CN109735632A - GUCY1A1 gene-specific SNP marker, detection method and application of lambing number traits in Hetian Qiaoda red sheep - Google Patents
GUCY1A1 gene-specific SNP marker, detection method and application of lambing number traits in Hetian Qiaoda red sheep Download PDFInfo
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Abstract
本发明公开了一种GUCY1A1基因特异SNP标记、和田乔达红羊产羔数性状的检测方法及其应用，涉及分子育种领域。本发明SNP标记来源于绵羊GUCY1A1基因，该SNP标记位于第17号染色体上第43266624位，原始碱基为C，变异碱基为T；该SNP标记核苷酸序列为SEQ ID NO.1。该SNP位点的CC基因型个体的产羔数显著高于TT基因型。本发明引物的核苷酸序列为SEQ ID NO.2‑4。本发明将上述SNP标记、引物和试剂盒应用于和田羊产羔性状检测，通过检测SNP标记基因型来确定待测和田羊的产羔性状如何；本发明的上述SNP、引物、试剂盒及检测方法可用于育种，筛选出产羔数较多的品种。
The invention discloses a GUCY1A1 gene-specific SNP marker, a method for detecting lambing number traits in Hotan Qiaoda red sheep and an application thereof, and relates to the field of molecular breeding. The SNP marker of the present invention is derived from the sheep GUCY1A1 gene, the SNP marker is located at the 43266624th position on chromosome 17, the original base is C, and the variant base is T; the SNP marker nucleotide sequence is SEQ ID NO.1. The number of lambs of individuals with CC genotype at this SNP locus was significantly higher than that with TT genotype. The nucleotide sequence of the primer of the present invention is SEQ ID NO.2-4. The present invention applies the above-mentioned SNP markers, primers and kits to the detection of lambing traits of Hetian sheep, and determines the lambing traits of the Hetian sheep to be tested by detecting the SNP marker genotype; the above-mentioned SNPs, primers, kits and detection methods of the present invention The method can be used for breeding to screen out varieties with more lambs.
Description
Technical field
The present invention relates to molecular breeding technology field more particularly to a kind of GUCY1A1 gene specific SNP marker and Tian Qiao
Up to the detection method and its application of red sheep litter size character.
Background technique
Sheep feeds earliest one of domestic animal as mankind's domestication, livestock products abundant is provided for the mankind, in animal husbandry
In occupy highly important status.The ecological environment of the topography and geomorphology of Xinjiang complexity, the geographic areas of the length and breadth of land and multiplicity, makes it
As one of China domestic sheep variety resource area the most abundant.The master formed by Xinjiang local people's long-term breeding
Representative kind is wanted, the red sheep of Tian Qiaoda is neutralized and is also important naked eyed test, be distributed mainly on hotan area Pishan
County, resistance is strong, premunition is also strong, extremely resistance to crude feed cultivation, and tolerance is strong under hot environment, also strong to the tolerance of arid,
It is famous to work out high-quality carpet wool.It is mostly group rearing, natural propagation with the red sheep of Tian Qiaoda, in most cases produces single tire, but this
Group is located at Pishan County, and feeding environment is hot severe, but population regularities power is high, and breast and mammiplasia are flourishing, and litter size is flat
It can reach 3-4 lamb, and lamb high survival rate, be the idealized population of tissue lamb production, it is great to have researching value.And Tian Qiao
Up to red sheep as Xinjiang representativeness excellent variety, has it unique, the effective protection of land race resource should be carried out, in addition,
Its excellent production meat characteristic is not developed and used reasonably, and the hair of local economy and Xinjiang meat sheep industry is greatly unfavorable for
Exhibition.Therefore, with advanced biotechnology, in conjunction with GENERALIZATION OF MODERN BREEDING TECHNIQUE and traditional breeding way, Development in Xinjiang advantage animal husbandry is mentioned
High sheep breeding degree cultivates high yield mutton sheep specific breed, mutton Animal product quality is improved, to accelerate Xinjiang Sheep industry
Development process.During in China, Sheep is fast-developing, key problem is to improve the quality of mutton sheep, increase going out for commodity
Column number amount, the core of the two are the optimization and utilization of the raising technology of mutton sheep.The core of the raising technology of mutton sheep is how to play
The performance potentiality of the reproductive trait of mutton sheep.Reproductive performance is one of important economical trait of sheep, and wherein litter size is to influence life
A key factor for producing benefit, is controlled by gene, and is Additive-dominance gene action mode.
