Methods for Identifying the Breeding Value of Cow Embryos

Abstract

The present invention discloses a method for identifying the breeding value of dairy cow embryos. The method comprises performing genomic selection on in vitro dairy cow embryo cells to be tested, obtaining the genomic breeding value of the dairy cow embryo to be tested, and identifying the breeding value of the dairy cow embryo based on the genomic breeding value. The present invention develops a new technology for rapid detection and evaluation of genetic performance of dairy cow breeding embryos before implantation, combines embryo micromanipulation sampling technology and genomic selection technology, and performs early and accurate genomic detection and genetic evaluation of embryos. The present invention applies whole genome selection technology to early detection of dairy cow embryos for the first time, transforming the original detection after calf birth to predicting the traits of reserve cows before embryo implantation, screening high-quality embryos, and rapidly expanding high-yielding dairy cows through embryo transplantation technology, constructing a high-yielding dairy cow breeding core group and screening excellent seed cows, thereby improving the directional and qualitative seed production capabilities of dairy cows, cultivating excellent breeding cows, improving selection accuracy, shortening the breeding cycle, accelerating genetic progress, and reducing breeding costs.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding, and relates to a method for identifying the breeding value of dairy cow embryos, and specifically to a method for early identification of the breeding value of dairy cow embryos by utilizing genome selection technology. Background Art

[0002] In traditional dairy cattle breeding, superior bulls are selected through progeny testing. This approach offers high accuracy, but the selection cycle is long (5-6 years), breeding costs are high, and efficiency is low. Elite individual cows are typically selected based on pedigree and phenotypic data for traits such as milk production and body conformation, but this approach is less accurate. Genomic selection (GS), the latest generation of breeding technology, significantly shortens generation intervals and achieves far greater breeding efficiency than traditional breeding methods. Currently, genomic selection is primarily used for the early selection of young bulls and core herd cows.

[0003] Embryo production and embryo transplantation are important technologies for the rapid expansion and multiplication of dairy cow breeding, and are currently widely used in dairy cow breeding and dairy farming. Excellent embryos with high breeding value can further accelerate the expansion and multiplication of excellent genes, improve the production level of dairy cow population, but how to identify the seed value of embryos remains an unresolved technical problem at present, usually based on the pedigree information (production performance or breeding value of father, mother, grandparents) of embryo donors (cows, bulls) or the milk production performance of donor cows, and accuracy is low, which greatly affects genetic progress and population quality. In view of this, it is necessary to provide a method for early identification of dairy cow embryo seed value, before embryo transplantation, i.e. before calf birth, genomic selection technology is utilized to identify and select excellent embryos to transplant, thereby rapidly expanding the excellent genetic material it carries, further shortening the breeding cycle, improving dairy cow directional and qualitative seed production ability, cultivating excellent breeding cattle, and accelerating population genetic progress. Thus, the present invention proposes a method for early accurate identification of dairy cow embryo seed value, including in vivo embryos, in vitro embryos and cloned embryos. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to accurately identify the breeding value of dairy cow embryos (including in vivo, in vitro, and / or cloned embryos) at an early stage, improve the targeted and qualitative breeding capabilities of dairy cows, shorten the dairy cow breeding cycle, and / or accelerate herd genetic progress. The technical problem to be solved is not limited to the technical subject matter described herein, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention first provides a method for early identification of the breeding value of dairy cow embryos (including in vivo embryos, in vitro embryos and / or cloned embryos). The method may include performing genomic selection on the in vitro dairy cow embryos to be tested, obtaining the genomic breeding value of the dairy cow embryos to be tested, and identifying the breeding value of the dairy cow embryos based on the genomic breeding value.

[0006] The genomic selection is performed before embryo implantation.

[0007] The cow embryos to be tested include in vivo embryos, in vitro embryos and / or cloned embryos.

[0008] Furthermore, the identification of the breeding value of dairy cow embryos may be an evaluation of the genetic performance of dairy cow breeding embryos before implantation.

[0009] Furthermore, the evaluation of genetic performance includes evaluating the genetic value of at least any of the following traits:

[0010] F1) milk production traits;

[0011] F2) health traits;

[0012] F3) Body shape traits.

[0013] Furthermore, the indicator of the milk production trait may be milk yield, milk fat content, milk fat percentage, milk protein content and / or milk protein percentage.

[0014] Furthermore, the health trait indicator may be a somatic cell score (an indicator of mastitis in dairy cows).

[0015] Furthermore, the indicators of the body shape traits may be a total body shape score, a lactation system score and / or a limb and hoof score.

[0016] In the above method, the genome selection method may include the following steps:

[0017] A1) obtaining a small amount of embryonic cells from a cow embryo to be tested using micromanipulation technology, and extracting a trace amount of genomic DNA from the small amount of embryonic cells;

[0018] A2) amplifying the trace amount of genomic DNA and performing a genomic DNA quality test to obtain quality-tested genomic DNA;

[0019] A3) performing embryonic genome detection on the quality-inspected genomic DNA using a dairy cow whole genome chip to obtain genomic chip genotype data, and performing quality control on the genomic chip genotype data to obtain quality-controlled genomic chip genotype data;

[0020] A4) Use the quality-controlled genomic chip genotype data to estimate genomic breeding values.

[0021] The quality-tested genomic DNA may be a sufficient amount of quality-tested genomic DNA.

[0022] The small number of embryonic cells mentioned herein may be 1 to 10 cells.

[0023] The 1 to 10 embryonic cells may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 embryonic cells.

