CN114231643A - Molecular marker influencing character of diameter variation coefficient of Eldos fine wool sheep wool, obtaining method, specific primer pair and application thereof - Google Patents
Molecular marker influencing character of diameter variation coefficient of Eldos fine wool sheep 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 influencing the character of the fiber diameter variation coefficient of an Erdos fine wool 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 TATT which are deleted on the No. 2 intron of a P1 fragment of an LMNB1 gene, and the molecular marker SNP1 is a changed base C or T on the No. 3 exon of the P1 fragment of the LMNB1 gene. According to the invention, through the first research on the correlation between the Ordos fine wool goat LMNB1 gene and wool character, the molecular marker which influences the correlation between the Ordos fine wool goat fiber diameter variation coefficient character is positioned to obtain the gene mutation site, and the gene type containing ATCT and TTTT is found to have advantages in the breeding of ultrafine groups, thereby providing a method and guidance for the breeding of high-yield and ultrafine groups of Ordos fine wool goats.
Description
Technical Field
The invention relates to the technical field of animal molecular markers, in particular to a molecular marker influencing the character of the diameter variation coefficient of an Erdos fine wool sheep wool fiber, an obtaining method, a specific primer pair and application thereof.
Background
The Orthoe fine wool sheep is a named breed of wool and meat dual-purpose fine wool sheep bred in 1985, the main production area is the MaoUsu sand area in Orthoe, and the Wujuqi is the core production area. In 2008, the Ordors fine wool sheep successfully acquired the agricultural department "geographical sign registration certificate for agricultural products". The Ordos fine wool sheep and the products thereof have larger competitive potential in the domestic and foreign markets. However, with the increasing material culture requirements and the improvement of textile technology, the wool textile products at home and abroad begin to develop towards being light, thin, soft, stiff and smooth and high-grade. Therefore, the breeding of high-yield and ultra-fine type groups of the Ordos fine wool sheep is the main task at present.
With the development of modern biotechnology, the main economic traits of livestock are researched on the molecular level, and the accuracy and efficiency of seed selection are improved. The gene of lamin (Lamin B1, LMNB1) maps to sheep chromosome 5, and 11 exons are available. LaminB1 has important regulation effect in organ development and tissue differentiation process, and is an important component of nuclear fiber layer. In the early stage of the T subject group, the expression of LMNB1 gene is found to be up-regulated in sheep skin of the superfine wool group relative to the fine wool group by a qRT-PCR method. The LMNB1 gene is suggested to participate in the regulation function in the wool trait. At present, the LMNB1 gene has less research on sheep wool traits, so polymorphism analysis of the LMNB1 gene of the Ordos fine wool sheep is needed, and the superfine group breeding process of the Ordos fine wool sheep is accelerated.
Disclosure of Invention
The invention provides a molecular marker influencing the character of the diameter variation coefficient of Ordos fine-hair sheep wool, an obtaining method, a specific primer pair and application thereof, wherein a gene mutation site is obtained by positioning the molecular marker influencing the character of the diameter variation coefficient of Ordos fine-hair sheep wool, so that genotyping is realized, the ATCT genotype and the TTTT genotype are found to have advantages in the breeding of ultrafine groups, and a method and a guide are provided for the breeding of high-yield and ultrafine groups of Ordos fine-hair sheep.
One of the technical schemes of the invention is realized by the following measures: a molecular marker influencing the character of the fiber diameter variation coefficient of an Eldos fine hair sheep comprises a molecular marker Indel1 and a molecular marker SNP1, wherein the molecular marker Indel1 is positioned on the No. 2 intron of an LMNB1 gene P1 fragment at 24654403bp-24654406bp of a sheep No. 5 chromosome, the molecular marker SNP1 is positioned on the No. 3 exon of an LMNB1 gene P1 fragment at 24654282bp of the sheep No. 5 chromosome, the molecular marker Indel1 is four bases TATT deleted on the No. 2 intron of an LMNB1 gene P1 fragment at 24654403bp-24654406bp of the sheep No. 5 chromosome, and the molecular marker SNP1 is G or A, allelic mutation C or T on the No. 3 exon of an LMNB1 gene P1 fragment at 24654282bp of the sheep No. 5 chromosome.