Genomics becomes one of the method for research domestic animal character at present, using genomics technologies to influence objective trait
Gene predicted, by molecular marking technique find influence objective trait specific mutational site, to improve herdsman's economy
Income, the lambing level for improving sheep comprehensively all have the meaning of reality.Both at home and abroad for the SNP marker research of sheep litter size
Research that is less, focusing mostly in terms of the functional gene of BMP15, BMPR-IB, GDF9；Wherein for influencing and the red silk floss of Tian Qiaoda
The research of sheep litter size SNP marker has not been reported.
Summary of the invention
In view of this, the embodiment of the invention provides a kind of GUCY1A1 gene specific SNP markers and the red sheep of Tian Qiaoda to produce
The detection method and its application of lamb number character.
In order to achieve the above objectives, invention broadly provides following technical solutions:
On the one hand, the embodiment of the invention provides a kind of GUCY1A1 gene specific SNP marker, the SNP marker is derived from
With the red sheep GUCY1A1 gene of Tian Qiaoda, the SNP marker be located at and No. 17 chromosome of field sheep on the 43266624th,
Original base is C, and variation base is T；Nucleotides sequence where the SNP marker is classified as SEQ ID NO.1.
Preferably, the litter size of the CC genotype individuals of the SNP site is significantly higher than the lambing of TT genotype individuals
Number.
On the other hand, the embodiment of the invention provides a kind of for detecting the primer of above-mentioned SNP marker, the core of the primer
Nucleotide sequence is SEQ ID NO.2-4.
In another aspect, the embodiment of the invention provides a kind of for detecting the kit of above-mentioned SNP marker, the kit
In include above-mentioned primer.
Another aspect, the embodiment of the invention provides above-mentioned SNP marker, above-mentioned primer or mentioned reagent box and Tian Mianyang
Application in breeding.
Another aspect, the embodiment of the invention provides the method for a kind of detection and field sheep litter size character, the methods
It include: by being detected the litter size character to determine the sheep to the above-mentioned SNP marker in sheep to be measured.
Preferably, the described method comprises the following steps:
Extract to be measured and field sheep genomic DNA；
It is amplification template with the DNA, using primer as claimed in claim 3 as amplimer, being at war with property equipotential base
Because of specific PCR parting, PCR genotyping result CC type, CT type and TT type are obtained, all of SNP marker are determined according to genotyping result
Genotype judges the litter size character of the sheep to be measured according to the genotype.
Preferably, the litter size of CC genotype individuals is significantly higher than TT genotype individuals in the genotype of the SNP
Litter size.
Preferably, the reaction condition of the PCR: 95 DEG C of initial denaturations 10min, 95 DEG C of denaturation 20s, circulation 10 times, 61-55
DEG C extend 1min, 95 DEG C of denaturation 20s, recycle 26 times, 55 DEG C of extension 1min；
The reaction system of the PCR:
The nucleotides sequence of the upstream primer 1 is classified as SEQ ID NO.2；
The nucleotides sequence of the upstream primer 2 is classified as SEQ ID NO.3；
The nucleotides sequence of the downstream primer 1 is classified as SEQ ID NO.4.
Another aspect, the embodiment of the invention provides a kind of and field sheep litter size character related SNP labels in scrapie of sheep
Application in educating selects genotype in the SNP marker for the individual of CC, selects more than litter size and field sheep variety.
