23K methylation chip of cattle and its design method and application

Abstract

The present invention provides a 23K methylation chip of cattle and a design method and application thereof. The methylation chip gene typing object is a methylation variant site combination including 23525 variant sites. The methylation chip can be applied to the detection of DNA methylation modification of cattle genome, the analysis of the epigenetic regulation mechanism of the environment on traits, the positioning of candidate genes of economic traits, genetic diversity analysis, germplasm resource evaluation and improvement, whole genome association analysis and whole genome selective breeding, etc. The methylation chip of the present invention can provide a good research foundation and data support for the fields of DNA methylation detection, epigenetic modification selective breeding and other research of cattle, and further reduce the cost of cattle genome methylation detection, accelerate the genetic progress of the improvement of high-quality cattle breeds in my country, and has good social value and promotion value.

Description

Technical Field

[0001] The invention belongs to the field of animal molecular biology technology and animal genetic breeding technology, and particularly relates to a cattle 23K methylation chip and a design method and application thereof. Background Art

[0002] Cattle are one of the most important livestock in the world, providing humans with basic living needs such as beef and milk. However, the production level of beef and dairy cattle in my country is relatively low compared with that in Europe and the United States. Rapidly improving the production level of beef and dairy cattle in my country is of great significance to the long-term stability and healthy development of my country's cattle industry. Genetics (genomic sequence) and environment are two key factors that determine the production level of cattle. The environment can change the production level of cattle through epigenetics. DNA methylation is the main form of epigenetics at the genome level. Changes in its modification level caused by the environment will directly affect the expression level of genes, thereby regulating the production capacity of cattle. Therefore, the study of DNA methylation in the cattle genome plays an important role in analyzing the key mechanisms regulating the production level of cattle and the mining of new genes. In addition, the study also found that early DNA methylation changes will affect the later development of individuals, thereby determining the production performance of cattle in adulthood, and some DNA methylation changes will be passed on to offspring, thereby passing the effects of DNA methylation changes to offspring. DNA methylation is considered to play a role similar to nucleic acid variations such as SNP at the epigenetic modification level. It can be used as an important molecular modification marker to predict the production level of cattle and used for cattle genome breeding.

[0003] There are currently a variety of methods for detecting genomic DNA methylation, including MeDIP, MIRE, MBD and other methods based on the principle of protein-DNA interaction, and WGBS, RRBS and other methods based on bisulfite treatment. The former has a lower detection cost, but cannot be accurate to the single base level, and the resolution is generally below 50bp, and cannot distinguish between CG and non-CG, two methylation modification sequence features with obvious differences; while the latter can be accurate to the resolution of a single base, but has the characteristics of high cost and cannot achieve large-scale detection of animal populations. The development of low-cost, high-resolution DNA methylation detection methods plays an important role in improving the production level of cattle in my country.

[0004] Gene chip, also known as DNA chip or biochip, is another high-throughput molecular marker technology. Its basic principle is to use hybridization sequencing to perform qualitative and quantitative analysis on the detected signals. It has the advantages of rapidity and high throughput, and can analyze a large amount of genomic information in parallel at the same time, which also makes it widely used in the fields of biological evolution, gene positioning, molecular breeding, etc. DNA methylation chip is a product derived from this. It has high specificity and can detect specific genes or sites, and can more accurately detect the methylation status of specific regions. At the same time, methylation chip uses solid phase probe technology, which can detect the methylation status of multiple genes at the same time. Therefore, under the condition of high sample throughput, the relative cost is low, and the operation of methylation chip is relatively simple, and the sample processing time is short, which is suitable for rapid screening and verification. In cattle, there is no research and development and application of DNA methylation chip, and only traditional methods can be used for detection. On the one hand, it cannot integrate the requirements of cost and resolution, which will cause a lot of capital waste; on the other hand, since the above technologies are all invented abroad, it will lead to capital outflow and be restricted by foreign countries; this has hindered the use of modern advanced technology to improve the production level of cattle in my country to a certain extent. Therefore, developing a DNA methylation detection chip for cattle is essential to promote the further healthy and stable development of my country's cattle industry and has significant scientific and application value. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a 23K methylation chip for cattle and a design method and application thereof. The chip can provide a good research basis and data support for the fields of DNA methylation detection, selective breeding and other research in cattle, and further reduce the cost of cattle genome selection, accelerate the genetic progress of improving my country's high-quality cattle breeds, and has good social value and promotion value.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an application of a combination of methylation variant sites of cattle in the detection of methylation modification of cattle genome DNA, analysis of the epigenetic regulatory mechanism of the environment on traits, positioning of candidate genes for economic traits, genetic diversity analysis, evaluation and improvement of germplasm resources, whole genome association analysis, and whole genome selection breeding. The combination of methylation variant sites of cattle includes 23,525 methylation variant sites, and the physical positions of the 23,525 methylation variant sites are determined based on the positive chain bases of the ARS-UCD1.2 reference genome. The physical position information of the 23,525 methylation variant sites is as follows:

