Cattle whole genome SNP chip, and use thereof

Abstract

Provided is a cattle whole genome SNP chip, the chip containing 50,000 SNP sites. The SNP chip comprises milk production traits, milk composition traits, reproductive traits and melatonin traits of cattle. The described molecular marker combination is used to construct a whole genome breeding chip, used for molecular breeding of cattle.

Description

A cattle whole genome SNP chip and its application

[0001] This application claims priority to the Chinese patent application with application number 2023116306711, application date November 30, 2023, and invention name “A cattle whole genome SNP chip and its application”. Technical Field

[0002] The present invention belongs to the field of agricultural biotechnology, and in particular relates to a cattle whole-genome SNP targeted capture liquid chip and an application thereof. Background Art

[0003] SNPs are polymorphisms in nucleic acid sequences caused by changes in a single nucleotide base at the genomic level. These variations include transitions, transversions, deletions, and insertions. They are numerous, widely distributed, and easily detectable. As genetic markers, SNPs contribute to genetic variation in quality traits, economic traits, and some complex disease traits, and are therefore widely used in genetic research.

[0004] The "targeted sequence capture" in the targeted capture liquid phase chip is also known as "Genotyping by target sequencing." The working principle of targeted sequence capture is based on site-specific capture sequencing based on the complementary binding of the target probe and the target sequence. First, a gDNA library is constructed for the material to be tested. At the same time, based on the principle of DNA complementarity, a probe covering the target SNP is designed at each site to be tested, and the target probe is modified with biotin labeling. Then, the biotin-modified probe is hybridized with the target region of the genome in a liquid to form a double strand. Finally, the captured target sequence is eluted, amplified, and sequenced to ultimately obtain the genotype of the target SNP.

[0005] Through targeted sequence capture, only the regions of interest need to be sequenced, which greatly reduces the sequencing amount and data output, thereby reducing sequencing costs.

[0006] However, for current cattle breeding chips, most markers have no effect due to the relatively single species and lack of genomic variation information.

[0007] Therefore, there is a need to develop a cattle whole-genome SNP chip. Summary of the Invention

[0008] In order to solve one of the above technical problems existing in the prior art, the present invention provides a cattle whole genome SNP chip and its application.

[0009] In a first aspect, the present invention provides a SNP molecular marker for the whole genome of cattle, wherein the SNP molecular marker is selected from at least one of the markers shown in Table 1.

[0010] In a second aspect, the present invention provides a molecular probe for specifically identifying the SNP molecular marker described in the first aspect.

[0011] In a third aspect, the present invention provides a bovine whole-genome SNP chip, wherein the SNP chip comprises probes targeting the SNP molecular markers described in the first aspect.

[0012] In some embodiments, the bovine whole-genome SNP chip of the present invention contains 50,000 SNP sites.

[0013] In some embodiments, the bovine whole-genome SNP chip is a liquid phase chip.

[0014] In some embodiments, the bovine whole-genome SNP chip is a bovine whole-genome SNP targeted capture liquid phase chip.

[0015] In some embodiments, the cattle include dairy cows and dual-purpose cattle.

[0016] In some embodiments, the dairy cow comprises a Holstein Dairy Cow, a Jersey Dairy Cow, a Brown Swiss Dairy Cow, or a Normande Dairy Cow.

[0017] In some embodiments, the dual-purpose dairy and beef cattle include Fleckvieh, Simmental Dairy Cow, Sanhe yellow cattle, Grassland red cattle, and Xinjiang brown cattle.

[0018] In some embodiments, the molecular marker is associated with one or more of a melatonin trait, a milk production trait, a milk composition trait, and a reproductive trait.

[0019] In some embodiments, the melatonin profile comprises: milk melatonin, serum melatonin.

[0020] In some embodiments, the milk production traits include: 305-day milk yield, daily milk yield, adult equivalent, lactation persistence, peak milk, peak day, milk loss, corrected milk, and herd level index.

[0021] In some embodiments, the milk component traits include: dry matter, urea nitrogen, milk protein, lactose, milk fat rate, first somatic cell fraction, and somatic cell fraction.

[0022] In some embodiments, the reproductive traits include parity, birth weight, weaning weight, parity spacing, number of repeat inseminations, interval from first mating to pregnancy after calving, and interval from first mating to last calving after calving.

[0023] In a fourth aspect, the present invention provides a kit comprising the molecular probe described in the second aspect, or the SNP chip described in the third aspect.

[0024] In a fifth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in whole genome breeding of cattle.

