CN117070642A - SNP molecular marker related to dairy cow production life and application thereof - Google Patents
SNP molecular marker related to dairy cow production life and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of dairy cow breeding markers, and particularly relates to a SNP molecular marker related to the production life of dairy cows and application thereof. The molecular marker is from an NDUFA9 gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, the 304bp position of the sequence is a mutation site, and the base is C or A. When the base of the mutation site is A, namely chr5:g105870613C > A, the mutation site is expressed as a character with long production life. According to the invention, 1 SNP locus is identified, the genotype of the NDUFA9 gene of the individual dairy cow is detected through a molecular biology related technology, and the frequency of the NDUFA9 gene long-production life genotype in the dairy cow group can be improved by selecting favorable genotype individual seed reserving through the correlation analysis of the estimated breeding value of the dairy cow production life trait, so that the service life of the dairy cow group is prolonged, the breeding cost is reduced, and the economic benefit of pastures is increased. The invention provides a new method for genetic improvement of the production life of cows.
Description
Technical Field
The invention belongs to the technical field of dairy cow breeding markers, and particularly relates to a SNP molecular marker related to the production life of dairy cows and application thereof.
Background
The production life of a cow refers to the time between calving and death or elimination of the cow at time 1. The production life of the dairy cows is closely related to the economic benefit of a cattle farm, and the longer the production life of the dairy cows is, the higher the milk yield of the dairy cows in the final production period is, so that the raising cost of the dairy cows in the replacement cattle stage can be compensated, and the raising income is improved. For a long time, the breeding of cows mainly emphasizes improving the productivity of cows, and the study on the production life of cows is relatively less. The production life of the dairy cows has no direct influence on production, but is closely related to the production cost, is a very important functional property, and is very important for increasing income and breeding work of dairy cows.
Studies show that the average service life of the German and American dairy cow groups is 3-4 lactation periods, and the maximum milk yield is expressed in the 4 th-7 th lactation period, so that most dairy cows are eliminated without fully exerting the maximum potential of lactation, thereby causing certain economic loss. When the utilization time of the Czochralski cows is increased from 3 to 4, the economic benefit is increased by 11 to 13 percent. However, the average number of cows in China is less than 3, so how to improve the production life of cows in China is a problem to be solved.
The genetic force reflects the magnitude relation of genetic action and environmental influence in the character expression in the specific character phenotype variation, and a proper breeding method can be determined by analyzing the genetic force, so that the estimation of the character genetic force in animal breeding work is particularly important. The scholars in China use different models such as a unisexual animal model, a unisexual male animal model, a multi-character animal model and the like to estimate that the production life and the genetic force of the Chinese Holstein cows are 0.047-0.069. In general, the genetic power of the production life of cows is low, and it is difficult for breeders to increase the production life of cows by a direct selection method. Therefore, the production life can be indirectly predicted through some characters which can be obtained earlier and have higher correlation with the life, so that the breeding value of the life is obtained in advance, and the genetic progress is effectively accelerated.
The whole genome selection can rapidly analyze the genetic value of individuals from the molecular level, so that the direct selection of genotypes is realized, the whole genome selection is combined with genotype, phenotype and genealogy data, the accuracy of genetic estimation can be improved, and the generation interval is reduced. Developed countries have applied whole genome selection to dairy cow breeding systems, but related studies on production life are currently less in our country or even developed countries.
Single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNP) refers mainly to DNA sequence polymorphism at the genomic level due to variation of a single nucleotide, including forms of single base transition, transversion, deletion, insertion, and the like. The distribution of SNPs in bovine genomes is quite extensive, and with the advancement of high-throughput sequencing technology, SNPs have become a new generation of molecular markers. The distribution positions of SNPs in a genome can be classified into gene coding region SNPs (cSNPs), intergenic SNPs (iSNPs) and peripheral SNPs (pSNPs), wherein cSNPs (functional markers) are novel molecular markers developed according to polymorphic motifs within functional genes that cause phenotypic trait variation. Such markers, due to functional motifs from within the gene, do not require further verification to determine the presence or absence of the target allele in different genetic contexts or in different bovine germplasm resources.
