CN114231643B - Molecular marker affecting wool fiber diameter variation coefficient character of Erdos fine wool, obtaining method, specific primer pair and application thereof - Google Patents
Molecular marker affecting wool fiber diameter variation coefficient character of Erdos fine wool, obtaining method, specific primer pair and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of animal molecular markers, in particular to a molecular marker affecting the diameter variation coefficient character of wool fibers of Hundos naps sheep, an obtaining method and a specific primer pair and application thereof, wherein the molecular marker comprises a molecular marker Indel1 and a molecular marker SNP1, the molecular marker Indel1 is four bases TAT deleted on the 2 nd intron of a P1 segment of an LMNB1 gene, and the molecular marker SNP1 is a variable base C or T on the 3 rd exon of the P1 segment of the LMNB1 gene. According to the invention, through researching the correlation between the LMNB1 gene and the wool character of the Erdos fine wool sheep for the first time, molecular markers related to the character of the coefficient of variation of the diameter of the wool fiber of the Erdos fine wool sheep are positioned, so that gene mutation sites are obtained, and the genotype containing ATCT and TTTT is found to have advantages in the ultra-fine group breeding, so that a method and a guidance are provided for the breeding of the Erdos fine wool sheep with high yield and ultra-fine group.
Description
Technical Field
The invention relates to the technical field of animal molecular markers, in particular to a molecular marker affecting the diameter variation coefficient character of wool fibers of Erdos naps sheep, an acquisition method, a specific primer pair and application thereof.
Background
The Hudoos fine wool sheep is a fine wool sheep breed which is bred and named in 1985 and has wool and meat dual-purpose, the main production area is a Husky area of Hudoos, and the Wuqi is a core production area. In 2008, the leopard sheep successfully acquired the agricultural department "agricultural product geographical sign registration certificate". The Erdos fine wool sheep and the products thereof have great competition potential in domestic and foreign markets. However, with the increasing material culture demands of people and the improvement of textile technology, wool spinning products at home and abroad start to develop towards the directions of lightness, thinness, softness, stiffness and smoothness and high grade. Therefore, the breeding of high-yield and superfine group of the Erdos fine wool sheep is the main task at present.
With the development of modern biotechnology, the main economic traits of livestock are researched from the molecular level, and the accuracy and efficiency of seed selection are improved. Lamin (LaminB 1, LMNB 1) gene maps to sheep chromosome 5, with 11 exons. LaminB1 plays an important role in regulating organ development and tissue differentiation, and is an important component of a nuclear fiber layer. The expression of LMNB1 gene was found to be up-regulated in sheep skin of ultrafine wool group relative to fine wool group by qRT-PCR method at the early stage of T-group. The LMNB1 gene is suggested to be involved in regulation and control in wool traits. At present, the LMNB1 gene has less research on sheep wool characteristics, so that the polymorphism analysis of the LMNB1 gene of the Erdos fine wool sheep is needed, and the ultra-fine population breeding process of the Erdos fine wool sheep is quickened.
Disclosure of Invention
The invention provides a molecular marker affecting the diameter variation coefficient character of a wool fiber of a Hudoos fine wool sheep, an obtaining method, a specific primer pair and application thereof, wherein a gene mutation site is obtained by locating the molecular marker affecting the diameter variation coefficient character of the wool fiber of the Hudoos fine wool sheep, and genotyping is realized.
One of the technical schemes of the invention is realized by the following measures: a molecular marker for influencing the variation coefficient character of the diameter of a wool fiber of a Hundos fine wool sheep comprises a molecular marker Indel1 and a molecular marker SNP1, wherein the molecular marker Indel1 is positioned on an introns 2 of a P1 fragment of an LMNB1 gene at 24654403bp-24654406bp of a sheep 5 chromosome, the molecular marker SNP1 is positioned on an exons 3 of the P1 fragment of the LMNB1 gene at 24654282bp of the sheep 5 chromosome, the molecular marker Indel1 is a four-base TATT deleted on the introns 2 of the P1 fragment of the LMNB1 gene at 24654403bp-24654406bp of the sheep 5 chromosome, the molecular marker SNP1 is a G or a on the exons 3 of the P1 fragment of the LMNB1 gene at 24654282bp of the sheep 5 chromosome, and the allele mutation C or T.
