CN106755321A - The method for screening hypoxia adaptability sheep - Google Patents

The method for screening hypoxia adaptability sheep Download PDF

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CN106755321A
CN106755321A CN201611055253.4A CN201611055253A CN106755321A CN 106755321 A CN106755321 A CN 106755321A CN 201611055253 A CN201611055253 A CN 201611055253A CN 106755321 A CN106755321 A CN 106755321A
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sheep
hypoxia adaptability
screening
hypoxia
adaptability
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姜雨
王喜宏
王文
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Inner Mongolia Zhongke Positive Biological Science And Technology Co Ltd
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Abstract

The invention discloses a kind of method for screening hypoxia adaptability sheep, comprise the following steps：1) genomic DNA of sheep to be detected is extracted；2) detect and one or more sites in the SNP site of the missing close linkage of 19272.8 19322.8kb regional genes on No. 18 chromosomes of sheep；3) by SNP site parting, sheep is divided into plateau type, plain type and heterozygous, wherein, plateau type sheep has hypoxia adaptability.The method can be applied to hypoxia adaptability sheep auxiliary and introduce a fine variety, selection cross, introduces a fine variety and breed breeding, required sample size is small, technical operation is simple, low cost, greatly improves breeding and the efficiency introduced a fine variety, and method is flexible, can be according to the different SNP site of different samples selections, and detected that accuracy is high using TaqMan probe method or ApoE gene method, it is reproducible.

Description

The method for screening hypoxia adaptability sheep
Technical field
The present invention relates to biological technical field, and in particular to the method for screening hypoxia adaptability sheep.
Background technology
The plateau of more than China height above sea level 3000m accounts for the 1/6 of national total area.In below height above sea level 5000m low latitudes, height above sea level Height often rises 100m, air pressure reduction about 1KPa.The plateau atmospheric oxygen content of height above sea level 3000m is only sea level area The atmospheric oxygen content in 73%, height above sea level 5000m area is only the 53% of sea level area.Therefore, the low pressure of highlands It is main ecological cause with low oxygen partial pressure, severely impacts the survival and development of animals and human beingses.
China area it is maximum pastoral area --- Qinghai-Tibet pastoral area there are about 2,000,000,000 mu of available high and cold grasslands, wherein continuous Sheep industry is the main department of local animal husbandry.However, most of sheep of Qinghai-Tibet Platean is breeds differentiation at present.Hide sheep constitution strong Strong, resistance to thick feeding is kind nomadic, but the speed of growth is slow, meat production is extremely low, reproductive capacity is weak.Some domestic and international famous naked eyed tests, such as Many areas are successfully introduced a fine variety, are promoted at home for Small-fat-tail sheep, Du Boyang, Texel sheep, Mutton Merino.However, high Former distinctive Alpine cold and hypoxia environment causes the kind introduced or introduced variety to be difficult to adapt to high with the filial generation of native breed Cold anaerobic environment.
Blood physiology is an importance of hypoxia adaptability, and used as the main carriers of gas in blood, red blood cell is special Property is directly related with hypoxia adaptability.Height above sea level can influence red blood cell characteristics, and hemoglobin is that oxygen and titanium dioxide are transported in blood The carrier of carbon, is transported to tissue, and carbon dioxide is transported into lung from tissue by oxygen by lung.So hemoglobin content increases favourable In gas transport.The generation of red blood cell is a heritable proterties, useful molecules mark prediction red blood cell characteristics, and then can be with Hypoxia adaptability sheep is screened.
Sheep hypoxia adaptability molecular genetic marker is located at 19272.8-19322.8kb on No. 18 chromosomes, will be including continuous Sheep reference gene group is designated as reference type in the interior genotype containing the region.In addition to reference type, strain or individuality in part In genome, the region shows missing in sequencing, and resulting polymorphism is designated as deletion form.Compared with deletion form, base Because group reference type individuality shows HC and less mean corpuscular volume higher, so as to be conducive in hypoxemia ring Survived in border.
