SNP (Single Nucleotide Polymorphism) molecular marker related to number of mature follicles of Holstein cattle, marker combination and application of SNP molecular marker

Abstract

The invention belongs to the technical field of molecular biology and molecular markers, and discloses an SNP molecular marker related to the number of mature follicles of Holstein cattle, a marker combination and application of the SNP molecular marker. The molecular marker related to the Holstein cattle mature follicle number character is screened on the basis of whole genome association analysis, the molecular marker is utilized, and the number of mature follicles of a group can be remarkably increased through molecular marker-assisted breeding, reservation of individuals with favorable genotypes and elimination of individuals with unfavorable genotypes, so that the number of mature follicles of the group is increased. Therefore, the purpose of improving the breeding efficiency of the excellent Holstein cows is achieved.

Description

[0001] This invention is a divisional application of a Chinese patent application with the application number CN202411877442.4 and the invention title "SNP Molecular Markers, Marker Combinations Related to the Number of Mature Follicles in Holstein Cows and Their Applications". Technical Field

[0002] This invention belongs to the technical fields of molecular biology and molecular marker technology. Specifically, it relates to SNP molecular markers, marker combinations related to the number of mature follicles in Holstein cows and their applications. Background Art

[0003] Holstein cows originated in the Netherlands and are the dairy cow breed with the longest history. 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 work of Holstein cows, embryo transfer technology can significantly improve the reproductive efficiency of excellent cows and is of great significance for the multiplication of improved Holstein cows. In embryo transfer technology, superovulation is a key technical link and is affected by various factors, with significant individual differences in response. Therefore, it is extremely important to select excellent Holstein cows with more mature follicles. It is very important to seek a rapid and effective method to improve the breeding means for the trait of the number of mature follicles in Holstein cows.

[0004] In traditional breeding, mostly based on phenotypic determination, the number of mature follicles is used to judge 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 viable follicles in dairy cows and found that their heritabilities were 0.38 and 0.34 respectively. Therefore, the number of mature follicles belongs to a trait with medium heritability. With the development of molecular biology, molecular marker-assisted selection has gradually been applied to breeding practice. Combining with genome-wide 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

[0005] The purpose of this invention is to provide SNP molecular markers, marker combinations related to the number of mature follicles in Holstein cows and their applications.

[0006] To achieve the purpose of this invention, in the first aspect, this invention provides SNP molecular markers related to the number of mature follicles in Holstein cows, and the markers are selected from any one of ①~⑥: ① The marker contains a nucleotide sequence with polymorphism of A / G at the 15144012 bp position on chromosome 9 of Holstein cows (chr9:15144012); ② The marker contains a nucleotide sequence with polymorphism of C / T at the 15943608 bp position on chromosome 9 of Holstein cows (chr9:15943608); ③ The polymorphism at the 82746084th bp of chromosome 12 in Holstein cattle of the said marker is a nucleotide sequence of T / C (chr12:82746084); ④ The polymorphism at the 51472324th bp of chromosome 17 in Holstein cattle of the said marker is a nucleotide sequence of A / G (chr17:51472324); ⑤ The polymorphism at the 52699731st bp of chromosome 17 in Holstein cattle of the said marker is a nucleotide sequence of C / G (chr17:52699731); ⑥ The polymorphism at the 18136765th bp of chromosome 21 in Holstein cattle of the said marker is a nucleotide sequence of G / T (chr21:18136765).

[0007] The above physical positions correspond to the bovine reference genome version Bos taurus ARS-UCD1.2.

[0008] Further, for the said marker ①, the Holstein cattle with the genotype GG at the polymorphic site have a higher number of mature follicles compared to those with the genotype AG; For the said marker ②, the Holstein cattle with the genotype CC at the polymorphic site have a higher number of mature follicles compared to those with the genotype TC; For the said marker ③, the Holstein cattle with the genotype TC at the polymorphic site have a higher number of mature follicles compared to those with the genotype CC; For the said marker ④, the Holstein cattle with the genotype GG at the polymorphic site have a higher number of mature follicles compared to those with the genotype AG; For the said marker ⑤, the Holstein cattle with the genotype CC at the polymorphic site have a higher number of mature follicles compared to those with the genotype GC; For the said marker ⑥, the Holstein cattle with the genotype GG at the polymorphic site have a higher number of mature follicles compared to those with the genotype TG.