[0024] The cow embryo to be tested may be a cow embryo at the morula stage, including an in vivo embryo, an in vitro embryo and / or a cloned embryo.

[0025] In the above method, the method for detecting the quality of the genomic DNA in A2) may include detecting the concentration, purity and / or length of the amplified genomic DNA, and detecting the integrity of the genomic DNA.

[0026] In the above method, the detection of genomic DNA integrity may include the following steps:

[0027] B1) selecting genes located on different chromosomes of dairy cows and designing primers, wherein the genes include SESN2, RPL11, DDIT3, GAPDH and DGAT1 genes;

[0028] B2) using the amplified genomic DNA as a template and performing PCR amplification using the primers in B1) to obtain an amplified product;

[0029] B3) using the amplified product to determine the integrity of the amplified genomic DNA.

[0030] Furthermore, the SESN2 gene (Gene ID: 509863) and the RPL11 gene (Gene ID: 512745) are functional genes on chromosome 2 of dairy cows; the DDIT3 gene (Gene ID: 777788) and the GAPDH gene (Gene ID: 281181) are functional genes on chromosome 5 of dairy cows; and the DGAT1 gene (Gene ID: 282609) is a functional gene on chromosome 14 of dairy cows.

[0031] Furthermore, the primers in B1) may include PCR amplification primers for SESN2, RPL11, DDIT3, GAPDH and DGAT1 genes.

[0032] Furthermore, the PCR amplification primers may include a primer combination shown in SEQ ID No. 1 to SEQ ID No. 10, wherein:

[0033] SESN2 gene primer F: 5′-GAAGTCCCGTTTCATCATGC-3′ (SEQ ID No. 1);

[0034] SESN2 gene primer R: 5′-TATCCGCTTGTCTGGCTTCT-3′ (SEQ ID No. 2);

[0035] RPL11 gene primer F: 5′-CGGGTTAACTGTCCTTCATCC-3′ (SEQ ID No. 3);

[0036] RPL11 gene primer R: 5′-AAGTGTAGGGTAACGGAAGGC-3′ (SEQ ID No. 4);

[0037] DDIT3 gene primer F: 5′-TCCGGGTCCAACACTAAGTC-3′ (SEQ ID No. 5);

[0038] DDIT3 gene primer R: 5′-GACGGGGTGTCTTTCTCGTA-3′ (SEQ ID No. 6);

[0039] GAPDH gene primer F: 5′-CAATGCTGAAACCTCCACGC-3′ (SEQ ID No. 7);

[0040] GAPDH gene primer R: 5′-AGCACTGCGGGAGAGTAGTA-3′ (SEQ ID No. 8);

[0041] DGAT1 gene primer F: 5′-TGTGAAACCAAGTAAGATCGGC-3′ (SEQ ID No. 9);

[0042] DGAT1 gene primer R: 5′-CAGGCTGACACCCAACGC-3′ (SEQ ID No. 10).

[0043] Furthermore, the reaction conditions for the PCR amplification in B2) may be: 95°C for 5 min; 95°C for 30 sec, 58°C for 30 sec, 72°C for 40 sec, for a total of 35 cycles; 72°C for 10 min; and storage at 4°C.

[0044] Furthermore, the reaction system for the PCR amplification in B2) may be: forward primer F (10 pmol / μL) 1.25 μL, reverse primer R (10 pmol / μL) 1.25 μL, 2×Taq Master Mix 12.5 μL, template DNA 2 μL, and ddH2O 8 μL.

[0045] In the above method, the time for amplifying the trace amount of genomic DNA in A2) can be 4 to 8 hours. Preferably, the time for amplifying the trace amount of genomic DNA in A2) can be 4 hours.

[0046] Furthermore, the reaction conditions for amplifying the trace amount of genomic DNA in A2) may be: amplifying the trace amount of genomic DNA at 30° C. for 4 hours.

[0047] Furthermore, the reaction system (40 μL) for amplifying the trace amount of genomic DNA in A2) is: REPLI-g scReaction Bufffer 29 μL, REPLI-g sc DNA Polymerase 2 μL, and H2O 9 μL.

[0048] In the above method, the method of performing quality control on the genomic chip genotype data in A3) may include eliminating SNP sites with a SNP site detection rate lower than 90% and a minimum allele frequency lower than 0.01.

[0049] Furthermore, the quality control can use plink software to perform quality control on the genotype data of the genome chip, and embryos with an individual detection rate lower than 90% can be re-extracted with DNA and re-genome chip detection.

[0050] In the above method, the method for estimating genomic breeding values described in A4) comprises the following steps:

[0051] C1) using a group of dairy cows whose conventional breeding value reliability based on phenotypic data and pedigree information is greater than or equal to 0.3 as a reference group, and calculating the inverse regression breeding value using an inverse regression iterative equation;

[0052] C2) filling missing genotypes in the genomic chip genotype data after quality control, and performing quality control on the filled genotype data to obtain chip genotype data after filling quality control;

[0053] C3) calculating the genomic relatedness between the embryos to be tested based on the chip genotype data after filling and quality control, and constructing a genomic relationship matrix;

[0054] C4) Using the inverse regression breeding value described in C1) as the response variable, the genomic breeding value (DGV) of each trait of the embryo to be tested is calculated based on the single-trait animal model GBLUP method. The mathematical model is as follows: y = μ + Zg + e

[0055] Where: y represents the inverse regression breeding value of milk production traits, health traits and body shape traits; μ represents the overall mean; Z represents the genomic kinship matrix; g represents the individual random additive genetic effect vector; e represents the random residual effect vector;

[0056] C5) calculating, based on the genomic breeding value, a genomic performance index of the embryo to be tested according to a genomic performance index formula;

[0057] C6) Ranking the embryos according to their genomic breeding values and / or genomic performance indices, and evaluating the breeding value of the dairy cow embryos based on the ranking.

