CN109182558A - It can indicate and identify the molecular labeling primer pair and application of sheep wool natural length - Google Patents
It can indicate and identify the molecular labeling primer pair and application of sheep wool natural length Download PDFInfo
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Abstract
The present invention discloses a kind of molecular labeling primer pair that can indicate and identify sheep wool natural length and application, primer pair, as shown in sequence table Seq ID No.1 and Seq ID No.2.Using as follows: design primer pair, and extract ovine genome DNA and expanded, then digestion obtains digestion products；Digestion products electrophoretic separation carries out genotype judgement according to electrophoretic separation result；It is associated analysis, and estimates the least squares means of character, result is that the wool natural length in 3 kinds of genotype with GG genotypic population is significantly higher than AA genotype and GA genotypic population, finally determines that the ewe for choosing GG genotype is cooked breeding population.The present invention can indicate and identify sheep wool natural length, provide a more efficiently molecule labelling method for the quality trait improvement of sheep wool, the sheep marker assisted selection field long applied to screening wool fiber natural length.Operation of the present invention is simple, expense is low.
Description
Technical field
The present invention relates to animal molecular genetics field, it is specifically related to a kind of to indicate and identify that sheep wool is naturally long
The molecular labeling primer pair and application of degree.
Background technique
Wool natural length is an important indicator in sheep wool quality character.Wool natural length is straight with ruler
Connect the length of measurement wool in its natural state.Fine, soft fur sheep variety is different, then wool natural length is not also identical, such as Australia beauty
The wool natural length of sharp slave sheep is just longer than many fine, soft fur sheep varieties in China.Under normal circumstances, wool natural length and wool are flat
Equal fibre diameter is positively correlated, i.e. wool natural length is longer, and wool avarage fiber diameter is bigger.Wool natural length is being weaved
Technologic meaning is only second to wool avarage fiber diameter, it not only affects the parameter setting of textile technology, also affects hair
The comfort level of textile.In addition, with the change of the market demand, influence of the wool natural length to the wool market price has been more than
Wool avarage fiber diameter becomes the biggest factor of the left and right wool market price.
China started to cultivate caddice wool chine (Xinjiang reclamation of wasteland by an army units type) in 1972, had cultivated six product even to this day
System, respectively reclamation of wasteland by an army units A type strain, reclamation of wasteland by an army units Type B strain, superfine type strain, meat polyembryony strain, hair polyembryony strain and U strain.
In order to cater to the market demand, in order to increase income to herdsman, wool natural length how is further increased, cultivates wool certainly
Right length long high quality merino sheep and rapid expansion population scale are still a major issue.Wool natural length is sheep
The quality trait and economic characters that gross weight is wanted, wool natural length directly affect the parameter setting and wool textile of textile technology
Comfort level, and be influence the wool market price the biggest factor, therefore, improve wool natural length, it will generate it is huge
Economic benefit.
Publication number: CN103276098A, a kind of denomination of invention: molecular labeling side of indication and identification sheep wool length
Method needs to introduce a G base in upstream primer sequence, and to meet the identification sequence of PvuI restriction endonuclease, term is " forcing enzyme
It cuts ".Since primer is there are due to Incomplete matching, so forcing the efficiency of digestion relatively low.
Publication number: denomination of invention: CN107619870A can indicate and identify molecular labeling and its spy of sheep wool length
Specific primer to and application, it is primary it is bright in need by PCR product through DNA mix pond sequencing, and using Fluidigm SNP detect skill
Art carries out parting, can identify the longer sheep individual of wool natural length according to genotyping result, whole process needs four and draws
Object, and it is cumbersome, and consuming time is long.
TOMM70A is a hypotype of mitochondrial outer membrane transhipment enzyme TOMM70, is the hydrophobin precursor of targetted mitochondria
Receptor.Studies have shown that TOMM70A expresses downward in mankind's thyroid papillary carcinoma, in addition, TOMM70A also take part in it is small
The scytitis of mouse Gsdma3 gene mutation induction and alopecia.
Summary of the invention
Based on the above shortcomings, it can indicate and identify sheep wool natural length the object of the present invention is to provide a kind of
Molecular labeling primer pair and application, can select wool natural length, not only product of sheep wool using the present invention
Matter character improvement provides a kind of effective molecular marker breeding means, also provides for marker assisted selection in Sheep Breeding work
One more effective, easy-to-use molecule labelling method.
