CN109182558B - Molecular marker primer pair capable of indicating and identifying natural length of sheep wool and application thereof - Google Patents
Molecular marker primer pair capable of indicating and identifying natural length of sheep wool and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker primer pair capable of indicating and identifying the natural length of sheep wool and application thereof, wherein the primer pair is shown in sequence tables Seq ID No.1 and Seq ID No. 2. The following applies: designing a primer pair, extracting sheep genome DNA for amplification, and then carrying out enzyme digestion to obtain an enzyme digestion product; carrying out electrophoretic separation on the enzyme digestion product, and judging the genotype according to the electrophoretic separation result; and performing correlation analysis, estimating the least square mean value of the characters, and finally determining that the natural length of wool with GG genotype group in 3 genotypes is obviously higher than that of AA genotype and GA genotype groups, and selecting a ewe with GG genotype as a breeding group. The invention can predict and identify the natural length of the sheep wool, provides a more effective molecular marking method for the quality character improvement of the sheep wool, and is applied to the field of sheep molecular assisted breeding for screening the natural length of wool fibers. The invention has simple operation and low cost.
Description
Technical Field
The invention relates to the field of animal molecular genetics, in particular to a molecular marker primer pair capable of indicating and identifying the natural length of sheep wool and application thereof.
Background
The natural length of wool is an important index in the quality traits of sheep wool. The natural length of wool is measured directly with a ruler in the natural state. The natural length of wool is different when the varieties of fine wool sheep are different, for example, the natural length of the wool of Australian merino sheep is longer than that of many fine wool sheep varieties in China. Generally, the natural length of wool is positively correlated to the average fiber diameter of wool, i.e., the longer the natural length of wool, the larger the average fiber diameter of wool. The meaning of the natural length of wool in the textile technology is only second to the average fiber diameter of wool, which not only influences the parameter setting of the textile technology, but also influences the comfort level of wool textiles. In addition, with the change of market demand, the influence of the natural length of wool on the market price of wool exceeds the average fiber diameter of wool, and becomes the largest factor of the market price of wool.
China begins to cultivate Chinese merino (Xinjiang military reclamation type) in 1972, and six strains, namely a military reclamation type A strain, a military reclamation type B strain, an ultra-fine strain, a meat polyembryony strain, a hair polyembryony strain and a U strain, have been cultivated up to now. In order to meet the market demand and increase the economic income for herdsmen, how to further improve the natural length of wool, cultivate high-quality fine wool sheep with long natural length of wool and quickly expand the population size is still an important problem. The natural length of wool is an important quality character and an economic character of wool, the natural length of wool directly influences the parameter setting of a textile process and the comfort level of wool textiles and is the largest factor influencing the market price of wool, and therefore, the natural length of wool is improved, and huge economic benefits can be generated.
Publication No.: CN103276098A, inventive name: a molecular marking method for predicting and identifying sheep wool length, wherein a G base is required to be introduced into an upstream primer sequence to satisfy a recognition sequence of a PvuI endonuclease, which is called as 'forced enzyme digestion'. The efficiency of forced cleavage is low due to incomplete matching of primers.
Publication No.: CN107619870A, inventive name: the invention discloses a molecular marker capable of predicting and identifying sheep wool length, a specific primer pair and application thereof.
TOMM70A is a subtype of the mitochondrial outer membrane transporter TOMM70, and is a receptor for mitochondrial hydrophobin precursors. The study shows that the expression of TOMM70A is down-regulated in human papillary thyroid carcinoma, and in addition, TOMM70A is also involved in skin inflammation and alopecia induced by mutation of mouse Gsdma3 gene.
Disclosure of Invention
Based on the defects, the invention aims to provide a molecular marker primer pair capable of predicting and identifying the natural length of sheep wool and application thereof.
The technology involved in the invention is as follows: a primer pair used in a molecular marking method capable of predicting and identifying the natural length of sheep wool is provided, wherein an upper primer TOMM70 AF: as shown in sequence table Seq ID No. 1; lower primer TOMM70 AR: as shown in sequence table SeqID No. 2.