[0009] In the second aspect, the present invention provides an SNP molecular marker combination related to the number of mature follicles in Holstein cattle, and the said marker combination comprises two or more of the said markers ① - ⑥.

[0010] Preferably, the said marker combination comprises the said markers ① - ⑥, chr9:15144012, chr9:15943608, chr12:82746084, chr17:51472324, chr17:52699731 and chr21:18136765.

[0011] In the third aspect, the present invention provides primers for amplifying the said marker or a primer set for amplifying the said marker combination.

[0012] In a fourth aspect, the present invention provides a detection reagent or kit containing the primer or primer set.

[0013] In a fifth aspect, the present invention provides any one of the following applications of the marker, the marker combination, the primer or primer set, or the detection reagent or kit: (1) For the identification, breeding and improvement of the number of mature follicles trait in Holstein cattle; (2) For the early prediction of the number of mature follicles trait in Holstein cattle; (3) For molecular marker-assisted breeding of the number of mature follicles trait in Holstein cattle.

[0014] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: Based on genome-wide association analysis, the present invention screens molecular markers related to the number of mature follicles trait in Holstein cattle. By using the molecular markers and through molecular marker-assisted breeding, individuals with favorable genotypes are retained and individuals with unfavorable genotypes are eliminated, which can significantly increase the number of mature follicles in the population, thereby achieving the purpose of improving the reproductive efficiency of excellent Holstein cows.

[0015] The present invention provides an SNP marker, a marker combination and a breeding method related to the number of mature follicles trait in Holstein cattle screened by genome-wide association analysis. By detecting SNP molecular markers, it is possible to quickly, low-cost and effectively predict the number of mature follicles in Holstein cattle, which is of great significance in improving the reproductive efficiency of Holstein cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the technical roadmap of the present invention.

[0017] Figure 2 It is the SNP density map in a preferred embodiment of the present invention.

[0018] Figure 3 It is the SNP distribution map in a preferred embodiment of the present invention.

[0019] Figure 4 It is the phenotypic distribution map of the number of mature follicles in a preferred embodiment of the present invention.

[0020] Figure 5 It is the 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 1 SNP locus.

[0021] Figure 6 It is the Q-Q plot of the genome-wide association analysis of SNPs related to the number of mature follicles trait in Holstein cattle in a preferred embodiment of the present invention.

[0022] Figure 7GO 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.

[0023] 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

[0024] 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.

[0025] The present invention adopts the following technical scheme (technical route see Figure 1 ): 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: 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.

[0026] 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.

[0027] Table 1 Sequence information Table 2 Sequence information Furthermore, the present invention provides 20 molecular marker combinations related to the trait of the number of mature follicles in Holstein cattle, including at least one or several combinations of the following 19 SNP markers and 1 InDel marker. The SNP loci of the molecular markers are: located at position 111326833 on chromosome 4 (chr4:111326833), located at position 37934428 on chromosome 7 (chr7:37934428), located at position 418838 on chromosome 8 (chr8:418838), position 657918 (chr8:657918), position 44605830 (chr8:44605830), located at position 14338175 on chromosome 9 (chr9:14338175), position 15144012 (chr9:15144012), position 15431515 (chr9:15431515), position 15462358 (chr9:15462358), position 15462370 (chr9:15462370), position 15943608 (chr9:15943608), located at position 12659153 on chromosome 11 (chr11:12659153), located at position 82746084 on chromosome 12 (chr12:82746084), located at position 71037557 on chromosome 13 (chr13:71037557), located at position 51472324 on chromosome 17 (chr17:51472324), position 52699731 (chr17:52699731), located at position 42734734 on chromosome 18 (chr18:42734734), located at position 18136765 on chromosome 21 (chr21:18136765), position 20533312 (chr21:20533312), located at position 49487727 on chromosome 23 (chr23:49487727). Among them, the polymorphisms of the SNP loci 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 polymorphism of the InDel locus is - and AAG.