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[0038] The left side of the site number indicates the chromosome where the site is located, and the right side indicates the position of the site on the chromosome.

[0039] In the second aspect, the present invention provides a set of probes for detecting the above-mentioned cattle methylation variation site combinations for use in the detection of cattle genome DNA methylation modification, analysis of the epigenetic regulatory mechanism of the environment on traits, localization of candidate genes for economic traits, genetic diversity analysis, germplasm resource evaluation and improvement, whole genome association analysis, and whole genome selection breeding.

[0040] In a third aspect, the present invention provides a 23K methylation chip of cattle, wherein the genotyping object of the methylation chip is the combination of methylation variation sites of the above cattle, and the methylation chip comprises a set of the above probes.

[0041] In the above technical solution, the methylation chip is obtained by the following method:

[0042] S1: Collect the original whole-genome methylation sequencing data of cattle, perform quality control on the data, align it to the reference genome ARS-UCD1.2 of cattle, extract the methylation information, and obtain the original methylation data information;

[0043] S2: Based on the methylation variant sites screened out, according to the sample tissue source, screen the methylation sites with large changes between tissues and within tissues, combine the location of the site on the chromosome, analyze its upstream and downstream sequences and design sequencing primers to select the methylation variant sites that can be used for chip development;

[0044] S3: The methylation chip is developed using methylation capture technology.

[0045] In a fourth aspect, the present invention provides the application of the above-mentioned methylation chip in the detection of DNA methylation modification in the cattle genome, the analysis of the epigenetic regulatory mechanism of the environment on traits, the positioning of candidate genes for economic traits, genetic diversity analysis, germplasm resource evaluation and improvement, whole genome association analysis and whole genome selection breeding.

[0046] In a fifth aspect, the present invention provides a method for designing the above-mentioned methylation chip, which specifically comprises the following steps:

[0047] S1, Collection of whole-genome methylation sequencing data of cattle;

[0048] S2, quality control of sequencing data, sequence comparison and methylation information extraction;

[0049] S3, comparison of differentially methylated intervals between and within tissues;

[0050] S4, integrated screening of all differentially methylated sites;

[0051] S5, customize the methylation array based on all candidate sites.

[0052] In the above technical solution, the specific process of S1 is: search for the sequencing information of cattle on NCBI, download the SRA file from the NCBI database using the prefetch tool in the SRAtoolkit software according to the accession number provided by NCBI, or use ascp to download the fastq file from the EBI database.

[0053] In the above technical scheme, the specific process of S2 is: trim_galore software is used to remove the adapter sequence and low-quality read length sequence (reads) from the original sequencing data fastq format file, and the execution parameters of the fastqc tool are specified using the --fastqc_args parameter to perform quality control checks immediately after trimming to obtain high-quality reads information;

[0054] The bismark tool was used to align the sequence information that passed quality control to the reference genome ARS-UCD1.2 of cattle. The deduplication_bismark method was used to deduplicate the comparison results. The bismark_methylation_extractor method was used to extract the methylation information of the sites from the deduplicated data.