The invention also has the following technical characteristics:
1. the primer pair is applied to preparation of a preparation or a kit for in vitro detection of a molecular marker capable of indicating and identifying the natural length of sheep wool.
2. The primer pair is applied to predicting and identifying the length character of the Chinese merino sheep wool.
3. The application of the method for predicting and identifying the length character of the Chinese merino sheep wool is disclosed, wherein the Chinese merino sheep is a Xinjiang military reclamation type ewe of 1-12 years old.
4. The application of the method for predicting and identifying the length character of the Chinese merino sheep wool comprises the following steps:
the method comprises the following steps: designing a pair of primers TOMM70AF and TOMM70AR according to the site G163139235A of the 7 th intron region of the sheep TOMM70A gene, wherein the primers are respectively shown as a sequence ID No.1 and a sequence ID No.2 in a sequence table, then carrying out PCR amplification on sheep genome DNA to obtain a PCR amplification product, and carrying out enzyme digestion on the PCR amplification product by using endonuclease Bae I to obtain an enzyme digestion product; wherein the sequence of the obtained PCR amplification product is shown as Seq ID No. 3;
step two: carrying out electrophoretic separation on the enzyme digestion product, and then carrying out genotype judgment according to the electrophoretic separation result;
the criteria for determining the genotype are as follows:
step two, firstly: two bands are displayed by electrophoresis, the size is 313bp and 152bp, when the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is not mutated, the PCR amplification product can be completely cut by Bae I enzyme, and the gene is named as GG genotype;
step two: electrophoresis presents a band with the size of 498bp, when the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is mutated, a PCR amplification product cannot be cut by Bae I enzyme, and the gene is named as AA genotype;
step two and step three: three bands are displayed by electrophoresis, the sizes are 498bp, 313bp and 152bp, the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is in a heterozygous state, a PCR amplification product cannot be completely cut by Bae I enzyme, and the gene is named as GA genotype;
step three: according to the characteristics of the test population of the Chinese merino sheep, a genotype effect statistical model is constructed: y ═ μ + G + L + a + gxl + gxa + axl + e, where Y is the observed value of the trait, μ is the population mean, G is the genotype effect, L is the line effect, a is the age effect, gxl is the interaction effect of the genotype and the line, gxa is the interaction effect of the genotype and the age, axl is the interaction effect of the line and the age, e is the residual effect, then the genotype is correlated with the continuity trait and the least squares mean of the trait is estimated;
step four: and finally, determining a ewe with the GG genotype as a breeding group.
5. The application of predicting and identifying the length character of the Chinese merino sheep wool is described as follows:
10×CutSmart Buffer+SAM 1μL
endonuclease Bae I1. mu.L
The enzyme digestion conditions are as follows: performing enzyme digestion at 25 ℃ for 30 min;
6. in the above application for predicting and identifying the length trait of the chinese merino sheep, the reaction system for PCR amplification in the first step is as follows:
the PCR amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53.8 ℃ for 30s, extension at 72 ℃ for 30s, 36 cycles, final extension at 72 ℃ for 10min, and reaction termination at 4 ℃;
the invention has simple operation, low cost and high precision, and can carry out automatic detection. When the marker genotype is used for selecting the natural length of the sheep wool, the natural length of the sheep wool is greatly improved genetically. The method can be effectively applied to the field of molecular assisted breeding for screening sheep with long natural length of wool, realizes early breeding selection of breeding sheep, and can carry out selective retention after birth, thereby accelerating the breeding process of sheep. In the invention, after the mutation of the site G163139235A of the 7 th intron region of the TOMM70A gene, the DNA fragment can be completely recognized by BaeI endonuclease, so that a primer sequence is completely matched with a template sequence, forced enzyme digestion is not required, the design of the primer is simpler and more convenient, and an enzyme digestion glue map is clearer and more distinguishable. According to the invention, only two primers are needed, the PCR product is subjected to enzyme digestion and then is subjected to agarose gel electrophoresis directly to identify sheep individuals with longer natural length of wool, and the experiment operation is simple and rapid.