But the detection on molecular biology to large fragment insertion/deletion lacks effective means at present.It is available in theory Detection method has：Genome sequencing, the hybridization of the Southern markings, immunofluorescence in situ hybridization (FISH) also have and develop in recent years Long-range PCR and multiplex ligation-dependent probe amplification (MLAP).These detection methods are to sample, technical equipment, experiment High cost.Difference test in addition directly by gene order in the gene cluster is also relatively difficult, and reason is that the region is by many The gene cluster of the gene family composition of individual high homology, it is difficult to design special primer.
The content of the invention
In view of this, it is an object of the invention to propose a kind of method for screening hypoxia adaptability sheep, needed for the method Sample size is small, and technical operation is simple, and low cost greatly improves breeding and the efficiency introduced a fine variety.
Large fragment of the present invention based on No. 18 chromosome 19272.8-19322.8kb of sheep loses variation and the average blood of sheep Hemoglobin concentration and mean corpuscular volume (MCV) are significantly correlated, and then closely related with sheep hypoxia adaptability.By analyzing Highly chain (the r of variation is lost to the large fragment with No. 18 chromosome 19272.8-19322.8kb2>0.6) 314 monokaryon glycosides Sour polymorphism (SNP) site, by detecting that SNP site predicts individual hypoxia adaptability characteristic, filter out hypoxia adaptability compared with The strain and hybrid individual of strong sheep.
The method of the screening hypoxia adaptability sheep provided based on the above-mentioned purpose present invention, is comprised the following steps：
1) genomic DNA of sheep to be detected is extracted；
2) SNP with the missing close linkage of 19272.8-19322.8kb regional genes on No. 18 chromosomes of sheep is detected One or more sites in site；
3) by the parting of SNP site, sheep is divided into plateau type, plain type and heterozygous, wherein, plateau type sheep tool There is hypoxia adaptability.
Wherein, the missing close linkage of 19272.8-19322.8kb regional genes on described and No. 18 chromosomes of sheep The linkage coefficient r of SNP site2>0.6。
Alternatively, step 2) in detection SNP site method be ApoE gene method.
Alternatively, step 2) in detection SNP site method be TaqMan probe method.
Alternatively, the ApoE gene method is or many in the close linkage SNP site chosen Individual site, is designed for expanding the primer of one or more SNP sites, and SNP site is judged by gene cloning, gene sequencing Parting, screen hypoxia adaptability sheep.
Alternatively, the TaqMan probe method is one or more sites in the close linkage SNP site chosen, Corresponding probe is designed, by the parting of the probe in detecting SNP site, the sheep of hypoxia adaptability is screened.
Alternatively, 5 ' ends of the probe are marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence group.
Alternatively, it is VIC or FAM that 5 ' ends of the probe are marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence Group is TAMRA or MGB.
Based on identical inventive concept, the method present invention also offers above-mentioned screening hypoxia adaptability sheep is suitable in hypoxemia Answering property sheep aids in the application in introducing a fine variety；Application in hypoxia adaptability sheep selection cross.
From the above it can be seen that the method for the screening hypoxia adaptability sheep that the present invention is provided, by analysis and 18 The large fragment of number chromosome 19272.8-19322.8kb loses the highly chain (r of variation2>0.6) SNP site, judges the region Lost with the presence or absence of large fragment and made a variation, and then predict the hypoxia adaptability characteristic of sheep.Sample size is small needed for the method, technology behaviour Make simple, low cost greatly improves breeding and the efficiency introduced a fine variety.
Specific embodiment
To make the object, technical solutions and advantages of the present invention become more apparent, below in conjunction with specific embodiment, to this hair Bright further description.
The data of the sequence of resurveying of 70 sheep according to international ovine genome tissue (ISGC), analyze sequencing result, find Variation is lost with the large fragment of sheep OAR3.1 version reference genes group No. 18 chromosome 19272.8-19322.8kb closely to connect 314 SNP sites (being shown in Table 1) of lock, wherein, r2Value represents the linkage degree of this SNP and large fragment deletion.314 SNP The parting in site is closely related with whether the region lacks, and can be used for screening hypoxia adaptability sheep, and wherein plateau type is represented With hypoxia adaptability.