[0055] In the above technical scheme, the specific process of S3 is: through the methylation data files processed by S2, group them according to the tissue source, combine them in pairs, filter the sites with read depth less than 10, and use methylKit software to detect the differential methylation intervals and differential methylation sites between the two tissues. In the same tissue, the average methylation level of the interval is calculated with 1000bp as the interval, and the intervals with significant differences in different samples of the same tissue in the same interval are extracted to obtain the differential methylation intervals in the tissue;

[0056] Among them, for the inter-tissue methylation intervals, the intervals that were differentially methylated in multiple tissues were selected. For the same interval of the intra-tissue methylation interval, the intervals with average methylation levels greater than 0.8 and less than 0.2 in different samples were recorded as high differential methylation intervals within the tissue. For the same interval of the intra-tissue methylation interval, the intervals with average methylation levels greater than 0.5 in different samples were recorded as relative differential methylation intervals within the tissue.

[0057] In the above technical solution, the specific steps of integrated screening in S4 are:

[0058] S41, based on the differential methylation intervals between and within tissues selected in S3, annotate with genomic intervals, functional elements, and QTLs, and mainly retain the intervals falling in the genomic intervals, functional elements, and QTL regions;

[0059] S42, for the inter-tissue difference intervals, according to the comparison between the inter-tissue difference sites and the difference intervals in different tissues, the top five difference sites are selected as candidate sites;

[0060] S43, extracting CG sites from the difference intervals within the tissue as candidate sites;

[0061] S44, performing depth filtering on the candidate sites to screen out sites with too small depth and too large depth;

[0062] S45, for each difference interval, select all samples in the interval, calculate the correlation coefficient between the candidate sites in the interval and the difference interval, use a correlation coefficient of 0.8 as a standard, screen candidate sites with too low correlation coefficients, and select the candidate site with the highest correlation coefficient in each difference interval;

[0063] S46, add some QTL intervals related to growth traits as supplements, and select high-correlation sites within the QTL interval according to the S44 and S45 methods;

[0064] S47, integrate the high correlation coefficient sites obtained in S45 and S46, screen the sites within 2 kb, and screen multiple sites in a gene according to the annotation status to retain the sites with good annotation status;

[0065] S48, probe design was performed for the differential methylation sites obtained in S47. The design rules were as follows: the probe performance was comprehensively evaluated from the aspects of GC content (20%-80%), Tm value (60-80°C), number of consecutive repeated bases (≤25nt), and number of alignments on the reference genome (≤5). The probe with the best evaluation index was selected for production. The probe length was 120nt. The differential methylation sites for which the probes were successfully designed were finally used as the final differential methylation sites for integration screening.

[0066] The beneficial effects of the present invention are:

[0067] 1. The 23K methylation chip of cattle of the present invention involves multiple tissues of multiple breeds, and the designed chip has breed diversity and tissue specificity. The chip not only contains the differential methylation sites detected in the present invention, but also adds CG sites in QTLs related to growth traits. These sites can be directly associated with important economic traits of cattle, reducing the number of redundant and irrelevant CGs, reducing costs, and having greater application value in molecular breeding of cattle.

[0068] 2. When the present invention screens the differential methylation intervals, both the inter-tissue differential intervals and the intra-tissue differential intervals are considered, different screening strategies are used for interval screening, and the functional interval annotations are taken into consideration during the screening process. In addition, the QTL regions related to growth traits supplement the differential methylation intervals. For the differential methylation intervals to be selected, representative methylation sites within the multiple-step selection intervals are selected as probe design sites, so as to achieve a comprehensive comparison of the DNA methylation levels of various tissues.

[0069] 3. The chip of the present invention can provide a good research foundation and data support for the detection of bovine genome DNA methylation modification, selective breeding and other research fields, and further reduce the cost of bovine genome selection, accelerate the genetic progress of my country's high-quality cattle breed improvement, and has good social value and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a partial flow chart of the site integration screening of the methylation chip of the present invention;

[0071] Figure 2 It is the chromosome distribution map of the methylation chip sites. DETAILED DESCRIPTION

[0072] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments described herein.

