CN109680070B - SNP (Single nucleotide polymorphism) marker and molecular marker remarkably related to Australia white sheep hoof color and application - Google Patents
SNP (Single nucleotide polymorphism) marker and molecular marker remarkably related to Australia white sheep hoof color and application Download PDFInfo
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Abstract
The invention belongs to the field of animal genetic breeding and SNP (single nucleotide polymorphism) markers and application, and particularly relates to an SNP marker which is remarkably related to the color of Australia white sheep hoofs, a molecular marker and application. The marker is located at 31603203 th nucleotide site (Chr19:31603203) on chromosome 19 of the international sheep genome (Oar _ v4.0), the site has two alleles of A and G, the site is located at intron 9 of the MITF gene, the site g.31603203A/G has polymorphism A/G, the hoof color of Australian white sheep of GG genotype is black, the hoof color of Australian white sheep of AG genotype is gray, and the hoof color of Australian white sheep of AA genotype is white. The SNP molecular marker can be used for breeding new Australia white sheep strains with uniform appearance and stable characters and breeding new Australia white sheep varieties.
Description
Technical Field
The invention belongs to the field of genetic engineering and molecular biology, relates to animal genetic breeding, SNP (single nucleotide polymorphism) markers and application, and particularly relates to an SNP marker which is remarkably related to the color of Australian white sheep hoofs, a molecular marker and application.
Background
The Australian white sheep is a coarse-wool type specialized mutton sheep variety which is bred by combining genes in a specific ratio and integrating excellent genes of varieties of white-headed Dorper sheep, Vanrie sheep, poll Dorset sheep and Teckessel sheep. The variety is officially registered in australia in 10 months in 2009 and marketed in 3 months in 2011. In 5 months 2011, Australian white sheep was introduced by Tianjin Olympic group animal husbandry, Inc. from Australian HVD breeder. Aiming at the problems encountered by introduction and local domestication, after the introduction adaptability test is completed, the performance measurement is enhanced while the breeding sheep feeding management method is gradually improved by combining the local situation, and the established small population breeding method is applied to develop strain breeding. After continuous breeding, efficient and rapid propagation and adjustment of feeding management technologies in different scales and feeding environments, a group with the unique genetic characteristic of black hoofs, stable heredity, reasonable group structure and certain scale is formed, and the characteristics of good adaptability, large size, fast growth, early maturity, full-year estrus, automatic hair replacement, good stress resistance, high lamb survival rate and the like are shown. With the introduction of white Australian sheep breeding by Tianjin Olympic group animal husbandry Co., Ltd, black hoof breeding sheep are widely popularized in sheep breeding areas such as northeast three provinces, Jingjin Ji, inner Mongolia, Gansu, Shandong, Shanxi, Henan, Hebei, Anhui and Xinjiang, and the genetic mechanism of the hoof color genetic characteristics is more and more concerned by people.
The hoof color of Australian White Sheep may appear black, gray or amber depending on the strain or origin of the blood system, or may be striped with a combination of different colors (Australian White Sheep bred Standards 2015). Generally, animal color traits including hoof color all belong to quality traits controlled by a few loci, and their color mainly depends on the type of melanin and its relative amount. The melanin types can be divided into eumelanin (eumelanin) and pheomelanin (pheomelanin), the former does not contain sulfur atom, is difficult to dissolve in various solvents and is mainly brown or black; the latter contain sulfur atoms, are round red particles soluble in alkali, and are yellow or reddish brown.
Melanin is an amorphous protein derivative produced by melanosomes within melanocytes that is insoluble in water and most organic solvents, and its true, pheochromomelanin ratio is closely related to the activity of Tyrosinase (Tyrosinase). The MITF gene is used as a transcription factor related to melanin generation, and the encoded MITF protein can act on a CATGTG sequence in a promoter of a tyrosine gene family (TYR, TYRP1 and TYRP2), catalyze tyrosine metabolism and promote the synthesis of eumelanin. The MITF protein belongs to a Tfe transcription factor super family member, can be combined with an E-box or M-box sequence taking 'CANNTG' as a core in a dimer form, and plays a role in regulating and controlling the transcription expression of a target gene. Previous studies have demonstrated that MITF is involved in the development, differentiation and functional regulation of melanocytes, ocular pigment epithelial cells, osteoclasts. Therefore, the MITF gene is closely related to the formation of the eumelanin, thereby indirectly regulating and controlling the color traits of animals.
