CN103146830A - Molecular marking method capable of simultaneously predicting and identifying fineness and crimpness of sheep wool - Google Patents
Molecular marking method capable of simultaneously predicting and identifying fineness and crimpness of sheep wool Download PDFInfo
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Abstract
The invention discloses a molecular marking method capable of simultaneously predicting and identifying fineness and crimpness of sheep wool and relates to a molecular marking method. The method comprises the following steps: 1, designing a primer for performing amplification on sheep genome DNA (Deoxyribonucleic acid), and performing enzyme digestion, thereby obtaining an enzyme digestion product; 2, performing electrophoresis separation on the enzyme digestion product, and determining the genotype according to the electrophoresis separation result; 3, performing correlation analysis, and estimating the least squares mean value of the properties, wherein the results are that the wool fineness of the AA, GA genotype group in three genotypes is obviously lower than that of the GG genotype group, and the wool crimpness with the AA genotype group is obviously higher than that of the GG genotype group; and 4, dividing the experimental group into three types, and finishing the operation. The fineness and crimpness of sheep wool of the sheep can be simultaneously predicted and identified, an effective, simple and feasible molecular marking method is provided for quality character improvement and auxiliary mark selection of the sheep wool, and the method can be effectively applied to the field of molecular assisted breeding for superfine wool sheep.
Description
Technical field
The present invention relates to the animal molecular genetics field, particularly relate to a kind of method that detects simultaneously sheep fineness of wool and crimpness with molecule marker.
Background technology
Fine-wool sheep occupies critical role in China's herding industry, the main products of fine-wool sheep is wool.Fine wool has higher economic worth as important textile raw material.The production of fine-wool sheep not only is related to Economic development and the social stability in producing region, and is related to development and the foreign trade balance of China's wool industry.Along with both at home and abroad to the increase of wool demand, the cultivation of high-quality fine-wool sheep become that the wool industry is produced and the Sheep Breeding field in problem demanding prompt solution.
Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) was cultivated since 1972, successively cultivated six strains, was 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.End 2002 and promote 120,000 of high-quality sheep to 23, whole nation province, autonomous region, for major contribution has been made in development and the improvement work of China's fine-wool sheep cause.But, how fineness of wool further is provided, cultivate ultra-fine hair and plant sheep, and enlarge fast population scale and remain a major issue.Fineness of wool is a kind of proterties of high heritability, and heritability reaches 0.59.Molecular marking technique can be avoided the interference of age, sex, environment in kind of sheep seed selection, realize selecting high quality merino sheep in early days, fast and accurately, sets up and enlarges the high quality merino sheep population.Therefore, seek fineness of wool genes involved and molecule marker, uses that modern molecular breeding new technology quickly breeding surpasses fine-wool sheep, to enlarge high quality merino sheep germplasm scale fast imperative.
The MTR genes encoding synthesizes methionine synthetase, this enzyme can synthesize methionine(Met) by catalysis homocysteine (Hcy), simultaneously also can make 5-methyltetrahydrofolate be converted into tetrahydrofolic acid (THFA), so in this gene and body, the cyclic metabolism of folic acid is closely related.Human inheritance's disease studies show that, MTR transgenation and methyl L-glutamic acid G type disappearance disease are closely related, and this is a kind of autosomal recessive hereditary diseases, often causes the relative diseases such as mental retardation, macrocytic anemia and homocysteine urine disease.Yet, rarely have report about the MTR gene in the research of livestock and poultry.
Summary of the invention
The object of the present invention is to provide a kind of molecule marking method that can indicate simultaneously and identify sheep fineness of wool and crimpness.