Compared with prior art, the beneficial effects of the present invention are:
The present invention is screened using selection signal detection method and the method for KASP technology using the method for simplifying genome
Gene relevant to sheep litter size character out, by KASP classifying method to the position Chr17:43266624 of GUCY1A1 gene
Point carry out parting, determine this site be mutated in the red sheep of Tian Qiaoda；The base positions for determining mutation, utilize biology
Learn statistic software SPSS 19.0, between different genotype and the litter size of the red sheep of Tian Qiaoda carry out significance test, through conspicuousness
Check analysis judges whether the SNP site significantly affects the litter size with the red sheep of Tian Qiaoda, final to determine and filter out influence
With the SNP marker of the red sheep litter size of Tian Qiaoda.The genotype determined in the method for the present invention is CC, CT, TT, selects CC and CT gene
Type individual eliminates TT genotype individuals, and breeding improves and the red sheep litter size of Tian Qiaoda, breeding polyembryony sheep variety, is Xinjiang herding
The sustainable development of industry provides scientific basis.
Detailed description of the invention
Fig. 1 is in present invention method using the Manhattan figure of the whole-genome association of general linear model；
Fig. 2 is in present invention method using the Manhattan figure of the whole-genome association of mixed linear model；
Fig. 3 is the QQ-plot detection figure of two kinds of models in present invention method；
Fig. 4 is in present invention method and field Qiao Dahong sheep DNA detects gel electrophoresis figure；
Fig. 5 is the parting structure of present invention method GUCY1A1 gene (site Chr17:g.43266624 C > T)
Figure.
Specific embodiment
For further illustrate the present invention to reach the technical means and efficacy that predetermined goal of the invention is taken, below with compared with
Good embodiment, to specific embodiment, technical solution, feature and its effect applied according to the present invention, detailed description is as follows.Under
Stating the special characteristic, structure or feature in multiple embodiments in bright can be combined by any suitable form.
Technical term involved in the embodiment of the present invention is explained as follows:
SNP marker of the present invention derives from and field sheep GUCY1A1 gene, i.e. guanylate cyclase is by sweet -5 '-three phosphorus of bird
Acid is converted into ring bird sweet -3 ', the enzyme of 5 '-one phosphoric acid, as adenylyl- cyclase, guanylate existing for film binding molecule
Enzyme exists with film combining form and cytoplasmatic forms；Soluble guanylate cyclase, the heterodimer enzyme being made of α and β subunit,
It is the unique receptor for the biological courier's nitric oxide (NO) identified so far, and participates in various signal transduction pathways closely；And it can
Dissolubility guanylate cyclase collection enzyme (GUCY1A1) then find it is related with hormone control, participate in oxytocins signal path, influence to breed
Power.
KASP: it is the abbreviation of competitive ApoE gene, can be right in extensive genome DNA sample
LnDels on SNPs and specific site carries out accurately diallele and judges, to carry out Genotyping；This technology is
Based on the specificity matching of prime end base come to SNP parting and detection lnDels；Need to prepare two ends in use
The Primer Mix that the different allele forward primer of base and a reverse primer are constituted, two end of forward primer 5 ' difference
It is connected with not homotactic detection primer sequence.KASP high throughput genotyping technique possesses higher flux, faster speed, lower
Cost and more accurate result.
SLAF-seq sequencing technologies: i.e. specific position amplified fragments are sequenced, and are a set of simplified genomic sequencing techniques,
SLAF technology represents primary on simplified gene order-checking the features such as effective reads long, flux are high together, conceptual design is flexible
Revolution, the effective gene group for bringing 2x100bp read length, provide unprecedented digestion scheme customization service, and primary development
Up to 10,000 labels obtain most complete variation image (SNPs, lnDels) within the scope of full-length genome, researcher can select
Most information content and reliable polymorphism mark are selected, to realize the exquisite ability of Main Agronomic Characters functional gene positioning.
Embodiment 1
Reagent and instrument and equipment: magnetic frame (Life Technologies DynaMagTM-2)；
Centrifuge (eppendorf Centrifuge 5424)；Douglas Scientific NEXAR；Douglas
Scientific Soellex；Douglas Scientific ARRAY；The biological Co., Ltd of visitor is stepped by hundred to provide；
(LGC company mentions 1536 FormulationV4.0 of mix:KASP 2 × Master Mix KBS-1016-011
For).