[0058] In the above method, the genome performance index (GCPI) formula in C5) can be as follows:

[0059]

[0060] Among them, GEBV Fat , GEBV Prot , GEBV SCS , GEBV Type , GEBV MS , GEBV F&L They are the genomic breeding values for milk fat content, milk protein content, somatic cell score, total body score, lactation system, and limb and hoof score.

[0061] Furthermore, the quality control method in C2) can be to eliminate SNP sites with a detection rate lower than 90% and a minimum allele frequency lower than 0.01.

[0062] The detection rate described in this article can be the ratio of samples in which a certain SNP site is successfully detected to all samples; the minimum allele frequency can be the frequency of occurrence of uncommon alleles in a given population.

[0063] Furthermore, the calculation process for constructing the genome relationship matrix (G matrix) in C3) is:

[0064]

[0065] Where M is an n×m matrix, i.e. n individuals and m SNPs. k is the minimum allele frequency of the i-th SNP. P is an n×m matrix with the k-th column element being 2p k .

[0066] In the above method, the indicator of the milk production trait in C4) may be milk yield, milk fat content, milk fat rate, milk protein content and / or milk protein rate, the indicator of the health trait may be the somatic cell score, and the indicator of the body shape trait may be the total body shape score, limb and hoof score and / or lactation system score.

[0067] Furthermore, the milk yield is the total milk production of the cow from the first day after calving to the 305th lactation day, expressed in kilograms. The milk fat percentage or milk protein percentage is the total milk fat or milk protein production of the cow from the first day after calving to the current measurement date / total milk production × 100, expressed in percentage (%). The milk fat content or milk protein content is the milk yield × milk fat percentage or milk protein percentage / 100, expressed in kilograms.

[0068] Furthermore, the somatic cell score is derived from the logarithmic conversion of the somatic cell count, calculated as log2(somatic cell count / 100)+3, with a score ranging from 0 to 9. Higher somatic cell counts correspond to higher scores, and it is an indicator of udder health in dairy cows. The somatic cell count refers to the number of somatic cells in milk from lactating cows on a given day, including immune cells such as neutrophils, lymphocytes, and macrophages, as well as epithelial cells shed from mammary tissue, and is measured in thousands per ml.

[0069] Furthermore, the total conformation score includes body volume, rump, limbs and hooves, lactation system, and milking characteristics; the lactation system includes udder morphology, front udder, and hind udder; and the limb and hoof score includes hoof angle, heel depth, bone texture, and hind limb lateral and hind limb posterior views. Cow conformation traits are assessed by professional dairy cow conformation assessors using a 9-point scoring system. Within 30 to 180 days after the first calving, conformation assessors first score multiple parts according to standards, and then integrate the scores using weighted coefficients.

[0070] In the above method, the whole genome chip in A3) contains a large number of molecular genetic markers, and the molecular genetic markers are high-density single nucleotide polymorphism sites (SNPs) covering the entire genome of the dairy cow.

[0071] Furthermore, the information of the molecular genetic markers may be as follows: genetic markers covering the entire genome of dairy cows, generally single nucleotide polymorphism sites, and commonly used commercial whole genome chips include GeneSeek 150K, GeneSeek100K, and Boridi 140K, which contain 139,376, 95,256, and 139,097 SNP sites, respectively.

[0072] In one embodiment of the present invention, the whole genome chip is the Boredi 140K whole genome chip (Cat. No. PHR0105_Bt140K).

[0073] The present invention also provides any of the following applications of any of the methods described herein:

[0074] D1) Application in early identification of the breeding value of dairy cow embryos;

[0075] D2) Application in building a core group of dairy cattle breeding and screening seed cows;

[0076] D3) Application in improving the directional and qualitative breeding ability of dairy cows;

[0077] D4) Application in independent breeding of cattle and herd genetic improvement;

[0078] D5) Application in shortening the selection cycle of dairy cattle breeding;

[0079] D6) Application in reducing dairy cattle breeding costs.

[0080] The present invention has developed a method for early identification of the breeding value of dairy cow embryos, namely a new technology for rapid detection and assessment of the genetic performance (breeding value) of dairy cow embryos before implantation. Combining embryo micromanipulation sampling technology with genomic selection technology, the embryos are subjected to early morula genome detection and genetic assessment, and high-quality embryos are selected for embryo transplantation as breeding stock, resulting in excellent replacement cows. Compared with the existing technology, the present invention has the following beneficial effects:

