CN-121992108-A - Sperm genome methylation detection method for biological breeding crop safety evaluation by using primate and application thereof

CN121992108ACN 121992108 ACN121992108 ACN 121992108ACN-121992108-A

Abstract

The invention discloses a sperm genome methylation detection method for evaluating crop safety by utilizing primate animals and application thereof, and relates to the technical field of crop safety evaluation, comprising the following steps of dividing non-human primate animals into three groups, collecting semen after long-term feeding, and extracting DNA; the method for detecting the methylation of the sperm genome by utilizing primate to carry out biological breeding crop safety evaluation and the application thereof can detect the slight epigenetic change which is difficult to find by traditional toxicology by utilizing a high-resolution WGBS technology, has important significance in cross-generation reproductive effect evaluation, is beneficial to perfecting a food safety evaluation system and establishes a more scientific and reliable safety interpretation basis.

Inventors

TANG DONGHONG
WANG CHENYUN
LI MINGHAO
LI ZHELI
Sun Kuanchao
Long Weihu

Assignees

中国医学科学院医学生物学研究所

Dates

Publication Date: 20260508
Application Date: 20260113

Claims (10)

1. A sperm genome methylation detection method for evaluating the safety of biological breeding crops by using primates, which is characterized by comprising the following steps: S1, randomly dividing sexually mature male non-human primate animals into an experimental group, a homologous control group and a basic control group, collecting semen or seminal plasma samples of the experimental animals after feeding is finished, and extracting genome DNA; S2, performing bisulfite treatment on the extracted DNA, constructing a whole genome sequencing library, simultaneously adding unmethylated DNA as a transformation efficiency contrast, and performing high-throughput sequencing to obtain original sequencing data; s3, performing quality control and filtration on the original sequencing data, comparing the original sequencing data to a reference genome, and evaluating the comparison rate, the coverage depth and the bisulfite conversion efficiency; S4, calculating and comparing methylation levels of the whole genome and the key functional areas of each group of samples, and performing inter-sample correlation analysis, principal component analysis based on windowed methylation data and methylation heterogeneity analysis; s5, performing differential methylation region analysis between the experimental group and each control group, identifying hypermethylated and hypomethylated DMR, and analyzing the length distribution and the distribution characteristics of the DMR in the genome functional region; s6, carrying out gene ontology and KEGG pathway enrichment analysis on the DMR related genes, thereby carrying out comprehensive safety evaluation on the biological breeding crops.
2. The method for detecting methylation of sperm genome in order to evaluate safety of a biologically bred crop using primate according to claim 1, wherein the non-human primate of S1 is a cynomolgus monkey.
3. The method for detecting methylation of sperm genome of a primate for evaluating safety of a biological breeding crop according to claim 1, wherein the experimental group is fed with a feed containing a target biological breeding crop for a long period of time, the homologous control group is fed with a homologous conventional crop feed without a target trait, and the basic control group is fed with a standard conventional feed.
4. A method of sperm genome methylation detection for biological breeding crop safety assessment using primate as described in claim 3, wherein said long term feeding is for a period of greater than 2 years.
5. The method for detecting methylation of sperm genome in a crop safety evaluation by use of primate according to claim 1, wherein the bisulfite conversion efficiency of S3 is greater than 99%.
6. The method for detecting methylation of sperm genome for evaluating safety of a biologically bred crop using primate according to claim 1, wherein the key functional region of S4 comprises CpG island, promoter, genome and repetitive sequence.
7. The sperm genome methylation detection method for biological breeding crop safety evaluation by using primates according to claim 1, wherein the methylation heterogeneity analysis in S4 specifically comprises calculating methylation entropy of single CpG sites based on shannon entropy, calculating coverage weighted average entropy in a preset window to obtain window-level heterogeneity values, and calculating coverage weighted average entropy of all windows in a sample layer to obtain sample-level DNA methylation heterogeneity index.
8. The method for detecting methylation of sperm genome for evaluating safety of a biologically bred crop by using primate as defined in claim 1, wherein the safety comprehensive evaluation in S6 is required to satisfy the following conditions: The methylation level of the experimental group and the control group in the whole genome and each key functional region has no obvious difference, and the obvious difference is P value <0.05; principal component analysis showed that the sample clusters were derived from individual differences, rather than group-to-group separation of experimental and control groups; the methylation heterogeneity index of the experimental group was not significantly increased compared to the control group, the significant increase was P-value <0.05; the DMR is distributed in a repeated sequence or an intron region, the distribution of the DMR in the CpG island and the promoter region is not remarkably enriched, and the distribution is remarkably enriched to be P value <1 multiplied by 10 -5 ; The enrichment pathway of DMR-related genes did not exhibit a specific pattern in the comparison of experimental and control groups.
9. Use of the method according to any one of claims 1-8 for long-term eating safety evaluation of a bio-bred crop.
10. The use according to claim 9, wherein the biological breeding crop comprises transgenic crop and genetically edited crop, and the safety assessment is an assessment of reproductive and epigenetic safety.

