SNP molecular markers, marker combinations and their applications related to the number of mature follicles in Holstein cattle

Abstract

The present invention belongs to the field of molecular biology and molecular marker technology, and discloses SNP molecular markers, marker combinations and applications thereof related to the number of mature follicles in Holstein cattle. The present invention screens molecular markers related to the trait of the number of mature follicles in Holstein cattle based on whole genome association analysis, and utilizes the molecular markers to assist breeding through molecular markers, retain individuals with favorable genotypes, and eliminate individuals with unfavorable genotypes, thereby significantly increasing the number of mature follicles in the population, thereby achieving the purpose of improving the reproductive efficiency of excellent Holstein cows.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and molecular markers, and in particular relates to SNP molecular markers, marker combinations and applications thereof related to the number of mature follicles of Holstein cattle. Background Art

[0002] Holstein cattle originated in the Netherlands and are the oldest dairy cattle breed. They are famous for their high milk production and have been exported to many countries, accounting for more than 80%-90% of the total number of dairy cows in the world. In the breeding of Holstein cattle, embryo transplantation technology can significantly improve the reproductive efficiency of excellent cows, which is of great significance for the expansion of Holstein cattle. In embryo transplantation technology, superovulation is a key technical link, which is affected by many factors and individual reactions vary greatly. Therefore, it is very important to breed excellent Holstein cows with more mature follicles. How to find a fast and effective method to improve the breeding method of the number of mature follicles in Holstein cattle is very important.

[0003] In traditional breeding, phenotypic determination is mostly based on the number of mature follicles to determine whether an individual is a high-quality Holstein cow. Based on previous studies, Machado et al. evaluated the heritability of the total number of follicles and the number of live follicles in dairy cows and found that their heritability was 0.38 and 0.34, respectively. Therefore, the number of mature follicles is a medium-heritability trait. With the development of molecular biology, molecular marker-assisted selection has gradually been applied to breeding practice. Combined with whole genome association analysis technology, it is expected to identify key mutation sites related to the number of mature follicles, which can effectively improve the efficiency of breeding work. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP molecular marker related to the number of mature ovarian follicles of Holstein cattle, a marker combination and an application thereof.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a SNP molecular marker associated with the number of mature follicles in Holstein cattle, wherein the marker is selected from any one of ① to ⑥:

[0006] ① The marker contains a nucleotide sequence with polymorphism A / G at 15144012 bp of chromosome 9 of Holstein cattle (chr9:15144012);

[0007] ② The marker contains a nucleotide sequence with a polymorphism of C / T at 15943608 bp of chromosome 9 of Holstein cattle (chr9:15943608);

[0008] ③ The marker contains a nucleotide sequence with a polymorphism of T / C at 82746084 bp of chromosome 12 of Holstein cattle (chr12:82746084);

[0009] ④ The marker contains a nucleotide sequence with polymorphism A / G at 51472324 bp of chromosome 17 of Holstein cattle (chr17:51472324);

[0010] ⑤ The marker contains a nucleotide sequence with a polymorphism of C / G at 52699731 bp of chromosome 17 of Holstein cattle (chr17:52699731);

[0011] ⑥ The marker contains a nucleotide sequence (chr21:18136765) with a polymorphism of G / T at 18136765 bp on chromosome 21 of Holstein cattle.

[0012] The above physical location corresponds to the cattle reference genome version number Bos taurus ARS-UCD1.2.

[0013] Furthermore, for the marker ①, the genotype of the polymorphic site contained is GG, and the number of mature follicles is higher in Holstein cattle with a genotype of AG;

[0014] For the marker ②, the number of mature follicles in Holstein cattle with the genotype CC of the polymorphic site is higher than that in Holstein cattle with the genotype TC;

[0015] For the marker ③, the number of mature follicles in Holstein cattle with the genotype of TC and CC at the polymorphic site is higher;

[0016] For the marker ④, the genotype of the polymorphic site contained is GG, and the number of mature follicles is higher in Holstein cattle with a genotype of AG;

[0017] For the marker ⑤, the genotype of the polymorphic site contained is CC, and the number of mature follicles is higher in Holstein cattle than in Holstein cattle with the genotype of GC;

[0018] Regarding the marker ⑥, the genotype of the polymorphic site contained is GG, and the number of mature follicles is higher in Holstein cattle with a genotype of TG.

[0019] In a second aspect, the present invention provides a SNP molecular marker combination associated with the number of mature follicles in Holstein cattle, wherein the marker combination comprises two or more of the markers ① to ⑥.

