CN114250309B - Molecular marker affecting fiber diameter properties of ARUM cashmere goat, specific primer pair and application thereof - Google Patents
Molecular marker affecting fiber diameter properties of ARUM cashmere goat, 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 fiber diameter property of ARUM cashmere goat cashmere, and a specific primer pair and application thereof, wherein the molecular marker affecting the fiber diameter property of ARUM cashmere goat cashmere is positioned on a KRAP 24-1 gene of ARUM cashmere goat chromosome 1, the KRAP 24-1 gene is positioned at 4033765bp-4034298bp of a chromosome 1 nucleotide sequence, the molecular marker is positioned at 4034201bp of the chromosome 1 nucleotide sequence, and a mutant base is C or T. The molecular marker can realize early seed selection of cashmere fiber diameter character good sheep, and can be selected and remained after birth, thereby reducing the breeding cost, accelerating the breeding process of high-quality superfine Jiangnan cashmere goats and providing theoretical basis and guidance for genetic improvement of the Jiangnan cashmere goats.
Description
Technical Field
The invention relates to the technical field of animal molecular markers, in particular to a molecular marker affecting the fiber diameter property of ARUM cashmere goat, a specific primer pair and application thereof, wherein the specific primer pair is applied to preparation of a reagent or a kit for in vitro detection of the molecular marker affecting the fiber diameter property of ARUM cashmere goat, and application of the molecular marker in selection of the fiber diameter property of ARUM cashmere goat.
Background
Cashmere is a main product of cashmere goats, has soft texture and extensibility, and is a special animal fiber of high-grade textile raw materials, and the textile is worn on the body, comfortable, sweat-absorbent, warm-keeping and breathable. The cashmere clothing is gorgeous, elegant in style and smooth in hand feeling.
The fineness of the cashmere is the most important index of the quality of the cashmere, the weight of the cashmere textile is lighter, the flexibility of the handfeel is related to the average diameter of the cashmere textile, the current technology level of the cashmere textile industry is urgently needed to be improved, the innovative brand is developed by using high-quality cashmere, and the problem of protecting the quality of the cashmere is important.
In 2020, the Jiangnan cashmere goat is a new cashmere goat breed formed by 40 years of cultivation, and has the excellent characteristics of high production performance, coarse feeding resistance and the like. The ecological environment of the ARUM Nannochlori goat is bad, and the core breeding base (ARUM Akesu comprehensive test station) is positioned on the arid desert and semi-desert grassland with the east longitude of 79 DEG 47', the North latitude of 41 DEG 23', and the altitude of 2000 meters. The annual average temperature is 6.5 ℃, and the rainfall is 200 mm. 90% of the grasslands are desert grasslands, and the vegetation coverage is about 10% to 30%.
However, in recent years, along with the impact of mutton price and the laching of basic breeding work, the phenomenon of original fine wool resource loss exists in the Jiang Nan cashmere goat, so that the quality of cashmere is seriously influenced, and the sales price of the cashmere is influenced. These cashmere goat breeds are the main economic source for local herding. The breeding industry is the basic stone developed by the modern sheep industry, and the genetic improvement is the key for improving the competitive power of the sheep industry. The breeding of superfine down goats suitable for extreme climate of Xinjiang is a local urgent task. Therefore, it is important to find a breeding means which is effective, convenient and capable of accelerating the breeding process of the down producing goats in a short time. Classical breeding methods are mainly through hybridization, but the method has slower genetic progress and longer breeding period. With the rapid development of molecular biology theory and technology, the combination of molecular genetics and genetic engineering methods and traditional marker assisted selection methods provides a better solution to the breeding problem. Finding genetic markers related to cashmere traits is the first aspect we focus on.
Classical breeding methods are mainly through hybridization, but the method has slower genetic progress and longer breeding period. With the rapid development of molecular biology theory and technology, the combination of molecular genetics and genetic engineering methods and traditional marker assisted selection methods provides a better solution to the breeding problem. Finding genetic markers related to cashmere traits is the first direction of attention.
At present, transcriptomics research is performed on main cashmere goat varieties in China, such as Liaoning cashmere goats, inner Mongolian cashmere goats and Tibetan cashmere goats. In the aspect of genome, the gene and molecular marker related to the cashmere fiber diameter are mainly excavated by adopting research strategies such as whole genome relativity analysis, candidate gene polymorphism analysis and the like. And after the new variety of the ARUM cashmere goat is approved, researches on the characteristics of cashmere are freshly reported.
