CN113684206B - Application of SNP molecular marker in identification of high-reproductive-performance dairy cows and auxiliary breeding - Google Patents
Application of SNP molecular marker in identification of high-reproductive-performance dairy cows and auxiliary breeding Download PDFInfo
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Abstract
The invention relates to application of SNP molecular markers in identifying high-reproductive-performance cows and auxiliary breeding, belongs to the technical field of biochemical genetic breeding, and particularly provides application of SIGLEC12 genes serving as cow gestation marker genes by a method of cow 50K SNP chip and whole genome association analysis, and provides information of a group of haplotypes of seventh exons of the SIGLEC12 genes by related molecular biology techniques, and can be used for selecting cow individuals with dominant gestation period genotypes. The breeding enterprises can improve the frequency of the dominant genotype of the SIGLEC12 gene gestation period in the dairy cow group by selecting dominant genotype individuals for seed reserving, thereby optimizing the gestation period of the dairy cow group, improving the reproduction efficiency of the dairy cows, reducing the breeding cost and increasing the economic benefit. The invention provides a new method for accelerating the gestation period genetic improvement of cows.
Description
Technical Field
The invention belongs to the technical field of dairy cow breeding markers, and particularly relates to an SNP molecular marker, a primer sequence for detecting the SNP molecular marker, a kit containing the primer sequence, and application of the SNP molecular marker, the primer and the kit in identifying dairy cows with high reproductive performance and assisting breeding.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
With the growth of the world population, the united nations grain and farming organization (FAO) predicts that the global demand for human consumption of milk in year 2050 will increase by 50%. For decades, dairy production systems have provided a series of selection methods for improving milk yield, reducing land cost, management cost and the like, and the breeding of cows tends to be intensified more and more. However, with the high milk yields of intensive breeding, there are increasing problems with animal health, especially with regard to reproduction, such as resting or no oestrus, reduced post-partum ovarian activity, reduced conception rates, increased elimination rates and mortality, and a progressive reduction in the reproductive capacity of cows as a whole (Gaddis et al 2016; gutierrez-Reinoso et al 2021).
Good reproductive performance is a critical factor in ensuring profitability in dairy cow production systems (shollo et al, 2014). Calving intervals are often used as an indicator for assessing cow reproductive performance (Roche et al, 2017). Calving interval is a complex trait with low heritability (Berry et al, 2014), meaning that only a small portion of phenotypic variation is explained by measurable additive genetic variances (Berry and Evans, 2014). In contrast, pregnancy cycles have moderate genetic power, with a genetic transmission of 0.33 to 0.62 (Hansen et al 2004;Norman et al, 2009), meaning that a relatively accurate prediction of phenotype can be achieved if a substantial proportion of additive genetic variation in the pregnancy cycle is predictable. Since the separation of homologous chromosomes during gametogenesis is relatively random, it is not possible to achieve a high accuracy prediction of genetic value based solely on pedigree information. Therefore, the genotype-based prediction produces a more accurate genetic estimate that can be used as an index for evaluating reproductive performance of cows.
The gestation period measures the critical stages of fetal development in a mammal from pregnancy to delivery. Events occurring during the gestation period have a significant impact on the health, productivity and fertility of the offspring. Abnormalities in the pregnancy cycle will lead to premature or outdated production, leading to acute and long-term health problems. Pregnancy cycle is highly correlated with health, productivity and reproductive performance. Prolonged pregnancy cycles can lead to increased fetal weight, reduced pregnancy rates, and dystocia (Vieira-Neto et al, 2017). The milk cow with short gestation period in a reasonable range has higher reproduction efficiency, which means lower raising cost and management cost, higher effective calving capacity and higher income for pastures. 25 SNP sites in the holstein cattle population, which are significantly correlated with gestation period and distributed over 6 QTL regions, were identified, with the most significant SNP sites downstream of the ZNF613 gene of chromosome 18, explaining a genetic variation of 1.37% (Purfield et al, 2019).