[0025] In some embodiments, the breeding involves selecting naturally high-melatonin cattle, wherein high-melatonin cattle refer to cattle with significantly higher melatonin production capacity than ordinary cattle, for example, 5-10 times higher.

[0026] Melatonin (MT) is a neuroendocrine hormone produced by the pineal gland in animals. It is involved in regulating biological rhythms and multiple physiological processes, and can improve sleep quality. Breeding naturally high-melatonin cattle can provide a basis and support for the research and development of natural melatonin-rich dairy products.

[0027] In a sixth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in identifying cattle kinship.

[0028] In a seventh aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in identifying cattle breeds.

[0029] In an eighth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in bovine genotyping detection.

[0030] In a ninth aspect, the present invention provides use of the molecular probe, or the SNP chip, or the kit in genetic diversity analysis of cattle populations.

[0031] In a tenth aspect, the present invention provides the use of the molecular probe, or the SNP chip, or the kit in the location analysis of QTL (quantitative trait loci) of target traits in cattle.

[0032] In an eleventh aspect, the present invention provides a method for preparing the bovine whole genome SNP chip, the method comprising the following steps:

[0033] (1) DNA extraction and quality control of bovine whole blood;

[0034] (2) Whole genome resequencing of samples that have passed quality control; providing all sequencing data (clean data) and performing quality assessment;

[0035] (3) Perform whole-genome variation detection (CALL SNP); based on the aligned files, perform deduplication and detect all potential polymorphic SNP sites in the whole genome of each sample to obtain a single-sample genome file, merge the single-sample genome files, and obtain all-sample variation files;

[0036] (4) Filtering cattle SNP sites;

[0037] (5) Analyze the correlation between SNPs and traits; select SNP sites with P < 0.0001 as candidate sites to prepare chips, and obtain a total of 50,000 SNP sites.

[0038] In some embodiments, in step (1), the quality control includes sequencing error rate of each base, base distribution detection and contamination detection.

[0039] In some embodiments, in step (2), the sequencing volume of a single sample of the whole genome resequencing is 54 GB, and the sequencing depth is 20×.

[0040] In some embodiments, in step (4), the filtering conditions are: removing individual samples with a genotype loss rate greater than 10%, removing sites with a minor allele frequency lower than 0.05, and retaining variant sites that are present in greater than 95% of individuals.

[0041] In some embodiments, in step (5), sire, weaning height, birth year, birth month, parity and days in milk production are combined as fixed effects.

[0042] The beneficial effects of the present invention include at least one of the following:

[0043] 1. While studying melatonin concentrations in raw milk from cows, the inventors discovered that melatonin concentrations in the milk of dual-purpose cows are generally higher than those in Holstein cows. Therefore, the inventors performed 20× resequencing on both Holstein cows and dual-purpose cows, conducted genome-wide association analysis on the SNP loci obtained from both types of cows, and designed a microarray. As a result, the SNP microarray of the present invention not only includes milk production, milk composition, and reproductive traits of both types of cows, but also melatonin traits. The SNP microarray of the present invention is suitable not only for dairy cow breeding, but also for dual-purpose cow breeding. It also lays the foundation for screening naturally high-melatonin cows and developing naturally high-melatonin milk products.

[0044] 2. The SNP chip of the present invention is applied to production practice, which can effectively shorten the generation interval of cattle and improve breeding efficiency and economic benefits.

[0045] 3. The present invention uses a molecular marker combination for whole-genome typing in cattle, targeting 50,000 single nucleotide polymorphisms (SNPs). The SNP chip of the present invention includes information on milk production, milk composition, reproductive traits, and melatonin. The present invention utilizes this molecular marker combination to construct a whole-genome breeding chip for molecular breeding in cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG1 shows a base quality distribution diagram of cow sequencing preprocessing data.

[0047] FIG2 shows the base quality distribution diagram of the sequencing preprocessing data of dual-purpose dairy and beef cattle.

[0048] FIG3 shows the distribution of base content in cow sequencing.

[0049] FIG4 shows the distribution of sequencing base content in dual-purpose dairy and beef cattle.

[0050] FIG5 shows the distribution of bovine SNP chip sites.

[0051] FIG6 shows a Manhattan plot of the melatonin trait.

[0052] FIG7 shows Manhattan plots of reproductive traits.

[0053] FIG8 shows a Manhattan plot of milk production traits.

[0054] FIG9 shows a Manhattan plot of milk component traits.

[0055] FIG10 shows the melatonin concentration of follicle fluid of different diameter follicles.