[0020] Preferably, the marker combination comprises markers ① to ⑥, chr9:15144012, chr9:15943608, chr12:82746084, chr17:51472324, chr17:52699731 and chr21:18136765.

[0021] In a third aspect, the present invention provides primers for amplifying the markers or a primer set for amplifying the marker combination.

[0022] In a fourth aspect, the present invention provides a detection reagent or a kit containing the primer or primer set.

[0023] In a fifth aspect, the present invention provides any of the following uses of the marker, the marker combination, the primer or primer set, or the detection reagent or kit:

[0024] (1) Used for identification, breeding and improvement of the number of mature follicles in Holstein cattle;

[0025] (2) Used for early prediction of the number of mature follicles in Holstein cattle;

[0026] (3) Used for molecular marker-assisted breeding of the trait of mature follicle number in Holstein cattle.

[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] The present invention screens molecular markers related to the trait of the number of mature follicles in Holstein cattle based on whole genome association analysis, utilizes the molecular markers, and through molecular marker-assisted breeding, retains individuals with favorable genotypes, eliminates individuals with unfavorable genotypes, and can significantly increase the number of mature follicles in the group, thereby achieving the purpose of improving the reproductive efficiency of excellent Holstein cows.

[0029] The present invention provides a SNP marker, a marker combination and a breeding method related to the trait of the number of mature follicles in Holstein cattle based on whole genome association analysis screening. By detecting SNP molecular markers, the number of mature follicles in Holstein cattle can be quickly read, low-cost and effectively predicted, which is of great significance in improving the reproductive efficiency of Holstein cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the technical roadmap of the present invention.

[0031] Figure 2 This is a SNP density map in a preferred embodiment of the present invention.

[0032] Figure 3 This is a SNP distribution diagram in a preferred embodiment of the present invention.

[0033] Figure 4 This is a phenotypic distribution diagram of the number of mature follicles in a preferred embodiment of the present invention.

[0034] Figure 5 This is a Manhattan plot of the genome-wide association analysis results of the number of mature follicles in Holstein cattle in a preferred embodiment of the present invention; the ordinate represents -lg of the association value, and each point represents a SNP site.

[0035] Figure 6 This is a QQ graph of the genome-wide association analysis of SNPs associated with the trait of mature follicle number in Holstein cattle in a preferred embodiment of the present invention.

[0036] Figure 7 GO and KEGG functional annotations of candidate genes associated with significant SNPs in the number of mature follicles in Holstein cattle in a preferred embodiment of the present invention.

[0037] Figure 8 This is a significant analysis of the genotype differences of SNPs related to the mature follicle number trait of Holstein cattle in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0038] The invention provides a SNP molecular marker related to the number of mature ovarian follicles of Holstein cattle, a marker combination and an application method thereof.

[0039] The present invention adopts the following technical scheme (technical route see Figure 1 ):

[0040] The present invention provides a method for screening candidate markers related to the trait of mature follicle number in Holstein cattle based on genome-wide association analysis, the method comprising the following steps:

[0041] Blood samples from multiple Holstein cattle were used for 10× resequencing (the data volume of each sample was not less than 41G). After preliminary genotype data processing, SNP markers were obtained. The collected data on the number of mature follicles in Holstein cattle were used for analysis based on the GMAT software whole genome association analysis method to screen significant SNP sites and annotated genes associated with the trait of mature follicles number, and integrate gene function annotations to screen SNP analysis markers related to the trait of mature follicles number in Holstein cattle.

[0042] Furthermore, after quality control of the whole genome association sequencing data, GMAT software was used to perform association analysis based on the LMM model to discover SNPs that were significantly associated with the number of mature follicles in Holstein cattle.

[0043] Table 1 Sequence information

[0044]

[0045] Table 2 Sequence information

[0046]

[0047] Furthermore, the present invention provides a combination of 20 molecular markers associated with the trait of the number of mature follicles in Holstein cattle, including at least one or a combination of the following 19 SNP markers and one InDel marker, wherein the SNP sites of the molecular markers are: located at position 111326833 on chromosome 4 (chr4:111326833), located at position 3793442 on chromosome 7 (chr7:37934428), located at position 418838 on chromosome 8 (chr8:4 18838), 657918 (chr8:657918), 44605830 (chr8:44605830), 14338175 (chr9:14338175), 15144012 (chr9:15144012), 15431515 (chr9:15431515), 15462358 (chr9:15462358), 15462370 15462370), 15943608 (chr9:15943608), 12659153 on chromosome 11 (chr11:12659153), 82746084 on chromosome 12 (chr12:82746084), 71037557 on chromosome 13 (chr13:71037557), 71037557 on chromosome 17 (chr17: :51472324), 52699731 (chr17:52699731), 42734734 (chr18:42734734) on chromosome 18, 18136765 (chr21:18136765) and 20533312 (chr21:20533312) on chromosome 21, and 49487727 (chr23:49487727) on chromosome 23. The polymorphisms of the SNP sites are G and A, C and T, C and A, G and A, T and G, A and G, A and G, A and T, G and A, C and T, T and C, T and C, C and G, A and G, C and G, G and A, G and T, A and G, C and T. The polymorphisms of the InDel site are - and AAG.