Disclosure of Invention
In order to overcome the problems, the invention provides SNP molecular markers related to the production life of cows and application thereof. In order to achieve the technical purpose, the molecular marker assisted breeding related to the production life of the dairy cows is designed and screened, so that the profitability of the dairy cows is improved. According to the invention, the NDUFA9 gene is used as a cow production life candidate gene, the correlation between the mutation site and the cow production life character is researched, and the following technical scheme is obtained:
in a first aspect of the invention, a SNP molecular marker related to the production life of dairy cows is provided, wherein the molecular marker is derived from an NDUFA9 gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, the 304bp position of the sequence is a mutation site, and the base is C or A.
Further the site is located at 105870613bp of bovine chromosome 5 sequence (bovine reference genome version Bos taurus UMD 3.3.1, gcf 000003055.6, chromosome number AC 000162.1).
Furthermore, when the base of the mutation site is A, that is, chr5:g105870613C > A, the mutation site is characterized by long production life.
In a second aspect of the present invention, there is provided a specific primer for detecting the SNP molecular marker described in the first aspect, comprising:
the upstream primer sequence is shown as SEQ ID NO. 2, and the downstream primer sequence is shown as SEQ ID NO. 3.
In a third aspect of the invention, there is provided a reagent or kit comprising the specific primer of the second aspect.
In a fourth aspect of the present invention, there is provided a method of identifying a long-life dairy cow, comprising:
(1) Extracting genome DNA of the dairy cows to be detected;
(2) Amplifying the 3' -UTR region of the dairy cow NDUFA9 gene by PCR reaction using the primer pair of the second aspect by using the genomic DNA of the step (1) as a template;
(3) Detecting the base type at the 304bp of the PCR amplified product fragment, judging whether the dairy cow to be detected belongs to an individual with long production life, and if the base at the 304bp of the amplified product fragment is A, determining that the dairy cow to be detected is the dairy cow with long production life.
In a fifth aspect of the present invention, there is provided a method for assisted breeding of cows, comprising breeding a cow individual having a dominant genotype obtained by the method for identifying a long-life cow according to the fourth aspect as a parent.
In a sixth aspect, the invention provides the use of the SNP molecular marker of the first aspect for identifying cows with long production life and for assisting in breeding.
In a seventh aspect, the invention provides the use of a reagent or kit of the specific primer of the third aspect for identifying cows with long production life and for assisting in breeding.
The invention provides a dairy cow production life property related NDUFA9 gene by a PCR method, and 1 SNP site is identified on the 3' -UTR of the gene by a Bovine SNP50 chip. In the Holstein cattle population, the genotype of 1 SNP on 3' -UTR of NDUFA9 gene and the genome breeding value of the production life trait of cattle in the population are subjected to correlation analysis, and the analysis result shows that the SNP is obviously related to the production life of cows. According to the identification result of the SNP locus, breeding personnel can select favorable genotype individuals for seed reservation, thereby improving the production life property of dairy cow groups and improving the economic benefit of pastures.
The SNP identification method adopted by the invention is as follows: genotyping was performed in the cow population by Bovine SNP50 chip, and found that 1 SNP (chr 5: g.105870613c > a) was present on 3' -UTR of NDUFA9 gene (fig. 1); through the association analysis of the dairy cow population genotype and the estimated breeding value of the dairy cow production life trait genome, the SNP locus is proved to be obviously related to the dairy cow production life trait.
Based on the research results, the SNP molecular marker on the 3' -UTR of the NDUFA9 gene can be used as a marker for identifying the production life character of cows and assisting breeding, can be used for screening parent individuals with longer production life, effectively improves the service life of cows, saves the breeding cost and improves the pasture income. 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 1 SNP locus on the 3' -UTR of the bovine NDUFA9 gene, adopts the related technology of molecular biology to identify the genotype of the dairy cow at the locus, and realizes the early selection of genotype individuals with long production life.