A kind ofly can indicate simultaneously and identify that the molecule marking method of sheep fineness of wool and crimpness carries out according to the following steps:
One, include G9542341A site, subarea design pair of primers MTRF1 and MTRR1 according to sheep MTR gene the 27th, then ovine genome DNA is carried out pcr amplification, obtain pcr amplification product, then cut pcr amplification product with restriction endonuclease MspI enzyme, obtain enzyme and cut product;
Two, working concentration is that 2%～3% sepharose is cut product to enzyme and carried out electrophoretic separation, then carrying out genotype according to the electrophoretic separation result judges, the standard of judging: 1. electrophoresis presents two bands, size is 269bp and 209bp, when sheep MTR gene the 27th includes G9542341A site, subarea and does not suddenly change, pcr amplification product can be cut fully by the MspI enzyme, with its called after GG genotype; 2. electrophoresis presents a band, and size is 478bp, and when sheep MTR gene the 27th included subarea G9542341A site mutation, pcr amplification product can not be cut by the MspI enzyme, with its called after AA genotype; 3. electrophoresis presents three bands, and size is 478bp, 269bp and 209bp, and sheep MTR gene the 27th includes G9542341A site, subarea and is in heterozygous state, and pcr amplification product can not be cut fully by the MspI enzyme, with its called after GA genotype;
three, characteristics according to Chinese Merino sheep experimental population, build genotype effect statistical model: Y=μ+G+L+A+G * L+G * A+L * A+e, wherein, Y is the observed value of proterties, μ is colony's average, G is the genotype effect, L is the strain effect, A is age effect, G * L be genotype and strain make mutually effect, G * A be genotype and age make mutually effect, L * A be strain and age make mutually effect, e is the residual value shi effect, then utilize the JMP4.0 statistical software that genotype and continuity proterties are carried out association analysis, and the least square average of estimation proterties, the association analysis result shows, sheep MTR gene the 27th includes genotype effect and the fineness of wool of Chinese Merino sheep experimental population and the significant correlation of crimpness that G9542341A site, subarea G/A single base mutation causes, the P value is respectively 0.0002 and 0.0351, carry out again multiple comparisons, have the fineness of wool of AA, GA genotype colony in 3 kinds of genotype significantly lower than GG genotype colony, wool crimping degree with AA genotype colony is significantly higher than GG genotype colony,
Four, according to genotype, Chinese Merino sheep experimental population is divided into three types, thereby realize indicating simultaneously and identifying the molecular mark of sheep fineness of wool and crimpness, namely complete the molecule marking method that indicates simultaneously and identify sheep fineness of wool and crimpness;
Wherein in step 1, the nucleotides sequence of MTRF1 is classified as: 5 '-TGGAGAACCAAACACTGTCCTGAGA-3 '; The nucleotides sequence of MTRR1 is classified as: 5 '-CTTACACCTTATCGTGAACACCTATGC-3 ';
The base sequence that in step 2, the MspI enzyme is identified is CCGG;
The characteristics of Chinese Merino sheep experimental population in step 3: 1., 711 Chinese Merino sheep are Xinjiang reclamation of wasteland by an army units type; 2., ultra-fine hair strain 173, maos are with 135 of polyembryony strains, 146 of A strains, 100 of B strains, 32 of U strains, 125 of meat polyembryony strains; 3., be ewe; 4., the age is 1～12 years old.
The present invention adopts method that pcr amplification separates with agarose gel electrophoresis to detect the simple point mutation in a site, sheep MTR gene intron district.Owing to the G9542341A site can being identified by restriction endonuclease MspI, primer MTRF1/MTRR1 for this site design can amplify the DNA fragmentation that comprises this site easily, uses the 2%-3% agarose gel electrophoresis to judge three kinds of genotype at an easy rate.
The present invention detects by the different genotype to MTR gene G9542341A site, and this site is positioned at MTR gene the 27th and includes the subarea, and near the sequence the primer pair G9542341A site of adopting that MTRF1 and MTRR1 represent increases.
The present invention is simple to operate, expense is low, tolerance range is high, can carry out automatization and detect.When using marker genetype of the present invention that sheep fineness of wool and crimpness are selected, will make fineness and the crimpness of sheep wool obtain very large genetic progress.Fineness of wool and crimpness are all important quality trait and economic characters, and crimpness and fineness are negative correlation, sheep tortuosity larger (many) simultaneously, hair thinner (diameter is less), the hair pliability is better, and textile performance is higher, and two kinds of proterties all play a decisive role to the wool price.Utilize the present invention to select simultaneously fineness of wool and two proterties of crimpness, not only the quality trait improvement for the sheep wool provides a kind of effective molecular marker breeding means, also provides more effective, a simple and easy to do molecule marking method for marker assisted selection in Sheep Breeding work.The present invention can effectively be applied to the marker assisted selection field of ultra-fine hair sheep, realizes the early stage seed selection of kind of sheep, can select and remain after birth, accelerates the breeding process of sheep.