The following is a further optimization or/and improvement of one of the above-mentioned technical solutions of the invention:
the method comprises the following steps: firstly, taking the blood genome DNA of an Erdos fine wool ewe to be detected; secondly, performing PCR amplification by using blood genome DNA as a template, wherein a PCR reaction system comprises: the PCR method comprises the following steps of (1) carrying out PCR reaction on an established PCR system in a PCR instrument, wherein the PCR reaction conditions comprise that (9) mu L of PCRMix 10 mu L, 0.5 mu L of each upstream primer and downstream primer with the concentration of 10mol/L, the sequence of the upstream primer is 5'-GCAGTTGAACCCACACTTCTG-3', the sequence of the downstream primer is 5'-TTGCCTACAGAATCCTTCCC-3', DNA, the template is 1.0 mu L, distilled water is added to 20 mu L: 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 storage at 4 ℃ to obtain a PCR product; and thirdly, performing bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and performing mutation genotyping by using a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the fiber diameter variation coefficient characters of the Eldos fine-hair sheep.
The genotype of the molecular marker Indel1 is linked with the genotype of the molecular marker SNP1, when the genotype of the molecular marker Indel1 is AA genotype, the genotype of the molecular marker SNP1 is CC genotype, and when the genotype of the molecular marker Indel1 is TT genotype, the genotype of the molecular marker SNP1 is TT genotype.
The second technical scheme of the invention is realized by the following measures: a method for obtaining a molecular marker influencing the character of the diameter variation coefficient of an Erdos fine-wool sheep wool fiber is carried out according to the following method: firstly, taking the blood genome DNA of an Erdos fine wool ewe to be detected; secondly, performing PCR amplification by using blood genome DNA as a template, wherein a PCR reaction system comprises: the PCR method comprises the following steps of (1) carrying out PCR reaction on an established PCR system in a PCR instrument, wherein the PCR reaction conditions comprise that (9) mu L of PCRMix 10 mu L, 0.5 mu L of each upstream primer and downstream primer with the concentration of 10mol/L, the sequence of the upstream primer is 5'-GCAGTTGAACCCACACTTCTG-3', the sequence of the downstream primer is 5'-TTGCCTACAGAATCCTTCCC-3', DNA, the template is 1.0 mu L, distilled water is added to 20 mu L: 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 storage at 4 ℃ to obtain a PCR product; and thirdly, performing bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and performing mutation genotyping by using a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the fiber diameter variation coefficient characters of the Eldos fine-hair 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, and the specific primer pair of the molecular marker Indel1 and the specific primer pair of the molecular marker SNP1 both comprise an upstream primer, a downstream primer and an extension primer, wherein the specific primer pair of the molecular marker Indel 1: the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3', the extension primer is 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3', and the specific primer pair of the molecular marker SNP 1: the forward primer was 5'-GACTCTCATACATTGACAAATACA-3', the reverse 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: a specific primer pair in a method for obtaining a molecular marker influencing the character of the fiber diameter variation coefficient of an Eldos fine-wool 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 both comprise an upstream primer, a downstream primer and an extension primer, and the specific primer pair of the molecular marker Indel1 comprises: the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3', the extension primer is 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3', and the specific primer pair of the molecular marker SNP 1: the forward primer was 5'-GACTCTCATACATTGACAAATACA-3', the reverse 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: an application of a specific primer pair in preparing a reagent or a kit for in vitro detecting molecular markers influencing the character of the diameter variation coefficient of the wool fibers of Erdos fine-wool sheep.
The fifth technical scheme of the invention is realized by the following measures: the application of a molecular marker influencing the character of the diameter variation coefficient of the wool fiber of an Ordos fine wool sheep in the breeding of high-yield and ultra-fine groups of the Ordos fine wool sheep.
According to the invention, the correlation between the polymorphism of the Ordos fine wool goat LMNB1 gene and the wool character is firstly researched, the molecular marker influencing the character of the diameter variation coefficient of Ordos fine wool goat hair fiber is positioned, the gene mutation site is obtained, the genotyping is realized, the ATCT genotype and the TTTT genotype are found to have advantages in the breeding of the superfine group, and a method and a guide are provided for the breeding of the high-yield and superfine group of Ordos fine wool goat.
Drawings
FIG. 1 shows the results of the genomic DNA assay of the present invention.
FIG. 2 shows the result of PCR detection of LMNB1 gene.
FIG. 3 shows the result of typing 2 mutant sites Snapshot of LMNB1 gene in the present invention.