[0048] The present invention also provides a method for molecular marker-assisted breeding of Holstein cattle with high number of mature follicles: using the polymorphism of chr9:15144012 molecular marker, the influence of different genotypes on the number of mature follicles is GG>AG, so the Holstein cattle individuals with G / G genotype are retained; using the polymorphism of chr9:15943608 molecular marker, the influence of different genotypes on the number of mature follicles is CC>TC, so the Holstein cattle individuals with C / C genotype are retained; using the polymorphism of chr12:82746084 molecular marker, the influence of different genotypes on the number of mature follicles is TC>CC, so the Holstein cattle individuals with T / C genotype are retained; using the polymorphism of chr17:51472324 molecular marker, the influence of different genotypes on the number of mature follicles is GG>AG, so the Holstein cattle individuals with G / G genotype are retained; using the polymorphism of chr17:52699731 molecular marker, the influence of different genotypes on the number of mature follicles is CC>GC Therefore, the Holstein cattle individuals with C / C genotype were retained. Using the polymorphism of the chr21:18136765 molecular marker, the effect of different genotypes on the number of mature follicles was GG>TG, so the Holstein cattle individuals with G / G genotype were retained.

[0049] The present invention also provides application of the molecular marker or marker combination in molecular marker-assisted selection of the trait of mature follicle number in Holstein cattle.

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] Example 1 Development of molecular markers associated with the number of mature ovarian follicles in Holstein cattle

[0052] 1. Materials and Methods

[0053] 1.1 Experimental animals and sample collection

[0054] The herd of 116 Holstein cattle in this study was provided by Shandong Aux Animal Husbandry and Seed Co., Ltd. Blood samples were collected from the herd and the phenotypic data of the number of mature follicles were recorded.

[0055] 1.2 Whole-genome resequencing

[0056] The genomic DNA was extracted by magnetic bead method. The genomic DNA was extracted according to the conventional extraction steps. The extracted DNA was tested for integrity and purity. The DNA that met the requirements was retained, and the DNA that did not meet the requirements was re-extracted. The samples that still did not meet the requirements after re-extraction were discarded. The experiment was performed according to the standard protocol provided by BGI. For the qualified genomic DNA samples, the fragments of appropriate size were selected by gel electrophoresis, and then the library was constructed by PCR enrichment. After the library was constructed, Qubit was used for quantitative quality control. The qualified library was sequenced with DNBSEQ-T7, which was completed by Beijing Compson Biotechnology Co., Ltd. After DNBSEQ-T7 sequencing, base sequencing quality distribution analysis, base content distribution analysis and filtering of the raw image data (Raw reads) files obtained by high-throughput sequencing were performed.

[0057] The final sequence obtained by sequencing is re-aligned to the reference genome for subsequent analysis. The reference gene species is cattle, and the reference genome link is: cattle reference genome ARS-UCD1.2.

[0058] 1.3 Quality Control of Genomic Data

[0059] In order to obtain reliable GWAS results, PLINK1.9 software was used to perform quality control on the genotype data. The quality control conditions were as follows:

[0060] (1) Retain the SNP sites on the autosomes;

[0061] (2) Eliminate sites with a deletion rate greater than 10%;

[0062] (3) Eliminate sites with minor allele frequencies less than 0.05;

[0063] (4) Eliminate those samples that do not meet the Hardy-Weinberg test P value less than 10 -6 The location of .

[0064] After screening, a total of 8,947,628 SNP sites covering chromosomes 1-29 were obtained.

[0065] 1.4 Genome-wide association analysis

[0066] The GMAT software was used to conduct a genome-wide association analysis on the mature follicle number trait of Holstein cattle. The advantages of this software are described as follows. The current GWAS analysis software only considers the additive effect of genes, ignoring the non-additive effect, especially the interaction effect, resulting in incomplete analysis of complex traits; the research traits also focus on cross-sectional data collected at a certain time point, while ignoring longitudinal traits. The GMAT software has six modules, including single trait analysis module, multi-trait analysis module, and longitudinal data analysis module. Compared with traditional GWAS analysis software, it is richer in model richness and trait applicability, significantly improves the statistical test power, and is more conducive to mining significant markers associated with traits. In terms of calculation, the GMAT software is written in C++ language, calling the Eigen and MKL libraries, using block reading and block calculation technology to save memory, and using technical means such as feature decomposition relationship matrix, linear transformation genome estimation, EM and AI algorithm weighting, which makes the GAMT software extremely efficient, and the calculation efficiency can be increased by thousands of times compared with the Fast-LMM algorithm.