Steps are as follows for specific experiment:
(1) screening of Sheep Reproductive Characters
Genome is referred to based on sheep, utilizes one after Plink1.9 software Quality Control using SLAF-seq sequencing technologies
As the method for linear model and mixed linear model carry out whole-genome association, as Figure 1-Figure 2, in two methods
The SNP site of top1% is selected, in Fig. 3, two methods filter out difference SNP site；By the site to selection into
Row gene annotation, filtering out FSHR gene may be the gene for influencing Fecundity Trait.
(2) extraction of ovine genome DNA
It is equipped with the heparin tube of sodium citrate anticoagulant, living body acquires and the red sheep jugular vein blood 5ml of Tian Qiaoda, and -20 DEG C
It saves；Genomic DNA is extracted using kit, -20 DEG C save stand-by, the gel electricity of genomic DNA testing result as shown in Figure 4
Swimming figure.
(3) design of primers and synthesis
According to the GUCY1A1 gene (GenBank ID:101110000) of sheep, referring to the nucleosides where the SNP marker
Acid sequence is that SEQ ID NO.1 utilizes prime3 software Design primers, is synthesized in Shanghai biotechnology Co., Ltd
Primer；Primer sequence is as follows:
Nucleotides sequence where the SNP marker is classified as SEQ ID NO.1:
GGCAGGTCCCCAGAGAGCCCTTGGGGGAAGCCACAGGGAGTGGGCCAGCAGCTA[C/T]CCCAGGGCA
GCCGAGCGTCTGTCCAGGTGTCCCTGACAAGAACCCGCCTG
Above-mentioned [C/T] indicates mutating alkali yl.
GUCY1A1 gene:
SEQ ID NO.2
Upstream primer 1:5 '-GAAGGTCGGAGTCAACGGATTGGAGTGGGCCAGCAGCTAC-3 '；
SEQ ID NO.3
Upstream primer 2:5 '-GAAGGTGACCAAGTTCATGCTGGAGTGGGCCAGCAGCTAT-3 '；
SEQ ID NO.4
Downstream primer 1:5 '-GTTCTTGTCAGGGACACCTGG-3 '.
PCR amplification is as shown in Tables 1 and 2.
Table 1.PCR amplification reaction system
| Reactant | Volume |
| 2×Master Mix | 2.5uL |
| Primer | 0.07uL |
| DNA | 30-50ug |
| Water | Supply 5uL |
Table 2.PCR amplified reaction program
(4) KASP parting
It is right using the Douglas platform of LGC company, Britain based on the special matching of prime end base come to SNP parting
SNPs carries out diallele judgement, to carry out Genotyping；It was found that the site Chr17:g.43266624 of GUCY1A1 gene
With sport 3 kinds of genotype in the red sheep of Tian Qiaoda；See Fig. 5.
(5) association analysis and application
Using software SPSS19.0,3 kinds of genotype are generated to the GUCY1A1 gene SNP site detected: CC, CT, TT with
Significance test of difference is carried out with the litter size of the red sheep of Tian Qiaoda, the results are shown in Table 3.
Table 3.PCR amplified reaction program
Note: Mean ± SD indicates mean+SD；Colleague's data when shoulder is designated as different lowercases, indicate difference
Significantly (P < 0.05).
From table 3 it is observed that the SNP site of GUCY1A1 gene with and the red sheep litter size of Tian Qiaoda it is closely related, CC base
Because type litter size ratio TT type is more, significant difference (P < 0.05) between the two.
It, can be by selecting genotype for the individual of CC, CT in production application, the individual that superseded genotype is TT,
The litter size with the red sheep of Tian Qiaoda is improved, the cultivation of the more lamb kinds of Xinjiang specialization is accelerated.
The present invention has determined above-mentioned SNP marker from sheep GUCY1A1 gene, tool by the method for embodiment 1
Position is set to the site Chr17:g.43266624 C > T (base C sports base T), wherein the individual lambing digital display of genotype CC
Write the individual litter size for being higher than genotype TT；The present invention is by having found that above-mentioned SNP marker can be used to identify and screen lambing
The relatively large number of sheep variety of number.