2. According to the invention, 1 SNP locus is identified, the genotype of the NDUFA9 gene of the individual dairy cow is detected through a molecular biology related technology, and the frequency of the NDUFA9 gene long-production life genotype in the dairy cow group can be improved by selecting favorable genotype individual seed reserving through the correlation analysis of the estimated breeding value of the dairy cow production life trait, so that the service life of the dairy cow group is prolonged, the breeding cost is reduced, and the economic benefit of pastures is increased. The invention provides a new method for genetic improvement of the production life 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 is a diagram of Sanger sequencing peaks at the chr5:g105870613C > A mutation site on the 3' -UTR sequence of the NDUFA9 gene.
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 production life is related to a plurality of indexes such as the production capacity of the dairy cows, and the increase of the production life of the dairy cows in a reasonable range can effectively prolong the production life of the dairy cows, increase the service life, improve the pasture income and reduce the raising cost. In order to realize genetic improvement breeding for prolonging the production life, the invention screens molecular markers related to the production life of cows, confirms that 1 SNP locus on 3' -UTR of NDUFA9 gene is obviously related to the production life of cows, can be used as an auxiliary breeding marker, and improves the production life and service life of cows. Based on the results, the invention provides application of the SNP molecular marker in identifying Chinese Holstein cows with long production life and auxiliary breeding.
As introduced by the background technology, the length of the production life is related to a plurality of indexes such as the production capacity of the dairy cows, and the increase of the production life of the dairy cows in a reasonable range can effectively prolong the production life of the dairy cows, increase the service life, improve the pasture income and reduce the raising cost. In order to realize genetic improvement breeding for prolonging the production life, the invention screens molecular markers related to the production life of cows, confirms that 1 SNP locus on 3' -UTR of NDUFA9 gene is obviously related to the production life of cows, can be used as an auxiliary breeding marker, and improves the production life and service life of cows. Based on the results, the invention provides application of the SNP molecular marker in identifying Chinese Holstein cows with long production life and auxiliary breeding.
In a first typical embodiment of the invention, a SNP molecular marker related to the production life of cows is provided, wherein the molecular marker is derived from a NDUFA9 gene, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 1, the 304bp of the sequence is a mutation site, and the base is C or A.
Wherein sequence 1 (SEQ ID NO: 1):
GCCTGACGGAAGGAAACTGAAATCCTCCAGCATTTCAGAGAACAGAAAGATGTATGTGTCACTATTATAAATAATAAGAAAATTAAAATCCTCCAGCATTTCAGAGAATAAAAGATGTACGTGGATTAAATATATACAATTAAGGAAAATTGTGCTCAAATTCTAGGTGGTTGAGTTTCTCCCTTTGTAAATCAAGAAAGAAAACGCGCCCACAGAATGTCATCTCAGGTTTTAATAGAGGCTCTCATTCACTATATACAGGATTCTCCGCCAAAGGTGGCCTGGTTGGTAGCAACCCAAACACCACCAAAAGCCGCGGGCAGCTCAGGGGCCACTAGTGGGAATTGTCTTGGCCGGCTGCACGTCCTCGATCTCAGAAGACAGCCAGCGGTAGGTGCGGTGACGCCGCAGCACCTCGATGGCCTTGAGTTCCAGGGGCGTGGCCTCAACGCCCAGGTCTTCCAAGCCTGGCAGGTGAGGCAGGATCTTGTCTGTGGTGTGAATCTGCGGATAGACAAGAGGCACAGAGAGCAC。
further the site is located at 105870613bp of bovine chromosome 5 sequence (bovine reference genome version Bos taurus UMD 3.3.1, gcf 000003055.6, chromosome number AC 000162.1).
Furthermore, when the base of the mutation site is A, that is, chr5:g105870613C > A, the mutation site is characterized by long production life.
Furthermore, the SNP molecular marker is an amplification product obtained by amplifying the 3' -UTR of the bovine NDUFA9 gene by a PCR method.
According to the research result of the invention, 1 SNP locus chr5:g.105870613C > A on 3' -UTR of NDUFA9 gene belongs to the SNP mutation of regulatory region.
In a second exemplary embodiment of the present invention, there is provided a specific primer for detecting the above SNP molecular marker, comprising:
the upstream primer sequence is shown as SEQ ID NO. 2, and the downstream primer sequence is shown as SEQ ID NO. 3.