The following are further optimizations and/or improvements to one of the above-described inventive solutions:
the method comprises the following steps: firstly, taking blood genome DNA of female sheep of the Hudoe fine sheep to be detected; secondly, taking blood genome DNA as a template to carry out PCR amplification, and a PCR reaction system is adopted: PCRMijture 10. Mu.L, 10mol/L upstream and downstream primers each 0.5. Mu.L, upstream primer sequence 5'-GCAGTTGAACCCACACTTCTG-3', downstream primer sequence 5'-TTGCCTACAGAATCCTTCCC-3', DNA template 1.0. Mu.L, distilled water to 20. Mu.L, and placing the established PCR system into a PCR instrument for reaction under the following conditions: pre-denaturing at 95 ℃ for 3min, denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min,34 cycles, extending at 72 ℃ for 5min, and preserving at 4 ℃ to obtain a PCR product; thirdly, carrying out bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and carrying out mutation genotyping by utilizing a snap shot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the wool fiber diameter variation coefficient characters of the Erdos fine wool sheep.
The molecular marker Indel1 genotype and the molecular marker SNP1 genotype are linked, when the molecular marker Indel1 genotype is AA genotype, the molecular marker SNP1 genotype is CC genotype, and when the molecular marker Indel1 genotype is TT genotype, the molecular marker SNP1 genotype is TT genotype.
The second technical scheme of the invention is realized by the following measures: the method for obtaining the molecular marker affecting the diameter variation coefficient property of the wool fibers of the Erdos fine wool sheep is carried out according to the following method: firstly, taking blood genome DNA of female sheep of the Hudoe fine sheep to be detected; secondly, taking blood genome DNA as a template to carry out PCR amplification, and a PCR reaction system is adopted: PCRMijture 10. Mu.L, 10mol/L upstream and downstream primers each 0.5. Mu.L, upstream primer sequence 5'-GCAGTTGAACCCACACTTCTG-3', downstream primer sequence 5'-TTGCCTACAGAATCCTTCCC-3', DNA template 1.0. Mu.L, distilled water to 20. Mu.L, and placing the established PCR system into a PCR instrument for reaction under the following conditions: pre-denaturing at 95 ℃ for 3min, denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min,34 cycles, extending at 72 ℃ for 5min, and preserving at 4 ℃ to obtain a PCR product; thirdly, carrying out bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and carrying out mutation genotyping by utilizing a snap shot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the wool fiber diameter variation coefficient characters of the Erdos fine wool sheep.
The specific primer pair comprises a specific primer pair of a molecular marker Indel1 and a specific primer pair of a molecular marker SNP1, wherein the specific primer pair of the molecular marker Indel1 and the specific primer pair of the molecular marker SNP1 comprise an upstream primer, a downstream primer and an extension primer, and the specific primer pair of the molecular marker Indel1 is as follows: an upstream primer of 5'-GACTCTCATACATTGACAAATACA-3', a downstream primer of 5'-ACTCCAAGTCCTCAGTAAG-3', an extension primer of 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3', a specific primer pair for molecular marker SNP 1: the upstream primer was 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer was 5'-ACTCCAAGTCCTCAGTAAG-3', and the extension primer was 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3'.