[0028] The present invention also provides a method for molecular marker-assisted breeding of Holstein cattle with a high number of mature follicles: by using the polymorphism of the chr9:15144012 molecular marker, the influence of different genotypes on the number of mature follicles is GG>AG, so individuals of Holstein cattle with the G / G genotype are retained; by using the polymorphism of the chr9:15943608 molecular marker, the influence of different genotypes on the number of mature follicles is CC>TC, so individuals of Holstein cattle with the C / C genotype are retained; by using the polymorphism of the chr12:82746084 molecular marker, the influence of different genotypes on the number of mature follicles is TC>CC, so individuals of Holstein cattle with the T / C genotype are retained; by using the polymorphism of the chr17:51472324 molecular marker, the influence of different genotypes on the number of mature follicles is GG>AG, so individuals of Holstein cattle with the G / G genotype are retained; by using the polymorphism of the chr17:52699731 molecular marker, the influence of different genotypes on the number of mature follicles is CC>GC, so individuals of Holstein cattle with the C / C genotype are retained; by using the polymorphism of the chr21:18136765 molecular marker, the influence of different genotypes on the number of mature follicles is GG>TG, so individuals of Holstein cattle with the G / G genotype are retained.

[0029] The present invention also provides the application of the said molecular marker or marker combination in molecular marker-assisted selection of the trait of the number of mature follicles in Holstein cattle.

[0030] The following examples are used to illustrate the present invention, but are not used 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.

[0031] Example 1 Development of molecular markers related to the number of mature follicles in Holstein cattle 1. Materials and methods 1.1 Experimental animals and sample collection The 116 Holstein cattle population in this study was provided by Shandong Oaks Animal Husbandry Seed Industry Co., Ltd. Blood samples of this population were collected, and the phenotypic data of the number of mature follicles were recorded.

[0032] 1.2 Whole-genome resequencing Genomic DNA was extracted by the magnetic bead method. Genomic DNA was extracted according to the conventional extraction steps. The extracted DNA was subjected to integrity and purity detection. The DNA that met the requirements was retained, and the DNA that did not meet the requirements was re-extracted. Samples that still did not meet the requirements after re-extraction were discarded. The experiment was carried out according to the standard protocol provided by BGI. For the qualified genomic DNA samples, fragments of appropriate size were selected by gel electrophoresis, and then PCR enrichment was used to construct a library. After the library construction was completed, Qubit was used for quantitative quality control. The qualified library was sequenced with DNBSEQ-T7, and this sequencing was completed by Beijing Compomics 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 carried out.

[0033] The final sequences obtained by sequencing were realigned to the reference genome, and then subsequent analysis was carried out. The reference gene species was cattle, and the reference genome link was: Bos taurus reference genome ARS-UCD1.2.

[0034] 1.3 Quality control of genomic data To obtain reliable GWAS results, PLINK1.9 software was used to perform quality control on the genotype data, and the quality control conditions were as follows: (1)Retain SNP sites on autosomes; (2)Exclude sites with a missing rate greater than 10%; (3)Exclude sites with a minor allele frequency less than 0.05; (4)Exclude sites that did not meet the Hardy-Weinberg test with a P-value less than 10 -6 .

[0035] After screening, a total of 8,947,628 SNP sites covering chromosomes 1-29 were finally obtained.

[0036] 1.4 Genome-wide association study The whole genome association analysis of the number of mature follicles in Holstein cattle was carried out using the GMAT software, and the advantages of this software are described as follows. Current GWAS analysis software only considers the additive effects of genes and ignores the non-additive effects, especially the interaction effects, resulting in incomplete analysis of complex traits. The studied traits also focus on cross-sectional data collected at a certain time point and ignore longitudinal traits. The GMAT software has six major modules, including single-trait analysis module, multi-trait analysis module, longitudinal data analysis module, etc. It is richer in model richness and trait applicability compared with traditional GWAS analysis software, significantly improving the statistical test power and being more conducive to mining significant markers associated with traits. In terms of calculation, the GMAT software is written in C++ language, calls the Eigen and MKL libraries, uses block reading and block calculation techniques to save memory, and also uses technical means such as eigen decomposition relationship matrix, linear transformation genomic estimates, EM and AI algorithm weighting, making the GAMT software have extremely high operation efficiency, and the calculation efficiency can be increased by thousands of times compared with the Fast-LMM algorithm.