Table 1：With 314 SNP that No. 18 large fragments of chromosome 19272.8-19322.8kb lose variation close linkage Point information
The method of the screening hypoxia adaptability sheep that the present invention is provided, comprises the following steps：
1) genomic DNA of sheep to be detected is extracted；
2) detection and 314 of the missing close linkage of 19272.8-19322.8kb regional genes on No. 18 chromosomes of sheep One or more sites in individual SNP site；
3) by SNP site parting, sheep is divided into plateau type, plain type and heterozygous, wherein, sheep has plateau type Hypoxia adaptability.
Choose the SNP of wherein numbering 15519342316Site, carries out method validation verification.
Embodiment 1：TaqMan probe method detects SNP19342316Parting and its forecasting efficiency checking in site
1st, experimental animal：
Experimental animal comes from Ida. Du Buwa experiment centres.Totally three kind Colombia (N=145) Polypay (N=438) and cloth made of orchid Lay (N=414) kind, are the purebred ewe of 1-5 Sui.Using similar feeding and management.
2nd, red blood cell characteristics analysis：
By jugular puncture, whole blood is collected using the coated vacuum test tubes of EDTA.All parameters by Phoenix Labs, Inc. measured according to testing standard, the reference value of all references is also provided by the laboratory.
Index measured directly：
Red blood cell count(RBC) (RBC, M/ml, term of reference：4.0-12.0M/ml)；
Content of hemoglobin (HGB, g/dl, term of reference：9.0-15.0g/dl)；
Packed cell volume (HCT, %, term of reference：27.0-45.0%).
Three derivative indexs：
Mean corpuscular volume (MCV) (MCV, with fL, term of reference：28.0-40.0fL), computing formula：
MCV(μm3)=packed cell volume (%) x10/ red blood cell count(RBC)s (million/mm3)；
Mean corpuscular hemoglobin (MCH, unit pg, term of reference:8.0-12.0pg), computing formula
MCH (pg)=HC (g/100ml) x10/ red blood cell count(RBC)s (million/mm3)；
(MCHC, unit is %, term of reference to mean corpuscular hemoglobin concentration (MCHC)：31.0-35.0%), computing formula： MCHC (g/100m)=HC (g/100ml) x100/ packed cell volumes (%).
3rd, SNP is contained19342316The acquisition of the fragment in site and parting are detected：
Conventional method extract sample gene group DNA, using TaqManH (Applied Biosystems, Foster City, CA) methods of genotyping detection SNP19342316Loci polymorphism and parting.
According to SNP19342316Following primer and probe are designed in site, and specific experiment step refers to TaqManH (Applied Biosystems, Foster City, CA) product description.
Amplimer 1：5’-CCGGTTCATCCTTGAGAAACTCTT-3’；
Amplimer 2：5’-CCCCACAGATGTGCTAATGGT-3’；
Probe 1 (detection A)：5’-CCTCCTCTTCTTCAACC-3’；
Probe 2 (detection G)：5’-CTCCTCCTCTTCAACC-3’.
5 ' ends of the probe 1 and probe 2 are marked with reporter fluorescence group VIC and FAM respectively, and 3 ' ends are marked with respectively quenches Go out fluorophor TAMRA and MGB.
SNP types plateau type (AA) in release judgement sample according to fluorescence signal, plain type (GG) or heterozygous (AG)。
4、SNP19342316The prediction effect checking of the parting in site
The SNP of step 3 detection19342316The parting in site is as shown in table 2 with the association analysis of red blood cell characteristics value：Plateau type Hemoglobin beyond allele (A) and normal range (NR) increases and cell volume reduces significantly correlated, and plain type equipotential base Because (G) shows normal scope.
Result shows SNP19342316The parting in site has HC higher for the idiotype of plateau type (AA) With less erythrocyte volume, thus low-oxygen environment is more adapted to.Show SNP19342316Site can be red to sheep as molecular labeling Cell characteristics are effectively predicted, screen hypoxia adaptability sheep.