Currently, the selection of the Australia white sheep hoof color mainly depends on subjective evaluation of the hoof color by observers, and differences exist among different observers in evaluation of dark gray, gray and light gray, so that the accuracy of seed selection is reduced. The molecular marker assisted selection only depends on the genotype information of the animal and does not depend on the subjective evaluation of an observer on the hoof color, so that the selection accuracy can be effectively improved, but the molecular marker for hoof color selection is lacked at present. Therefore, the screening and identification of the molecular markers related to the hoof color are beneficial to accelerating the breeding of Australian white sheep strains with consistent hoof color.
Through a search for a patent publication, no patent publication that is the same as the present patent application is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an SNP marker which is remarkably related to the color of Australia white sheep hoof, a molecular marker and application.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
an SNP marker which is remarkably related to Australia white sheep hoof color and is characterized in that: the marker presents both a and G alleles, located in intron 9 of the MITF gene.
And, the SNP marker is located at 31603203 th nucleotide site on the chromosome 19 of the reference sequence of version 4.0 of the international sheep genome, and has A/G polymorphism, the SNP marker is located in the 9 th intron of the transcription factor MITF gene related to melanin generation, and the SNP marker is extremely obviously related to Australia white sheep hoof color, and P is less than 0.01.
Furthermore, the polymorphism A/G was g.31603203A/G.
Furthermore, the hoof color of the Australian white sheep with GG genotype at the g.31603203A/G site is shown as gray, the hoof color of the Australian white sheep with AG genotype at the g.31603203A/G site is shown as gray, and the hoof color of the Australian white sheep with AA genotype at the g.31603203A/G site is shown as white.
A molecular marker based on SNP markers which are remarkably related to the hoof color of Australian white sheep, which is characterized in that: the molecular marker sequence is SEQ ID NO 1.
A preparation method of a molecular marker based on an SNP marker remarkably related to the hoof color of Australian white sheep is characterized in that: the method comprises the steps of taking a nucleotide sequence containing the SNP marker as a basic sequence, designing a primer pair, and carrying out PCR amplification by taking Australia white sheep genome DNA as a template to convert the SNP marker into a molecular marker, thus obtaining the molecular marker based on the SNP marker.
Moreover, the upstream primer of the primer pair is: 2, the downstream primer is: 3, SEQ ID NO.
Moreover, the primer is applied to the breeding of the Australia white sheep pure line with uniform appearance and stable characters.
Furthermore, M at position 173 of the SNP marker in the molecular marker sequence is indicated as an A/G polymorphism.
The SNP marker which is obviously related to the color of the Australian white sheep hoof is used for assisting in breeding a new Australian white sheep strain with uniform appearance and stable characters and breeding a new variety by utilizing the Australian white sheep.
The invention has the advantages and positive effects that:
the SNP marker provided by the invention is extremely obviously related to the hoof color of Australian white sheep, Australian white sheep individuals with black and gray hoof colors can be screened by identifying the SNP marker, the selection efficiency is improved by utilizing the molecular marker for auxiliary selection, and the method has important significance for breeding new Australian white sheep strains and breeding new varieties by utilizing Australian white sheep.
Drawings
FIG. 1 is a Manhattan diagram of SNP markers of the present invention which are extremely significantly related to the color of Australian white cotton sheep hooves, wherein the markers indicated by red circles and arrows are molecular markers selected by the present invention, and the markers are located on the nucleotide sequence of the MITF gene on the sheep chromosome 19;
FIG. 2 is an agarose gel electrophoresis of the primers of the present invention, wherein the M lane: DNA Marker DL 100; 2 AA genotypes, GA genotypes and GG genotypes are arranged on the right side of the lane M in sequence;
FIG. 3 is a graph showing the peak of G-A mutation at nucleotide g.31603203A/G on chromosome 19 of Australian white sheep, i.e., three different genotypes (AA, AG, GG) in the present invention.