FIG. 4 shows the 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 may be determined according to the technical solutions and practical situations of the present invention. The various chemical reagents and chemical articles mentioned in the invention are all the chemical reagents and chemical articles which are well known and commonly used in the prior art, unless otherwise specified; the percentages in the invention are mass percentages unless otherwise specified; the solution in the present invention is an aqueous solution in which the solvent is water, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution, unless otherwise specified; the normal temperature and room temperature in the present invention generally mean a temperature of 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to the following examples:
example 1: the molecular marker influencing the character of the fiber diameter variation coefficient of the Eldos fine hair sheep comprises a molecular marker Indel1 and a molecular marker SNP1, wherein the molecular marker Indel1 is positioned on the No. 2 intron of an LMNB1 gene P1 fragment at the position of 24654403bp-24654406bp of a sheep No. 5 chromosome, the molecular marker SNP1 is positioned on the No. 3 exon of an LMNB1 gene P1 fragment at the position of 24654282bp of the sheep No. 5 chromosome, the molecular marker Indel1 is four bases TATT deleted on the No. 2 intron of an LMNB1 gene P1 fragment at the position of 24654403bp-24654406bp of the sheep No. 5 chromosome, and the molecular marker SNP1 is a changed base G or A, an allelic gene mutation C or T on the No. 3 exon of an LMNB1 gene P1 fragment at the position of 24654282bp of the sheep No. 5 chromosome.
Example 2: as an optimization of the above embodiment, it was obtained as follows: firstly, taking the blood genome DNA of an Erdos fine wool ewe to be detected; secondly, performing PCR amplification by using blood genome DNA as a template, wherein a PCR reaction system comprises: PCRMix 10 mu L, upstream and downstream primers with the concentration of 10mol/L are respectively 0.5 mu L, the sequence of the upstream primer is 5'-GCAGTTGAACCCACACTTCTG-3' (shown as a sequence table SEQ ID No: 7), the sequence of the downstream primer is 5'-TTGCCTACAGAATCCTTCCC-3' (shown as a sequence table SEQ ID No: 8), 1.0 mu L of DNA template is added with distilled water to 20 mu L, the well-established PCR system is put into a PCR instrument for reaction, and the reaction conditions are as follows: 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 storage at 4 ℃ to obtain a PCR product; and thirdly, performing bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and performing mutation genotyping by using a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the fiber diameter variation coefficient characters of the Eldos fine-hair sheep.
Example 3: as an optimization of the above example, there is a linkage between the genotype of the molecular marker Indel1 and the genotype of the molecular marker SNP1, when the genotype of the molecular marker Indel1 is AA genotype, the genotype of the molecular marker SNP1 is CC genotype, and when the genotype of the molecular marker Indel1 is TT genotype, the genotype of the molecular marker SNP1 is TT genotype.
Molecular marker technology can represent differences between individual organisms or populations by detecting variations in the genes or genotypes of animals. In the invention, deletion of four bases of TATT is found on the 2 nd intron (Indel1) of the P1 fragment of the LMNB1 gene, a mutant base C or T is found on the 3 rd exon (SNP1) of the LMNB1 gene, and linkage between Indel1 and SNP1 can be known through Snapshot detection and typing. Therefore, the fiber diameter variation coefficient character of the 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), thereby providing experimental basis for breeding the Ordos fine wool sheep ultrafine population.
Example 4: the method for obtaining the molecular marker influencing the character 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 the blood genome DNA of an Erdos fine wool ewe to be detected; secondly, performing PCR amplification by using blood genome DNA as a template, wherein a PCR reaction system comprises: PCRMix 10 mu L, upstream and downstream primers with the concentration of 10mol/L are respectively 0.5 mu L, the sequence of the upstream primer is 5'-GCAGTTGAACCCACACTTCTG-3' (shown as a sequence table SEQ ID No: 7), the sequence of the downstream primer is 5'-TTGCCTACAGAATCCTTCCC-3' (shown as a sequence table SEQ ID No: 8), 1.0 mu L of DNA template is added with distilled water to 20 mu L, the well-established PCR system is put into a PCR instrument for reaction, and the reaction conditions are as follows: 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 storage at 4 ℃ to obtain a PCR product; and thirdly, performing bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and performing mutation genotyping by using a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the fiber diameter variation coefficient characters of the Eldos fine-hair sheep.
