whether the reads originate from the LW or SE genome. This process generates read counts for LW and SE haplotypes respectively, which are used for subsequent calculations of gene expression levels and chromatin accessibility.

[0065] 5.1. Differential site annotation: For each aligned read, check all differential sites it covers;

[0066] 5.2 Allele determination: If the bases at the differential site of the read are identical to those of the LW allele (base quality Q≥30), it is marked as LW evidence; if it is identical to the SE allele, it is marked as SE evidence.

[0067] 5.3 Read source allocation rules: If all evidence consistently points to LW, the read is assigned to the LW allele; if all evidence consistently points to SE, the read is assigned to the SE allele; if there is conflicting evidence, the read is discarded; reads that do not cover any informative SNPs are marked as non-informative reads.

[0068] 5.4 Gene / Peak Level Counting:

[0069] RNA data: Gene expression levels from LW and SE source reads were counted using featureCounts based on Ensembl gene annotations, generating LW and SE expression matrices respectively;

[0070] ATAC data: Peaks were detected using MACS2, and bedtools was used to calculate the coverage of LW and SE source reads at each peak, generating LW accessibility matrices and SE accessibility matrices.

[0071] S6. Single-cell data quality control:

[0072] Use the R package Seurat to perform quality control on the data and screen out high-quality cells:

[0073] RNA data quality control criteria: The number of genes detected per cell is 200 - 6000, and the proportion of mitochondrial genes is less than 15%;

[0074] ATAC data quality control criteria: The number of ATAC peaks per cell is 500 - 20000, the TSS enrichment score is greater than 2.0, the nucleosome signal ratio is less than 2.0, and the fragment length distribution shows a nucleosome periodic pattern.

[0075] After quality control, 118015 high-quality single cells were screened out from 140803 original cells (pass rate 83.8%), with an average of 9078 cells per embryo. Validation showed that the ATAC signal was enriched 8 - 12 times around the transcription start site, confirming the reliability of the data quality.

[0076] S7. Use the XCI activity score method to evaluate whether the development of hybrid embryos is abnormal:

[0077] Perform XCI activity scoring based on the RNA data in the allele-specific data obtained in S5:

[0078] For each female hybrid embryo, count the gene expression levels of the X chromosomes from the LW source and the SE source for each cell separately, and calculate the X chromosome activity:

[0079]

[0080] A value of 1 indicates that one X chromosome is active (XCI completed), a value of 2 indicates that both X chromosomes are active (XCI not completed), and a value between 1 and 2 indicates that XCI is in progress.

[0081] Classify cells according to the X chromosome activity:

[0082] Uninitiated XCI: 1.8 < X chromosome activity ≤ 2.0, indicating that both X chromosomes are active; <o000178>Ongoing XCI: 1.2 < X chromosome activity ≤ 1.8, indicating that the inactivation of one X chromosome is in progress;

[0084] Completed XCI: 1.0 ≤ X chromosome activity ≤ 1.2, indicating that one X chromosome is active.

[0085] As Figure 2 shown, Figure 2 the abscissa represents different embryo individuals, each point represents a cell, and the ordinate is the XCI activity score. From Figure 2It is evident that approximately two-thirds of the cells in the FSL1 embryo showed either no initial XCI or ongoing XCI, and the cells that completed XCI lacked cells with inactivated X chromosomes that completed LW, indicating abnormal embryonic development.

[0086] 7.1 Male embryos: Male embryos express only one X chromosome (from the mother, LW, or SE). Figure 2 The XCI activity scores of male embryos showed that only one X chromosome was active, which verified the reliability of the dataset.

[0087] 7.2 Normal female embryos: Female embryos show only a small number of cells in the uninitiated and ongoing XCI stages, such as... Figure 2 Normal female embryos (except FSL1) show that only a small number of cells are not the cells that complete XCI;

[0088] 7.3 Abnormal Embryos: From Figure 2 One female embryo (FSL1) was found to have approximately two-thirds of its cells in the uninitiated and ongoing XCI states, and the embryo lacked cells that had completed LW with inactivated X chromosomes, indicating that it was an abnormal XCI embryo.

[0089] S8. Verify the accuracy of the abnormal embryos identified through the XCI viability score in S7:

[0090] 8.1 Validation through analysis of chromatin accessibility data: ATAC data from the allele-specific data obtained in S5 were analyzed. Cells that completed SE (X chromosome inactivation, as no cells completed LW X chromosome inactivation were found in abnormal embryos) and cells that did not initiate XCI were counted separately. The ATAC peaks of these cells were then used to assess chromosome accessibility. For example... Figure 3 As shown, the horizontal axis represents the 30-90 Mb range of the X chromosome, and the vertical axis represents the ATAC peak levels of the LW or SE genomes in different embryonic individuals. Inactivated X chromosomes should exhibit significant chromatin closure (few ATAC peaks), with ATAC signals significantly lower than those of active X chromosomes. From... Figure 3 It can be observed that the ATAC peak of the X chromosome genome of cells that have completed X chromosome inactivation of SE is significantly smaller than that of the X chromosome genome of LW. Furthermore, for FSL1, an XCI-abnormal embryo, both the X chromosome genomes of LW and SE in cells that have not initiated XCI show high ATAC peaks, verifying that the abnormal embryos identified by the XCI activity score of S7 are accurate.