Studies have shown that the 274-281 day gestation period is optimal for the Holstein cattle population, and that milk yield is maximized, so that calving difficulty, perinatal mortality and the like are minimized. There is still room for optimizing gestation period in Holstein cattle population in our country. However, there is little research on the genetic mechanism of the gestation period of cattle, and related development work of molecular markers is urgent to be enhanced. The whole genome association analysis method (GWAS) is a powerful means for analyzing complex trait genetic mechanisms and locating key candidate molecular marker loci, and plays an important role in analyzing quality trait and quantitative trait genetic mechanisms of model species and non-model species. Sialic acid binding immunoglobulin-like lectin 13 (SIGLEC 12) belongs to the cell surface protein family of the immunoglobulin superfamily. They mediate protein-carbohydrate interactions by selectively binding to different sialic acid fragments on glycolipids and glycoproteins. The SIGLEC12 gene is obviously related to calving traits of Holstein cows, and the existing research shows that the SIGLEC12 gene is expressed in human placenta.
Disclosure of Invention
Based on the research background, the invention aims to provide a genetic improvement method for shortening the gestation period of cattle, and in order to achieve the technical aim, the invention designs and screens molecular marker-assisted breeding related to the gestation period of cattle so as to improve the reproduction efficiency. The prior study shows that the SIGLEC12 gene is obviously related to the calving character of Holstein cows and possibly participates in the initiation of delivery. The invention takes SIGLEC12 gene as cow gestation period candidate gene, researches the relativity of mutation site and cow breeding character, and obtains the following technical scheme:
the invention firstly provides an SNP molecular marker, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO:4, wherein the single nucleotide site at 481bp of the sequence is T or G, the single nucleotide site at 485bp is G or A, and the single nucleotide site at 531bp is C or T.
The method for improving the gestation period length of the group of cows according to the technical scheme of the invention comprises the following steps: the molecular marker related to the periodic character of the dairy cows in gestation period is obtained by a PCR method. In further researches, the invention provides a cow pregnancy cycle character related SIGLEC12 gene by a PCR method, and 3 SNP loci are identified on the 7 th exon of the gene by a Bovine SNP50 chip to form 1 haplotype. In the Holstein cattle population, the genotype of 3 SNP aiming at the 7 th exon of the SIGLEC12 gene is subjected to correlation analysis with the gestation period long and short genome breeding value of cattle in the population, and the analysis result shows that the haplotype is obviously related to the gestation period of the dairy cows. According to the identification result of the SNP locus, breeding personnel can select favorable haplotype individuals for seed reservation, thereby improving the gestation period character of the dairy cow population and improving the reproduction efficiency.
The SNP identification method adopted by the invention is as follows: genotyping is carried out in a dairy cow population by using a Bovine SNP50 chip, and 3 SNP loci (chr18: g.57591390T > G, chr18: g.57591394G > A, chr18: g.57591440C > T) exist on the 7 th exon of the SIGLEC12 gene (the coordinate position of the SNP loci is standard with Bovine reference genome UMD 3.1) (figure 1), and mutation loci are completely linked to form a haplotype combination TGC/GAT; the correlation analysis of the genotype of the dairy cow population and the estimated breeding value of the dairy cow gestation period genome proves that the haplotype combination is obviously related to the dairy cow gestation period.
Based on the research results, the SNP molecular marker in the 7 th exon of the SIGLEC12 gene can be used as a marker for identifying high-reproduction-character cows and assisting in breeding, can be used for screening parent individuals with shorter gestation period, effectively shortens the breeding period and saves breeding resources. In addition, the related detection reagent of the molecular marker can be developed into related products for Chinese Holstein cow breeding.