Detailed Description
The embodiments of the invention are described in further detail below with reference to the following figures:
the innovation of the SNP marker which is remarkably related to the color of Australia white sheep hoof is that: the marker presents both a and G alleles, located in intron 9 of the MITF gene.
The SNP marker is located at 31603203 th nucleotide site on chromosome 19 of reference sequence version 4.0 of international sheep genome and has A/G polymorphism, the SNP marker is located in 9 th intron of transcription factor MITF gene related to melanin generation, and the SNP marker is extremely obviously related to Australian white sheep hoof color with P < 0.01.
The polymorphism A/G is g.31603203A/G.
The hoof color of the Australian white sheep with the GG genotype at the g.31603203A/G site is represented as gray, the hoof color of the Australian white sheep with the AG genotype at the g.31603203A/G site is represented as gray, and the hoof color of the Australian white sheep with the AA genotype at the g.31603203A/G site is represented as white.
The molecular marker based on the SNP marker which is remarkably related to the hoof color of Australian white sheep is characterized in that: the molecular marker sequence is SEQ ID NO 1.
A molecular marker preparation method based on SNP markers remarkably related to the hoof color of Australian white sheep is characterized by comprising the following steps: the method comprises the steps of taking a nucleotide sequence containing the SNP marker as a basic sequence, designing a primer pair, and carrying out PCR amplification by taking Australia white sheep genome DNA as a template to convert the SNP marker into a molecular marker, thus obtaining the molecular marker based on the SNP marker.
The upstream primer of the primer pair is as follows: 2, the downstream primer is: 3, SEQ ID NO.
The primer is applied to breeding the pure Australia white sheep strain with uniform appearance and stable characters.
The SNP marker is positioned at M173 th position of the molecular marker sequence and is expressed as A/G polymorphism.
The SNP marker which is obviously related to the color of the Australian white sheep hoof is used for assisting in breeding a new Australian white sheep strain with uniform appearance and stable characters and breeding a new variety by utilizing the Australian white sheep.
Specifically, the method comprises the following steps:
1. source of experimental animal
TIANJIN AOQUN ANIMAL HUSBANDRY Co.,Ltd.
2. Australia white sheep hoof color phenotype assay
Hoof color depth was scored at 1, 2 and 3 points for amber, dark gray and black hoof colors, respectively, based on an observed evaluation of hoof color in 571 Australian white sheep. The original phenotype of 10 sheep selected is recorded in table 1.
TABLE 1
| Number of person to be checked | Sex | Number of same births | Date of birth | Color scoring of hooves |
| AW0249 | Ram | 2 | 2015-10-16 | 3 |
| AW0256 | Ram | 1 | 2015-11-03 | 1 |
| AW0259 | Ram | 1 | 2015-10-31 | 3 |
| AW0269 | Ram | 1 | 2015-11-05 | 3 |
| AW0375 | Ewe | 1 | 2015-10-10 | 3 |
| AW0377 | Ewe | 1 | 2015-10-29 | 3 |
| AW0493 | Ram | 1 | 2015-08-29 | 1 |
| AW0506 | Ram | 1 | 2015-05-05 | 1 |
| AW0507 | Ewe | 2 | 2015-05-05 | 3 |
| AW0777 | Ewe | 2 | 2015-10-13 | 3 |
3. Extraction of genomic DNA
Ear tissue samples of 571 Australian white sheep were collected and placed in a centrifuge tube containing 70% alcohol and stored in a freezer at-20 ℃ for further use.
Extracting the genomic DNA of the ear tissue by using a magnetic bead method, wherein the required reagents comprise:
lysate BL (Changchun city zhiang biotechnology limited)
Washing solution BW (Changchun city zhiang biotechnology limited)
Magnetic beads BC (Changchun city zhiang biotechnology limited)
Proteinase K (Kingpunoan Biotechnology Co., Ltd.)