Keratin and keratin-associated proteins are the major proteins that make up hair. The genes encoding keratin-related proteins are often rich in mutations that may be related to the down producing properties of the down producing goat. At present, KRAP 15-, KRAP 13.1, KRAP 27-1 and KRAP 24-1 isogenic polymorphisms have been demonstrated to affect cashmere traits in sheep and goats. However, it is not clear whether these genes can affect the cashmere fiber diameter of Jiangnan cashmere goats. Therefore, the research analyzes the influence of KRTAP24-1 gene polymorphism of the Jiangnan cashmere goat on the diameter of cashmere fibers, and aims to provide experimental basis for cultivating superfine cashmere goats.
Disclosure of Invention
The invention provides a molecular marker affecting the fiber diameter property of the cashmere of the Jiangnan cashmere goat, a specific primer pair and application thereof, and discloses a molecular marker different from the existing molecular marker affecting the fiber diameter property of the cashmere of the goat for the first time.
One of the technical schemes of the invention is realized by the following measures: a molecular marker (SNP 28 locus of the invention) for influencing the fiber diameter property of the cashmere fiber of the Jiangnan cashmere goat is positioned on the KRAP 24-1 gene of the 1 st chromosome of the Jiangnan cashmere goat, the KRAP 24-1 gene is positioned at 4033765bp-4034298bp of the 1 st chromosome nucleotide sequence, the molecular marker is positioned at 4034201bp of the 1 st chromosome nucleotide sequence, and the mutation base is C or T.
The second technical scheme of the invention is realized by the following measures: a specific primer pair for a molecular marker affecting the fiber diameter property of ARUM cashmere goat cashmere, which is one of the amplification technical schemes, is characterized by comprising an upstream primer and a downstream primer,
upstream primer F:5'-tggtgtgacatagaccaaagagcta-3';
the downstream primer R:5'-ctcttggataagcagttaagttgcc-3'.
The third technical scheme of the invention is realized by the following measures: the application of the specific primer pair in the second technical scheme in preparing a reagent or a kit for in vitro detection of molecular markers affecting the fiber diameter properties of ARUM cashmere goat.
The fourth technical scheme of the invention is realized by the following measures: an application of a molecular marker affecting the diameter property of the cashmere fiber of the Jiangnan cashmere goat in the selection of the diameter property of the cashmere fiber of the Jiangnan cashmere goat in one of the technical schemes.
The following is a further optimization and/or improvement of the fourth technical scheme of the invention:
the method comprises the following steps: firstly, taking genomic DNA of a to-be-detected Jiangnan cashmere goat; the second step, taking genome DNA as a template, and carrying out first round of amplification by utilizing a specific primer pair, wherein a first round of PCR system is a reaction system of 25 mu l, and the first round of PCR system consists of the following components:
the first round of PCR amplification conditions were: pre-denaturation at 98 degrees for 3 min, followed by 8 cycles of 94 degrees for 30 seconds, 50 degrees for 30 seconds, 72 degrees for 30 seconds; immediately after 25 cycles, the conditions were 98 degree denaturation for 30 seconds, 66 degree annealing for 30 seconds, 72 degree extension for 30 seconds; finally, the extension is carried out at 72 ℃ for 5 minutes, after the PCR reaction is finished, the temperature is kept at 4 ℃, 1% agarose gel electrophoresis is used for detecting the PCR product, the size of the product is determined to be correct, the AMPure XP magnetic beads are used for purifying and recycling the PCR product,
upstream primer pool: 5'-tggtgtgacatagaccaaagagcta-3';
downstream primer pool: 5'-ctcttggataagcagttaagttgcc-3';
third, the first round PCR product is used as a template to execute the second round PCR reaction to obtain a library with molecular tags for sequencing,
the second round of PCR system is a reaction system of 30 μl, and the second round of PCR system consists of the following components:
the second round of PCR amplification conditions were: 98 degrees pre-denaturation for 5 minutes, then 5 cycles were performed under conditions of denaturation for 94 degrees 30 seconds, 55 degrees annealing for 20 seconds, 72 degrees extension for 30 seconds, and finally 72 degrees extension for 5 minutes, after the PCR reaction was completed, 4 degrees incubation was performed, and the final PCR products were purified and recovered using AMPure XP magnetic beads, and after equal amounts of each PCR product were mixed, were sequenced using a Hiseq XTen sequencer (Illumina, san Diego, calif.); genotyping was performed according to the sequencing result,
for the cashmere fiber diameter property, in the genotypes corresponding to the molecular markers, the Jiangnan cashmere goats containing TT or CT genes of T genes belong to dominant individuals with finer cashmere fiber diameter property.
In the invention, multiple PCR (two rounds of PCR) and high-throughput sequencing are utilized to identify the mutation site of the exon region of the KRAP 24-1 gene of Mount; aiming at the research of target gene polymorphism of large groups, compared with the previous research methods, such as PCR-SSCP and PCR-RFLP technology, the method of combining multiple PCR with high-throughput sequencing can detect more abundant mutation sites. Compared with other gene chips and whole genome re-sequencing technologies, the method is more economical. In addition, the sequencing result is uploaded to NCBI public database (PRJNA 738549), and the accumulation of genome raw data is helpful for developing the research on the germplasm characteristics of the local ARUM Nannocher goats in China and protecting and utilizing resources.