Description

Sperm genome methylation detection method for biological breeding crop safety evaluation by using primate and application thereof Technical Field The invention relates to a crop safety evaluation technology, in particular to a sperm genome methylation detection method for biological breeding crop safety evaluation by using primates and application thereof. Background Biological breeding crops, such as transgenic crops, have great potential in terms of yield improvement, stress resistance enhancement and the like, but long-term edible safety, particularly potential influence on reproductive systems and cross-representative genetics, are important scientific problems requiring long-term and systematic evaluation. Epigenetic modifications, particularly DNA methylation, are key mechanisms for the regulation of gene expression and may be affected by environmental factors (e.g., diet). The methylation status of sperm affects not only fertilization and embryo development, but may also convey environmental information to offspring. At present, toxicology evaluation on crop safety focuses on traditional pathological and physiological indexes, and a systematic and high-resolution evaluation method on the aspect of epigenetic aspects, particularly on the aspect of germ cells is not perfect. Although the whole genome bisulfite sequencing technology can provide a methylation map with single base resolution, how to apply the methylation map to long-term feeding safety evaluation of primate models and establish a complete system from experimental design, sample processing and data analysis to result interpretation is still a technical blank. Disclosure of Invention The invention aims to provide a sperm genome methylation detection method for evaluating the safety of biological breeding crops by using primates and application thereof, so as to solve the defects in the prior art. In order to achieve the above purpose, the invention provides a sperm genome methylation detection method for evaluating the safety of biological breeding crops by using primates, which comprises the following steps: S1, randomly dividing sexually mature male non-human primate animals into an experimental group, a homologous control group and a basic control group, collecting semen or seminal plasma samples of the experimental animals after feeding is finished, and extracting genome DNA; S2, performing bisulfite treatment on the extracted DNA, constructing a whole genome sequencing library, simultaneously adding unmethylated DNA as a transformation efficiency contrast, and performing high-throughput sequencing to obtain original sequencing data; s3, performing quality control and filtration on the original sequencing data, comparing the original sequencing data to a reference genome, and evaluating the comparison rate, the coverage depth and the bisulfite conversion efficiency; S4, calculating and comparing methylation levels of the whole genome and the key functional areas of each group of samples, and performing inter-sample correlation analysis, principal component analysis based on windowed methylation data and methylation heterogeneity analysis; s5, performing differential methylation region analysis between the experimental group and each control group, identifying hypermethylated and hypomethylated DMR, and analyzing the length distribution and the distribution characteristics of the DMR in the genome functional region; s6, carrying out gene ontology and KEGG pathway enrichment analysis on the DMR related genes, thereby carrying out comprehensive safety evaluation on the biological breeding crops. Further, the non-human primate of S1 is a cynomolgus monkey. Further, the experimental group is fed with feed containing target biological breeding crops for a long time, the homologous control group is fed with homologous conventional crop feed without target characters, and the basic control group is fed with standard conventional feed. Further, the long term feeding is for a period of greater than 2 years. Further, the bisulfite conversion efficiency of S3 is greater than 99%. Further, the key functional region of S4 includes CpG islands, promoters, genome and repetitive sequences. Further, the methylation heterogeneity analysis in S4 specifically comprises the steps of calculating methylation entropy of single CpG sites based on shannon entropy, calculating coverage weighted average entropy in a preset window to obtain window-level heterogeneity values, and calculating coverage weighted average entropy of all windows in a sample layer to obtain sample-level DNA methylation heterogeneity indexes. Further, the comprehensive security evaluation in S6 needs to satisfy the following conditions: The methylation level of the experimental group and the control group in the whole genome and each key functional region has no obvious difference, and the obvious difference is P value <0.05; principal component analysis showed that the sample clusters were