Anhydrous ethanol (Xiong science corporation)
Eluent CE (Changchun city zhiang biotechnology limited)
The method comprises the following specific steps:
1) taking a soybean tissue sample, shearing the soybean tissue sample as much as possible, and putting the soybean tissue sample into a 1.5mL centrifuge tube;
2) adding 400 μ L lysate and 20 μ L proteinase K (20mg/mL), and mixing by vortex oscillation;
3) putting the sample in a water bath kettle at 65 ℃ for incubation overnight until the cracking liquid in the tube is clear and transparent;
4) centrifuging at 4 ℃ and 12000r/min for 2min, standing at room temperature for 10min, and placing the supernatant in a 1.5mL centrifuge tube;
5) adding 450 mu L of absolute ethyl alcohol, reversing and mixing uniformly, adding 30 mu L of magnetic beads, carrying out vortex oscillation and mixing uniformly, standing at room temperature for 10min for adsorption, and carrying out vortex oscillation and mixing uniformly for 1 time in the 3 rd minute and the 6 th minute in the standing process;
6) after adsorption, performing vortex oscillation to uniformly mix the magnetic beads, placing the centrifugal tube on a magnetic frame, repeatedly reversing the magnetic frame, washing the magnetic beads possibly remained on the tube cover part into the solution in the tube, standing the magnetic frame for magnetic absorption for 1min or clarifying the solution, discarding the tube cover and supernatant in the tube, and paying attention to not touch the magnetic beads;
7) adding 700 mu L of washing liquid BW added with absolute ethyl alcohol, carrying out high-speed vortex oscillation for 1min, placing the centrifugal tube on a magnetic frame, repeatedly reversing the magnetic frame, washing magnetic beads possibly remaining on the part of the tube cover into the solution in the tube, standing the magnetic frame for magnetic absorption for 1min or clarifying the solution, discarding the tube cover and supernatant in the tube, and paying attention to not touch the magnetic beads;
8) 800. mu.L of washing solution BW to which absolute ethanol has been added are added. Washing for 1 time according to the method in the step (7);
9) adding 800 μ L of 80% ethanol solution, and washing for 1 time according to the method in step (7);
10) placing the centrifugal tube on a magnetic frame, uncapping and drying at room temperature for 5-10 min until no ethanol remains;
11) adding 50 μ L of CE eluent, mixing, incubating at 56 deg.C for 10min for DNA elution, and vortexing at 5min for 1 time. And after the elution is finished, mixing the magnetic beads uniformly, carrying out magnetic attraction for 1min or until the solution is clarified, carefully absorbing the supernatant, and placing the supernatant into a new 1.5mL centrifuge tube for storage at the temperature of-20 ℃ for later use.
4. Australian white sheep whole genome sequencing typing
Genomic DNA extracted from sheep ear tissue samples was paired-end sequenced using the Hiseq X ten sequencing platform using Restriction-site associated DNA sequencing (RAD-seq) technique (PE 150).
After obtaining sequencing data, extracting individual data according to tag sequences, filtering out low-quality sequencing fragments, then using BWA software (Li, Durbin,2009) to align high-quality fragments to sheep reference genome (exemplar Oar _ v4.0, https:// www.ncbi.nlm.nih.gov/genome/.
And performing quality control on the preliminarily obtained SNP data, wherein the quality control standard is as follows: callrate >0.7, snp mass value >20, minimum allele frequency >0.01, heterozygosity <0.9, giving a total number of snps of 175902.
Filling the deletion genotype by adopting fastphase software (Scheat, Stephens,2006), and filtering the filled SNP markers again by adopting the same quality control standard to obtain 171842 high-quality SNP markers.
5. Genome-wide association analysis (GWAS) of Australian white sheep hoof color trait
The GWAS analysis was performed on the hoof color scores using the EMMAX program (http:// genetics. cs. ucla. edu/EMMAX/index. html). The analytical model is as follows:
y＝Xb+Zu+m+e
in the model, y represents hoof color scoring, X represents a fixed effect incidence matrix, b represents a fixed effect vector, the fixed effect comprises year-season, number of same births, gender and 3 principal component effects, Z represents an additive genetic effect incidence matrix, u represents an individual additive genetic effect vector, e represents a residual error,
g represents a genome association matrix, I represents an identity matrix,
respectively represent additive genetic effect variance and residual variance, and m represents SNP marker effect.