[0067] The model adopts a mixed linear model (LMM), which is widely recognized as the best GWAS analysis model at present because it can well correct the population structure and complex kinship within the population. In addition, the present invention adopts the repeatability model in the mixed linear model, adds permanent environmental effects to the model, and is more suitable for repeated measurement traits such as the number of mature follicles in Holstein cattle.

[0068]

[0069] is the phenotype vector, is the group structure effect, To test the marker effect, For polygenic effects, For permanent environmental effects, is the residual effect. K in the polygenic effect is the kinship matrix inferred by the markers.

[0070] 1.5 Candidate gene identification and functional annotation

[0071] The reference genome information of the corresponding species was downloaded from the ENSEMBL website, and the genes near the significant SNPs were annotated using ANNOVAR software.

[0072] Then the clusterProfiler package was used to perform gene function enrichment analysis on the annotated candidate genes according to the GO and KEGG databases.

[0073] 1.6 Association analysis between significant loci and traits and multiple comparisons

[0074] The association test between marker genotype and phenotype was performed using R4.2 software. The model is as follows:

[0075]

[0076] y is the phenotype vector, γ is the marker effect to be tested, Z is the association matrix of γ, is the residual effect. LSD method was used for multiple comparisons between different genotypes.

[0077] 2. Results and Analysis

[0078] 2.1 Sequencing data quality control and comparison with reference genome

[0079] The sample sequencing quality control data are shown in Table 3. The base type distribution test is mainly used to check whether there is AT and CG separation. As shown in Table 3, the average percentage of G and C in the sample is 45.09% of the total bases, the number of bases with a quality value greater than or equal to 20 accounts for 98.9% of the total bases, and the number of bases with a quality value greater than 30 accounts for 96.91% of the total bases. The average efficiency of the comparison between sample DNA and genomic DNA is 99.78%. This shows that the library construction and sequencing of the samples of this group are normal.

[0080] Table 3 Quality control statistics of sample sequencing data

[0081]

[0082] 2.2 Statistical analysis of the number of mature follicles in Holstein cattle

[0083] As shown in Table 4, the mean, maximum, minimum and coefficient of variation of the mature follicle number phenotype of 116 Holstein cows were statistically analyzed, and the corresponding individual number of mature follicles is shown in Table 4. Figure 2 The phenotypic distribution of mature follicle numbers is shown in Figure 4 .

[0084] Table 4 Statistical analysis of the number of mature follicles in Holstein cattle

[0085]

[0086] 2.3 GWAS analysis results

[0087] After quality control, 8,947,628 SNPs were retained for association analysis. The distribution of SNPs on different chromosomes is shown in Figure 3 , the general mixed linear model was used to associate the significant SNPs sites associated with the mature follicle number trait. The information of each significant site is shown in Table 5. Figure 5 and Figure 6 .

[0088] Table 5 Information on significant SNPs associated with the number of mature follicles in Holstein cattle

[0089]

[0090] 2.4 Identification and GO functional annotation of candidate genes related to the number of mature follicles

[0091] To further identify candidate genes related to the mature follicle number trait of Holstein cows, 25 candidate functional genes near the 20 significant loci SNPs were found: ABHD2, AGBL1, CYP26B1, NTRK3, AGBL1, ANXA10 etc. (Table 3, Figure 7 and Figure 8 ).

[0092] Membrane progesterone receptor α / β hydrolase domain protein 2 ( ABHD2 ) is located on chromosome 21 of Holstein cows. The protein it encodes is an acylglycerol lipase that catalyzes the hydrolysis of endogenous cannabinoid arachidonoylglycerol from cell membranes and belongs to the α / β hydrolase family. It plays an important role in fatty acid metabolism and cell signaling. ABHD2 It can hydrolyze triglycerides and other lipids, affecting cell energy metabolism, inflammatory response and endocrine function. Studies have shown that ABHD2 High expression in mammalian ovaries indicates that this protein plays a regulatory role in follicle maturation and the female reproductive cycle. ABHD2 The gene causes dysrhythmia of the estrous cycle, with females showing a shortened luteal phase and a longer estrus phase. ABHD2 The ovaries of the knockout mice showed polycystic ovary morphology with a large number of degenerating follicles, which could be restored by injection of gonadotropin. ABHD2 Females with the gene significantly increased the number of mature and fertile eggs released compared to their wild-type counterparts. ABHD2 Plays an important role in nongenomic steroid regulation of the female reproductive cycle.