[0056] FIG. 11 shows the expression of genes significantly related to melatonin and reproductive traits in follicles of different diameters. DETAILED DESCRIPTION

[0057] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0058] definition

[0059] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0060] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0061] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0062] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0063] Example 1. Sequencing data output and quality summary

[0064] The selection criteria for dairy and dual-purpose cattle are: Chinese Holstein cattle and Fleckvieh dual-purpose cattle, multiparous cattle with complete production performance, reproduction and pedigree records.

[0065] Fifty-nine Chinese Holstein dairy cows and 53 Fleweherbred dairy and beef cattle were selected for whole-genome sequencing. Ear margin tissue was collected to extract total DNA. DNA libraries were constructed using standard Illumina methods, followed by 20× resequencing. Raw data quality was assessed prior to information analysis. Quality control results are presented as base quality distribution plots, with the horizontal axis representing the base position of clean reads and the vertical axis representing the average base quality. The base quality distributions for Holstein and Fleweherbred sequencing data are shown in Figures 1 and 2. The results show that base quality values ​​were all above 30, indicating good sequencing quality. Under normal circumstances, due to the principle of complementary base pairing and the random nature of sequencing, the ratio of A to T bases, and the ratio of G to C bases, are equal at each sequencing position. The corresponding distribution diagrams were obtained using the base position in the Clean Reads as the horizontal axis and the ratio of ATCGN bases at each position as the vertical axis. The ATCGN base quality distributions of the sequencing data of Holstein cows and Flavech cows are shown in Figures 3 and 4. The results show that both conform to the principle of complementary base pairing, indicating that there are no problems with the sequencing results.

[0066] Example 2. SNP variation detection and statistics

[0067] Based on the aligned BAM files, we used Picard-MarkDuplicates to remove duplicates. We then used GATK to detect all potential polymorphic SNPs across the entire genome for each sample to generate a single-sample GVCF. Finally, we merged the 112 single-sample GVCFs using GATK-CombineGVCF. These merged SNPs were then initially filtered using the following criteria: "QD < 2.0 || FS > 60.0 || MQ < 35.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0 || DP > 6950."

[0068] Example 3. SNP site filtering

[0069] PLINK was used to filter SNPs from 112 dairy cows using the following filtering criteria: remove individual samples with a genotype dropout rate greater than 10% (--mind 0.10), remove sites with a minor allele frequency less than 0.05 (--maf 0.05), and retain only variants shared by more than 95% of individuals (--geno 0.05). After filtering, no individual samples with a genotype dropout rate greater than 10% were found, sites with a minor allele frequency less than 0.05 were removed, and sites were removed due to genotype loss.

[0070] Example 4. Genome-wide association study (GWAS)

[0071] The association between SNPs and traits was analyzed using mixed linear models in PLINK. The traits mainly included melatonin, reproduction, milk production, and lactation. Pedigree, birth year, herd stratification, and breed were combined as fixed effects, and 91,360 P < 10 -4 The SNPs are used as the sites to be developed on the chip.

[0072] Example 5. Chip Design

[0073] Probes were designed for unexplored SNP sites with a P value less than 0.0001. Among them, 2,536 probes were successfully designed for two adjacent sites, 66 for three adjacent sites, and 5 for five adjacent sites. However, adjacent SNP sites were excluded due to the risk of site detection failure or low typing accuracy. Closely adjacent SNP sites were eliminated based on LD decay distance, resulting in a final set of 50,000 SNP sites.

[0074] The distribution of SNP chip sites is shown in Figure 5.

[0075] Example 6. Chip Sites

[0076] The SNP chip location information is shown in Table 1.

[0077] Table 1. SNP chip location information

[0078] Example 7. Application of the cattle whole genome SNP chip of the present invention in whole gene association analysis

[0079] The detection efficiency (Effective Rate) of this chip is greater than 99.5%, the Q20 is greater than 98%, the Q30 is greater than 95%, and the GC content is around 45%, indicating that the sequencing quality is good. The mapping rate (Mapped ratio) is greater than 99.9%, and the mapping results are normal, which can be used for subsequent variation detection and related analysis. The average data volume of this chip is 4.37G clean, and the average Q30 is 95.88%, which meets the requirements. The average 5× sequencing depth of this chip is 99.71%, and the detection rate is good. By typing 3 groups of repeated samples, the average consistency rate reached 99.74%, indicating that the typing of this chip is stable and reliable.