The beneficial effects of the above technical scheme are:
1. the invention utilizes the information of 3 SNP loci on 7 th exons of bovine SIGLEC12 genes, and adopts related molecular biology technology to identify genotypes of dairy cows at the 3 loci, thereby realizing early selection of genotype individuals with short gestation period.
2. The invention identifies 1 group of haplotypes formed by 3 SNP combinations, detects the genotype of the SIGLEC12 gene of the individual dairy cow at the locus through a molecular biology related technology, selects favorable haplotype individual seed reserving through the correlation analysis of the estimated breeding value of the gestation periodic character of the dairy cow, and can improve the frequency of the SIGLEC12 gene genotype in the dairy cow group in the short gestation period, thereby improving the breeding efficiency of the dairy cow group, reducing the breeding cost of pastures and improving the breeding benefit. The invention provides a new method for the gestation period genetic improvement of cows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows the first generation sequencing result of 3 SNP sites on the 7 th exon sequence of SIGLEC12 gene;
FIG. 2 is a schematic diagram of the amino acid sequence and predicted secondary structure of the wild type bovine SIGLEC12 gene;
FIG. 3 is a schematic diagram of amino acid sequence and predicted secondary structure of bovine SIGLEC12 gene mutant.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As introduced by the background technology, the length of the gestation period is related to the health, the productivity and other indexes of offspring, and the reasonable shortening of the gestation period can effectively improve the reproduction efficiency of cows and reduce the feeding cost. In order to realize genetic improvement breeding for shortening the gestation period of the cow, the invention screens molecular markers related to the gestation period of the cow, confirms that haplotypes consisting of 3 SNP on the 7 th exon of the SIGLEC12 gene are related to the gestation period of the cow, can be used as auxiliary breeding markers, and improves the reproductive efficiency of the cow. Based on the results, the invention provides application of the SNP molecular marker in identifying Chinese Holstein cows with high reproductive performance and auxiliary breeding.
In a first aspect of the invention, a SNP molecular marker is provided, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 4, the single nucleotide site at the 132 th bp of the sequence is T or G, the single nucleotide site at the 136 th bp is G or A, and the single nucleotide site at the 182 th bp is C or T.
According to the results of the present invention, SNP site chr18:g57591390T > G on exon 7 of SIGLEC12 gene belongs to synonymous mutation, where both the wild-type codon TCT and mutant codon TCG encode serine (S). SNP locus chr18:g57591394G > A belongs to missense mutation, the wild codon GAG where it is located codes for glutamic acid (E), and mutant codon AAG codes for lysine (K); SNP locus chr18:g.57591440C > T belongs to a missense mutation, the wild codon GCG where it is located codes for alanine (A), and the mutant codon GTG codes for valine (V).
In a second aspect of the present invention, the primer for detecting the SNP molecular marker of the first aspect, wherein the upstream primer sequence is shown as SEQ ID NO.2, and the downstream primer sequence is shown as SEQ ID NO. 3.
The SNP molecular marker of the first aspect is positioned at the 7 th exon of the SIGLEC12 gene, and the nucleotide sequence of the 7 th exon is shown as SEQ ID NO. 1. The SNP molecular marker is an amplification product obtained by amplifying the 7 th exon of the bovine SIGLEC12 gene by a PCR method, and the sequence of the amplification product is shown as SEQ ID NO. 4 or SEQ ID NO. 5, and the size of the amplification product is 421bp.
In the SNP molecular marker, the sequence shown in SEQ ID NO. 4 represents a dominant trait, and the sequence shown in SEQ ID NO. 5 represents a disadvantaged trait.
In a third aspect of the invention, a kit comprising a primer according to the second aspect.
Preferably, the kit further comprises one or more of sampling tools, genomic DNA extraction reagents or PCR reaction reagents.
According to a fourth aspect of the invention, there is provided the use of the SNP molecular marker according to the first aspect for identifying cows with high reproductive performance.