Example 5: the specific primer pair in the method for obtaining the molecular marker influencing the character of the fiber diameter variation coefficient of the Eldos fine-wool 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 both comprise an upstream primer, a downstream primer and an extension primer, and the specific primer pair of the molecular marker Indel1 comprises: the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3' (shown as SEQ ID No:1 in the sequence table), the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3' (shown as SEQ ID No:2 in the sequence table), the extension primer is 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3' (shown as SEQ ID No:3 in the sequence table), and the specific primer pair of the molecular marker SNP 1: the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3' (shown as SEQ ID No:4 in the sequence table), the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3' (shown as SEQ ID No:5 in the sequence table), and the extension primer is 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3' (shown as SEQ ID No:6 in the sequence table).
Example 6: the application of the specific primer pair in preparing a reagent or a kit for in vitro detection of molecular markers influencing the character of the diameter variation coefficient of the wool fibers of the Erdos fine-wool sheep.
Example 7: the molecular marker influencing the character of the diameter variation coefficient of the wool fibers of the Ordos fine wool sheep is applied to the breeding of high-yield and ultra-fine groups of the Ordos fine wool sheep.
The following are experimental studies of the molecular markers of the invention related to the characteristics of the diameter and coefficient of variation of the Ordos fine-wool cashmere fibre:
1 materials and methods
1.1 sample Collection
A total of 462 underwent erdos fine hair ewes were collected as experimental animals from five towns, the grand towns (n: 97), the threatened sappan (n: 134), the pictk towns (n: 91), and the wu ju towns (n: 99), from among the grand ewes, the deldos city, inner mongolian municipality. The experimental ewe is subjected to venous blood collection to a 5ml anticoagulation tube, and the tube is placed into a refrigerator at the temperature of-20 ℃ for storage and used for subsequent extraction of blood genome DNA. Meanwhile, a wool sample is collected at the position 10cm above the posterior edge of the scapula above the midline of the left side of the experimental sheep. The wool sample is sent to a quality supervision, inspection and test center for sheep and wool cashmere in rural areas, and the average fiber diameter and the property of the fiber diameter variation coefficient of the wool sample are detected by an optical fiber diameter analyzer (OFDA 2000). And finally, collecting 2020-year identification records of experimental flocks, wherein the identification records mainly comprise wool production, wool fiber length and the like.
1.2 construction of DNA Mixed pool
Blood genomic DNA was extracted using a blood genomic DNA extraction kit (Tiangen Biotech Co., Ltd.). 4ul of DNA was taken, DNA quality was checked by 1.0% agarose gel electrophoresis, and 1ul of DNA was taken, and DNA concentration was checked by a nucleic acid protein detector. In the measured wool fiber diameter data, 20 extremes were selected respectivelyIndividual (very thin n 10, very thick n 10) DNA samples. The average fiber diameter of the very fine group was 16.36 μm, and the average fiber diameter of the very coarse group was 21.50. mu.m. The extreme individual DNA concentrations were detected by a nucleic acid protein detector and ddH was added2O is adjusted to be uniform. 10ul of each of the 20 extreme individuals were taken, and 2 DNA mixing pools (ultrafine DNA mixing pool, extra-coarse DNA mixing pool) were constructed according to the ultrafine and extra-coarse groups
1.3PCR amplification and sequencing
Primers were designed using PremerPremer5.0 based on the sequence of LMNB1 gene (accession number: ENSOART00000019747.1) in Ensembl nucleic acid database, and the primer sequences are shown in Table 1. The primers were synthesized by Shanghai bioengineering technology, Inc. Performing PCR amplification by using 2 mixed pool DNAs as templates, wherein a PCR reaction system comprises the following steps: mu.L of PCRMixture10, 0.5. mu.L of each of the upstream and downstream primers (10mol/L), 1.0. mu.L of DNA template, and distilled water to 20. mu.L. And putting the established PCR system into a PCR instrument for reaction. Reaction conditions are as follows: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 1min, 34 cycles, extension at 72 deg.C for 5min, and storage at 4 deg.C. The PCR product was detected by electrophoresis on a 1.5% agarose gel. The PCR products with bright bands were sent directly to Biotechnology engineering (Shanghai) GmbH for bidirectional sequencing. Sequencing results sequence alignment and correction were performed using the SeqMan program of DNASTAR software, and peak mapping was performed using the BioEdit software.