Table 2：Red blood cell key property value and SNP19342316The relation of site parting
AA AG GG Standardization P values
MCHC (%) 36.252 34.402 33.991 6.2*10-14
Mean corpuscular volume (MCV) (fl) 32.398 33.923 34.563 2.5*10-6
Embodiment 2：ApoE gene detects SNP19342316The parting in site and prediction effect checking
Experimental animal, red blood cell characteristics analysis are with embodiment 1.According to SNP19342316Design following allele specific in site All samples are expanded by property PCR primer group：
Deletion form (plateau type)：
5’-TGCAAGACTGGTTCACCCCAGAT-3’
5’-CCGGCTTTATGTCAGTGATACGTG-3’
Product is 330bp.
Reference type (plain type)：
5’-TAGAAGTACTCCTACCTGAACGA-3’
5’-CCAAACAAATCCCTGAGCCATTTC-3’
Product is 719bp.
Using TaqManH universal PC R mixtures, performing PCR is entered to specifications.Denaturation 95 DEG C 10 minutes, often afterwards It is individual circulation 95 DEG C 30 seconds, 64 DEG C 30 seconds, 68 DEG C 1 minute, 40 circulation.PCR primer carries out gel electrophoresis and imaging.
Reference type is only capable of obtaining band when primer is reference type primer, and deletion form is only capable of when primer is deletion form primer Band is obtained, two kinds of primers of heterozygous have band.
SNP19342316The parting in site is as shown in table 3 with the association analysis of red blood cell characteristics value：Deletion form (plateau type) with Hemoglobin beyond normal range (NR) increases and cell volume reduction is significantly correlated, and reference type (plain type) shows normally Scope.Result shows that deletion form is individual has HC and less erythrocyte volume higher, thus more adapts to low Oxygen environment.The large fragment deletion mark of 19272.8-19322.8kb can be effectively predicted sheep red blood cell (SRBC) characteristic, and then is sieved Select low oxyphile sheep.
Table 3：Red blood cell key property value detects SNP with ApoE gene19342316The relation of site parting
Deletion form Heterozygous Reference type Standardization P values
MCHC (%) 36.1 34.3 33.9 1.1*10-13
Mean corpuscular volume (MCV) (fl) 32.3 33.6 34.3 6.2*10-6
Embodiment 3：SNP19342316Distribution of the parting in site in different population
Several country are have chosen from Hapmap databases and has been introduced by main lines, and hidden sheep and live in Ke Shi meter A smooth sheep on your plateau.Comparative studies SNP19342316The parting and frequency in site.As shown in table 4, sheep is hidden all to show Homozygosis AA partings (plateau type), a smooth sheep from Kashmir 4000 meters or so of smooth regional height above sea level and high from Northern Europe Finland The Finland sheep on ground also shows A (plateau type) frequency (0.996,0.854) very high.Relative, from European plains region Individual major part shows the GG partings (plain type) of higher proportion.Result shows the sheep almost all survived in altitude environment It is plateau type.
Table 4：SNP19342316Distribution of the site parting frequency in several principal items
Strain/the place of production Sample number AA AG GG A gene frequencies Original producton location
Australian Merino 50 0 2 48 0.02 Southwestern Europe
Caddice wool chine 23 1 9 13 0.239 Southwestern Europe
Australia Dorset Down Sheep 108 7 39 62 0.245 Eastern Europe
New Zealand's Romney Marsh sheep 24 1 10 13 0.25 Eastern Europe
Iranian Suffolk 55 4 25 26 0.3 Eastern Europe
Scotland Te Kesaier sheep 80 18 40 22 0.475 Eastern Europe
New Zealand Te Kesaier sheep 24 6 13 5 0.521 Eastern Europe
Africa Du pool sheep 21 10 9 2 0.69 Africa
Finland sheep 99 74 21 4 0.854 Northern Europe
Open smooth sheep 29 27 2 0 0.966 Asia
Hide sheep 37 37 0 0 1 Asia
Embodiment 4：ApoE gene method and TaqMan probe method detection world's Different Altitude, different cultivars sheep In SNP19342316Site parting
The sequencing of world ovine genome tissue has been downloaded from NCBI 40 the 70 of kind sheep it is individual~10X genes Group weight sequencing sequence.Then all sequences are corresponded on sheep reference gene group OARv3.1 using BWA softwares, by statistics Whether the sequence coverage in this region of the marked region (No. 18 chromosome 19272.8-19322.8kb) sequencing depth assessment is about The 100%, 50% and 0% of mean coverage, thus judges whether this region is that fragment has homozygous, fragment presence and loses The heterozygous lost and deposit, and fragment loss is homozygous.