Preferably, the SNP molecular marker is a combination of the three SNP loci, and the SNP loci are positioned at the base at 3401bp, 3405bp or 3451bp of the SIGLEC12 gene sequence; wherein the base at 3401bp is G, the base at 3401bp is A or the base at 3451bp is T, namely g.+3401T > G, g.+3405G > A, g.+3451C > T; in the scheme with better effect, the combination of the sites is haplotype GAT serving as a marker with high breeding performance. When the SIGLEC12 gene sequence is expressed as GAT haplotype, the GAT haplotype is the dominant haplotype of the gestation period character of the dairy cows.
Preferably, the cow is one of Chinese Holstein cow, sanhe cow, xinjiang brown cow or grassland red cow. In a further preferred embodiment, the SNP molecular marker is used for identifying Chinese Holstein cows with high breeding performance.
The breeding performance of the livestock in the field comprises a distribution rate, a conception rate, a no-return rate, a mating index, a calving rate, a calving index, a calf survival rate, a breeding survival rate and the like. In the fourth aspect, the high reproductive performance refers specifically to the length of gestation period of the Chinese Holstein cows, and it should be clear that the SNP molecular markers are used for identifying cows with reasonable and shorter gestation period, and the cows with GAT haplotype phenotype have shorter gestation period.
In a fifth aspect, the invention provides the detection primer of the SNP molecular marker in the second aspect and the application of the kit in the fourth aspect in identifying Chinese Holstein cows with high breeding performance or Chinese Holstein cow assisted breeding.
In a sixth aspect of the present invention, there is provided a method for identifying high-breeder chinese Holstein cows, characterized by:
(1) Extracting genome DNA of the Chinese Holstein cows to be detected;
(2) Amplifying the 7 th exon of the Chinese Holstein cow SIGLEC12 gene by using the primer pair in the second aspect by using the genomic DNA in the step (1) as a template;
(3) Detecting PCR amplification products, and if the amplification sequence comprises the SNP molecular marker in the first aspect, the cattle to be detected belong to individuals with high reproductive performance.
In a preferred scheme, in the amplified sequence, when the genotype of the 481bp, 485bp and 531bp is GAT, the amplified sequence is judged to be an individual with high breeding performance.
In a seventh aspect, the present invention provides an auxiliary breeding method for chinese holstein cows, which includes breeding a holstein cow individual, which is a dominant haplotype obtained by the identification method of the sixth aspect, as a parent.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
The 7 th exon, the molecular marker and the application of the invention affecting the cow gestation period SIGLEC12 gene are described in detail below.
The invention relates to a 7 th exon affecting a cow gestation period SIGLEC12 gene, and a molecular marker and application thereof, wherein the specific scheme is as follows:
1. screening SIGLEC12 gene as cow gestation period candidate gene
(1) Blood or hair follicle samples from 2709 Holstein cows were collected in 11 large scale farms
Holstein Niu Yangpin was collected in 11 pastures across the country, with the place of the pasture and the number of samples collected per pasture being shown in Table 1.
TABLE 1 pasture where samples were taken and number of samples taken
| Pasture site | Number of samples | Pasture site | Number of samples |
| Gansu Jinchang (golden Chang) | 294 | Hebei Chen Tai | 279 |
| Inner Mongolia Tongliao | 399 | Jiangsu dormitory migration | 285 |
| |
200 | |
200 |
| Shandong Linyi | 398 | Shandong Jinan province | 94 |
| Shandong Taian (Thailand mountain tea) | 96 | Eastern mountain camp | 268 |
| Guangdong Guangzhou province | 196 |
(2) The genotype of the collected samples was determined using a Illumina BovineSNP50 chip. Genotyping was performed by neotame, and genotypes of 47843 SNP marker loci were obtained in total.
(3) A gestation period genome breeding value of each individual is estimated based on the genotype. Genome-estimated breeding values were performed by new commute company.