Description of drawings
Fig. 1 is that in embodiment, enzyme is cut the electrophorogram that product carries out electrophoretic separation.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is a kind of can indicate simultaneously and identify that the molecule marking method of sheep fineness of wool and crimpness carries out according to the following steps:
One, include G9542341A site, subarea design pair of primers MTRF1 and MTRR1 according to sheep MTR gene the 27th, then ovine genome DNA is carried out pcr amplification, obtain pcr amplification product, then cut pcr amplification product with restriction endonuclease MspI enzyme, obtain enzyme and cut product;
Two, working concentration is that 2%～3% sepharose is cut product to enzyme and carried out electrophoretic separation, then carrying out genotype according to the electrophoretic separation result judges, the standard of judging: 1. electrophoresis presents two bands, size is 269bp and 209bp, when sheep MTR gene the 27th includes G9542341A site, subarea and does not suddenly change, pcr amplification product can be cut fully by the MspI enzyme, with its called after GG genotype; 2. electrophoresis presents a band, and size is 478bp, and when sheep MTR gene the 27th included subarea G9542341A site mutation, pcr amplification product can not be cut by the MspI enzyme, with its called after AA genotype; 3. electrophoresis presents three bands, and size is 478bp, 269bp and 209bp, and sheep MTR gene the 27th includes G9542341A site, subarea and is in heterozygous state, and pcr amplification product can not be cut fully by the MspI enzyme, with its called after GA genotype;
three, characteristics according to Chinese Merino sheep experimental population, build genotype effect statistical model: Y=μ+G+L+A+G * L+G * A+L * A+e, wherein, Y is the observed value of proterties, μ is colony's average, G is the genotype effect, L is the strain effect, A is age effect, G * L be genotype and strain make mutually effect, G * A be genotype and age make mutually effect, L * A be strain and age make mutually effect, e is the residual value shi effect, then utilize the JMP4.0 statistical software that genotype and continuity proterties are carried out association analysis, and the least square average of estimation proterties, the association analysis result shows, sheep MTR gene the 27th includes genotype effect and the fineness of wool of Chinese Merino sheep experimental population and the significant correlation of crimpness that G9542341A site, subarea G/A single base mutation causes, P value difference 0.0002 and 0.0351, carry out again multiple comparisons, have the fineness of wool of AA, GA genotype colony in 3 kinds of genotype significantly lower than GG genotype colony, wool crimping degree with AA genotype colony is significantly higher than GG genotype colony,
Four, according to genotype, Chinese Merino sheep experimental population is divided into three types, thereby realize indicating simultaneously and identifying the molecular mark of sheep fineness of wool and crimpness, namely complete the molecule marking method that indicates simultaneously and identify sheep fineness of wool and crimpness;
Wherein in step 1, the nucleotides sequence of MTRF1 is classified as: 5 '-TGGAGAACCAAACACTGTCCTGAGA-3 '; The nucleotides sequence of MTRR1 is classified as: 5 '-CTTACACCTTATCGTGAACACCTATGC-3 ';
The base sequence that in step 2, the MspI enzyme is identified is CCGG;
The characteristics of Chinese Merino sheep experimental population in step 3: 1., 711 Chinese Merino sheep are Xinjiang reclamation of wasteland by an army units type; 2., ultra-fine hair strain 173, maos are with 135 of polyembryony strains, 146 of A strains, 100 of B strains, 32 of U strains, 125 of meat polyembryony strains; 3., be ewe; 4., the age is 1～12 years old.
In present embodiment, fineness of wool refers to the fiber diameter of wool, and crimpness refers to along the amount of crimp of every centimetre on the wool length direction.
Ovine genome sequence in present embodiment in pcr amplification product is as shown in SEQ ID No.3.
Relate in present embodiment that primer is synthetic to be completed by Shanghai English fine horse biological company limited.
Present embodiment relates to the equal by specification operation of test kit of use in concrete operations.