The third technical scheme of the invention is realized by the following measures: the specific primer pair in the method for obtaining the molecular marker affecting the characteristic of the diameter variation coefficient of the wool fibers of the Erdos fine wool comprises a specific primer pair of a molecular marker Indel1 and a specific primer pair of a molecular marker SNP1, wherein the specific primer pair of the molecular marker Indel1 and the specific primer pair of the molecular marker SNP1 comprise an upstream primer, a downstream primer and an extension primer, and the specific primer pair of the molecular marker Indel1 is prepared by the following steps: an upstream primer of 5'-GACTCTCATACATTGACAAATACA-3', a downstream primer of 5'-ACTCCAAGTCCTCAGTAAG-3', an extension primer of 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3', a specific primer pair for molecular marker SNP 1: the upstream primer was 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer was 5'-ACTCCAAGTCCTCAGTAAG-3', and the extension primer was 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3'.
The fourth technical scheme of the invention is realized by the following measures: application of a specific primer pair in preparing a reagent or a kit for in vitro detection of molecular markers affecting the wool fiber diameter variation coefficient property of the Erdos fine wool sheep.
The fifth technical scheme of the invention is realized by the following measures: the application of molecular markers affecting the variation coefficient character of the diameter of the wool fiber of the Erdos fine wool sheep in the breeding of high-yield and superfine population of the Erdos fine wool sheep.
According to the invention, through researching the correlation between the LMNB1 gene polymorphism of the Erdos fine wool sheep and wool traits for the first time, molecular markers affecting the wool fiber diameter variation coefficient traits of the Erdos fine wool sheep are positioned, gene mutation sites are obtained, genotyping is realized, the ATCT genotype and TTTT genotype are found to have advantages in superfine population breeding, and a method and guidance are provided for breeding of high-yield superfine population of the Erdos fine wool sheep.
Drawings
FIG. 1 shows the results of genomic DNA detection according to the present invention.
FIG. 2 shows the detection results of PCR products of LMNB1 gene in the present invention.
FIG. 3 shows the result of the Snapshot typing of 2 mutation sites of LMNB1 gene in the invention.
FIG. 4 is a schematic representation of typing verification of 2 mutation sites of LMNB1 gene in the present invention.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments can be determined according to the technical scheme and practical situations of the present invention. The various chemical reagents and chemical supplies mentioned in the invention are all commonly known and used in the prior art unless specified otherwise; the percentages in the invention are mass percentages unless specified otherwise; the solutions in the invention are aqueous solutions in which the solvent is water unless otherwise specified, for example, the hydrochloric acid solution is hydrochloric acid aqueous solution; the room temperature and the room temperature in the present invention generally refer to temperatures ranging from 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to examples:
example 1: the molecular marker affecting the wool fiber diameter coefficient of variation property of the Erdos fine wool comprises a molecular marker Indel1 and a molecular marker SNP1, wherein the molecular marker Indel1 is positioned on an introns 2 of the LMNB1 gene P1 fragment at 24654403bp-24654406bp of a sheep 5 chromosome, the molecular marker SNP1 is positioned on an exons 3 of the LMNB1 gene P1 fragment at 24654282bp of the sheep 5 chromosome, the molecular marker Indel1 is a four-base TATT deleted on the introns 2 of the LMNB1 gene P1 fragment at 24654403bp-24654406bp of the sheep 5 chromosome, the molecular marker SNP1 is a variant base G or A on the exons 3 of the LMNB1 gene P1 fragment at 24654282bp of the sheep 5 chromosome, and the allele is mutated C or T.
Example 2: as an optimization of the above embodiment, it was obtained as follows: firstly, taking blood genome DNA of female sheep of the Hudoe fine sheep to be detected; secondly, taking blood genome DNA as a template to carry out PCR amplification, and a PCR reaction system is adopted: PCRMijture 10 mu L, 10mol/L of each of the upstream and downstream primers, 5'-GCAGTTGAACCCACACTTCTG-3' (shown as SEQ ID No: 7) and 5'-TTGCCTACAGAATCCTTCCC-3' (shown as SEQ ID No: 8) of the downstream primer, 1.0 mu L of DNA template, adding distilled water to 20 mu L, and placing the established PCR system into a PCR instrument for reaction under the following reaction conditions: pre-denaturing at 95 ℃ for 3min, denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min,34 cycles, extending at 72 ℃ for 5min, and preserving at 4 ℃ to obtain a PCR product; thirdly, carrying out bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and carrying out mutation genotyping by utilizing a snap shot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the wool fiber diameter variation coefficient characters of the Erdos fine wool sheep.