Result is as shown in table 5：In addition to sheep is hidden in an east, other individual SNP19342316Site parting with deletion form phase Symbol (GG correspondence reference types, AA correspondence deletion forms, AG correspondences heterozygous), illustrates SNP19342316The parting in site has with gene delection Good correlation.In addition, this group of Dynamic data exchange demonstrates the SNP in embodiment 119342316And gene delection polymorphism is not With the distribution frequency in species.That is, plateau strain A and reference type frequency are higher.
Table 5：The missing and SNP of whole world different lines some individuals19342316Parting compares
Individuality numbering Sequencing coverage rate Whether lack SNP partings Original producton location
1 merino 1 0 Reference type GG Southwestern Europe
2 merinos 2 0.000576 Reference type GG Southwestern Europe
3 merinos 3 0.001079 Reference type GG Southwestern Europe
4 Dorset Down Sheeps 0.00223 Reference type GG Eastern Europe
5 Du pool sheep 1 0.557122 Heterozygous AG Africa
6 Du pool sheep 2 1.228633 Deletion form AA Africa
7 Te Kesaier sheep 1.328273 Deletion form AA Eastern Europe
8 smooth sheep 1 1.141295 Deletion form AA The Asia Pamir Mountain Area
9 smooth sheep 2 1.194676 Deletion form AA The Asia Pamir Mountain Area
10 northern Tibetan sheep 1.460935 Deletion form AA Asia Qinghai-Tibet Platean
Hide sheep in 11 east 0.481583 Heterozygous AA Asia Qinghai-Tibet Platean
Embodiment 5：Hide the SNP in sheep19342316Site parting
From Ali Area, Xizang environment of high-altitude area (>4000m) tibetan sheep of medium-term and long-term adaptation life picks 20 at random, The genome for carrying out 10X is resurveyed sequence, carries out ibid data processing and analysis, as a result 20 SNP of Tibetan sheep19342316Site is all AA, i.e. plateau type, with hypoxia adaptability.The experiment further demonstrates screening hypoxia adaptability sheep provided by the present invention Method validity.
Embodiment 6：
The experiment that remaining 313 SNP site repeats embodiment 1-5 is chosen, hypoxia adaptability sheep can be accurately screened, it is accurate True rate is up to more than 90%.
Embodiment 7：
Choose r2<0.6 SNP site repeats the experiment of embodiment 1-5, finds the accuracy rate of screening hypoxia adaptability sheep Appearance is decreased obviously.
For example, for r2=0.552 SNP site, accuracy rate is 74%；For r2=0.496 SNP site, screening is accurate True rate is 51%；For r2=0.33 SNP site, screening accuracy rate drops to 43%.
Therefore, using the accuracy rate and linkage coefficient r of close linkage SNP site screening hypoxia adaptability sheep2In the presence of pass Connection property, according to experimental result, only r2>0.6 SNP site just can guarantee that with No. 18 chromosome 19272.8-19322.8kb's Large fragment loses the height linksystem of variation, so as to ensure to screen accuracy rate.
Work as r2<When 0.6, linkage disequilibrium relevance is gradually weakened, although r2>0.33 is generally accepted still to belong to tight association, But 0.33<r2<0.6 SNP site with No. 18 large fragments of chromosome 19272.8-19322.8kb it is difficult to ensure that lose The uniformity of variation, it is difficult to meet the requirement of screening accuracy.