Above-mentioned SNP marker is used to identify litter size character by the present invention, while also being developed and being can be used for detecting above-mentioned SNP mark
The primer of note, two forward primers (SEQ ID NO.2-3) and a reverse primer (SEQ ID NO.4):
Upstream primer 1:5 '-GAAGGTCGGAGTCAACGGATTGGAGTGGGCCAGCAGCTAC-3 '；
Upstream primer 2:5 '-GAAGGTGACCAAGTTCATGCTGGAGTGGGCCAGCAGCTAT-3 '；
Downstream primer 1:5 '-GTTCTTGTCAGGGACACCTGG-3 '.
The present invention can prepare a kind of for detecting the kit of litter size character according to above-mentioned SNP marker.
Above-mentioned SNP marker of the invention, primer, kit can be applied to detection litter size character or with field Sheep Breeding
Field.
Above-mentioned SNP marker, primer or kit can be used to detect and the Fecundity Trait of field sheep in the present invention, therefrom screens
Genotype CC individual is as voluminous kind out, so be applied to and the breeding of field sheep in.
Place, those skilled in the art can not select from the prior art to the greatest extent in the embodiment of the present invention.
Disclosed above is only a specific embodiment of the invention, but scope of protection of the present invention is not limited thereto, is appointed
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with above-mentioned scope of protection of the claims
It is quasi-.
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Claims (10)
1. a kind of GUCY1A1 gene specific SNP marker, which is characterized in that the SNP marker derives from and the red sheep of Tian Qiaoda
GUCY1A1 gene, the SNP marker be located at and No. 17 chromosome of field sheep on the 43266624th, original base be C, become
Isobase is T；Nucleotides sequence where the SNP marker is classified as SEQ ID NO.1.
2. a kind of GUCY1A1 gene specific SNP marker as described in claim 1, which is characterized in that the CC of the SNP site
The litter size of genotype individuals is significantly higher than the litter size of TT genotype individuals.
3. a kind of primer for SNP marker described in detecting as claimed in claim 1 or 22, which is characterized in that the nucleotide of the primer
Sequence is SEQ ID NO.2-4.
4. a kind of for detecting the kit of as claimed in claim 1 or 22 SNP markers, which is characterized in that include in the kit
Primer as claimed in claim 3.
5. SNP marker of any of claims 1 or 2, primer as claimed in claim 3 or kit as claimed in claim 4 exist
With the application in the sheep breeding of field.
6. a kind of method of detection and the red sheep litter size character of Tian Qiaoda, which is characterized in that the described method includes: by treating
It surveys SNP marker in sheep and is detected the litter size character to determine the sheep；Wherein, the SNP marker is right
It is required that SNP marker described in 1 or 2.
7. the method for a kind of detection and the red sheep litter size character of Tian Qiaoda as claimed in claim 6, which is characterized in that described
Method the following steps are included:
Extract to be measured and field sheep genomic DNA；
It is amplification template with the DNA, using primer as claimed in claim 3 as amplimer, being at war with property allele is special
Anisotropic PCR parting obtains PCR genotyping result CC type, CT type and TT type, all genes of SNP marker is determined according to genotyping result
Type judges described to be measured and field sheep litter size character according to the genotype.
8. the method for a kind of detection and the red sheep litter size character of Tian Qiaoda as claimed in claim 7, which is characterized in that described
In the genotype of SNP, the litter size of CC genotype individuals is significantly higher than the litter size of TT genotype individuals.
9. the method for a kind of detection and the red sheep litter size character of Tian Qiaoda as claimed in claim 6, which is characterized in that described
The reaction condition of PCR: 95 DEG C of initial denaturations 10min, 95 DEG C of denaturation 20s, circulation 10 times, 61-55 DEG C of extension 1min, 95 DEG C of denaturation
20s, circulation 26 times, 55 DEG C of extension 1min；
The reaction system of the PCR:
The nucleotides sequence of the upstream primer 1 is classified as SEQ ID NO.2；
The nucleotides sequence of the upstream primer 2 is classified as SEQ ID NO.3；
The nucleotides sequence of the downstream primer 1 is classified as SEQ ID NO.4.
10. one kind of any of claims 1 or 2 SNP marker relevant with the red sheep litter size character of Tian Qiaoda is in sheep breeding
In application, which is characterized in that select genotype in the SNP marker for the individual of CC, select more than litter size and field sheep
Kind.
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