The technology that the present invention is participated in is as follows: a kind of molecule labelling method that can indicate and identify sheep wool natural length
The primer pair, upper primer TOMM70AF: as shown in sequence table Seq ID No.1；Lower primer TOMM70AR: such as sequence table Seq
Shown in ID No.2.
The present invention also has following technical characteristic:
1, primer pair as described above can indicate and identify the molecule of sheep wool natural length as preparation vitro detection
The preparation of label or the application in kit.
2, primer pair as described above is indicating and is identifying the application in caddice wool chine wool length character.
3, the application in indication and identification caddice wool chine wool length character as described above, the middle Guomei benefit
The Xinjiang reclamation of wasteland by an army units type ewe that slave sheep is 1~12 years old.
4, the application in indication and identification caddice wool chine wool length character as described above, includes the following steps:
Step 1: design pair of primers in the site sub-district G163139235A is included according to sheep TOMM70A gene the 7th
TOMM70AF and TOMM70AR, respectively as shown in sequence table Seq ID No.1 and Seq ID No.2, then to ovine genome
DNA carries out PCR amplification, obtains pcr amplification product, then uses restriction endonuclease Bae I digestion pcr amplification product, acquisition digestion products；Its
The pcr amplification product of middle acquisition, sequence is as shown in Seq ID No.3；
Step 2: being separated by electrophoresis digestion products, then carries out genotype judgement according to electrophoretic separation result；
Wherein the standard of the judgement of genotype is as follows:
Step 2 one: two bands are presented in electrophoresis, and size is 313bp and 152bp, then the 7th introne of sheep TOMM70A gene
When the area site G163139235A is unmutated, pcr amplification product can be cut completely through by Bae I enzyme, be named as GG gene
Type；
Step 2 two: a band is presented in electrophoresis, and size 498bp, then sheep TOMM70A gene the 7th includes sub-district
When G163139235A site mutation, pcr amplification product cannot be named as AA genotype by Bae I digestion；
Step 2 three: three bands, size 498bp, 313bp and 152bp, then sheep TOMM70A gene the 7th is presented in electrophoresis
It includes the site sub-district G163139235A and is in heterozygous state, pcr amplification product cannot be cut completely through by Bae I enzyme, be named
For GA genotype；
Step 3: the characteristics of testing group according to caddice wool chine constructs genotype effects statistical model: Y=μ+G+L
+ A+G × L+G × A+A × L+e, wherein Y is the observation of character, and μ is group's mean value, and G is genotype effects, and L is strain effect
It answers, A is age effect, and G × L is the reciprocal effects of genotype and strain, and G × A is the reciprocal effects of genotype and age, A × L
For the reciprocal effects of strain and age, e is remaining value effect, and genotype and continuity character are then associated analysis, and
Estimate the least squares means of character；
Step 4: finally determine that the ewe for choosing GG genotype is cooked breeding population.
5, the application in indication and identification caddice wool chine wool length character as described above, in the step one
Digestion system is as follows:
10×CutSmart Buffer+SAM 1μL
1 μ L of restriction endonuclease Bae I
10 μ L of PCR product
Digestion condition are as follows: 25 DEG C of digestion 30min；
6, the application in indication and identification caddice wool chine wool length character as described above, in the step one
The reaction system of PCR amplification is as follows:
PCR amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 53.8 DEG C of annealing 30s, 72 DEG C of extension 30s, altogether
36 circulations, 72 DEG C extend 10min, 4 DEG C of termination reactions eventually；
Operation of the present invention is simple, expense is low, accuracy is high, can be automated detection.Use marker gene of the invention
When type selects sheep wool natural length, the natural length of sheep wool will be made to obtain very big genetic progress.This hair
The early stage choosing of kind of sheep is realized in the bright marker assisted selection field that can be effectively applied to the long sheep of screening wool natural length
Kind, it can be selected and remain after birth, accelerate the breeding process of sheep.In the present invention, TOMM70A gene the 7th includes sub-district
After the site G163139235A mutates, it can be identified completely by BaeI restriction endonuclease, therefore primer sequence and template sequence complete
Match, does not need to force digestion, design of primers is simpler, convenient in the present invention, and digestion glue figure is more clear distinguishable.In the present invention
Two primers are only needed, PCR product directly can identify the longer silk floss of wool natural length through agarose gel electrophoresis after digestion
Sheep individual, experimental implementation is simple, fast.