The 31603203 th nucleotide site on chromosome 19 reached a level of 5% significance throughout the genome (p-9.40E-36).
Validation of SNP site (i.e., SNP marker) g.31603203A/G in 6 Australian white sheep:
1. extraction of Australian white sheep genome DNA
Ear tissue samples of 6 Australian white sheep with hoof color scores, 2 each of 3 scores, were collected and placed in a centrifuge tube containing 70% alcohol and stored in a freezer at-20 ℃ until needed. The method is used for extracting the genome DNA of the ear tissue, and the ear tissue is preserved at the temperature of minus 20 ℃ for standby after the quality and the concentration detection.
2. PCR amplification and sequencing of target fragment
Using the extracted DNA as a template, and carrying out PCR amplification according to the designed primer: taking 2.5 mu L of DNA template, 1.25 mu L of primers shown in SEQ ID NO. 2 and SEQ ID NO. 3 respectively, 25 mu L of PCR Mix reagent and 20 mu L of double distilled water; setting a PCR amplification system: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 15 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 30 s; 35 cycles; then extended for 5min at 72 ℃.
Detecting the PCR product in 1.5% agarose gel electrophoresis, detecting the amplified target segment with 529bp size, and obtaining an electrophoretogram shown in figure 2, sequencing the rest amplified products, comparing and analyzing the sequencing result with the sequence of the sheep related gene segment in GenBank by using SnapGene software, and judging the genotype of g.31603203A/G. The sequencing sequence conforms to the sequence information of SEQ ID NO. 1, and a SNP site g.31603203A/G exists at the 173 th base of the sequence. And the genotype of 1-point individual is AA, and the genotype of 2-point 3-point individual is AG or GGGG.
SNP site g.31603203A/G hoof color scores of different genotype individuals were compared in the 212 Australian white sheep validation cohort:
the 212 Australian white sheep in the group was verified to be a population of 571 sheep that were genotyped by RAD sequencing, scored for hoof color, but not included in the whole genome association analysis.
As can be seen from Table two, the amount of G allele at the g.31603203A/G locus was very significantly correlated with the hoof color score (rank correlation test p <0.01), with the higher the number of G alleles, the higher the hoof color score.
Table 2 verifies that group 212 sheep scored individuals for different genotypes at the g.31603203A/G locus
A method for screening Australia white sheep strain with black hoof color comprises the steps of detecting the genotype of an Australia white sheep g.31603203A/G nucleotide locus, and breeding a GG type individual of the g.31603203A/G nucleotide locus to serve as a breeding sheep. (the hoof nails of pure Australian white sheep had pigmentation and appeared dark gray black.)
Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Sequence listing
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Claims (1)
1. The application of a detection reagent of a typing SNP molecular marker g.31603203A/G in breeding of a new Australian white sheep strain with uniform hoof color is assisted, the SNP molecular marker g.31603203A/G is SNP which is located at 31603203 th nucleotide site on chromosome No. 19 of reference sequence version 4.0 of international sheep genome and has A/G polymorphism, the breeding g.31603203A/G nucleotide site is a GG type individual and is used as a breeding sheep, and the hoof color of the GG type Australian white sheep is dark gray black.
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| CN111485026B (en) * | 2020-04-20 | 2023-08-18 | 天津奥群牧业有限公司 | SNP locus related to sheep birth weight, application, molecular marker and primer |
| CN112553345B (en) * | 2020-12-02 | 2022-11-22 | 天津奥群牧业有限公司 | Application of sheep PRNP gene insertion/deletion in breeding character early selection |
| CN112695102B (en) * | 2021-02-07 | 2023-01-10 | 西北农林科技大学 | Application of goat PRNT gene single nucleotide polymorphism in early selection of lambing traits |
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