[0093] ATP / GTP-binding protein-like 1 ( AGBL1 ) is located on chromosome 21 of Holstein cows. The protein it encodes is a glutamate decarboxylase that catalyzes the deglutamylation of polyglutamylated proteins. Ccp4 , involved in a variety of physiological processes within cells. As a hydrolase, AGBL1 It plays a key role in protein degradation and regulation of cell signaling. Studies have shown that AGBL1 It is particularly important in the reproductive system and plays an important role in the non-seasonal lambing of sheep, suggesting that it is related to fertility and may affect the development and maturation of eggs. In addition, it may also play a role in other biological processes such as cell cycle and metabolic regulation.

[0094] Cytochrome P450, family 26, subfamily B, polypeptide 1 ( CYP26B1 ) is located on chromosome 11 of Holstein cows and encodes a P450 cytochrome enzyme that degrades retinoic acid (RA). Cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism and the synthesis of cholesterol, steroids, and other lipids. Cyp26b1 It has been shown to play an important role in male germ cell meiosis, but its expression in the ovary is largely lost around embryonic day 12.5. Studies have shown that Cyp26b1 mRNA is expressed in granulosa cells of follicles at all stages of postnatal development.

[0095] Neurotrophic tyrosine receptor kinase 3 ( NTRK3 ) is located on chromosome 21 of Holstein cows and encodes a neurotrophic tyrosine receptor kinase ( NTRK ) family. This kinase is a membrane-bound receptor that, upon binding of neurotrophins, phosphorylates itself and members of the MAPK pathway. Signaling through this kinase leads to cell differentiation and may play a role in the development of proprioceptive neurons that sense body position. Studies have shown NTRK3 The copy number loss may be associated with prominent reproductive traits. NTRK3 It plays a vital role in follicular development, ovarian quality and infertility in humans and chickens.

[0096] 2.5 Validation and multiple comparisons of association analysis between significant loci and traits

[0097] Through the association test between marker genotype and phenotype, the significance test of these SNP marker genotypes can be further performed. By using the LSD method for multiple comparisons between different genotypes, combined with phenotypic data, the dominant allele genotype of each SNP marker can be determined. The results showed that there were two genotypes (AG and GG) in the Holstein cattle population at the chr9:15144012 site, among which the number of mature follicles in the GG Holstein cattle was 1.23 higher than that in the AG type (significant difference). There were two genotypes (TC and CC) in the Holstein cattle population at the chr9:15943608 site, among which the number of mature follicles in the CC Holstein cattle was 1.35 higher than that in the TC type (significant difference). There were two genotypes (TC and CC) in the Holstein cattle population at the chr12:82746084 site, among which the number of mature follicles in the TC Holstein cattle was 1.00 higher than that in the CC type (significant difference). There are two genotypes (AG and GG) in the Holstein cattle population at the chr17:51472324 site, among which the number of mature follicles in the AG Holstein cattle is 1.49 higher than that in the GG type (significant difference). There are two genotypes (GC and CC) in the Holstein cattle population at the chr17:52699731 site, among which the number of mature follicles in the CC Holstein cattle is 1.42 higher than that in the GC type (significant difference). There are two genotypes (TG and GG) in the Holstein cattle population at the chr21:18136765 site, among which the number of mature follicles in the GG Holstein cattle is 1.55 higher than that in the TG type (significant difference). The specific analysis results are shown in Table 6.

[0098] Table 6 Trait association analysis and multiple comparison results of different genotypes

[0099]

[0100] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of a SNP molecular marker associated with the number of mature follicles in Holstein cattle, or a primer for amplifying the marker, or a detection reagent or kit containing the primer: (1) Used for identification, breeding and improvement of the number of mature follicles in Holstein cattle; (2) Used for early prediction of the number of mature follicles in Holstein cattle; (3) Used for molecular marker-assisted breeding of the number of mature follicles in Holstein cattle; The marker contains a nucleotide sequence with polymorphism A / G at 15144012 bp of chromosome 9 of Holstein cattle; The above physical location corresponds to the cattle reference genome version number Bos taurus ARS-UCD1.

2.

2. The use according to claim 1, characterized in that: Holstein cows with the GG genotype of the polymorphic loci have a higher number of mature follicles than those with the AG genotype.

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