Embodiment two: present embodiment is different from embodiment one is that the reaction system of pcr amplification in step 1 is 10 μ L reaction systems, is comprised of following ingredients:
The pcr amplification condition is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 65 ℃ of annealing 25s, 72 ℃ are extended 30s, totally 33 circulations, then 72 ℃ of extension 7min, 4 ℃ of insulations.Other is identical with embodiment one.
Embodiment three: what present embodiment was different from embodiment one is that the system that in step 1, enzyme is cut is as follows:
The enzyme tangent condition is: 37 ℃ of enzymes are cut 1-2h or spend the night.Other is identical with embodiment one.
Embodiment:
A kind ofly can indicate simultaneously and identify that the molecule marking method of sheep fineness of wool and crimpness carries out according to the following steps:
One, include G9542341A site, subarea design pair of primers MTRF1 and MTRR1 according to sheep MTR gene the 27th, then ovine genome DNA is carried out pcr amplification, obtain pcr amplification product, then cut pcr amplification product with restriction endonuclease MspI enzyme, obtain enzyme and cut product;
Two, working concentration is that 2%～3% sepharose is cut product to enzyme and carried out electrophoretic separation, then carrying out genotype according to the electrophoretic separation result judges, the standard of judging: 1. electrophoresis presents two bands, size is 269bp and 209bp, when sheep MTR gene the 27th includes G9542341A site, subarea and does not suddenly change, pcr amplification product can be cut fully by the MspI enzyme, with its called after GG genotype; 2. electrophoresis presents a band, and size is 478bp, and when sheep MTR gene the 27th included subarea G9542341A site mutation, pcr amplification product can not be cut by the MspI enzyme, with its called after AA genotype; 3. electrophoresis presents three bands, and size is 478bp, 269bp and 209bp, and sheep MTR gene the 27th includes G9542341A site, subarea and is in heterozygous state, and pcr amplification product can not be cut fully by the MspI enzyme, with its called after GA genotype;
three, characteristics according to Chinese Merino sheep experimental population, build genotype effect statistical model: Y=μ+G+L+A+G * L+G * A+L * A+e, wherein, Y is the observed value of proterties, μ is colony's average, G is the genotype effect, L is the strain effect, A is age effect, G * L be genotype and strain make mutually effect, G * A be genotype and age make mutually effect, L * A be strain and age make mutually effect, e is the residual value shi effect, then utilize the JMP4.0 statistical software that genotype and continuity proterties are carried out association analysis, and the least square average of estimation proterties, the association analysis result shows, sheep MTR gene the 27th includes genotype effect and the fineness of wool of Chinese Merino sheep experimental population and the significant correlation of crimpness that G9542341A site, subarea G/A single base mutation causes, the P value is respectively 0.0002 and 0.0351, carry out again multiple comparisons, have the fineness of wool of AA, GA genotype colony in 3 kinds of genotype significantly lower than GG genotype colony, wool crimping degree with AA genotype individuality is significantly higher than GG genotype colony,
Four, according to genotype, Chinese Merino sheep experimental population is divided into three types, thereby realize indicating simultaneously and identifying the molecular mark of sheep fineness of wool and crimpness, namely complete the molecule marking method that indicates simultaneously and identify sheep fineness of wool and crimpness;
Wherein in step 1, the nucleotides sequence of MTRF1 is classified as: 5 '-TGGAGAACCAAACACTGTCCTGAGA-3 '; The nucleotides sequence of MTRR1 is classified as: 5 '-CTTACACCTTATCGTGAACACCTATGC-3 ';
The base sequence that in step 2, the MspI enzyme is identified is CCGG;
The characteristics of Chinese Merino sheep experimental population in step 3: 1., 711 Chinese Merino sheep are Xinjiang reclamation of wasteland by an army units type; 2., ultra-fine hair strain 173, maos are with 135 of polyembryony strains, 146 of A strains, 100 of B strains, 32 of U strains, 125 of meat polyembryony strains; 3., be ewe; 4., the age is 1～12 years old.
Gather the sheep ear tissue in the present embodiment ,-20 ℃ save backup.Adopt conventional phenol/chloroform method to extract ovine genome DNA.Wool traits is measured according to national examination of fibers standard and with reference to international wool manufacturing tissue (IWTO) fiber examination criteria, sheep side section galley proof is carried out the mensuration of fineness and crimpness.Fineness of wool often represents with diameter, 300 of homogeneity full scale amounts, and heterogeneous fleece is measured 400, averages, unit: μ m; Crimpness represents the degree of wool crimping, represent with the crimpness of every centimetre, unit be (curling/2.5cm).