Wherein sequence 2 (SEQ ID NO: 2):
5’-GCCTGACGGAAGGAAACTGA-3’；
sequence 3 (SEQ ID NO: 3):
5’-GTGCTCTCTGTGCCTCTTGT-3’。
in a third exemplary embodiment of the invention, a reagent or kit is provided comprising the specific primer according to the second aspect.
In one or more embodiments, the kit further comprises one or more of a sampling tool, a genomic DNA extraction reagent, or a PCR reaction reagent.
In a fourth exemplary embodiment of the present invention, there is provided a method for identifying a long-life dairy cow, comprising:
(1) Extracting genome DNA of the dairy cows to be detected;
(2) Amplifying the 3' -UTR region of the dairy cow NDUFA9 gene by PCR reaction using the primer pair of the second aspect by using the genomic DNA of the step (1) as a template;
(3) Detecting the base type at the 304bp of the PCR amplified product fragment, judging whether the dairy cow to be detected belongs to an individual with long production life, and if the base at the 304bp of the amplified product fragment is A, determining that the dairy cow to be detected is the dairy cow with long production life.
In one or more embodiments, the cows are one of Chinese Holstein cows, sanhe cows, xinjiang brown cows or grassland red cows, more preferably Chinese Holstein cows.
In a fifth exemplary embodiment of the present invention, there is provided a method for assisted breeding of cows, which comprises breeding a cow individual having a dominant genotype by using the method for identifying a long-life cow according to the fourth aspect as a parent.
In a sixth exemplary embodiment of the present invention, there is provided the use of the SNP molecular marker described in the first aspect for identifying cows with long production life and for assisting in breeding.
In a seventh exemplary embodiment of the present invention, there is provided the use of a reagent or kit of the specific primer according to the third aspect for identifying cows with long production life and for assisting in breeding.
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 following describes the 3' -UTR of the NDUFA9 gene affecting the production life of cows and the molecular markers and application thereof in detail.
The invention relates to a 3' -UTR affecting dairy cow production life NDUFA9 gene and a molecular marker and application thereof, which comprises the following specific scheme:
1. screening NDUFA9 gene as cow production life candidate gene
(1) Collecting blood or hair follicle samples of 2000 Holstein cows in 4 large-scale farms
Holstein Niu Yangpin was collected in 4 pastures across the country, with the location 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 |
| Shandong Dezhou | 800 | Hebei Shijizhuang | 400 |
| Ningxia Yinchuan | 400 | Beijing changping | 400 |
(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) The production life genomic breeding value of each individual is estimated from the genotypes. Genome-estimated breeding values were performed by new commute company.
(4) Based on genotype data using GEMMA software, whole genome correlation analysis was performed for production life genome breeding values, with the analysis model using a hybrid linear model, pasture and bovine month age as covariates. Multiple test corrections were made using the FDR method. The correlation analysis results showed that the most significant 1 SNP site was located on the 3' -UTR on the NDUFA9 gene, with a significance P value of 0.0052.
Identification of dominant genotypes on the 3' -UTR of the NDUFA9 Gene
Among the 2000 Holstein cattle collected, 1145 individuals with AA pure and genotype were identified, the average of the estimated breeding values for the production life genome was 2.24, the individuals with AC genotype were 755, the average of the estimated breeding values for the production life genome was 2.22, the individuals with CC genotype were 100, the average of the estimated breeding values for the production life genome was 1.89, and in the genome genetic evaluation, the higher the estimated breeding values for the production life genome were, the better the estimated breeding values for the production life genome were, and thus the AA genotype was the dominant genotype.
3. Bovine NDUFA9 gene 3' -UTR partial sequence amplification
(1) Bovine tail venous blood collection
Cattle with long production life and cattle with short production life 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 3M NaAc (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 NDUFA9 gene (GenBank accession number 404188). Wherein,
the forward primer is NDUFA9-5F:5'-GCCTGACGGAAGGAAACTGA-3';
the reverse primer is NDUFA9-3F:5'-GTGCTCTCTGTGCCTCTTGT-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. The genotype of the PCR product was determined by Sanger sequencing.