1.4Snapshot detection
Designing an upstream primer, a downstream primer and an extension primer according to mutation sites screened by bidirectional sequencing, wherein the primer information is shown in Table 2. 462 individuals were detected by the Snapshot genotyping technique from beijing, oto, ding, biotechnology ltd, and typed with the ABI3730XL sequencer. And finally, randomly selecting 3 samples from each site to perform first-generation sequencing, and verifying the Snapshot typing detection result.
1.5 statistical analysis
The gene frequency, genotype frequency, effective allele factor, gene heterozygosity, polymorphic information content, and Hardy-Weinberg equilibrium of SNPs were calculated using Popgene software. SAS9.2 software is used for analyzing the association of different genotypes of SNPs and characters for Ordos fine wool. The results are expressed as least squares means ± standard error, and the linear model is:
Yick＝μ+Gi+Fe+eick
in the formula, Yick: the individual phenotype value of the fine wool sheep; μ: group mean value; gi: genotype SNP effect; fe: a field effect; e.g. of the typeick: random error.
2 results and analysis
2.1DNA and PCR detection results
The extracted genomic DNA was detected by electrophoresis on a 1% agarose gel, and the DNA band was bright, as shown in FIG. 1. The DNA is detected by a nucleic acid protein detector, the OD ratio under 260nm and 280nm is 1.8-2.1, and the quality and purity of the extracted DNA meet the requirements of subsequent experiments. The result of PCR product detection of LMNB1 gene is shown in FIG. 2, wherein M.2000 gene is used as reference. As can be seen from FIG. 2, the PCR product was consistent with the expected fragment size, and was free of bands, meeting the requirements of the subsequent experiments.
2.2 Mixed pool results analysis
2 mutations were found by pool-mixing sequencing of the E.E.fine wool gene PCR fragment. 2 mutations (Indel1 and SNP1) were found in the P1 fragment of LMNB1 gene. The information of 2 mutations in LMNB1 gene is shown in table 3. Wherein Indel1 is a deletion of TATT4 bases in intron 2 of the P1 fragment of the LMNB1 gene; SNP1 is a synonymous mutation on an exon of LMNB1 gene.
2.3Snapshot detection typing and verification results
The peak pattern of 2 mutation sites of 462 Eldos fine wool sheep LMNB1 gene detected by Snapshot typing technique is shown in FIG. 3. In Snapshot detection, 2 mutation sites were successfully typed, and all had three genotypes. Wherein the extension products of Indel1 and SNP1 are forward extensions. In addition, Indel1 and SNP1 genotypes share the same typing pattern, i.e., SNP1 is the CC genotype when Indel1 is a four base deletion (AA genotype) of TATT; when Indel1 is TATT four-base insertion (TT genotype), SNP1 is TT genotype, two mutations of Indel1 and SNP1 are presumed to be linked, and therefore two mutation sites Indel1-SNP1 are combined and analyzed subsequently. According to the Snapshot typing detection result, 2 individuals with different mutation genotypes are randomly selected for 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 completely matches the Snapshot typing results, which indicates that the Snapshot typing results are reliable.
2.4 analysis of genetic polymorphisms
The results of 2 mutant genotype frequencies, allele frequencies and χ 2 calculation of Ordos fine wool sheep LMNB1 gene are shown in Table 4. As can be seen from Table 4, the dominant genotype of Indel1-SNP1 is TTTT genotype. Meanwhile, the chi 2 values of 2 mutation sites do not reach a remarkable level, and Hardy-Weinberg is balanced. In addition, the analysis of the genetic homozygosity (Ho), genetic heterozygosity (He), effective allele factor (Ne), and Polymorphism Information Content (PIC) of 2 mutant sites of the orndos fine wool goat LMNB1 gene is shown in table 5. As can be seen from Table 5, the 2 mutation sites have high degree of purity, and the number of effective alleles is 1.807 respectively. According to the mutation site polymorphism standard: PIC >0.5 is highly polymorphic, 0.25< PIC <0.5 is moderately polymorphic, and PIC <0.25 is lowly polymorphic. As can be seen from Table 5, the Indel1-SNP1 mutation site was moderately polymorphic.