Extract ovine genome DNA in the present embodiment:
(1) get Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) ear tissue 5g, reject reticular tissue, tissue block with 70% alcohol wash, sterilization, is inserted in the Eppendorf pipe, shred with scissors, or grind broken;
(2) after the alcohol in the Eppendorf pipe volatilizees fully, add dissociating buffer 700 μ l, suspend after the tissue that shreds, add Proteinase K (20mg/ml) 5.0 μ l, 55 ℃ of effect 8-12h are until the inorganization piece;
(3) will digest good tissue juice and take out, add the saturated phenol of equivalent tissue juice, mixing 10min, 4 ℃, 12000rpm, 10min is centrifugal;
(4) get supernatant liquor, add the phenol/chloroform of equivalent, mixing 10min gently, 4 ℃, 12000rpm, 10min is centrifugal;
(5) get supernatant liquor, add the chloroform of equivalent, mixing 10min, 4 ℃, 12000rpm, 10min is centrifugal;
(6) get supernatant liquor, add the dehydrated alcohol precipitation of 2 times of amounts, after putting upside down mixing, standing 10-20min under room temperature, the DNA precipitation forms white floss;
(7) supernatant discarded, then add 70% ethanol cleaning, supernatant discarded sucks unnecessary liquid on thieving paper, after natural air drying, add appropriate TE dissolving ,-20 ℃ of preservations.
(8) if not dissolved particles is arranged in DNA solution, can be of short duration centrifugal at 5000rpm, get supernatant; As removing RNA wherein, can add 5 μ l RNaseA (10 μ g/ μ l), 37 ℃ of insulation 30min after the phenol extracting, precipitate DNA again by step 4-7.
In the present embodiment step 1, the reaction system of pcr amplification is 10 μ L reaction systems, is comprised of following ingredients:
The pcr amplification condition is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 65 ℃ of annealing 25s, 72 ℃ are extended 30s, totally 33 circulations, then 72 ℃ of extension 7min, 4 ℃ of insulations.
The system that in the present embodiment step 1, enzyme is cut is as follows:
The enzyme tangent condition is: 37 ℃ of enzymes are cut 1-2h or spend the night.
Utilize primer of the present invention (MTRF1 and MTRR1) to carry out pcr amplification to the genomic dna of Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) 711 individualities of experimental population in the present embodiment, amplified production is carried out enzyme cut, then carry out the agarose gel electrophoresis analysis.3 kinds of genotype detected altogether in Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) experimental population.When sheep MTR gene the 27th included G9542341A site, subarea and do not suddenly change, pcr amplification product can be cut fully by the MspI enzyme, and electrophoresis presents two bands, and size is respectively 269bp and 209bp, with its called after GG genotype; When sheep MTR gene the 27th included subarea G9542341A site mutation, pcr amplification product can not be cut by the MspI enzyme, and electrophoresis presents a band, and size is 478bp, with its called after AA genotype; When sheep MTR gene the 27th included G9542341A site, subarea and is in heterozygous state, it was three bands that the enzyme of pcr amplification product is cut product, i.e. 478bp, 269bp and 209bp are with its called after GA genotype (seeing Fig. 1).Distribution in 6 Sheep Populations is analyzed to 3 kinds of different genotype, and the GA type is at most individual, and frequency is 0.405 (table 1).
The distribution of table 1MTR gene G9542341A site different genotype in Sheep Populations
| Strain | AA | GA | GG | Amount to |
| Ultra-fine hair strain | 90 | 68 | 15 | 173 |
| Hair polyembryony strain | 5 | 46 | 84 | 135 |
| The A strain | 15 | 73 | 58 | 146 |
| The B strain | 18 | 43 | 39 | 100 |
| The U strain | 7 | 14 | 11 | 32 |
| Meat polyembryony strain | 16 | 44 | 65 | 125 |
| Amount to | 151 | 288 | 272 | 711 |
| Genotype frequency | 0.212 | 0.405 | 0.383 | 1 |
MTR gene the 27th is included 3 kinds of genotype of G9542341A site, subarea G/A single base mutation and the fineness of wool of Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) 711 individualities of experimental population, the fineness standard deviation, discrete and the crimpness proterties of fineness etc. is carried out the least square analysis, result shows that sheep MTR gene the 27th includes the fineness of wool of G9542341A site, subarea different genotype polymorphism and Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) experimental population, the fineness standard deviation, discrete and the crimpness of fineness is significant correlation all, wherein the P value is followed successively by 0.0002,＜0.0001, 0.0030 and 0.0351.