[0081] In the existing technology, the technical difficulties in applying whole genome selection technology to early detection of dairy cow embryos include: micro-manipulation sampling technology of a small number of living cells in early embryos, extraction and genome detection of trace genomic DNA from a small number of embryonic cells, and genome chip data quality control. These technical links have not been effectively solved, making it difficult to identify the breeding value before embryo transplantation. The present invention optimizes and establishes for the first time a technical process for obtaining a small number of cells by micromanipulation at the morula stage of dairy cow embryos, extracting and amplifying trace genomic DNA from a small number of cells, performing chip data quality control, and performing genome evaluation. This process can obtain a small number of living embryonic cells, sufficient and complete genomic DNA, a genome chip data detection rate of not less than 90%, a chip data filling accuracy of not less than 96%, and an average genetic evaluation accuracy of not less than 60% for major traits. Compared with the selection accuracy of conventional pedigree indexes, the present invention improves the selection accuracy by 20% to 30% (Zhang Yuan, Livestock Breeding (Second Edition), 2018, China Agriculture Press, Table 4-8 on page 89 shows that when the heritability of the traits is 0.10, 0.25, and 0.50, the selection accuracy using conventional pedigree indexes is 23%, 35%, and 50%, respectively. Gong Weijia, Research on Genetic Analysis of Chinese Holstein Cattle Populations, PhD Dissertation of China Agricultural University, 2010: The heritability of milk production, health, and body conformation traits of Chinese Holstein dairy cows is 0.09 to 0.50).

[0082] Secondly, applying whole-genome selection technology to early-stage testing of dairy cow embryos enables early selection of high-quality embryos with high breeding value, allowing for the production of high-quality replacement heifers through embryo transfer. This shifts from post-calf testing to pre-implantation prediction of replacement heifer traits. Compared to conventional post-calf testing, this shortens the breeding cycle by 12 months (the cow's gestation period is 9 months, and the calf's hair or blood samples are collected for genomic testing and evaluation for 3 months after birth), further shortening the breeding cycle and accelerating genetic progress. Furthermore, the application of early-stage testing of dairy cow embryos allows for the selection of high-quality embryos for transplantation and the elimination of embryos with poor genetic performance, saving both embryo transfer costs and the costs of cow gestation and calf rearing, thereby reducing breeding costs. Thus, by transplanting high-quality embryos, it is possible to rapidly expand the number of high-yielding, high-quality dairy cows, establish a core group of high-yielding dairy cow breeding stock, select excellent seed cows, improve the directional and qualitative seed production capabilities of dairy cows, cultivate excellent breeding cattle, further shorten the generation interval in dairy cow breeding, accelerate genetic progress, and reduce breeding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Micromanipulation of dairy cow embryos to obtain 1 to 10 embryonic cells.

[0084] Figure 2 The graph shows the 1.0% agarose gel electrophoresis results of embryonic genomic DNA at three amplification times: 4h, 6h, and 8h.

[0085] Figure 3 The integrity of embryonic genomic DNA amplification (SESN2 gene) at three amplification times of 4h, 6h, and 8h.

[0086] Figure 4 The integrity of embryonic genomic DNA amplification (RPL11 gene) at three amplification times of 4h, 6h, and 8h.

[0087] Figure 5 The integrity of embryonic genomic DNA amplification (DDIT3 gene) at three amplification times of 4h, 6h, and 8h.

[0088] Figure 6 The integrity of embryonic genomic DNA amplification (GAPDH gene) at three amplification times of 4h, 6h, and 8h.

[0089] Figure 7 The integrity of embryonic genomic DNA amplification (DGAT1 gene) at three amplification times of 4h, 6h, and 8h.

[0090] Figure 8 This is the genotype data result of the embryo genome chip with an amplification time of 4 hours.

[0091] Figure 9This is the genotype data result of the embryo genome chip with an amplification time of 6 hours.

[0092] Figure 10 This is the genotype data result of the embryo genome chip with an amplification time of 8 hours.

[0093] Figure 11 The figure shows the results of 1.0% agarose gel electrophoresis of genomic DNA of 24 embryos to be tested.

[0094] Figure 12 The integrity of genomic DNA amplification of 24 embryos tested (SESN2 gene).

[0095] Figure 13 The integrity of genomic DNA amplification of 24 embryos to be tested (RPL11 gene).

[0096] Figure 14 The integrity of genomic DNA amplification of 24 embryos to be tested (DDIT3 gene).

[0097] Figure 15 The integrity of genomic DNA amplification of 24 embryos to be tested (GAPDH gene).

[0098] Figure 16 The integrity of genomic DNA amplification of 24 embryos to be tested (DGAT1 gene).

[0099] Figure 17 The quality control results of the genomic chip genotype data of 24 embryos to be tested.

[0100] Figure 18 A technical process for identifying the breeding value of dairy cow embryos.

[0101] Figure 19 The genomic evaluation results of 24 embryos were tested. DETAILED DESCRIPTION

[0102] 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.

[0103] 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.

[0104] The IVC solution in the following examples is an in vitro embryo development culture medium purchased from IVF Bioscience, product number 71005.

[0105] The holding solution in the following examples is a product of ICP bio, product number MPHM-500.

[0106] The dairy cow embryos at the morula stage in the following examples were sourced from Inner Mongolia Saikexing Livestock Breeding and Reproduction Biotechnology Research Institute Co., Ltd.

[0107] Example 1: Obtaining a small number of cells from early-stage dairy cow embryos

[0108] In this example, cow embryonic cells were collected from 33 cow embryos using micromanipulation technology. 1 to 10 embryonic cells were collected from each cow embryo. The specific steps were as follows:

[0109] 1. Retrieve 33 dairy cow embryos at the morula stage from the ranch, wash away the holding solution with IVC solution, and place the morulas in IVC solution for culture.

[0110] 2. Make drops on the bottom of a 60 mm dish, divided into two rows. Make three drops in the first row, each containing 50 μL of 7% PVP (see Table 1 for the formula); make three drops in the second row, each containing 100 μL of HM (see Table 2 for the formula), and add 6 mL of mineral oil until the droplets are covered.