[0073] like Figure 1-2 As shown, the present invention provides a bovine 23K methylation chip and a design method and application thereof. The specific design method of the methylation chip comprises the following steps:

[0074] S1, Collection of cattle second-generation whole-genome sequencing data.

[0075] A total of 279 samples of 28 tissues from 15 cattle breeds were collected and the original whole-genome methylation sequencing data were obtained from NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ) to download the collection.

[0076] Among them, the 15 cattle breeds include: Angus, Asturian Valley x Holstein, Asturian Valleyx Limousin, Brangus, Brangus-Angus, Charolais, Chinese Red Steppes, Hereford, Holstein, Japaneseblack (Wagyu), Sanjiang cattle, Shorthorn crossbred, Shuxuan, Simmental, and Tibetan cattle.

[0077] The 28 tissues include: fat, adrenal gland, blood, bone, breast, cerebral cortex, ear, embryonic stem cells, fetal skeletal muscle, fibroblasts, heart, hypothalamus, ileum, kidney, liver, lung, muscle, ovary, pituitary gland, placenta, rumen, somatic cells, sperm, spleen, stromal vascular cells, testicle, tongue, and uterus.

[0078] Search for cattle sequencing information on NCBI. According to the accession number provided by NCBI, use the prefetch tool in the SRAtoolkit software to download the SRA file from the NCBI database, or use ascp to download the fastq file from the EBI database.

[0079] S2, Quality control and sequence comparison of sequencing data.

[0080] The trim_galore software was used to remove the adapter sequences and low-quality read sequences (reads) from the original sequencing data fastq format files, and the --fastqc_args parameter was used to specify the execution parameters of the fastqc tool for quality control checks after trimming to obtain high-quality reads information.

[0081] Bismark uses Bowtie2 to perform alignment analysis of methylation data with the reference genome. Bismark converts both the sequencing results and the reference genome from C to T and G to A (reverse complement), aligns the converted sequencing results and genomes in pairs, and outputs all alignment results. The specific operation is to use the bismark tool to align the sequence information that has passed quality control to the reference genome ARS-UCD1.2 of cattle, use the deduplication_bismark method to deduplicate the alignment results, and use the bismark_methylation_extractor method to extract methylation sites from the deduplication data.

[0082] S3, Comparison of differentially methylated intervals between and within tissues.

[0083] The methylation data files obtained by S2 processing are grouped according to tissue origin. This means that data from the same tissue source are grouped together for subsequent comparison and analysis. The grouped methylation data are combined in pairs to form different tissue comparison pairs. This allows the methylation differences between different tissues to be compared. Before performing differential methylation analysis, sites with a read depth of less than 10 need to be filtered out. This is because sites with a smaller depth may cause unstable analysis results due to insufficient sequencing data, ensuring the reliability of the analysis results. The methylKit software can be used to detect differential methylation intervals and differential methylation sites between two tissues. The software can detect regions and microsingles with significant differences in genomic methylation levels in two groups, and is used to discover methylation patterns and characteristics between different tissues. For inter-tissue methylation intervals, intervals that are differentially methylated in multiple tissues are selected as inter-tissue differential methylation intervals.

[0084] In order to further explore the methylation differences within the tissue, a deeper analysis was performed on the data within the same tissue. Taking 1000bp as the interval, the average methylation level of each interval was calculated. This step helps us understand the methylation status in different intervals. For the same interval of the methylation interval within the tissue, the intervals with average methylation levels greater than 0.8 and less than 0.2 in different samples were recorded as high-difference methylation intervals within the tissue. For the same interval of the methylation interval within the tissue, the average methylation levels in different samples differed by more than 0.5 and were recorded as relative differential methylation intervals within the tissue.

[0085] S4, integrated screening of all differentially methylated sites. The main screening process includes the following steps:

[0086] S41, based on the differential methylation intervals between and within tissues selected in S3, annotate the genomic intervals, functional elements, and QTLs, and mainly retain the intervals falling in the genomic intervals, functional elements, and QTL regions as the methylation differential intervals for preliminary exploration.