Least square average between 3 kinds of genotype is carried out multiple comparisons, result show have AA, the fineness of wool of GA genotype colony is significantly lower than GG genotype colony; Have the fineness of wool standard deviation of AA genotype colony significantly lower than GA genotype colony, and the fineness of wool standard deviation of GA genotype colony is significantly lower than GG genotype colony; Fineness of wool with AA, GA genotype colony is discrete significantly lower than GG genotype colony; Wool crimping degree with AA genotype colony is significantly higher than GG genotype colony (P＜0.05) (table 2).
Table 2MTR gene G9542341A site different genotype is to Chinese Merino sheep (Xinjiang reclamation of wasteland by an army units type) fineness of wool, thin
The degree standard deviation, fineness is discrete and the impact of crimpness
| Proterties | AA | GA | GG |
| Fineness | 20.270938±0.21055646 a | 20.559213±0.11905353 a | 21.181972±0.13819445 b |
| The fineness standard deviation | 3.9039388±0.07179087 a | 4.0732458±0.04059223 b | 4.3577356±0.04711848 c |
| Fineness is discrete | 19.285963±0.32122159 a | 19.832183±0.18162618 a | 20.508073±0.21082725 b |
| Crimpness | 12.556957±0.23860822 a | 12.269694±0.13493026 ab | 11.877859±0.15662485 b |
Average relatively the time with delegation without same letter person's significant difference (
a-bP＜0.05).
Above result shows, the MTR gene can be simultaneously as one of main candidate of sheep fineness of wool and crimpness, the AA type can be used as molecular genetic marker and is used for prediction sheep fineness of wool and two proterties of crimpness.Can set up AA type individuality is main fineness of wool and crimpness proterties breeding population, effectively cultivates low fineness, high curling fine, soft fur sheep strain.
Claims (3)
1. molecule marking method that can indicate simultaneously and identify sheep fineness of wool and crimpness is characterized in that it carries out according to the following steps:
One, include G9542341A site, subarea design pair of primers MTRF1 and MTRR1 according to sheep MTR gene the 27th, then ovine genome DNA is carried out pcr amplification, obtain pcr amplification product, then cut pcr amplification product with restriction endonuclease MspI enzyme, obtain enzyme and cut product;
Two, working concentration is that 2%～3% sepharose is cut product to enzyme and carried out electrophoretic separation, then carrying out genotype according to the electrophoretic separation result judges, the standard of judging: 1. electrophoresis presents two bands, size is 269bp and 209bp, when sheep MTR gene the 27th includes G9542341A site, subarea and does not suddenly change, pcr amplification product can be cut fully by the MspI enzyme, with its called after GG genotype; 2. electrophoresis presents a band, and size is 478bp, and when sheep MTR gene the 27th included subarea G9542341A site mutation, pcr amplification product can not be cut by the MspI enzyme, with its called after AA genotype; 3. electrophoresis presents three bands, and size is 478bp, 269bp and 209bp, and sheep MTR gene the 27th includes G9542341A site, subarea and is in heterozygous state, and pcr amplification product can not be cut fully by the MspI enzyme, with its called after GA genotype;
three, characteristics according to Chinese Merino sheep experimental population, build genotype effect statistical model: Y=μ+G+L+A+G * L+G * A+L * A+e, wherein, Y is the observed value of proterties, μ is colony's average, G is the genotype effect, L is the strain effect, A is age effect, G * L be genotype and strain make mutually effect, G * A be genotype and age make mutually effect, L * A be strain and age make mutually effect, e is the residual value shi effect, then utilize the JMP4.0 statistical software that genotype and continuity proterties are carried out association analysis, and the least square average of estimation proterties, the association analysis result shows, sheep MTR gene the 27th includes genotype effect that G9542341A site, subarea G/A single base mutation causes and fineness of wool and the crimpness proterties significant correlation of Chinese Merino sheep experimental population, the P value is respectively 0.0002 and 0.0351, carry out again multiple comparisons, have the fineness of wool of AA, GA genotype colony in 3 kinds of genotype significantly lower than GG genotype colony, wool crimping degree with AA genotype colony is significantly higher than GG genotype colony,