Detailed description of the invention
Fig. 1 is the electrophoretic separation figure of digestion products.
Fig. 2 is TOMM70A gene template sequence and TOMM70A patent upstream primer sequence comparison result figure.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Embodiment 1
The acquisition of caddice wool chine genomic DNA
Sheep ear tissue is acquired, -20 DEG C save backup.Ovine genome DNA is extracted using conventional phenol/chloroform method.
The specific method is as follows:
(1) caddice wool chine (Xinjiang reclamation of wasteland by an army units type) ear tissue 5g is taken, connective tissue is rejected, by 70% alcohol of tissue block
Cleaning, disinfection, are placed in Eppendorf pipe, are shredded with scissors, or grinding is broken；
(2) after volatilizing completely to the alcohol in Eppendorf pipe, 700 μ L of dissociating buffer is added, suspend the group shredded
After knitting, Proteinase K (20mg/mL) 5.0 μ L, 55 DEG C of effect 8-12h, until inorganization block is added；
(3) by the tissue fluid digested take out, be added equivalent tissue fluid saturated phenol, mix 10min, 4 DEG C, 12000r/
M is centrifuged 10min；
(4) supernatant is taken, the phenol/chloroform of equivalent is added, 10min is mixed gently, 4 DEG C, 12000r/m, is centrifuged 10min；
(5) supernatant is taken, the chloroform of equivalent is added, 10min is mixed, 4 DEG C, 12000r/m, is centrifuged 10min；
(6) supernatant is taken, adds the dehydrated alcohol precipitating of 2 times of amounts, after being mixed by inversion, stand 10-20min at room temperature, DNA is heavy
Shallow lake forms White Flocculus；
(7) discard supernatant, then plus 70% ethyl alcohol cleaning, discard supernatant, surplus liquid, natural wind drawn on blotting paper
After dry, suitable TE is added to dissolve, -20 DEG C of preservations；
(8) if there is insoluble particle in DNA solution, supernatant can be taken in the of short duration centrifugation of 5000r/m；It such as to remove therein
RNA, can be added 5 μ L RNaseA (10 μ g/ μ L), and 37 DEG C of heat preservation 30min after being extracted with phenol, precipitate DNA by step 4-7 again.
Embodiment 2
The acquisition of sheep TOMM70A gene digestion products
According in ovine genome the site gene G163139235A TOMM70A design pair of primers TOMM70AF and
Then TOMM70AR carries out PCR amplification to ovine genome DNA, obtain pcr amplification product, then with restriction endonuclease Bae I digestion
Pcr amplification product obtains digestion products.
As shown in Fig. 2, TOMM70A gene the 7th includes after the site sub-district G163139235A mutates, it can quilt completely
The identification of BaeI restriction endonuclease, therefore primer sequence and template sequence exactly match, and do not need to force digestion.
Primer sequence is as follows:
TOMM70AF:5 '-ATGTGGCTTCTTGGAGTG-3 '
TOMM70AR:5 '-CACAAATAAGCGCAGTTC-3 '
PCR amplification system is as follows:
PCR amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 53.8 DEG C of annealing 30s, 72 DEG C of extension 30s, altogether
36 circulations, 72 DEG C extend 10min, 4 DEG C of termination reactions eventually.
Endonuclease reaction system is as follows:
10×CutSmart Buffer+SAM 1μL
1 μ L of restriction endonuclease Bae I
10 μ L of PCR product
Digestion condition are as follows: 25 DEG C of digestion 30min.
Embodiment 3
The judgement of sheep TOMM70A genotype
The digestion products in embodiment 1 are separated by electrophoresis using the Ago-Gel that concentration is 2%~3%, according to
Result is separated by electrophoresis and carries out genotype judgement, the standard of judgement: (1) two bands are presented in electrophoresis, and size is 313bp and 152bp, then
Sheep TOMM70A gene the 7th include the site sub-district G163139235A it is unmutated when, pcr amplification product can be complete by Bae I enzyme
Slitting-up is named as GG genotype；(2) band, size 498bp, then sheep TOMM70A gene the 7th is presented in electrophoresis
When including sub-district G163139235A site mutation, pcr amplification product cannot be named as AA genotype by Bae I digestion；
(3) three bands are presented in electrophoresis, and size 498bp, 313bp and 152bp, then sheep TOMM70A gene the 7th includes sub-district
The site G163139235A is in heterozygous state, and pcr amplification product cannot be cut completely through by Bae I enzyme, is named as GA gene
Type.