[0091] 8.2 Verification of Heterozygous Expression Ratio at SNP Loci on the X Chromosome: The heterozygosity of expression reads in male, incomplete XCI, and whole female embryonic cells was statistically analyzed. Cells that had not completed XCI were expected to show more heterozygous expression (biale expression), while cells that had completed XCI should primarily show homozygous expression (monelele expression, similar to that of male individuals). Figure 4 As shown, the horizontal axis represents the position coordinates of the X chromosome, and the vertical axis represents the proportion of heterozygous reads. Figure 4 The results showed that cells that did not complete XCI had a significantly higher proportion of heterozygous expression of reads, while cells that completed XCI had a very low proportion of heterozygous expression.

[0092] The validation results from S8 show that chromatin accessibility analysis and SNP heterozygous expression rate analysis of embryos with abnormal XCI activity scores revealed consistent abnormal results from the three methods on the same embryo, confirming the accuracy and reliability of using XCI activity scores to detect developmental abnormalities in hybrid embryos.

[0093] S9. Evaluation of abnormal development in hybrid embryos using the dose-compensation state method:

[0094] At the single-cell level, gene expression levels on the X chromosome and autosomes are compared, and the dose ratio (X:A ratio) of the X chromosome to autosomes is calculated to assess dose compensation.

[0095] 9.1 Calculation method:

[0096] For female or male hybrid embryos, the average gene expression levels on the X chromosome and autosomes were calculated for each cell.

[0097]

[0098] 9.2 Abnormal Embryo Dosage Compensation Status:

[0099] Embryos with abnormal XCI were observed to have higher X:A ratios in both gene expression and chromatin accessibility than other embryos, indicating a failure of dose-compensation regulation. Figure 5 As shown, the X:A ratio of the abnormal embryo FSL1 was significantly higher than that of the other embryos. The mean X:A ratio of the FSL1 embryo was 0.40, while the mean values ​​of the other 12 embryos were 0.36, 0.35, 0.31, 0.33, 0.32, 0.33, 0.34, 0.36, 0.34, 0.35, 0.34, and 0.35, respectively. The mean X:A ratio of the FSL1 embryo was more than 10% higher than that of the other normal embryos.

[0100] S10. Parent-Specific Dosage Imbalance Analysis: By analyzing the dose ratio of the LW and SE genomes in abnormal embryos, the source of the dosage imbalance can be located, and it can be determined whether the dosage imbalance is caused by the LW or SE genome.

[0101] 10.1 Genome-Specific Dosage Analysis:

[0102] Genome-specific dose analysis based on RNA data obtained from S5:

[0103] LW genome-wide dose ratio = LW's X chromosome gene expression level / LW's autosome gene expression level;

[0104] SE intragenomic dose ratio = gene expression level on the X chromosome of SE / gene expression level on the autosomes of SE;

[0105] The transgenomic dose ratio 1 = gene expression level on the X chromosome of LW / gene expression level on the autosome of SE;

[0106] Transgenomic dose ratio 2 = gene expression level on the X chromosome of SE / gene expression level on the autosome of LW;

[0107] like Figure 6 As shown, because in developmentally abnormal FSL1 embryos, only cells that have completed XCI and whose X chromosome has been inactivated (SE) are present in the cells that have completed SE, this study compared X chromosome inactivated cells that have completed SE from all female embryos. The abnormal FSL1 dose compensation was found to be caused by severe underexpression of the autosomal genome of SE.

[0108] In summary, using the method of this invention, one developmentally abnormal embryo (FSL1) was successfully detected among 13 Eurasian pig hybrid embryos. This abnormal embryo exhibited the following characteristics:

[0109] (1) From S10 and Figure 6 It is evident that this abnormal embryo exhibits an extreme imbalance in allele effects, manifested as an extreme LW bias with a significantly insufficient contribution from the SE genome; and a transgenomic dosage imbalance, with the dosage ratio of the LW X chromosome to the SE autosome significantly higher in this abnormal embryo than in normal embryos.

[0110] (2) From S7 and Figure 2 It is evident that this abnormal embryo exhibits systemic X chromosome inactivation failure, with approximately two-thirds of its cells in an incomplete XCI state, whereas normal embryos have only a small number of cells incomplete XCI.