Drawings
FIG. 1 is an electrophoretic separation chart of the cleavage products.
FIG. 2 is a diagram showing the alignment result of the TOMM70A gene template sequence and the upstream primer sequence of the TOMM70A patent.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
Example 1
Acquisition of genomic DNA of Chinese merino sheep
Sheep ear tissue was collected and stored at-20 ℃ for future use. Sheep genomic DNA was extracted by a conventional phenol/chloroform method. The specific method comprises the following steps:
(1) taking 5g of ear tissue of Chinese merino (Xinjiang military reclamation type), removing connective tissue, cleaning and disinfecting tissue blocks by using 70% alcohol, placing the tissue blocks in Eppendorf tubes, and shearing or grinding the tissue blocks by using scissors;
(2) after the alcohol in the Eppendorf tube is completely volatilized, 700 mu L of separation buffer solution is added, after the minced tissue is suspended, 5.0 mu L of proteinase K (20mg/mL) is added, and the mixture is acted for 8-12h at 55 ℃ until no tissue block exists;
(3) taking out the digested tissue fluid, adding saturated phenol with the same amount of tissue fluid, mixing for 10min, centrifuging at 4 deg.C and 12000r/m for 10 min;
(4) taking the supernatant, adding equal amount of phenol/chloroform, gently mixing for 10min, centrifuging at 4 deg.C and 12000r/m for 10 min;
(5) collecting supernatant, adding equal amount of chloroform, mixing well for 10min, at 4 deg.C and 12000r/m, centrifuging for 10 min;
(6) taking the supernatant, adding 2 times of absolute ethyl alcohol for precipitation, reversing and uniformly mixing, standing at room temperature for 10-20min, and forming a white floccule by DNA precipitation;
(7) discarding the supernatant, adding 70% ethanol, cleaning, discarding the supernatant, absorbing the excessive liquid on absorbent paper, naturally drying, adding appropriate amount of TE, dissolving, and storing at-20 deg.C;
(8) if insoluble particles exist in the DNA solution, the DNA solution can be centrifuged for a short time at 5000r/m, and the supernatant is taken; to remove RNA, 5. mu.L RNaseA (10. mu.g/. mu.L) was added, incubated at 37 ℃ for 30min, extracted with phenol, and the DNA was re-precipitated as in steps 4-7.
Example 2
Obtaining of sheep TOMM70A gene enzyme cutting product
Designing a pair of primers TOMM70AF and TOMM70AR according to the G163139235A locus of a TOMM70A gene in a sheep genome, then carrying out PCR amplification on the sheep genome DNA to obtain a PCR amplification product, and carrying out enzyme digestion on the PCR amplification product by using an endonuclease Bae I to obtain an enzyme digestion product.
As shown in FIG. 2, after the mutation occurs at the site G163139235A of the 7 th intron of TOMM70A gene, it can be completely recognized by BaeI endonuclease, so the primer sequence and the template sequence are completely matched, and no forced digestion is required.
The primer sequences are shown below:
TOMM70AF：5’-ATGTGGCTTCTTGGAGTG-3’
TOMM70AR：5’-CACAAATAAGCGCAGTTC-3’
the PCR amplification system is as follows:
the PCR amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53.8 ℃ for 30s, and extension at 72 ℃ for 30s for 36 cycles, final extension at 72 ℃ for 10min, and termination at 4 ℃.
The enzyme digestion reaction system is as follows:
10×CutSmart Buffer+SAM 1μL
endonuclease Bae I1. mu.L
The enzyme digestion conditions are as follows: the enzyme was cleaved at 25 ℃ for 30 min.