The invention discloses a 4034201bp locus (SNP 28 locus) of a No. 1 chromosome nucleotide sequence as a molecular marker for influencing the fiber diameter property of the ARUM Nannoch goat, and discloses a 4034201bp locus of the No. 1 chromosome nucleotide sequence for the first time in the selection of the fiber diameter property of the ARUM Nannoch goat; when the molecular marker affecting the fiber diameter property of the cashmere of the Jiangnan cashmere goat is adopted to select the fiber diameter property of the Jiangnan cashmere goat, the molecular marker detection layer starts, so that the accuracy of variety selection can be improved, the detection efficiency is improved, and the automatic detection can be realized; in addition, the early-stage seed selection of the cashmere fiber diameter character good product breeding sheep can be realized, and the breeding sheep can be selected and remained after birth, so that the breeding cost is reduced, the breeding process of the high-quality superfine type Jiangnan cashmere goat is accelerated, and theoretical basis and guidance are provided for the genetic improvement of the Jiangnan cashmere goat.
Drawings
FIG. 1 is an electrophoresis detection chart of genomic DNA of ARUM Nannochlori goat.
FIG. 2 shows the result of KRAP 24-1 gene mutation site heterozygous generation sequencing.
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 software mentioned in the present invention is all the software known and commonly used in the art.
The invention is further described below with reference to examples:
example 1: the molecular marker (SNP 28 locus of the invention) influencing the fiber diameter property of the cashmere of the Jiang Nan cashmere goat is positioned on the KRAP 24-1 gene of the No. 1 chromosome of the Jiang Nan cashmere goat, the KRAP 24-1 gene is positioned at 4033765bp-4034298bp of the nucleotide sequence of the No. 1 chromosome, the molecular marker is positioned at 4034201bp of the nucleotide sequence of the No. 1 chromosome, and the mutation base is C or T.
Example 2: a specific primer pair for amplifying the molecular marker affecting the fiber diameter property of ARUM cashmere goat cashmere of example 1, characterized by comprising an upstream primer and a downstream primer,
upstream primer F (shown as SEQ ID No. 1 of the sequence Listing):
5’-tggtgtgacatagaccaaagagcta-3’；
downstream primer R (shown as SEQ ID No. 2 of the sequence Listing):
5’-ctcttggataagcagttaagttgcc-3’。
example 3: application of the specific primer pair described in the embodiment 2 in preparation of a reagent or a kit for in vitro detection of molecular markers affecting the fiber diameter properties of ARUM cashmere goat.
Example 4: the use of the molecular marker of example 1 affecting the fiber diameter properties of the ARUM cashmere goat, in the selection of the ARUM cashmere fiber diameter properties, comprising the steps of: firstly, taking genomic DNA of a to-be-detected Jiangnan cashmere goat; the second step, taking genome DNA as a template, and carrying out first round of amplification by utilizing a specific primer pair, wherein a first round of PCR system is a reaction system of 25 mu l, and the first round of PCR system consists of the following components:
the first round of PCR amplification conditions were: pre-denaturation at 98 degrees for 3 min, followed by 8 cycles of 94 degrees for 30 seconds, 50 degrees for 30 seconds, 72 degrees for 30 seconds; immediately after 25 cycles, the conditions were 98 degree denaturation for 30 seconds, 66 degree annealing for 30 seconds, 72 degree extension for 30 seconds; finally, the extension is carried out at 72 ℃ for 5 minutes, after the PCR reaction is finished, the temperature is kept at 4 ℃, 1% agarose gel electrophoresis is used for detecting the PCR product, the size of the product is determined to be correct, the AMPure XP magnetic beads are used for purifying and recycling the PCR product,
upstream primer pool: 5'-tggtgtgacatagaccaaagagcta-3';
downstream primer pool: 5'-ctcttggataagcagttaagttgcc-3';
third, the first round PCR product is used as a template to execute the second round PCR reaction to obtain a library with molecular tags for sequencing,
the second round of PCR system is a reaction system of 30 μl, and the second round of PCR system consists of the following components:
the second round of PCR amplification conditions were: 98 degrees pre-denaturation for 5 minutes, then 5 cycles were performed under conditions of denaturation for 94 degrees 30 seconds, 55 degrees annealing for 20 seconds, 72 degrees extension for 30 seconds, and finally 72 degrees extension for 5 minutes, after the PCR reaction was completed, 4 degrees incubation was performed, and the final PCR products were purified and recovered using AMPure XP magnetic beads, and after equal amounts of each PCR product were mixed, were sequenced using a Hiseq XTen sequencer (Illumina, san Diego, calif.); genotyping was performed according to the sequencing result,
for the cashmere fiber diameter property, in the genotypes corresponding to the molecular markers, the Jiangnan cashmere goats containing TT or CT genes of T genes belong to dominant individuals with better cashmere fiber diameter property.