Example 3: as an optimization of the above embodiment, there is linkage between the molecular marker Indel1 genotype and the molecular marker SNP1 genotype, and when the molecular marker Indel1 genotype is the AA genotype, the molecular marker SNP1 genotype is the CC genotype, and when the molecular marker Indel1 genotype is the TT genotype, the molecular marker SNP1 genotype is the TT genotype.
Molecular marker techniques can represent differences between individuals or populations of organisms by detecting variations in the genes or genotypes of the animals. According to the invention, deletion of four bases of TATT is found on an intron 2 (Indel 1) of a P1 fragment of an LMNB1 gene, mutant base C or T is found on an exon 3 (SNP 1) of the LMNB1 gene, and linkage between Indel1 and SNP1 is known through Snapshot detection typing. Therefore, the fiber diameter variation coefficient character of an AACC genotype individual is obviously lower than that of an ATCT genotype and a TTTT genotype (P < 0.05), and the LMNB1 gene Indel1-SNP1 mutation site has obvious influence on the fiber diameter variation coefficient character (P < 0.05), so that an experimental basis is provided for breeding of a Hummer nape ultrafine population.
Example 4: the method for obtaining the molecular marker affecting the characteristic of the diameter variation coefficient of the wool fiber of the Erdos fine wool sheep is carried out according to the following method: firstly, taking blood genome DNA of female sheep of the Hudoe fine sheep to be detected; secondly, taking blood genome DNA as a template to carry out PCR amplification, and a PCR reaction system is adopted: PCRMijture 10 mu L, 10mol/L of each of the upstream and downstream primers, 5'-GCAGTTGAACCCACACTTCTG-3' (shown as SEQ ID No: 7) and 5'-TTGCCTACAGAATCCTTCCC-3' (shown as SEQ ID No: 8) of the downstream primer, 1.0 mu L of DNA template, adding distilled water to 20 mu L, and placing the established PCR system into a PCR instrument for reaction under the following reaction conditions: pre-denaturing at 95 ℃ for 3min, denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min,34 cycles, extending at 72 ℃ for 5min, and preserving at 4 ℃ to obtain a PCR product; thirdly, carrying out bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and carrying out mutation genotyping by utilizing a snap shot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the wool fiber diameter variation coefficient characters of the Erdos fine wool sheep.
Example 5: the specific primer pair in the method for obtaining the molecular marker affecting the characteristic of the diameter variation coefficient of the wool fibers of the Erdos nap sheep comprises a specific primer pair of a molecular marker Indel1 and a specific primer pair of a molecular marker SNP1, wherein the specific primer pair of the molecular marker Indel1 and the specific primer pair of the molecular marker SNP1 comprise an upstream primer, a downstream primer and an extension primer, and the specific primer pair of the molecular marker Indel1 is as follows: the specific primer pair of the molecular marker SNP1 comprises an upstream primer 5'-GACTCTCATACATTGACAAATACA-3' (shown as a sequence table SEQ ID No: 1), a downstream primer 5'-ACTCCAAGTCCTCAGTAAG-3' (shown as a sequence table SEQ ID No: 2) and an extension primer 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3' (shown as a sequence table SEQ ID No: 3): the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3' (shown as SEQ ID No. 4 of the sequence table), the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3' (shown as SEQ ID No. 5 of the sequence table), and the extension primer is 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3' (shown as SEQ ID No. 6 of the sequence table).
Example 6: the application of the specific primer pair in the preparation of a reagent or a kit for in vitro detection of molecular markers affecting the wool fiber diameter variation coefficient property of the Erdos fine wool sheep.