(4) Based on genotype data using GEMMA software, whole genome association analysis was performed for gestational period genome breeding values, with the analysis model using a hybrid linear model, pasture and bovine month of gestation as covariates. Multiple test corrections were made using the FDR method. The association analysis result shows that the most obvious 3 SNP loci are positioned on the 7 th exon on the SIGLEC12 gene, and form a haplotype, and the significance P value is 2.63E-04.
Identification of dominant haplotype of exon 7 of SIGLEC12 Gene
Of the 2709 Holstein cattle collected, 2586 individuals with GAT/GAT pure and haplotypes were identified, with a mean of-0.699 for gestation period estimated breeding values, 123 individuals with TGC/GAT heterozygous haplotypes, with a mean of-0.024 for gestation period estimated breeding values, whereas in genomic genetic evaluation, the lower the gestation period genomic breeding value the better, therefore GAT is the dominant haplotype.
3. Partial sequence amplification of 7 th exon of bovine SIGLEC12 gene
(1) Bovine tail venous blood collection
Cattle with long gestation period and cattle with short gestation period are selected as test materials, and tail vein blood of the cattle is collected.
(2) Genomic DNA extraction
500. Mu.L of whole blood was taken, 500. Mu.L of STE lysis buffer was added, 50. Mu.L of 10% SDS, 5. Mu.L of proteinase K (20 mg/ml) were added in this order, and the whole blood was lysed at 56℃for about 3 hours until the lysate became clear. The same volume of saturated phenol (250. Mu.L), chloroform/isoamyl alcohol (24:1) (250. Mu.L) was added, gently swirled for 20min and centrifuged at 12000rpm for 10min. Taking the supernatant, repeating the steps until no protein layer exists between the aqueous phase and the organic phase. The supernatant was taken, added with chloroform/isoamyl alcohol in the same volume, gently shaken for 20min, and centrifuged at 12000rpm for 10min. Taking the supernatant, adding 1/10 volume of 3MNAAc (pH 5.2) and 2 times volume of cold absolute ethyl alcohol, shaking uniformly, standing at-20 ℃ for 20min, and centrifuging at 12500rpm for 20min. Nucleic acid was precipitated at the bottom of the tube. The supernatant was discarded and the precipitate was washed with 70% ethanol. The precipitate was collected and air dried until all ethanol was evaporated. Add 20. Mu.L TE (containing RNaseA) to dissolve DNA, leave it at 37℃for about 30min and store at 4 ℃. Detecting the DNA sample by 1% agarose gel electrophoresis, and detecting the concentration and the purity by an ultraviolet spectrophotometer.
(3) Primer design
A pair of specific primers was designed based on the gene sequence of bovine SIGLEC12 gene (GenBank accession number LOC 618463). Wherein, the liquid crystal display device comprises a liquid crystal display device,
the forward primer is SIGLEC12-5F:5'-GGGCGTGGGACAACTAAC-3'
The reverse primer is SIGLEC12-3F:5'-AGGGCTCAGCAGGAAAGG-3';
(4) Complex enzyme chain reaction
PCR amplification was performed using this primer, and the reaction system was as follows: 10 XBuffer 1. Mu.L, 2.5mM dNTP 0.8. Mu.L, 2.5mM MgCl 2 0.6. Mu.L, primer F (10. Mu.M) 0.1. Mu.L, primer R (10. Mu.M) 0.5. Mu.L, taq enzyme (5U/. Mu.L) 0.1. Mu.L, template 0.5. Mu.L, LCGreen saturated fluorescent dye 0.7. Mu.L, H 2 O was made up to 10. Mu.L. The amplification reaction was completed on the Applied Biosystem PCR system under the following reaction conditions: 95 ℃ for 5min;95 ℃ for 30s,59 ℃ for 30s and 72 ℃ for 1min;35 cycles; and at 72℃for 5min. Haplotypes of the PCR products were determined by Sanger sequencing.