2.5 analysis of correlation between mutant sites and wool traits
The SAS9.2 software is used for analyzing the correlation between 2 mutation site polymorphisms of the LMNB1 gene of Erdos fine hair sheep and wool traits as shown in Table 6. The fiber diameter variation coefficient character of AACC genotype at the mutation site of LMNB1 gene Indel1-SNP1 is 21.72, the fiber diameter variation coefficient character genotype of ATCT genotype is 22.33, and the fiber diameter variation coefficient character of TTTT genotype is 22.43. As can be seen from Table 6, the fiber diameter coefficient of variation properties of the ATCT genotype and the TTTT genotype are significantly higher than those of the AACC genotype (P <0.05), and the difference between the ATCT genotype and the TTTT genotype is not significant (P > 0.05).
In conclusion, according to the invention, correlation between polymorphism of the Ordos fine wool goat LMNB1 gene and wool character is firstly researched, molecular markers influencing the character of the diameter variation coefficient of Ordos fine wool goat hair are positioned, gene mutation sites are obtained, genotyping is realized, the ATCT genotype and the TTTT genotype are found to be superior in ultrafine group breeding, and a method and guidance are provided for breeding high-yield and ultrafine groups of Ordos fine wool goats.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs 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 the LMNB1 gene
TABLE 5 polymorphism analysis of LMNB1 Gene population
TABLE 6 correlation analysis of mutation sites of LMNB1 gene and wool traits
The difference between the average values of different lower case letters of shoulder marks is significant (P < 0.05); the average values of different capital letters marked on shoulders are very different (P < 0.01); the shoulder marks the mean of the same letter or no letter without significant difference.
Sequence listing
<110> institute of livestock and poultry of Sinkiang academy of livestock sciences and the scientific institute of farming and animal husbandry of Ordos city (Oldos institute of agricultural sciences of inner Mongolia)
<120> molecular marker influencing E' erdos fine wool sheep wool fiber diameter variation coefficient, and specific primer pair and application thereof
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Claims (8)
1. A molecular marker influencing the character of the fiber diameter variation coefficient of an Eldos fine hair 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 the 24654403bp-24654406bp position of a sheep 5 chromosome, the molecular marker SNP1 is positioned on the 3 rd exon of an LMNB1 gene P1 fragment at the 24654282bp position of the sheep 5 chromosome, the molecular marker Indel1 is four bases AATA deleted on the 2 nd intron of an LMNB1 gene P1 fragment at the 24654403bp-24654406bp position of the sheep 5 chromosome, and the molecular marker SNP1 is a changed base G or A on the 3 rd exon of an LMNB1 gene P1 fragment at the 24654282bp position of the sheep 5 chromosome, and allelic gene mutation C or T.
2. The molecular marker affecting the character of the E' erdos fine wool goat hair fiber diameter coefficient of variation according to claim 1, which is obtained by the following method: firstly, taking the blood genome DNA of an Erdos fine wool ewe to be detected; secondly, performing PCR amplification by using blood genome DNA as a template, wherein a PCR reaction system comprises: the PCR method comprises the following steps of (1) carrying out PCR reaction on an established PCR system in a PCR instrument, wherein the PCR reaction conditions comprise that (9) mu L of PCRMix 10 mu L, 0.5 mu L of each upstream primer and downstream primer with the concentration of 10mol/L, the sequence of the upstream primer is 5'-GCAGTTGAACCCACACTTCTG-3', the sequence of the downstream primer is 5'-TTGCCTACAGAATCCTTCCC-3', DNA, the template is 1.0 mu L, distilled water is added to 20 mu L: 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 storage at 4 ℃ to obtain a PCR product; and thirdly, performing bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and performing mutation genotyping by using a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the fiber diameter variation coefficient characters of the Eldos fine-hair sheep.
3. The molecular marker affecting the character of the index of variation of the fiber diameter of the fine-wool ewe's hair according to claim 1 or 2, wherein the molecular marker Indel1 genotype is linked with the molecular marker SNP1 genotype, 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.
4. A method for obtaining a molecular marker influencing the character of the diameter variation coefficient of an Erdos fine-wool sheep wool fiber is characterized by comprising the following steps: firstly, taking the blood genome DNA of an Erdos fine wool ewe to be detected; secondly, performing PCR amplification by using blood genome DNA as a template, wherein a PCR reaction system comprises: the PCR method comprises the following steps of (1) carrying out PCR reaction on an established PCR system in a PCR instrument, wherein the PCR reaction conditions comprise that (9) mu L of PCRMix 10 mu L, 0.5 mu L of each upstream primer and downstream primer with the concentration of 10mol/L, the sequence of the upstream primer is 5'-GCAGTTGAACCCACACTTCTG-3', the sequence of the downstream primer is 5'-TTGCCTACAGAATCCTTCCC-3', DNA, the template is 1.0 mu L, distilled water is added to 20 mu L: 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 storage at 4 ℃ to obtain a PCR product; and thirdly, performing bidirectional sequencing on the PCR product, screening mutation sites according to the bidirectional sequencing, designing a specific primer pair, and performing mutation genotyping by using a Snapshot detection technology to obtain a molecular marker Indel1 and a molecular marker SNP1 which are obviously related to the fiber diameter variation coefficient characters of the Eldos fine-hair sheep.