568 caddice wool chines in the present invention are the ewe of Xinjiang reclamation of wasteland by an army units type 1~12 years old, detect 3 kinds of bases altogether
Because of type, distribution of 3 kinds of different genotypes in 6 Sheep Populations to be analyzed, at most, the results are shown in Table 1 for GA type individual,
Frequency is.
Distribution of the 1 TOMM70A gene different genotype of table in Sheep Populations
Embodiment 4
The building of genotype effects statistical model
The characteristics of testing group according to caddice wool chine constructs genotype effects statistical model: Y=μ+G+L+A+G × L
+ G × A+A × L+e, wherein Y is the observation of character, and μ is group's mean value, and G is genotype effects, and L is strain effect, and A is year
Age effect, G × L be genotype and strain reciprocal effects, G × A be genotype and age reciprocal effects, A × L be strain and
The reciprocal effects at age, e are remaining value effect.
Wool natural length measurement of the present invention is fine according to national examination of fibers standard and international wool manufacturing tissue (IWTO)
Examination criteria is tieed up, and keeps " wool and wool quality " book that benevolence academician writes with reference to Liu, wool natural length ruler is direct
Measure the length of wool in its natural state.
The present invention includes 3 kinds of genotype of the site sub-district G163139235A G/A single base mutation to TOMM70A gene the 7th
Individual wool avarage fiber diameter, wool average fiber are straight with caddice wool chine (Xinjiang reclamation of wasteland by an army units type) test group 568
Diameter standard deviation, wool crimping degree and wool natural length character etc. carry out Least square analysis, the results showed that sheep TOMM70A base
Cause the 7th includes the site sub-district G163139235A different genotype polymorphism and caddice wool chine (Xinjiang reclamation of wasteland by an army units type) trial flock
The significant correlation of wool natural length degree of body, P value are 0.0111；The least squares means 3 kinds of genotype are carried out again more
Compare again, the results showed that the wool natural length with GG genotypic population is significantly higher than AA genotype and GA genotypic population (P
< 0.05), the results are shown in Table 2.
2 sheep TOMM70A gene the 7th of table includes sub-district G163139235A site different genotype to wool natural length
Influence
When mean value compares with a line without same letter person significant difference (a-bP<0.05)
The above result shows that TOMM70A gene can be used as one of the main candidate of sheep wool natural length, GG base
Because type can be used as molecular genetic marker for predicting sheep wool natural length.The silk floss based on GG genotype individuals can be set up
Sheep breeding population effectively cultivates the longer sheep breeds of natural length.
Embodiment 5
The establishment of the molecule labelling method of sheep wool natural length
Caddice wool chine test group is divided into three types according to genotype, to realize indication and identification sheep sheep
The molecular mark of hair natural length completes the molecule labelling method of indication and identification sheep wool natural length.
Sequence table
<110>Sun Yat-sen Memorial Hospital
<120>it can indicate and identify the molecular labeling primer pair and application of sheep wool natural length
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgtggcttc ttggagtg 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cacaaataag cgcagttc 18
<210> 3
<211> 498
<212> DNA
<213>sheep (sheep)
<400> 3
atgtggcttc ttggagtgca ttagagtgga aagtatggac taatccatat aataacagaa 60
aagttcaagt cctttctaat ggaaaatttt acctaatatt tctgtctgga gcatcttaag 120
aagtatcgtg ataaccttaa aattcttgat atttttatcc ttactggtgt atcttaagat 180
taatatgaaa tgtaattatt taatgtctga tctatcccta ctttgaagtt tgaaagtaca 240
gagacagttc atattctagg agaaaaatca cttaagagtg attaaacact gataggttta 300
taatgcttta aaaaaaaatt gaacactagt tctaagtttt agcacctttc ctttaaaatt 360
ataagatttt ctggttcact gtttctagaa agtaattcta tattagtaat tccatgctta 420
gctttatgct gattatgcaa aaaggcacag tatgtgctca actggtgtag tttttgtttt 480
gaactgcgct tatttgtg 498
Claims (7)
1. a kind of molecular labeling the primer pair that can indicate and identify sheep wool natural length, which is characterized in that upper primer
TOMM70AF: as shown in sequence table Seq ID No.1；Lower primer TOMM70AR: as shown in sequence table Seq ID No.2.
2. primer pair according to claim 1 can indicate and identify sheep wool natural length as preparation vitro detection
The preparation of molecular labeling or the application in kit.