[0087] S42, for the inter-tissue difference intervals, according to the comparison of the inter-tissue difference sites and the difference intervals in different tissues, the top five difference sites are selected as candidate sites. The comparison is specifically manifested as the distribution consistency of the differential methylation sites and the differential methylation intervals in different inter-tissue comparison combinations.

[0088] S43, for the difference intervals within the tissue, extract the CG sites therein as candidate sites.

[0089] S44, depth filtering is performed on the candidate sites to screen sites with too small depth and too large depth. The minimum depth is 10 and the maximum depth is 200.

[0090] S45, for each difference interval, select all samples in the interval, calculate the correlation coefficient between the candidate sites in the interval and the difference interval, use a correlation coefficient of 0.8 as a standard, screen the candidate sites with too low correlation coefficients, and select the candidate site with the highest correlation coefficient in each difference interval.

[0091] S46, adds some QTL intervals related to growth traits as supplements, and selects highly correlated sites within the QTL interval according to the S44 and S45 methods. The obtained sites are supplements to the differential methylation sites and are also for better application in genetic breeding practices.

[0092] S47, integrate the high correlation coefficient sites obtained in S45 and S46, screen sites within 2kb, and screen multiple sites in a gene according to the annotation status to retain sites with good annotation. Avoid selecting sites that are too close and over-concentrated in some genes, which will help to make the final selected sites widely and evenly distributed and better detect DNA methylation.

[0093] S48, probe design was performed for the differential methylation sites obtained in S47. The design rules were as follows: the probe performance was comprehensively evaluated from the aspects of GC content (20%-80%), Tm value (60-80°C), number of consecutive repeated bases (≤25nt), and number of alignments on the reference genome (≤5). The probe with the best evaluation index was selected for production. The probe length was 120nt. The differential methylation sites for which the probes were successfully designed were used as the final differential methylation sites for integration screening. The specific information of the sites is shown in the table above.

[0094] S5, customize the methylation array based on all candidate sites.

[0095] The methylation array was customized based on all 23,525 candidate sites, and the final candidate sites screened were submitted to Beijing Compson Agriculture Co., Ltd. for confirmation and customization.

[0096] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A probe for detecting a combination of methylation variation sites in cattle is used in the detection of methylation modification of cattle genome DNA, analysis of the epigenetic regulation mechanism of the environment on traits, localization of candidate genes for economic traits, genetic diversity analysis, evaluation and improvement of germplasm resources, genome-wide association analysis, and genome-wide selective breeding, characterized by: The combination of methylation variant sites of the cattle includes 23,525 methylation variant sites. The physical positions of the 23,525 methylation variant sites are determined based on the positive chain bases of the ARS-UCD1.2 reference genome. The physical position information of the 23,525 methylation variant sites is as follows: The left side of the site number indicates the chromosome where the site is located, and the right side indicates the position of the site on the chromosome.

2. A bovine 23K methylation chip, characterized in that: The methylation chip genotyping object is the methylation variation site combination of the cattle according to claim 1, and the methylation chip comprises a set of probes according to claim 1.

3. The methylation chip according to claim 2, characterized in that: The methylation chip is obtained by the following method: S1: Collect the original whole-genome methylation sequencing data of cattle, perform quality control on the data, align it to the reference genome ARS-UCD1.2 of cattle, extract the methylation information, and obtain the original methylation data information; S2: Screen out methylation variant sites based on S1, and screen methylation sites with large inter-tissue and intra-tissue variations based on the sample tissue source. Combine the location of the site on the chromosome, analyze its upstream and downstream sequences, design sequencing primers, and select methylation variant sites that can be used for chip development; S3: The methylation chip is developed using methylation capture technology.

4. Application of the methylation chip described in claim 2 in the detection of DNA methylation modification in the cattle genome, analysis of the epigenetic regulatory mechanism of the environment on traits, localization of candidate genes for economic traits, genetic diversity analysis, evaluation and improvement of germplasm resources, whole genome association analysis, and whole genome selection breeding.

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