[0111] (3) From S9 and Figure 5 It is evident that this abnormal embryo exhibits a dose-compensation imbalance, with an X:A ratio in gene expression that is higher than that of normal embryos.

[0112] These molecular-level abnormalities indicate that the embryo suffers from dose compensation imbalance and XCI failure due to weak allelic effects, predicting developmental defects. This demonstrates that the XCI activity scoring method or dose compensation status method of this invention can effectively evaluate whether the hybrid embryo is developing abnormally and locate the source of dose imbalance. In modern large-scale pig breeding systems, timely detection of the developmental status of hybrid embryos can guide subsequent parental hybridization mating programs, reduce unexplained abortions caused by parental genomic problems, and improve the economic benefits of the pig industry.

[0113] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0114] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for detecting abnormal development of hybrid embryos for non-disease diagnostic purposes, characterized by, The method comprises the following steps: obtaining a hybrid embryo, preparing a single cell suspension, obtaining the transcriptome data of a single cell by single cell multi-omics sequencing technology, the data containing allele-specific information capable of distinguishing the origins of the parents, assigning the obtained sequencing data to the respective parental genomes to obtain allele-specific data; based on the allele-specific data, using the XCI activity score method or the dosage compensation state method to evaluate whether the development of the hybrid embryo is abnormal, wherein, the XCI activity score method is: for a female hybrid embryo, counting the gene expression amount of the X chromosome from the father and the mother respectively for each cell, and calculating the X chromosome activity: X chromosome activity = 1 + minimum value of gene expression amount of two X chromosomes / maximum value of gene expression amount of two X chromosomes; according to the X chromosome activity, the XCI activity score of the cell is calculated: no initiation of XCI: 1.8 < X chromosome activity ≤ 2.0, indicating that both X chromosomes are active; ongoing XCI: 1.2 < X chromosome activity ≤ 1.8, indicating that one X chromosome inactivation is in progress; completed XCI: 1.0 ≤ X chromosome activity ≤ 1.2, indicating that one X chromosome is active; the cells with no initiation of XCI and ongoing XCI are evaluated as incomplete XCI, and when the proportion of the cells with incomplete XCI in the female hybrid embryo exceeds 20%, the female hybrid embryo is evaluated as abnormal development; the dosage compensation state method is: for a female or male hybrid embryo, the average gene expression amount ratio of the X chromosome and the autosomes is calculated for each cell, that is: X:A ratio = gene expression amount of X chromosome / gene expression amount of autosome; when the average of X:A ratio of all cells of an embryo is higher or lower than the average of the normal embryo population by more than 10%, it indicates that dosage compensation fails, and the embryo is evaluated as abnormal development.

2. The method of claim 1, wherein the non-diagnostic purpose is to detect abnormal embryonic development in a hybrid embryo. The hybrid embryo is a hybrid embryo whose X chromosome inactivation process has been completed in theory.

3. The method for detecting developmental abnormalities in hybrid embryos for non-disease diagnostic purposes according to claim 2, characterized in that, When the hybrid embryo is from a pig, the hybrid embryo is an embryo above 27 days after fertilization.

4. The method for detecting developmental abnormalities in hybrid embryos for non-disease diagnostic purposes according to claim 1, characterized in that, When obtaining the allele-specific data, mask processing is performed on the sequencing data to eliminate alignment bias, and the mask processing method is to replace all parent genome difference sites in the reference genome with ambiguous bases N, and to reconstruct the alignment index for the masked reference genome.

5. Use of the method of detecting abnormal hybrid embryo development according to any one of claims 1 to 4 for the purpose other than disease diagnosis, characterized in that, For the hybrid embryo with an evaluation result of abnormal development, the dosage ratio of the parental genomes is analyzed to locate the source of dosage imbalance, and the parents that do not produce abnormal development of hybrid embryos are screened for hybridization; the method for analyzing the dosage ratio of the parental genomes is as follows: intra-parental 1 genome dosage ratio = gene expression amount of X chromosome of parent 1 / gene expression amount of autosome of parent 1; intra-parental 2 genome dosage ratio = gene expression amount of X chromosome of parent 2 / gene expression amount of autosome of parent 2; inter-genome dosage ratio 1 = gene expression amount of X chromosome of parent 1 / gene expression amount of autosome of parent 2; Trans-genomic dosage ratio 2 = the gene expression amount of the X chromosome of parent 2 / the gene expression amount of the autosomes of parent 1; When the dosage ratio within the genome of a parent is normal but the trans-genomic dosage ratio is abnormal, it is determined that the genome of the parent has dosage imbalance, which will produce hybrid embryos with developmental abnormalities.

6. The use of the method for detecting developmental abnormalities of hybrid embryos for non-disease diagnostic purposes according to any one of claims 1 to 5 in guiding the breeding of pigs.

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