Example 3
Determination of sheep TOMM70A genotype
The enzyme-digested product in example 1 was subjected to electrophoretic separation using agarose gel with a concentration of 2% to 3%, and genotype determination was performed according to the electrophoretic separation result, and the determination criteria were: (1) two bands are displayed by electrophoresis, the size is 313bp and 152bp, when the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is not mutated, the PCR amplification product can be completely cut by Bae I enzyme, and the gene is named as GG genotype; (2) electrophoresis presents a band with the size of 498bp, when the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is mutated, a PCR amplification product cannot be cut by Bae I enzyme, and the gene is named as AA genotype; (3) three bands are displayed by electrophoresis, the sizes are 498bp, 313bp and 152bp, the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is in a heterozygous state, and a PCR amplification product cannot be completely cut by Bae I enzyme, so that the gene is named as GA genotype.
568 Chinese merino sheep were all the ewes of Xinjiang military reclamation type 1-12 years old, 3 genotypes were detected, the distribution of 3 different genotypes in 6 sheep groups was analyzed, the number of GA-type individuals was the most, the results are shown in Table 1, and the frequency was.
TABLE 1 distribution of different genotypes of the TOMM70A gene in the sheep population
Example 4
Construction of genotype effect statistical model
According to the characteristics of the test population of the Chinese merino sheep, a genotype effect statistical model is constructed: y ═ μ + G + L + a + gxl + gxa + axl + e, where Y is the observed value of the trait, μ is the population mean, G is the genotype effect, L is the line effect, a is the age effect, gxl is the interaction effect of the genotype and the line, gxa is the interaction effect of the genotype and the age, axl is the interaction effect of the line and the age, and e is the residual effect.
The natural length of the wool is measured according to the national fiber inspection standard and the International Wool Textile Organization (IWTO) fiber detection standard, and the natural length of the wool is directly measured by using a ruler according to the book of wool and wool quality written by Liu-Sai ren academy.
The invention carries out least square analysis on 3 genotypes of G/A single base mutation of a G163139235A locus of a 7 th intron region of a TOMM70A gene and 568 individuals of a test population of Chinese merino (Xinjiang military reclamation type) on the average fiber diameter of wool, the standard deviation of the average fiber diameter of wool, the crimp degree of wool, the natural length characters of wool and the like, and the result shows that polymorphism of different genotypes of the G163139235A locus of the 7 th intron region of the TOMM70A gene of a sheep is obviously related to the natural length degree of wool of the test population of the Chinese merino (Xinjiang military reclamation type) and the P value is 0.0111; the least squares means of the 3 genotypes were then subjected to multiple comparisons, and the results showed that the natural length of wool with the GG genotype group was significantly higher than that of AA genotype and GA genotype groups (P <0.05), with the results shown in Table 2.
TABLE 2 influence of different genotypes of the 7 th intron region G163139235A of the sheep TOMM70A gene on the natural length of wool
The difference is significant when the same row has no same letter in the mean comparison (a-bP<0.05)
The results show that the TOMM70A gene can be used as one of the main candidate genes of the natural length of sheep wool, and the GG genotype can be used as a molecular genetic marker for predicting the natural length of sheep wool. Can establish a sheep breeding group with GG genotype individuals as main parts and effectively breed sheep strains with longer natural length.
Example 5
Establishment of molecular marking method for natural length of sheep wool
Chinese merino sheep test groups are divided into three types according to genotypes, so that molecular marker-assisted breeding for predicting and identifying the natural length of the sheep wool is realized, and the molecular marker method for predicting and identifying the natural length of the sheep wool is completed.
Sequence listing
<110> grand era memorial Hospital of Zhongshan university
<120> molecular marker primer pair capable of indicating and identifying natural length of sheep wool and application
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<170>SIPOSequenceListing 1.0
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<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
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<213> sheep (sheet)
<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
Claims (4)
1. The application of a PCR amplification primer pair for identifying the length of Chinese merino sheep wool in selective breeding population of Chinese merino sheep is characterized in that an upper primer TOMM70 AF: as shown in sequence table Seq ID No. 1; lower primer TOMM70 AR: as shown in sequence table Seq ID No. 2.