[0037] The model adopts the 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. And the present invention adopts the repeatability model in the mixed linear model, adding the permanent environmental effect, which is more applicable to repeated measurement traits such as the number of mature follicles in Holstein cattle.

[0038] is the phenotypic vector, is the population structure effect, is the marker effect to be tested, is the polygenic effect, is the permanent environmental effect, is the residual effect. K in the polygenic effect is the kinship matrix inferred by the markers.

[0039] 1.5 Candidate gene identification and functional annotation Download the reference genome information of the corresponding species on the ENSEMBL website and use the ANNOVAR software to annotate the genes near the significant SNPs.

[0040] Then use the clusterProfiler package to perform gene function enrichment analysis on the annotated candidate genes according to the GO and KEGG databases.

[0041] 1.6 Association analysis and multiple comparisons between significant loci and traits Use the R4.2 software to perform the association test between marker genotypes and phenotypes. The model is as follows: Let \(y\) be the phenotypic vector, \(\gamma\) be the marker effect to be tested, and \(Z\) be the incidence matrix of \(\gamma\), and \(\epsilon\) be the residual effect. The least significant difference (LSD) method was used for multiple comparisons between different genotypes.

[0042] 2. Results and Analysis 2.1 Sequencing data quality control and alignment results with the reference genome The sequencing quality control data of the samples are shown in Table 3. The detection of base type distribution is mainly used to check for AT and CG segregation. As shown in Table 3, the average percentages of G and C in the total bases of the samples are 45.09%, the number of bases with a quality value greater than or equal to 20 accounts for 98.9% of the total number of bases, and the number of bases with a quality value greater than 30 accounts for 96.91% of the total number of bases. The average alignment efficiency between the sample DNA and the genomic DNA is 99.78%. This indicates that the library construction and sequencing of the samples in this population are normal.

[0043] Table 3 Sequencing data quality control statistics of samples

[0044] 2.2 Statistical analysis of the number of mature follicles in Holstein cows As shown in Table 4, the mean, maximum, minimum, and coefficient of variation of the phenotypes of the number of mature follicles in 116 Holstein cows were statistically analyzed, and the corresponding number of individuals for the number of mature follicles is shown in Figure 2 . The phenotypic distribution map of the number of mature follicles is shown in Figure 4 .

[0045] Table 4 Statistical analysis of the phenotypes of the number of mature follicles in Holstein cows

[0046] 2.3 GWAS analysis results A total of 8,947,628 SNPs retained after quality control were used for association analysis. The distribution of these SNP loci on different chromosomes is shown in Figure 3 . Using the general linear mixed model, significant SNP loci associated with the trait of the number of mature follicles were identified. The information of each significant locus is shown in Table 5, Figure 5 and Figure 6 .

[0047] Table 5 Information of significant SNP loci related to the number of mature follicles in Holstein cows

[0048] 2.4 Identification of candidate genes related to the trait of the number of mature follicles and GO functional annotation To further identify candidate genes related to the trait of the number of mature follicles in Holstein cows, 25 candidate functional genes located near SNPs at 20 significant loci were found, namely: ABHD2, AGBL1, CYP26B1, NTRK3, AGBL1, ANXA10 etc. (Table 3, Figure 7 and Figure 8 ).