[0080] The whole-genome SNP chip provided by the present invention was used to perform genome-wide association analysis to explore key genes for melatonin traits, reproductive traits, milk production traits, and milk composition traits. The specific steps are as follows:

[0081] The results of the 50K microarray test on 688 dairy cows were filtered using the following filtering conditions: removing individual samples with a genotype loss rate greater than 10% (--mind 0.10), removing sites with a minor allele frequency less than 0.01 (--mar 0.01), and retaining only variant sites that were present in more than 95% of individuals (--geno 0.05). A total of 53,603 variant sites were detected on the microarray. After filtering, no individual samples with a genotype loss rate greater than 10% were found. 2,770 sites with a minor allele frequency less than 0.01 were removed, and 5,524 sites were removed due to genotype loss. Therefore, 45,309 sites passed the screening. Statistical analysis was performed using the GWAS analysis model described in Example 4.

[0082] P<10 -5 A total of 813 genes were annotated for reproductive traits, 161 genes for milk production traits, 141 genes for milk composition traits, and 112 genes for melatonin-related traits. Manhattan plots for melatonin, reproductive traits, milk production morphology, and milk composition traits are shown in Figures 6, 7, 8, and 9, respectively. Based on the Manhattan plots, genes significantly associated with melatonin and reproduction were identified as OPCML, KAT6B, LDLRAD3, ERC1, ITPK1, CNTNAP2, and BMP6.

[0083] Melatonin is present in large quantities in the local reproductive system. The melatonin concentration in bovine follicular fluid is closely related to the number and quality of oocytes. Melatonin can increase the number of mature oocytes and improve oocyte quality. Therefore, we used granulosa cells in follicles of different diameters as research objects, defining follicles with a diameter greater than 6 mm as large follicles, follicles with a diameter between 3-6 mm as medium follicles, and follicles with a diameter less than 3 mm as small follicles. The experiment collected three batches (approximately 25 pairs per batch) of bovine ovaries to collect fluid from large, medium and small follicles, and collected granulosa cells from the fluid of large, medium and small follicles. The melatonin concentration of follicle fluid of follicles of different diameters was detected. The results are shown in Figure 10. AANAT and ASMT are regulatory genes that affect the key rate-limiting enzymes in melatonin synthesis. The higher their expression, the higher the melatonin concentration. Using the expression of these two genes as markers, fluorescence quantitative PCR was used to detect the expression of genes significantly associated with melatonin and reproductive traits in the granulosa cells of large, medium and small follicles. The results are shown in Figure 11. It can be seen that the expression of genes significantly related to melatonin and reproductive traits in medium follicles (3-6 mm in diameter) was significantly higher than that in large follicles (diameter > 6 mm) and small follicles (diameter < 3 mm), which was consistent with the expression of marker genes (P < 0.05). Therefore, OPCML, KAT6B, LDLRAD3, ERC1, ITPK1, CNTNAP2, and BMP6 genes can be used as candidate genes for melatonin and reproductive traits.

[0084] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker for the whole genome of cattle, characterized in that: The SNP molecular marker is selected from at least one of the markers shown in Table 1.

2. A molecular probe, characterized in that The molecular probe is used to specifically identify the SNP molecular marker described in claim 1.

3. A cattle whole genome SNP chip, characterized in that: The SNP chip comprises probes targeting the SNP molecular markers according to claim 1.

4. The cattle whole genome SNP chip according to claim 3, characterized in that: The molecular marker is associated with one or more of a melatonin trait, a milk production trait, a milk composition trait, and a reproductive trait; Preferably, the melatonin properties include: milk melatonin, serum melatonin; Preferably, the milk production traits include: 305-day milk production, daily milk production, adult equivalent, lactation persistence, peak milk, peak day, milk loss, corrected milk, and herd level index; Preferably, the milk component traits include: dry matter, urea nitrogen, milk protein, lactose, milk fat rate, first somatic cell fraction, somatic cell fraction; Preferably, the reproductive traits include: parity, birth weight, weaning weight, parity spacing, number of repeated inseminations, interval from first mating to pregnancy after calving, and interval from first mating to last calving after calving.

5. A kit, characterized in that: The kit comprises the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4.

6. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in cattle whole genome breeding; Preferably, the breeding is to select cattle with naturally high melatonin.

7. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in identification of cattle kinship.

8. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in cattle breed identification.

9. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in bovine genotyping detection.

10. Use of the molecular probe according to claim 2, or the SNP chip according to claim 3 or 4, or the kit according to claim 5 in genetic diversity analysis of cattle populations or QTL location analysis of target traits.

Images