[0111] 3. Place the embryo into any droplet in the second row, perform micromanipulation with a micromanipulator, and use an injection needle with an inner diameter of 20 μm to break the zona pellucida. After the zona pellucida is broken, aspirate 1 to 10 embryonic cells ( Figure 1 ) to obtain embryonic cell samples for the extraction and amplification of trace genomic DNA from a small number of cells in the late embryo, perform genomic testing and genomic genetic evaluation on the obtained embryonic genomic DNA, and select high-quality and excellent embryos for breeding.

[0112] Table 1, 7% PVP formula

[0113] composition Ratio PVP (polyvinyl pyrrolidone) 7g Medium 199(1×) 93mL

[0114] Table 2. HM formula

[0115] composition Ratio PS (phosphatidylserine) 40 μL FBS (fetal bovine serum) 1mL Medium 199(1×) 9mL

[0116] Example 2: Extraction of trace genomic DNA from a small number of cells in dairy cow embryos

[0117] The genomic DNA of a small number of cells from the 33 dairy cow embryos obtained in Example 1 was extracted respectively, and the specific steps were as follows:

[0118] 1. Place 1 to 10 embryonic cells from each dairy cow embryo obtained by micromanipulation into a 200 μL centrifuge tube containing 4 μL PBS.

[0119] 2. Prepare cell lysis buffer according to Table 3. Add 3 μL of cell lysis buffer to the 200 μL centrifuge tube described in step 1. Gently flick the centrifuge tube to mix, and centrifuge at 7000 rpm for 1 min. Place the centrifuge tube in a PCR amplifier (model: BIO-RADT100) and incubate at 65°C for 10 min to lyse the embryonic cells and release genomic DNA. Add 3 μL of Stop Solution to terminate the cell lysis reaction. Gently flick the centrifuge tube to mix, centrifuge at 7000 rpm for 1 min, and place on ice to stop cell lysis. Trace genomic DNA from 33 cow embryos was harvested.

[0120] Table 3. Cell lysis buffer system

[0121]

[0122] Example 3: Optimization of the method for amplifying trace genomic DNA from a small number of cells in dairy cow embryos

[0123] 1. To obtain sufficient embryonic genomic DNA that meets the requirements for genomic genetic evaluation, amplify the trace genomic DNA obtained in Example 2. Prepare the reaction system according to Table 4. Add 40 μL of the DNA amplification system to the 200 μL centrifuge tube described in Step 2 of Example 2 (i.e., the centrifuge tube containing the trace genomic DNA). Gently flick the tube to mix thoroughly and centrifuge at 7000 rpm for 1 minute. Place the tube in a PCR amplifier (Model: BIO-RAD T100) and amplify the trace genomic DNA at 30°C for 4-8 hours. Then, incubate the tube in the PCR amplifier (Model: BIO-RAD T100) at 65°C for 3 minutes to inactivate the REPLI-g sc DNA Polymerase enzyme. Store the amplified genomic DNA at 4°C or -20°C.

[0124] Table 4. Embryo genomic DNA amplification reaction system

[0125]

[0126] In the above steps, considering that the amplification time affects the genome detection rate of embryonic genomic DNA, the present invention optimizes the amplification time of the trace genomic DNA in step 1. Nine of the 33 dairy cow embryos in Example 1 were selected from the same donor cow and donor bull. The amplification time of the trace genomic DNA of these nine embryos was set to three different gradients: 4 hours, 6 hours, and 8 hours, respectively. Each time parameter contained three embryos, i.e., three biological replicates.

[0127] 2. Further compare and analyze the effects of the three amplification time parameters of 4h, 6h, and 8h on the concentration, purity, and length of the obtained embryonic genomic DNA.

[0128] The concentration and purity of genomic DNA from nine embryos were determined using a nucleic acid analyzer (NanoDrop 2000). The quality control results for embryonic genomic DNA amplified at 4, 6, and 8 h are shown in Table 5. The DNA concentrations for all nine embryos ranged from 193.6 ng / uL to 217.9 ng / uL, and the DNA purity ratios (A260 / A280) were all between 1.7 and 2.0. These results indicate that the concentration and purity of genomic DNA from embryos amplified at 4, 6, and 8 h met the requirements for whole-genome array testing.

[0129] The length of genomic DNA from 9 embryos was detected by 1.0% agarose gel electrophoresis. The results of 1.0% agarose gel electrophoresis of genomic DNA from embryos at three amplification times of 4h, 6h, and 8h are shown in the figure. Figure 2 As shown, the results showed that the length of the embryonic genomic DNA obtained at the three amplification times of 4h, 6h, and 8h were all diffuse bands of 2kb to 10kb, which were consistent with the results of embryonic genomic DNA amplification.