Example 7: the molecular marker affecting the characteristics of the diameter variation coefficient of the wool fibers of the Erdos fine wool sheep is applied to the breeding of high-yield and superfine populations of the Erdos fine wool sheep.
The following is the experimental study of the molecular marker related to the characteristics of the diameter and the coefficient of variation of the Hudoos fine wool fibers:
1 materials and methods
1.1 sample collection
Five towns from ullan Tao Legai towns (n=41), ga Lu Tuzhen (n=97), surlyn sappan wood (n=134), tukey towns (n=91), wu-zhen towns (n=99) in the inner mongolian municipality, the erdos, a total of 462 female goats of the perigee erdos nape were collected as experimental animals. The experimental ewe is subjected to vein blood sampling into a 5ml anticoagulation tube, and is put into a refrigerator at the temperature of minus 20 ℃ for preservation, and is used for the subsequent extraction of blood genome DNA. Meanwhile, a wool sample is collected at the position 10cm above the middle line of the left side body of the experimental sheep at the rear edge of the scapula. The wool sample is sent to the quality supervision and inspection test center of sheep and wool cashmere in agricultural rural areas, and the average fiber diameter and the variation coefficient character of the fiber diameter of the wool sample are detected by a fiber diameter optical analyzer (OFDA 2000). Finally, the identification record of the experimental flock in 2020 is collected and mainly comprises the wool yield, the length of wool fibers and the like.
1.2 construction of DNA mixing pool
Blood genomic DNA was extracted using a blood genomic DNA extraction kit (Tiangen Biotech Co., ltd.). 4ul of DNA was taken, the DNA mass was detected by 1.0% agarose gel electrophoresis, 1ul of DNA was taken, and the DNA concentration was detected by a nucleic acid protein detector. In the measured wool fiber diameter data, 20 extreme individual (extremely fine n=10, extremely coarse n=10) DNA samples were individually selected. Wherein the average fiber diameter of the very fine group was 16.36. Mu.m, and the very coarse group was 21.50. Mu.m. Extreme individual DNA concentrations were detected by a nucleic acid protein detector and ddH was added 2 O is adjusted to unity. From 20 extreme individuals, 10ul of each was taken, and 2 DNA mixing pools (extremely fine DNA mixing pool, extremely coarse DNA mixing pool) were constructed from extremely fine and extremely coarse groups
1.3PCR amplification and sequencing
Primers were designed based on the LMNB1 gene (accession number: ENSOART 00000019747.1) sequence in the Ensembl nucleic acid database using premerPremer5.0, and the primer sequences are shown in Table 1. Primers were synthesized by Shanghai Bioengineering technology Limited company. 2 mixed pool DNAs are used as templates for PCR amplification, and a PCR reaction system is adopted: PCRMijture 10. Mu.L, upstream and downstream primers (10 mol/L) each 0.5. Mu.L, DNA template 1.0. Mu.L, and distilled water to 20. Mu.L. And (5) placing the established PCR system into a PCR instrument for reaction. Reaction conditions: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 1min,34 cycles, extension at 72℃for 5min, and preservation at 4 ℃. The PCR products were detected by electrophoresis on a 1.5% agarose gel. The brightly banded PCR products were sent directly to the engineering (Shanghai) Co., ltd for two-way sequencing. Sequencing results sequence splicing and correction were performed using the SeqMan program of DNASTAR software and peak plots were aligned using BioEdit software.
1.4Snapshot detection
The upstream and downstream primers and the extension primers were designed according to the two-way sequencing screening to the mutation site, and the primer information is shown in Table 2. 462 individuals were detected by the Beijing Oridae Dingsheng biotechnology Co.Ltd using Snapshot genotyping technology and typed using an ABI3730XL sequencer. And finally, randomly selecting 3 samples at each position to perform first-generation sequencing, and verifying the Snapshot typing detection result.