Large fragment deletion is the fundamental cause for causing erythrocyte phenotype to make a variation, but the more difficult detection of large fragment deletion.In addition, Present molecular biology is difficult to find the mutational site for determining proterties, thus, the most of molecular labeling for finding at present be all with The SNP site of unknown decisive mutation linkage.The method of the screening hypoxia adaptability sheep that the present invention is provided, by analysis and 18 The large fragment of number chromosome 19272.8-19322.8kb loses the highly chain (r of variation2>0.6) 314 SNP sites, judge Variation is lost in the region with the presence or absence of large fragment, and then predicts the hypoxia adaptability characteristic of sheep.Sample size is small needed for the method, Technical operation is simple, low cost, greatly improves breeding and the efficiency introduced a fine variety, and method flexibly, can be different according to different samples selections SNP site.And can be detected that accuracy is high using TaqMan probe method or ApoE gene method, repeatability It is good.
Those of ordinary skill in the art should be understood：The discussion of any of the above embodiment is exemplary only, not It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under thinking of the invention, above example Or can also be combined between the technical characteristic in different embodiments, step can be realized with random order, and be existed such as Many other changes of upper described different aspect of the invention, for simplicity, they are provided not in details.Therefore, it is all Within the spirit and principles in the present invention, any omission, modification, equivalent, improvement for being made etc. should be included in of the invention Within protection domain.

Claims (9)

1. it is a kind of screen hypoxia adaptability sheep method, it is characterised in that comprise the following steps：
1) genomic DNA of sheep to be detected is extracted；
2) SNP site with the missing close linkage of 19272.8-19322.8kb regional genes on No. 18 chromosomes of sheep is detected In one or more sites；
3) by the parting of SNP site, sheep is divided into plateau type, plain type and heterozygous, wherein, the sheep of plateau type has Hypoxia adaptability.
2. the method for screening hypoxia adaptability sheep according to claim 1, it is characterised in that No. 18 dyes with sheep The linkage coefficient r of the SNP site of the missing close linkage of 19272.8-19322.8kb regional genes on colour solid2>0.6。
3. it is according to claim 1 screening hypoxia adaptability sheep method, it is characterised in that step 2) in detection SNP The method in site is ApoE gene method or TaqMan probe method.
4. it is according to claim 3 screening hypoxia adaptability sheep method, it is characterised in that the allele specific Property PCR methods be one or more sites in the close linkage SNP site chosen, be designed for amplification this one or more The primer of SNP site, the parting of SNP site is judged by gene cloning, gene sequencing, so as to screen the silk floss of hypoxia adaptability Sheep.
5. it is according to claim 3 screening hypoxia adaptability sheep method, it is characterised in that the TaqMan probe method It is one or more sites in the close linkage SNP site chosen, corresponding probe is designed, by the probe in detecting The parting of SNP site, screens the sheep of hypoxia adaptability.
6. it is according to claim 5 screening hypoxia adaptability sheep method, it is characterised in that described probe 5 ' end Reporter fluorescence group is marked with, 3 ' ends are marked with quenching fluorescence group.
7. it is according to claim 6 screening hypoxia adaptability sheep method, it is characterised in that described probe 5 ' end It is VIC or FAM to be marked with reporter fluorescence group, and it is TAMRA or MGB that 3 ' ends are marked with quenching fluorescence group.
8. the method for the screening hypoxia adaptability sheep as described in any one in claim 1-7 is auxiliary in hypoxia adaptability sheep The application helped in introducing a fine variety.
9. the method for the screening hypoxia adaptability sheep as described in any one in claim 1-7 is miscellaneous in hypoxia adaptability sheep Hand over the application in seed selection.
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CN107400713A (en) * 2017-08-18 2017-11-28 公安部物证鉴定中心 A kind of method and system that the Qinghai-Tibet Tibetan Population individual of China is identified in 27 colonies
CN107400713B (en) * 2017-08-18 2020-06-30 公安部物证鉴定中心 Method and system for identifying individuals of Tibetan nationality of Tibet plateau in 27 groups
CN110093406A (en) * 2019-05-27 2019-08-06 新疆农业大学 A kind of argali and its filial generation gene research method

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Application publication date: 20170531