Four, according to genotype, Chinese Merino sheep experimental population is divided into three types, thereby realize indicating simultaneously and identifying the molecular mark of sheep fineness of wool and crimpness, namely complete the molecule marking method that indicates simultaneously and identify sheep fineness of wool and crimpness;
Wherein in step 1, the nucleotides sequence of MTRF1 is classified as: 5 '-TGGAGAACCAAACACTGTCCTGAGA-3 '; The nucleotides sequence of MTRR1 is classified as: 5 '-CTTACACCTTATCGTGAACACCTATGC-3 ';
The base sequence that in step 2, the MspI enzyme is identified is CCGG;
The characteristics of Chinese Merino sheep experimental population in step 3: 1., 711 Chinese Merino sheep are Xinjiang reclamation of wasteland by an army units type; 2., ultra-fine hair strain 173, maos are with 135 of polyembryony strains, 146 of A strains, 100 of B strains, 32 of U strains, 125 of meat polyembryony strains; 3., be ewe; 4., the age is 1～12 years old.
2. a kind of molecule marking method that can indicate simultaneously and identify sheep fineness of wool and crimpness according to claim 1, the reaction system that it is characterized in that pcr amplification in step 1 is 10 μ L reaction systems, is comprised of following ingredients:
The pcr amplification condition is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 65 ℃ of annealing 25s, 72 ℃ are extended 30s, totally 33 circulations, then 72 ℃ of extension 7min, 4 ℃ of insulations.
3. a kind of molecule marking method that can indicate simultaneously and identify sheep fineness of wool and crimpness according to claim 1 is characterized in that the system that in step 1, enzyme is cut is as follows:
The enzyme tangent condition is: 37 ℃ of enzymes are cut 1-2h or spend the night.
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| CN103276098A (en) * | 2013-06-14 | 2013-09-04 | 东北农业大学 | Molecular marking method for predicting and identifying length of sheep wool |
| CN105063213A (en) * | 2015-08-10 | 2015-11-18 | 东北农业大学 | Molecular marker method capable of indicating and identifying curling degree of sheep wools and primer pair for molecular marker method |
| CN107630095A (en) * | 2017-10-23 | 2018-01-26 | 新疆畜牧科学院畜牧研究所 | The molecular labeling related to sheep wool number of bends character and its specific primer pair and application |
| 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 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276098A (en) * | 2013-06-14 | 2013-09-04 | 东北农业大学 | Molecular marking method for predicting and identifying length of sheep wool |
| CN103276098B (en) * | 2013-06-14 | 2014-07-16 | 东北农业大学 | Molecular marking method for predicting and identifying length of sheep wool |
| CN105063213A (en) * | 2015-08-10 | 2015-11-18 | 东北农业大学 | Molecular marker method capable of indicating and identifying curling degree of sheep wools and primer pair for molecular marker method |
| CN105063213B (en) * | 2015-08-10 | 2018-08-28 | 东北农业大学 | It can indicate and identify the molecule labelling method and its primer pair of sheep wool crimping degree |
| CN107630095A (en) * | 2017-10-23 | 2018-01-26 | 新疆畜牧科学院畜牧研究所 | The molecular labeling related to sheep wool number of bends character and its specific primer pair and application |
| CN107630095B (en) * | 2017-10-23 | 2021-03-05 | 新疆畜牧科学院畜牧研究所 | Molecular marker related to sheep wool bending number character and specific primer pair and application thereof |
| 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 |
| CN109825598B (en) * | 2018-11-01 | 2022-05-31 | 天津奥群牧业有限公司 | SNP (Single nucleotide polymorphism) marker remarkably related to Australian white sheep hair thickness, molecular marker and application |
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| CN103146830B (en) | 2014-05-07 |
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