1.5 statistical analysis
The gene frequency, genotype frequency, effective allele, gene heterozygosity, polymorphism information content, and Hardy-Weinberg equilibrium of SNPs were calculated using Popgene software. The correlation of different genotypes of SNPs and the character of the jaw-Erdos fine wool was analyzed by using SAS9.2 software. Results are expressed in terms of least squares mean ± standard error, the linear model is:
Y ick ＝μ+G i +F e +e ick
in the formula, Y ick : a fine wool sheep individual body surface shape value; mu: population means; g i : genotype SNP effect; f (F) e : a field effect; e, e ick : random errors.
2 results and analysis
2.1DNA and PCR detection results
The extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA band was bright, and the result was shown in FIG. 1. The OD ratio of 260nm to 280nm is 1.8-2.1 by using a nucleic acid protein detector to detect DNA, which shows that the quality and purity of the extracted DNA meet the requirements of subsequent experiments. The results of the LMNB1 gene PCR product detection are shown in FIG. 2, wherein M.2000 is a gene reference. As can be seen from FIG. 2, the PCR products were consistent with the expected fragment size, and were free of bands, meeting the requirements of subsequent experiments.
2.2 pool mixing results analysis
2 mutations were found by pool sequencing of PCR fragments of the Erdos fine wool sheep gene. Wherein 2 mutations (Indel 1 and SNP 1) were found on the P1 fragment of the LMNB1 gene. The information of 2 mutations in the LMNB1 gene is shown in Table 3. Wherein Indel1 is a deletion of TATT4 bases from intron 2 of the P1 fragment of the LMNB1 gene; SNP1 is a synonymous mutation on the exon of the LMNB1 gene.
2.3Snapshot detection typing and verification result
The typing peak diagram of 2 mutation sites of 462 Erdos fine wool sheep LMNB1 genes detected by using Snapshot typing technology is shown in FIG. 3. In Snapshot detection, 2 mutation sites were successfully typed and all had three genotypes. Wherein Indel1 and SNP1 extension products are forward extension. In addition, indel1 and SNP1 genotypes have the same typing pattern, i.e., SNP1 is CC genotype when Indel1 is a TATT four base deletion (AA genotype); when Indel1 is a TATT four base insertion (TT genotype), SNP1 is TT genotype, it is presumed that there is linkage between two mutations of Indel1 and SNP1, so that two mutation sites Indel1-SNP1 are subsequently pooled for analysis. According to the Snapshot typing detection result, randomly selecting 2 individuals with different mutation genotypes for carrying out first-generation sequencing, wherein Indel1 and SNP1 are reverse sequencing, and the verification result is shown in figure 4. As can be seen from fig. 4, the first generation sequencing is completely matched with the snapplot typing result, which indicates that the snapplot typing result is reliable.
2.4 analysis of genetic polymorphisms
The results of the 2 mutant genotype frequencies, allele frequencies and χ2 calculation of the LMNB1 gene of Erdos nape are shown in Table 4. As is clear from Table 4, indel1-SNP1 dominant genotype is TTTT genotype. At the same time, the χ2 values at the 2 mutation sites did not reach significant levels, hardy-Weinberg equilibrium. In addition, analysis of genetic homozygosity (Ho), genetic heterozygosity (He), effective allele (Ne), and Polymorphism Information Content (PIC) of 2 mutation sites of the LMNB1 gene of erdos fine wool sheep are shown in table 5. As shown in Table 5, 2 mutation sites were highly homozygous, and the number of effective alleles was 1.807. According to mutation site polymorphism criteria: PIC >0.5 is highly polymorphic, 0.25< PIC <0.5 is moderately polymorphic, and PIC <0.25 is lowly polymorphic. As is clear from Table 5, indel1-SNP1 mutation sites are moderately polymorphic.