[0130] Table 5. Quality inspection results of embryonic genomic DNA at three amplification times: 4h, 6h, and 8h

[0131]

[0132]

[0133] 3. Further comparative analysis was conducted on the effects of the three amplification time parameters of 4h, 6h, and 8h on the integrity of the obtained embryonic genomic DNA. Five functional genes located on different chromosomes of dairy cows were selected, including the SESN2 gene (Gene ID: 509863) and the housekeeping gene RPL11 (Gene ID: 512745) on chromosome 2, the DDIT3 gene (Gene ID: 777788) and the housekeeping gene GAPDH (Gene ID: 281181) on chromosome 5, and the DGAT1 gene (Gene ID: 282609) on chromosome 14. PCR amplification primers for each of the five genes were designed using Primer 3.0 (http: / / primer3.wi.mit.edu / ). Using the embryonic genomic DNA obtained at the three amplification times of 4h, 6h, and 8h as templates, PCR amplification was performed using primers of each gene to obtain amplified products. The length of the PCR amplified product was compared with the length of the target fragment by 1.2% agarose gel electrophoresis to determine whether the integrity of the embryonic genomic DNA obtained at the three amplification times of 4h, 6h, and 8h was determined. The results of the integrity of the embryonic genomic DNA obtained at the three amplification times of 4h, 6h, and 8h are shown in Figure 2. Figures 3 to 7 As shown, the results showed that the lengths of the SESN2, RPL11, DDIT3, GAPDH and DGAT1 gene amplification products of the embryonic genomic DNA obtained at the three amplification times of 4h, 6h and 8h were consistent with the target band length, and the five selected genes were completely amplified. The results showed that the embryonic genomic DNA obtained at the three amplification times of 4h, 6h and 8h had good integrity.

[0134] The primer sequences, product lengths, and annealing temperatures for PCR amplification of the five genes are shown in Table 6 , and the PCR reaction system and PCR reaction conditions are shown in Tables 7 and 8 .

[0135] Table 6. PCR amplification primer sequences, product lengths, and annealing temperatures for five genes

[0136]

[0137] Table 7. PCR reaction system

[0138]

[0139]

[0140] Table 8. PCR reaction conditions

[0141]

[0142] 4. The embryonic genomic DNA obtained by the three amplification time parameters of 4h, 6h, and 8h described in step 1 was used to perform embryonic genome detection using the Boredi 140K whole genome chip (catalog number PHR0105_Bt140K) to obtain the genome chip genotype data of each embryo.

[0143] 5. Use Plink software to analyze the detection rate of embryonic genome chip genotype data obtained at three amplification times of 4h, 6h, and 8h. The detection rate of embryonic genome chip at three amplification times of 4h, 6h, and 8h is as follows: Figures 8 to 10 , as shown in Table 9. With a 4-hour amplification time, the individual detection rate of microarray data from embryonic cells was 0.929-0.975; with a 6-hour amplification time, the individual detection rate was 0.864-0.913; and with an 8-hour amplification time, the individual detection rate was 0.865-0.910. These results indicate that the individual detection rate of microarray data from embryonic cells with a 4-hour amplification time for trace genomic DNA was significantly higher than that with 6 and 8 hours, which better meets the requirements of genomic genetic assessment.

[0144] Table 9 Individual detection rates of embryonic genome chip data at three amplification times: 4h, 6h, and 8h

[0145] Embryo number Amplification time Individual detection rate 4-1 4 hours 0.975 4-2 4 hours 0.929 4-3 4 hours 0.956 6-1 6 hours 0.913 6-2 6 hours 0.894 6-3 6 hours 0.864 8-1 8 hours 0.865 8-2 8 hours 0.910 8-3 8 hours 0.906

[0146] In this embodiment, the optimization of the method for amplifying trace genomic DNA from a small number of cells in dairy cow embryos based on genomic genetic evaluation is the first innovative achievement of the present invention. Through the above comparative analysis of the effects of the three amplification times of 4h, 6h, and 8h on the concentration, purity, integrity, and genome chip detection rate of the obtained embryonic genomic DNA, the results showed that the embryonic genomic DNA chip detection rate with an amplification time of 4 hours was significantly higher than that with an amplification time of 6 hours and 8 hours. At the same time, the concentration and purity met the requirements for genome chip detection, and the obtained embryonic genomic DNA had good integrity.

[0147] Example 4: Amplification of trace genomic DNA from a small number of cells in the embryo to be tested and genomic DNA quality detection

[0148] 1. Based on the optimized amplification time parameter of 4 hours for the trace genomic DNA of cow embryos in Example 3, the trace genomic DNA of the remaining 24 embryos to be tested among the 33 cow embryos was amplified. The other steps were the same as those described in step 1 of Example 3 to obtain the amplified genomic DNA, and the amplified genomic DNA was further quality tested.

[0149] 2. Detect the concentration, purity and length of the genomic DNA obtained from the 24 embryos to be tested (i.e., the amplified genomic DNA obtained in step 1). The specific steps are the same as those described in step 2 of Example 3. The quality inspection results are shown in Table 10. The DNA concentration is 102.1ng / uL~252.6ng / uL, and the DNA purity A260 / A280 is 1.7~2.0. The results show that the concentration and purity of the genomic DNA of the 24 embryos to be tested meet the requirements of the whole genome chip detection. The results of 1.0% agarose gel electrophoresis of the genomic DNA of the 24 embryos to be tested are as follows: Figure 11 As shown, the genomic DNA length is a diffuse band of 2kb to 10kb, which is consistent with the results of embryonic genomic DNA amplification.

[0150] 3. Detect the integrity of the genomic DNA obtained from the 24 embryos to be tested. The steps are the same as step 3 in Example 3, and finally obtain the genomic DNA after quality inspection. The results of the integrity of the embryonic genomic DNA obtained from the 24 embryos to be tested are as follows: Figures 12 to 16 As shown, the results showed that the lengths of the amplified products of SESN2, RPL11, DDIT3, GAPDH and DGAT1 genes of the embryonic genomic DNA obtained from the 24 embryos to be tested were consistent with the target band lengths, and the five selected genes were completely amplified. The results showed that the embryonic genomic DNA obtained from the 24 embryos to be tested had good integrity.