5. The method for obtaining the molecular marker affecting the E.deltoid fine wool sheep fiber diameter variation coefficient character according to claim 4, wherein the specific primer pair comprises a specific primer pair of a molecular marker Indel1 and a specific primer pair of a molecular marker SNP1, the specific primer pair of the molecular marker Indel1 and the specific primer pair of the molecular marker SNP1 both comprise an upstream primer, a downstream primer and an extension primer, wherein the specific primer pair of the molecular marker Indel 1: the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3', the extension primer is 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3', and the specific primer pair of the molecular marker SNP 1: the forward primer was 5'-GACTCTCATACATTGACAAATACA-3', the reverse primer was 5'-ACTCCAAGTCCTCAGTAAG-3', and the extension primer was 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3'.
6. A specific primer pair in a method for obtaining a molecular marker influencing the character of the fiber diameter variation coefficient of an Eldos fine-wool sheep is characterized by comprising 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 both comprise an upstream primer, a downstream primer and an extension primer, and the specific primer pair of the molecular marker Indel1 comprises: the upstream primer is 5'-GACTCTCATACATTGACAAATACA-3', the downstream primer is 5'-ACTCCAAGTCCTCAGTAAG-3', the extension primer is 5'-CTGACTGACTGACTGACTGACTTGATTCAATCCATCGGTATC-3', and the specific primer pair of the molecular marker SNP 1: the forward primer was 5'-GACTCTCATACATTGACAAATACA-3', the reverse primer was 5'-ACTCCAAGTCCTCAGTAAG-3', and the extension primer was 5'-CTGACTGACTGACTGACTGACTAGATGAAACTTTACTTAAAGTGGA-3'.
7. An application of the specific primer pair of claim 6 in preparing a reagent or a kit for in vitro detection of a molecular marker affecting the character of the variation coefficient of the diameter of the wool fibers of the Aldos fine-wool sheep.
8. Use of the molecular marker affecting the character of the variation coefficient of the diameter of the wool fibers of an Ordos fine-wool sheep as claimed in claim 1, 2 or 3 in the breeding of high-yielding, ultra-fine groups of Ordos fine-wool sheep.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009100029A1 (en) * | 2008-02-01 | 2009-08-13 | The General Hospital Corporation | Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions |
| CN107604078A (en) * | 2017-10-23 | 2018-01-19 | 新疆畜牧科学院畜牧研究所 | The molecular labeling related to sheep wool fiber diameter character and its specific primer pair and application |
| EP3476940A2 (en) * | 2017-10-25 | 2019-05-01 | Università Degli Studi Di Torino | Rna interference mediated therapy for neurodegenerative diseases |
| CN111996265A (en) * | 2020-09-20 | 2020-11-27 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing wool fiber diameter of fine wool sheep and application thereof |
-
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- 2022-01-07 CN CN202210015141.5A patent/CN114231643B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009100029A1 (en) * | 2008-02-01 | 2009-08-13 | The General Hospital Corporation | Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions |
| CN107604078A (en) * | 2017-10-23 | 2018-01-19 | 新疆畜牧科学院畜牧研究所 | The molecular labeling related to sheep wool fiber diameter character and its specific primer pair and application |
| EP3476940A2 (en) * | 2017-10-25 | 2019-05-01 | Università Degli Studi Di Torino | Rna interference mediated therapy for neurodegenerative diseases |
| CN111996265A (en) * | 2020-09-20 | 2020-11-27 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing wool fiber diameter of fine wool sheep and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| DONALDSON L等: "Construction and validation of a Bovine Innate Immune Microarray.", 《BMC GENOMICS.》 * |
| 赵冰茹;黄锡霞;田可川;付雪峰;徐新明;柏妍;田月珍;: "细毛羊LAMB1基因外显子多态性及其与羊毛纤维直径的关联性分析", 中国畜牧兽医 * |
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