2.5 analysis of the correlation of mutation loci with wool Properties
Correlation analysis of 2 mutation site polymorphisms of the LMNB1 gene of Erdos nape sheep with wool traits using SAS9.2 software is shown in Table 6. The LMNB1 gene Indel1-SNP1 mutation site AACC genotype fiber diameter variation coefficient character is 21.72, the ATCT genotype fiber diameter variation coefficient character genotype is 22.33, and the TTTT genotype fiber diameter variation coefficient character is 22.43. As can be seen from Table 6, the fiber diameter coefficient of variation traits of the ATCT genotype and TTTT genotype were significantly higher than those of the AACC genotype (P < 0.05), and the difference between the ATCT genotype and the TTTT genotype was not significant (P > 0.05).
In conclusion, the invention firstly researches the correlation between the LMNB1 gene polymorphism of the Erdos fine wool sheep and the wool character, locates the molecular markers affecting the wool fiber diameter variation coefficient character of the Erdos fine wool sheep, obtains the gene mutation sites, realizes genotyping, finds that the ATCT genotype and TTTT genotype are contained to have advantages in superfine population breeding, and provides a method and guidance for breeding of high-yield and superfine population of the Erdos fine wool sheep.
The technical characteristics form the embodiment of the invention, have stronger adaptability and implementation effect, and can increase or decrease unnecessary technical characteristics according to actual needs so as to meet the requirements of different situations.
TABLE 1 primer information Table
TABLE 2 detection of amplification primers and extension primers
TABLE 3 LMNB1 Gene mutation site information Table
TABLE 4 genotype frequencies and allele frequencies of LMNB1 genes
TABLE 5 LMNB1 Gene population polymorphism analysis
TABLE 6 analysis of association of LMNB1 Gene mutation loci with wool Properties
The average value of the different lowercase letters of the shoulder marks is obviously different (P < 0.05); the differences between the average values of the different capital letters of the shoulder marks are extremely remarkable (P < 0.01); the differences between the average values of the same letters or no letters of the shoulder marks are not significant.
Sequence listing
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<212>mRNA
<213> artificial sequence
<400>1
<210>4
<211>24
<212>mRNA
<213> artificial sequence
<400>1
Gactctcatagattgacaaaataca 24
<210>5
<211>19
<212>mRNA
<213> artificial sequence
<400>1
Actccaagtcctcagtaag 19
<210>6
<211>46
<212>mRNA
<213> artificial sequence
<400>1
Ctgactgactgactgactgactagatgaaactttacttaaaggtgga 46
<210>7
<211>21
<212>mRNA
<213> artificial sequence
<400>1
gcagttgaac ccacacttct g 21
<210>8
<211>20
<212>mRNA
<213> artificial sequence
<400>1
ttgcctacag aatccttccc 20
Claims (6)
1. The application of a molecular marker affecting the wool fiber diameter variation coefficient property of the Erdos fine wool sheep in breeding of high-yield and ultrafine population of the Erdos fine wool sheep is characterized by comprising a molecular marker Indel1 and a molecular marker SNP1, wherein the molecular marker Indel1 is positioned on the 2 nd intron of an LMNB1 gene P1 fragment at 24654403bp-24654406bp of sheep 5, the molecular marker SNP1 is positioned on the 3 rd exon of an LMNB1 gene P1 fragment at 24654282bp of sheep 5, the molecular marker Indel1 is four AATA deleted on the 2 nd intron of an LMNB1 gene P1 fragment at 24654403bp-24654406bp of sheep 5, the molecular marker SNP1 is a variable base G or A on the 3 rd exon of an LMNB1 gene P1 fragment at 24654282bp of sheep 5, and the molecular marker SNP1 is an allelic mutation C or T.