[0151] Table 10. Results of genomic DNA quality inspection of 24 embryos tested

[0152]

[0153]

[0154] Example 5: Acquisition and quality control of genotype data from 24 embryos to be tested using genome chips

[0155] 1. The genomic DNA of 24 embryos to be tested (i.e., the genomic DNA obtained after quality inspection in Example 4) was used to perform embryo genome detection using the Boredi 140K whole genome chip (Cat. No. PHR0105_Bt140K) to obtain the genomic chip genotype data of each embryo.

[0156] 2. Use plink software to perform quality control on the genomic chip genotype data of the 24 embryos to be tested, and obtain the genomic chip genotype data after quality control. The quality control method is to eliminate SNP sites with a detection rate lower than 90% and a minimum allele frequency lower than 0.01. Embryos with an individual detection rate lower than 90% in chip data can be re-extracted for DNA extraction and chip testing. The quality control results are as follows: Figure 17 shown.

[0157] Example 6: Genomic genetic evaluation of 24 embryos

[0158] Based on the genomic chip data and inverse regression breeding value data of the large-scale Chinese Holstein cattle genome selection reference population constructed by the inventors in the early stage (Zhang Qi, Research on the genetic evaluation of dairy cattle genomes integrating GWAS prior information, Master's thesis of China Agricultural University, 2022), and using the quality-controlled genomic chip genotype data, the genomic breeding values of 24 embryos to be tested were estimated. The specific method is as follows:

[0159] 1. Based on the Chinese Holstein cattle genomic reference population, individuals with conventional breeding values (EBV) reliability greater than or equal to 0.3 were selected as the reference population (a total of 17,940 heads), and the inverse regression breeding value (DRP) was calculated using the inverse regression iterative equation.

[0160] 2. Based on a reference population of 17,940 heads, Beagle 5.0 software was used to fill missing genotypes in the genome chip genotype data of 24 embryos to be tested (i.e., the genome chip genotype data after quality control). The filled genotype data were then quality controlled using the method described in step 2 of Example 5 to obtain the chip genotype data after filling and quality control.

[0161] 3. Based on the chip genotype data after filling quality control (including the reference group and the embryos to be tested), the genomic relationship between the 24 embryos to be tested is calculated and the genomic relationship matrix (G matrix) is constructed. The calculation process is as follows:

[0162]

[0163] Where M is an n×m matrix, i.e. n individuals and m SNPs. k is the minimum allele frequency of the i-th SNP. P is an n×m matrix with the k-th column element being 2p k .

[0164] 4. Using the inverse regression breeding value of the reference population as the response variable, based on the single-trait animal model GBLUP method, the direct genomic breeding value (DGV) of each trait of the 24 embryos to be tested was calculated using DMU software. The mathematical model is as follows:

[0165] y=u+Zg+e

[0166] Where y is the inverse regression breeding value of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content or milk protein percentage), health traits (somatic cell score) and body conformation traits (total body score, limb and hoof score or lactation system score); μ is the overall mean; Z is the genomic kinship matrix; g is the individual random additive genetic effect vector; and e is the random residual effect vector.

[0167] 5. Based on the direct genomic breeding value, the GCPI of the 24 embryos to be tested was obtained according to the Chinese Dairy Cow Genomic Performance Index (GCPI) formula. The GCPI formula is as follows:

[0168]

[0169] Among them, GEBV Fat , GEBV Prot , GEBV SCS , GEBV Type , GEBV MS , GEBV F&L They are the genomic breeding values for milk fat content, milk protein content, somatic cell score, total body score, lactation system, and limb and hoof score.

[0170] 6. Rank the 24 embryos based on their genomic breeding values and / or GCPI values for each trait, assess their breeding value, and select high-quality, superior embryos for breeding. Transplanting high-quality, superior embryos allows for rapid expansion of high-yielding, high-quality dairy cows, building a core breeding herd of high-yielding dairy cows, and screening for superior seed cows. This improves the ability to produce both directional and qualitative dairy cows, cultivates superior breeding stock, further shortens the generation interval in dairy cow breeding, accelerates genetic progress, and reduces breeding costs.

[0171] The present invention establishes a complete technical process for early identification of the breeding value of dairy cow embryos (such as Figure 18 The genomic evaluation results of the 24 embryos to be tested are as follows Figure 19 The results show that the present invention has established a technical process for extracting and amplifying trace genomic DNA from 1 to 10 cells of dairy cow embryos at the morula stage using micromanipulation, as well as a small amount of embryonic cells, followed by genome chip data quality control and genome evaluation. This process can obtain 1 to 10 living morula cells, sufficient and complete genomic DNA, a genome chip data detection rate of no less than 90%, a chip data filling accuracy of no less than 96%, and an average genome evaluation accuracy of no less than 60% for major traits.

[0172] Compared with the selection accuracy of conventional pedigree index, the present invention improves the selection accuracy by 20% to 30% (Zhang Yuan, Livestock Breeding (Second Edition), 2018, China Agriculture Press, Table 4-8 on page 89 shows that when the heritability of the trait is 0.10, 0.25, and 0.50, the selection accuracy using the conventional pedigree index is 23%, 35%, and 50%, respectively. Gong Weijia, Research on Genetic Analysis of Chinese Holstein Cattle Population, Doctoral Dissertation of China Agricultural University, 2010: the heritability of milk production, health, and body shape traits is 0.09 to 0.50).