2. A method for selecting a breeding population for the length character of Chinese merino sheep by adopting a molecular marker is characterized by comprising the following steps:
the method comprises the following steps: designing a pair of primers TOMM70AF and TOMM70AR according to the site G163139235A of the 7 th intron region of the sheep TOMM70A gene, wherein the primers are respectively shown as a sequence ID No.1 and a sequence ID No.2 in a sequence table, then carrying out PCR amplification on sheep genome DNA to obtain a PCR amplification product, and carrying out enzyme digestion on the PCR amplification product by using endonuclease Bae I to obtain an enzyme digestion product; wherein the sequence of the obtained PCR amplification product is shown as Seq ID No. 3;
step two: carrying out electrophoretic separation on the enzyme digestion product, and then carrying out genotype judgment according to the electrophoretic separation result;
the criteria for determining the genotype are as follows:
step two, firstly: two bands are displayed by electrophoresis, the size is 313bp and 152bp, when the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is not mutated, the PCR amplification product can be completely cut by Bae I enzyme, and the gene is named as GG genotype;
step two: electrophoresis presents a band with the size of 498bp, when the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is mutated, a PCR amplification product cannot be cut by Bae I enzyme, and the gene is named as AA genotype;
step two and step three: three bands are displayed by electrophoresis, the sizes are 498bp, 313bp and 152bp, the site G163139235A of the 7 th intron region of the sheep TOMM70A gene is in a heterozygous state, a PCR amplification product cannot be completely cut by Bae I enzyme, and the gene is named as GA genotype;
step three: according to the characteristics of the test population of the Chinese merino sheep, a genotype effect statistical model is constructed: y ═ μ + G + L + a + gxl + gxa + axl + e, where Y is the observed value of the trait, μ is the population mean, G is the genotype effect, L is the line effect, a is the age effect, gxl is the interaction effect of the genotype and the line, gxa is the interaction effect of the genotype and the age, axl is the interaction effect of the line and the age, e is the residual effect, then the genotype is correlated with the continuity trait and the least squares mean of the trait is estimated;
step four: and finally, determining a ewe with the GG genotype as a breeding group.
3. The method for selecting a breeding population for the length trait of the Chinese merino sheep wool by using a molecular marker according to claim 2, wherein the enzyme digestion system in the first step is as follows:
10×CutSmart Buffer+SAM 1μL
endonuclease Bae I1. mu.L
PCR product 10. mu.L
The enzyme digestion conditions are as follows: the enzyme was cleaved at 25 ℃ for 30 min.
4. The method for selecting a breeding population for the length trait of the chinese merino sheep wool using molecular markers as claimed in claim 2, wherein the reaction system for PCR amplification in the first step is as follows:
the PCR amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53.8 ℃ for 30s, and extension at 72 ℃ for 30s for 36 cycles, final extension at 72 ℃ for 10min, and termination at 4 ℃.
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| 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 |
| CN113278716B (en) * | 2021-07-23 | 2021-11-09 | 中国农业大学 | Gene chip for analyzing characters for sheep wool, molecular probe combination, kit and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1253179A (en) * | 1998-11-09 | 2000-05-17 | 中国人民解放军军事医学科学院放射医学研究所 | Structure of antisense oligonucleotide to inhibit activity of telomerase and its application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1253179A (en) * | 1998-11-09 | 2000-05-17 | 中国人民解放军军事医学科学院放射医学研究所 | Structure of antisense oligonucleotide to inhibit activity of telomerase and its application |
Non-Patent Citations (2)
| Title |
|---|
| Ovis aries breed Texel chromosome 1 genomic scaffold, Oar_v3.1 OAR1, whole genome shotgun sequence，NW_004080164.1;Archibald,A.L.;《NCBI Genbank》;20150403;序列表 * |
| PREDICTED: Ovis aries translocase of outer mitochondrial membrane 70 homolog A (S. cerevisiae) (TOMM70A), mRNA, XM_004002879.1;Archibald,A.L.;《NCBI Genbank》;20121202;全文 * |
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