2. The application of the molecular marker affecting the characteristics of the diameter variation coefficient of the wool fibers of the Erdos fine wool sheep in the breeding of high-yield and superfine-type groups of the Erdos fine wool sheep according to claim 1, which is characterized by being obtained by the following method: firstly, taking blood genome DNA of female sheep of the Hudoe fine sheep to be detected; secondly, taking blood genome DNA as a template to carry out PCR amplification, and a PCR reaction system is adopted: PCRMijture 10. Mu.L, 10mol/L upstream and downstream primers each 0.5. Mu.L, upstream primer sequence 5'-GCAGTTGAACCCACACTTCTG-3', downstream primer sequence 5'-TTGCCTACAGAATCCTTCCC-3', DNA template 1.0. Mu.L, distilled water to 20. Mu.L, and placing the established PCR system into a PCR instrument for reaction under the following conditions: pre-denaturing at 95 ℃ for 3min, denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min,34 cycles, extending at 72 ℃ for 5min, and preserving at 4 ℃ to obtain a PCR product; thirdly, carrying out bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a primer pair, and carrying out mutation genotyping by utilizing a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the wool fiber diameter variation coefficient characters of the Erdos fine wool sheep.
3. A method for obtaining a molecular marker affecting the characteristic of the coefficient of variation of the diameter of the wool fibers of the jaw fine wool sheep according to claim 1, characterized by comprising the following steps: firstly, taking blood genome DNA of female sheep of the Hudoe fine sheep to be detected; secondly, taking blood genome DNA as a template to carry out PCR amplification, and a PCR reaction system is adopted: PCRMijture 10. Mu.L, 10mol/L upstream and downstream primers each 0.5. Mu.L, upstream primer sequence 5'-GCAGTTGAACCCACACTTCTG-3', downstream primer sequence 5'-TTGCCTACAGAATCCTTCCC-3', DNA template 1.0. Mu.L, distilled water to 20. Mu.L, and placing the established PCR system into a PCR instrument for reaction under the following conditions: pre-denaturing at 95 ℃ for 3min, denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min,34 cycles, extending at 72 ℃ for 5min, and preserving at 4 ℃ to obtain a PCR product; thirdly, carrying out bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a primer pair, and carrying out mutation genotyping by utilizing a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the wool fiber diameter variation coefficient characters of the Erdos fine wool sheep.
4. The method for obtaining a molecular marker affecting the character of the coefficient of variation of the diameter of the wool fibers of a sheep in Erdos, according to claim 3, wherein the primer pair comprises a primer pair of a molecular marker Indel1 and a primer pair of a molecular marker SNP1, and the primer pair of the molecular marker Indel1 and the primer pair of the molecular marker SNP1 each comprise an upstream primer, a downstream primer and an extension primer, wherein the primer pair of the molecular marker Indel 1: primer pair for molecular marker SNP1 with upstream primer 5'-GACTCTCATACATTGACAAATACA-3', downstream primer 5'-ACTCCAAGTCCTCAGTAAG-3' and extension primer 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3': the upstream primer was 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer was 5'-ACTCCAAGTCCTCAGTAAG-3', and the extension primer was 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3'.
5. The primer pair in the method for obtaining the molecular marker affecting the characteristic of the diameter variation coefficient of the wool fibers of the Erdos fine wool sheep is characterized by comprising a primer pair of a molecular marker Indel1 and a primer pair of a molecular marker SNP1, wherein the primer pair of the molecular marker Indel1 and the primer pair of the molecular marker SNP1 comprise an upstream primer, a downstream primer and an extension primer, and the primer pair of the molecular marker Indel1 is characterized in that: primer pair for molecular marker SNP1 with upstream primer 5'-GACTCTCATACATTGACAAATACA-3', downstream primer 5'-ACTCCAAGTCCTCAGTAAG-3' and extension primer 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3': the upstream primer was 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer was 5'-ACTCCAAGTCCTCAGTAAG-3', and the extension primer was 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3'.
6. Use of the primer pair according to claim 5 in the preparation of a reagent or kit for in vitro detection of a molecular marker affecting the characteristic of the coefficient of variation of the diameter of wool fibers of a jaw-dos fine wool sheep.
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