[0173] Applying whole-genome selection technology to early embryo testing in dairy cows enables the early selection of high-quality embryos with high breeding value, allowing for the production of high-quality replacement heifers through embryo transfer. This shifts from post-calf testing to pre-implantation prediction of replacement heifer traits. Compared to conventional post-calf testing, this shortens the breeding cycle by 12 months (the cow's gestation period is nine months, and the calf's hair or blood samples are collected for genomic testing and evaluation three months after birth), further shortening the breeding cycle and accelerating genetic progress. Furthermore, the application of early embryo testing in dairy cows allows for the selection of high-quality embryos for transfer and the elimination of embryos with poor genetic performance, saving on embryo transfer costs as well as the costs of cow gestation and calf rearing, thereby reducing breeding costs. By transferring high-quality embryos, high-yielding, high-quality dairy cows can be rapidly propagated, establishing a core breeding herd, screening seed cows, improving the ability to produce both targeted and qualitative seed, and cultivating high-quality breeding stock. This further shortens the generation interval in dairy cow breeding, accelerates genetic progress, and reduces breeding costs.

[0174] 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 method for identifying the breeding value of dairy cow embryos, characterized in that: The method comprises performing genomic selection on an in vitro dairy cow embryo to be tested, obtaining a genomic breeding value of the dairy cow embryo to be tested, and identifying the breeding value of the dairy cow embryo according to the genomic breeding value; The method of genome selection comprises the following steps: A1) obtaining a small amount of embryonic cells from a cow embryo to be tested using micromanipulation technology, and extracting a small amount of genomic DNA from the small amount of embryonic cells; A2) amplifying the trace amount of genomic DNA and performing a quality test on the genomic DNA to obtain quality-tested genomic DNA; A3) performing embryonic genome detection on the quality-tested genomic DNA using a cow whole genome chip to obtain genome chip genotype data, and performing quality control on the genome chip genotype data to obtain quality-controlled genome chip genotype data; A4) Estimating genomic breeding values using the quality-controlled genomic chip genotype data; the method for estimating genomic breeding values comprises the following steps: C1) The group of dairy cows whose conventional breeding value reliability based on phenotypic data and pedigree information is greater than or equal to 0.3 is used as the reference group, and the inverse regression breeding value is calculated using the inverse regression iterative equation; C2) filling missing genotypes in the quality-controlled genomic chip genotype data, performing quality control on the filled genotype data, and obtaining the quality-controlled filled chip genotype data; C3) Calculating the genomic relationship between the embryos to be tested based on the chip genotype data after filling and quality control, and constructing a genomic relationship matrix; the calculation process of constructing the genomic relationship matrix is as follows: Where M is an n×m matrix, i.e. n individuals and m SNPs; p k is the minimum allele frequency of the i-th SNP; P is an n×m matrix with the k-th column element being 2p k ; C4) Using the inverse regression breeding value described in C1) as the response variable, the genomic breeding value of each trait of the embryo to be tested is calculated based on the single-trait animal model GBLUP method. The mathematical model is as follows: y = μ + Zg + e Where: y represents the inverse regression breeding value of milk production traits, health traits and body shape traits; μ represents the overall mean; Z represents the genomic kinship matrix; g represents the individual random additive genetic effect vector; e represents the random residual effect vector; C5) Based on the genomic breeding value, the genomic performance index of the embryo to be tested is calculated according to a genomic performance index formula; the genomic performance index formula is as follows: in, 、 、 、 、 、 These are the genomic breeding values for milk fat content, milk protein content, somatic cell score, total body score, lactation system, and limb and hoof score traits; C6) The embryos to be tested are ranked according to their genomic breeding values and / or genomic performance indices, and the breeding value of dairy cow embryos is identified based on the ranking.

2. The method according to claim 1, characterized in that The method for detecting the quality of genomic DNA described in A2) includes detecting the concentration, purity and / or length of the amplified genomic DNA, and detecting the integrity of the genomic DNA.

3. The method according to claim 2, characterized in that The detection of the integrity of the genomic DNA comprises the following steps: B1) Designing primers for genes located on different chromosomes of dairy cows, including SESN2, RPL11, DDIT3, GAPDH and DGAT1 Gene; B2) using the amplified genomic DNA as a template and performing PCR amplification using the primers in B1) to obtain an amplified product; B3) using the amplified product to determine the integrity of the amplified genomic DNA.

4. The method according to any one of claims 1 to 3, characterized in that The time for amplifying the trace amount of genomic DNA in A2) is 4 to 8 hours.

5. The method according to any one of claims 1 to 3, characterized in that: The method for quality control of the genomic chip genotype data in A3) includes eliminating SNP sites with a SNP site detection rate lower than 90% and a minimum allele frequency lower than 0.

01.

6. The method according to any one of claims 1 to 3, characterized in that: The indicators of milk production traits in C4) are milk yield, milk fat content, milk fat percentage, milk protein content and / or milk protein percentage, the indicators of health traits are somatic cell score, and the indicators of body shape traits are total body score, limb and hoof score and / or lactation system score.

7. Any of the following uses of the method according to any one of claims 1 to 6: D1) Application in early identification of the breeding value of dairy cow embryos; D2) Application in building a breeding nucleus for dairy cattle and selecting seed cow herds; D3) Application in improving the directional and qualitative breeding ability of dairy cows; D4) Application in independent breeding of cattle and herd genetic improvement; D5) Application in shortening the selection cycle of dairy cattle breeding; D6) Application in reducing dairy cattle breeding costs.

Images