The method comprises the steps of selecting a cow core group individual, detecting the gene chr18:g.57591390T > G, chr18:g.57591394G > A and chr18:g.57591440C > T genotypes of three sites by using the related molecular biology technology, selecting favorable haplotype individual seed reserving, improving the gestation cycle character of the cow group, improving the reproduction efficiency, reducing the cultivation cost, increasing the cultivation income, and laying a foundation for cultivating a new excellent cow line with high reproduction efficiency.
Example 2
The genotype and allele frequency distribution of 3 SNP loci of exon 7 of SIGLEC12 in a Holstein cattle population was examined by Sanger sequencing and the results are shown in Table 2, which includes Holstein cattle in multiple pastures in China. The detection result shows that 2 genotypes exist in Holstein cow groups in China; in all populations examined, the GAT/GAT haplotype combination frequency was 97.8%, the TGC/GAT haplotype combination frequency was 2.2%, and the GAT haplotype was the dominant haplotype (see Table 2).
TABLE 2 distribution results of bovine SIGLEC12 genes in Holstein cattle populations
Table 2 illustrates: the group materials are Holstein cattle groups in China, wherein: group 1 is the Holstein cow group in Gansu pasture, group 2 is the Holstein cow group in Hebei pasture, and group 3 is the Holstein cow group in inner Mongolia pasture.
To determine whether bovine SIGLEC12 haplotypes are associated with differences in gestation periods in cattle, 2709 holstein cattle were selected for association analysis of SIGLEC12 haplotypes with estimated breeding values of gestation periods. The average genomic breeding value of TGC/GAT haplotype individuals was-0.024, and the average genomic breeding value of GAT/GAT haplotype individuals was-0.699. Correlation of different haplotypes with bovine gestation cycle traits was analyzed using PLINK v1.90 software, and showed a significant correlation between haplotypes and genomic breeding values (P value 5.052 e-11).
The invention identifies 1 group of haplotypes formed by 3 SNP combinations, detects the haplotype of the SIGLEC12 gene of the individual dairy cow at the locus through a molecular biology related technology, selects favorable haplotype individual seed reserving through the correlation analysis of the estimated breeding value of the gestation period character of the dairy cow, and can improve the frequency of the dominant haplotype of the gestation period of the SIGLEC12 gene in the dairy cow group, thereby improving the reproduction efficiency of the dairy cow group, reducing the raising cost of pastures, improving the raising benefit and providing a new method for the gestation period genetic improvement of the dairy cows.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong Oryza animal husbandry Co.Ltd at Shandong agricultural sciences animal husbandry institute
<120> application of SNP molecular marker in identification of high reproductive performance dairy cows and auxiliary breeding
<130> 202123885
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aggaagaagt ctgtggagag agcgtcgagc caagatggcg actgcccagc gtcgcgtccc 180
gcgtcccggg tgagtgacgt gggcggcttg ccgtccagtg tccccaccgg acccctctgc 240
ctacacaggg gccgcggcag cctacttagc acctaagaaa aacgtgatct cccccacccc 300
gcccccgcct ccaagtttgc ttttcctctg gagtttcttg ttgcactcaa atcaccctct 360
cgtctttctg cactcgccgg agcctagaat tcaccgagtc ctgtccatct tccgtttagc 420
a 421
Claims (2)
1. The application of the SNP molecular marker in identifying Chinese Holstein cows with high reproductive performance is characterized in that the SNP molecular marker is chr18:g.57591390T > G, chr18:g.57591394G > A, chr18:g.57591440C > T, and the combination of three SNP loci is GAT haplotype, which is the dominant haplotype of the gestation period character of the cows;
the GAT haplotype phenotype cows have shorter gestation periods.
2. The use according to claim 1, wherein the primer for detecting the SNP molecular marker according to claim 1, wherein the upstream primer sequence is shown in SEQ ID NO.2 and the downstream primer sequence is shown in SEQ ID NO. 3.
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