[0049] Membrane progesterone receptor alpha / beta hydrolase domain-containing protein 2 ( ABHD2 ) is located on chromosome 21 of Holstein cows. The protein it encodes is an acylglycerol lipase that can catalyze the hydrolysis of the endogenous cannabinoid arachidonoyl glycerol from cell membranes and belongs to the alpha / beta hydrolase family. It plays an important role in fatty acid metabolism and cell signaling. ABHD2 It can hydrolyze triglycerides and other lipids, affecting the energy metabolism, inflammatory response, and endocrine function of cells. Research shows that ABHD2 high expression in the mammalian ovary indicates that this protein plays a regulatory role in follicle maturation and the female reproductive cycle. Knockout of ABHD2 gene leads to disrupted estrous cycle rhythm, with females showing a shortened luteal phase and a longer duration in the estrus phase. The ovaries of knockout ABHD2 gene present characteristics similar to polycystic ovary morphology, accompanied by a large number of degenerative follicles, and these follicles can be restored by injecting gonadotropins. In addition, females with knockout ABHD2 gene significantly increase the number of mature and fertile eggs discharged compared to their wild-type counterparts. Therefore, ABHD2 plays an important regulatory role in the non-genomic steroid regulation of the female reproductive cycle.

[0050] ATP / GTP-binding protein-like 1 ( AGBL1 ) is located on chromosome 21 of Holstein cows. The protein it encodes is a glutamate decarboxylase that can catalyze the deglutamylation of polyglutamylated proteins, also known as Ccp4 , and is involved in various physiological processes within the cell. As a hydrolase, AGBL1 plays a key role in protein degradation and regulation of cell signaling. Research shows that AGBL1 is particularly important in the reproductive system and plays an important role in non-seasonal lambing in sheep, indicating its association with fertility and potentially affecting 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.

[0051] Cytochrome P450, family 26, subfamily B, polypeptide 1 ( CYP26B1) Located on chromosome 11 of Holstein cows, the encoded protein is a P450 cytochrome enzyme that can degrade 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 mostly lost around embryonic day 12.5. Studies have shown that Cyp26b1 mRNA is expressed in granulosa cells of follicles at all postnatal developmental stages.

[0052] Neurotrophic tyrosine receptor kinase 3 ( NTRK3 ) is located on chromosome 21 of Holstein cows, and the encoded protein is a member of the neurotrophic tyrosine receptor kinase ( NTRK ) family. This kinase is a membrane-bound receptor that phosphorylates itself and members of the MAPK pathway after binding to neurotrophins. 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 that NTRK3 copy number deletions may be associated with prominent reproductive traits, NTRK3 and play a crucial role in follicle development, ovarian quality, and infertility in humans and chickens.

[0053] 2.5 Verification of the association between significant loci and traits and multiple comparisons By testing the association between the marked genotypes and phenotypes, the significance test of these SNP marker genotypes can be further carried out. By using the LSD method for multiple comparisons between different genotypes and combining the phenotypic data, the dominant allele genotypes 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 locus. The number of mature follicles in GG-type Holstein cattle was 1.23 higher than that in AG-type (the difference was significant). There were two genotypes (TC and CC) in the Holstein cattle population at the chr9:15943608 locus. The number of mature follicles in CC-type Holstein cattle was 1.35 higher than that in TC-type (the difference was significant). There were two genotypes (TC and CC) in the Holstein cattle population at the chr12:82746084 locus. The number of mature follicles in TC-type Holstein cattle was 1.00 higher than that in CC-type (the difference was significant). There were two genotypes (AG and GG) in the Holstein cattle population at the chr17:51472324 locus. The number of mature follicles in AG-type Holstein cattle was 1.49 higher than that in GG-type (the difference was significant). There were two genotypes (GC and CC) in the Holstein cattle population at the chr17:52699731 locus. The number of mature follicles in CC-type Holstein cattle was 1.42 higher than that in GC-type (the difference was significant). There were two genotypes (TG and GG) in the Holstein cattle population at the chr21:18136765 locus. The number of mature follicles in GG-type Holstein cattle was 1.55 higher than that in TG-type (the difference was significant). The specific analysis results are shown in Table 6.

[0054] Table 6 Results of trait association analysis and multiple comparisons of different genotypes

[0055] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

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 51472324 bp of chromosome 17 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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