3. primer pair according to claim 1 is indicating and is identifying the application in caddice wool chine wool length character.
4. the application in indication according to claim 3 and identification caddice wool chine wool length character, feature exist
In the Xinjiang reclamation of wasteland by an army units type ewe that the caddice wool chine is 1~12 years old.
5. the application in indication according to claim 4 and identification caddice wool chine wool length character, feature exist
In including the following steps:
Step 1: design pair of primers TOMM70AF in the site sub-district G163139235A is included according to sheep TOMM70A gene the 7th
And then TOMM70AR carries out ovine genome DNA respectively as shown in sequence table Seq ID No.1 and Seq ID No.2
PCR amplification, obtains pcr amplification product, then with restriction endonuclease Bae I digestion pcr amplification product, obtains digestion products；Wherein obtain
Pcr amplification product, sequence is as shown in Seq ID No.3；
Step 2: being separated by electrophoresis digestion products, then carries out genotype judgement according to electrophoretic separation result；
Wherein the standard of the judgement of genotype is as follows:
Step 2 one: two bands are presented in electrophoresis, and size is 313bp and 152bp, then sheep TOMM70A gene the 7th includes sub-district
When the site G163139235A is unmutated, pcr amplification product can be cut completely through by Bae I enzyme, be named as GG genotype；
Step 2 two: a band is presented in electrophoresis, and size 498bp, then sheep TOMM70A gene the 7th includes sub-district
When G163139235A site mutation, pcr amplification product cannot be named as AA genotype by Bae I digestion；
Step 2 three: three bands are presented in electrophoresis, and size 498bp, 313bp and 152bp, then sheep TOMM70A gene the 7th includes
The site sub-district G163139235A is in heterozygous state, and pcr amplification product cannot be cut completely through by Bae I enzyme, is named as GA
Genotype；
Step 3: the characteristics of testing group according to caddice wool chine constructs genotype effects statistical model: Y=μ+G+L+A+G
× L+G × A+A × L+e, wherein Y is the observation of character, and μ is group's mean value, and G is genotype effects, and L is strain effect, A
For age effect, G × L is the reciprocal effects of genotype and strain, and G × A is the reciprocal effects of genotype and age, and A × L is product
The reciprocal effects of system and age, e are remaining value effect, genotype and continuity character are then associated analysis, and estimate
The least squares means of character；
Step 4: finally determine that the ewe for choosing GG genotype is cooked breeding population.
6. the application in indication according to claim 5 and identification caddice wool chine wool length character, feature exist
In digestion system is as follows in the step one:
10×CutSmart Buffer+SAM 1μL
1 μ L of restriction endonuclease Bae I
10 μ L of PCR product
Digestion condition are as follows: 25 DEG C of digestion 30min.
7. the application in indication according to claim 5 and identification caddice wool chine wool length character, feature exist
In the reaction system of PCR amplification is as follows in the step one:
PCR amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 53.8 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 36
Circulation, 72 DEG C extend 10min, 4 DEG C of termination reactions eventually.
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| CN109825598A (en) * | 2018-11-01 | 2019-05-31 | 天津奥群牧业有限公司 | It is a kind of to the extremely significant relevant SNP marker of the white sheep hair thickness in Australia, molecular labeling and application |
| CN113278716A (en) * | 2021-07-23 | 2021-08-20 | 中国农业大学 | Gene chip for analyzing characters for sheep wool, molecular probe combination, kit and application |
| CN113293220A (en) * | 2021-07-23 | 2021-08-24 | 中国农业大学 | Gene chip for analyzing ear size of sheep, molecular probe combination, kit and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109825598A (en) * | 2018-11-01 | 2019-05-31 | 天津奥群牧业有限公司 | It is a kind of to the extremely significant relevant SNP marker of the white sheep hair thickness in Australia, molecular labeling and application |
| CN113278716A (en) * | 2021-07-23 | 2021-08-20 | 中国农业大学 | Gene chip for analyzing characters for sheep wool, molecular probe combination, kit and application |
| CN113293220A (en) * | 2021-07-23 | 2021-08-24 | 中国农业大学 | Gene chip for analyzing ear size of sheep, molecular probe combination, kit and application |
| CN113293220B (en) * | 2021-07-23 | 2022-06-10 | 中国农业大学 | Gene chip for analyzing ear size of sheep, molecular probe combination, kit and application |
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