The method comprises the steps of selecting a dairy cow core group individual, detecting the genotype of the NDUFA9 gene chr5:gS 105870613C > A locus by using the related molecular biology technology, selecting an advantageous genotype individual for seed reserving, improving the production life character of the dairy cow group, improving the reproduction efficiency, reducing the cultivation cost, increasing the cultivation income, and laying a foundation for cultivating a new good dairy cow line with long production life.
Example 2
Genotype and allele frequency distribution of 1 SNP site of 3' -UTR of NDUFA9 in Holstein cattle population was examined by Sanger sequencing method, and the results are shown in Table 2, which includes Holstein cattle in multiple pastures in our country. The detection result shows that 3 genotypes exist in Holstein cow groups in China; in all the populations examined, the AA gene frequency was 62.6%, the AC genotype frequency was 33.5%, the CC genotype frequency was 3.9% and the AA genotype was the dominant genotype (see table 2).
TABLE 2 distribution results of bovine NDUFA9 Gene in Holstein cattle population
Table 2 illustrates: the group materials are Holstein cattle groups in China, wherein: group 1 is a Holstein cow group in a pasture in Shandong, group 2 is a Holstein cow group in a pasture in Beijing, and group 3 is a Holstein cow group in a pasture in Ningxia.
To determine whether the bovine NDUFA9 genotype is correlated with bovine production longevity, 955-head holstein cattle were selected for correlation analysis of NDUFA9 genotype with estimated breeding values of production longevity genome. The average genomic breeding value of AA genotype individuals was 1.95, the average genomic breeding value of ac genotype individuals was 1.86, and the average genomic breeding value of cc genotype individuals was 1.47. Correlation of different genotypes with bovine production longevity traits was analyzed using PLINK v1.90 software, which showed a significant correlation between genotype and genomic breeding value (P value of 0.0012).
According to the invention, 1 SNP locus is identified, the genotype of an individual dairy cow NDUFA9 gene at the locus is detected by a related technology of molecular biology, and a favorable genotype individual seed reserving is selected by correlation analysis with an estimated breeding value of the dairy cow production life trait, so that the frequency of the dominant genotype of the production life of the NDUFA9 gene in a dairy cow group can be improved, the production life of the dairy cow group is prolonged, the milk yield of the whole life cycle of the dairy cow is improved, the breeding cost is reduced, the breeding benefit is improved, and a novel method is provided for genetic improvement of the production life of the dairy cow.
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.
Claims (10)
1. A SNP molecular marker related to the production life of cows is characterized in that the molecular marker is derived from an NDUFA9 gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, the 304bp position of the sequence is a mutation site, and the base is C or A.
2. The SNP molecular marker according to claim 1, wherein, when the base of the mutation site is A, the gene exhibits a long production life.
3. A specific primer for detecting the SNP molecular marker as set forth in claim 1 or claim 2, characterized by comprising:
the sequence of the upstream primer is shown as SEQ ID NO. 2;
the sequence of the downstream primer is shown as SEQ ID NO. 3.
4. A reagent comprising the specific primer according to claim 3.
5. A kit comprising the specific primer according to claim 3.
6. A method of identifying a long-life dairy cow comprising:
(1) Extracting genome DNA of the dairy cows to be detected;
(2) Amplifying the 3' -UTR region of the dairy cow NDUFA9 gene by PCR reaction using the primer pair of claim 3 by using the genomic DNA of the step (1) as a template;
(3) Detecting the base type at the 304bp of the PCR amplified product fragment, judging whether the dairy cow to be detected belongs to an individual with long production life, and if the base at the 304bp of the amplified product fragment is A, determining that the dairy cow to be detected is the dairy cow with long production life.
7. The method of identifying long-life cows of claim 6, wherein the cows are one of chinese holstein cows, trionyx, sinkiang brown cows or grassland red cows, and more preferably chinese holstein cows.
8. An assisted breeding method for cows, which comprises breeding a cow individual having a dominant genotype obtained by the method for identifying a long-life cow of claim 6 as a parent.
9. Use of the SNP molecular marker of claim 1 or claim 2 for identifying long-life cows and for assisted breeding.
10. The use of the kit of claim 5 for identifying cows with long production life and for assisting in breeding.
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