The following is the experimental study of the molecular marker affecting the fiber diameter property of the ARUM cashmere goat cashmere:
1 Experimental materials and methods
1.1 laboratory animals
354 sheep goats (JN) were selected from the breeding center of the species sheep in the prefecture, temperature dormitory, arctic, and the two ages, 144 (AL, n=144), sha Kequn (SK, n=79) for salsa, 53 (TZ, n=54) for vomit, and 79 (YM, n=79) for emma.
1.2 sample collection
And collecting cashmere samples at the position 10cm above the middle line of the left side body of the Jiangnan cashmere goat at Apcaput, washing according to a conventional washing process, naturally drying, and measuring average fiber diameter, fiber diameter variation coefficient and fiber diameter standard deviation by using a fiber diameter optical analyzer OFDA2000 under the conditions of constant temperature and humidity of 20+/-2 ℃ and 65+/-4%.
Corresponding to cashmere samples, 5mL of experimental sheep blood is collected in an anticoagulant tube and stored in a refrigerator at-20 ℃. The goat down DNA was extracted using a blood genomic DNA extraction kit (TIANGENG, USA) and the quality of the DNA was detected by electrophoresis on a 1.0% agarose gel. The DNA concentration was detected using a nucleic acid protein detector.
1.3 multiplex amplification and high throughput sequencing
The KRAP 24-1 gene exon region was selected according to the published sequence in NCBI goat chromosome 1 NC_030808.1 accession number. A Primer pool containing the exon region of the target gene was designed using Primer 5.0 and synthesized by Shanghai, and the Primer list was as shown in Table S1. Then the amplification of the target SNP site sequence and the preparation of a compatible Illumina sequencing library are completed by a two-step PCR method on a PCR instrument (BIO-RAD, T100 TM). The two-step PCR system is shown in tables S2 and S4, and the reaction procedures are shown in tables S3 and S5. The final PCR product was recovered using AMPure XP magnetic bead purification. After equal amounts of each PCR product were mixed, sequencing was performed using a HiSeq XTen sequencer (Illumina, san Diego, canada).
1.4 sequencing data quality control
Off-press data cut off any partial sequence containing the sequencing adapter sequence using the cutadapt (v 1.2.1) software, and cut off the first 10 bases of the reads 5 'and 3' ends; the remaining sequences were quality controlled using PRINSEQ-lite (v 0.20.3) software, deleting bases with quality threshold below 20 in the order of 3 'to 5' of the sequence.
1.5 sequencing data analysis and validation
The remaining sequence after quality control is regarded as a qualified sequence. And then, using BWA (v 0.7.13-r 1126) software to align the quality-qualified sequences into target region sequences in a pair-end mapping mode. Based on the alignment results, genotype results for the target site were calculated by samtools software (version 0.1.18). Finally, annovar software was used to make gene annotation of SNP sites.
By a first generation sequencing technology, heterozygous individuals with four mutation sites are selected for each gene, and high-throughput sequencing results are verified. The sequence of the first generation was spliced and aligned using the SeqMan program of DNASTAR software and the peak plots were aligned using BioEdit software.
1.6 statistical analysis
The frequency of minor alleles of SNPs was calculated using the Popgene software. And (3) analyzing the correlation of different genotypes of SNPs and characteristics for cashmere by combining the cashmere fiber diameter measurement result and utilizing a GLM model in SAS 9.2 software. Results are expressed in terms of least squares mean ± standard error, the linear model is:
Y ick ＝μ+G i +F c +e ick
in the formula, Y ick : a cashmere goat individual body surface model value; mu: population means; g i : genotype SNP effect; f (F) c : a field effect; e, e ick : random errors.
2.2 sequencing data quality control
Genomic DNA of ARUM goat was extracted and 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 to 2.1, which shows that the quality and purity of the extracted DNA meet the requirements of the subsequent library construction.
The quality control is carried out on the data after the HiSeq XTen sequencer is used for sequencing, the average Coverage ratio (Coverage) of each fragment to the target region sequence is 96.09%, the average Coverage depth (mean_depth) is 4838.75, and the sequencing Error percentage (error_ratio) is 4.52%, so that the quality of the sequencing data is higher, and the subsequent genotyping analysis is satisfied.
2.3 typing result annotation and verification
In combination with the multiple PCR technique and high throughput sequencing, 6 mutation sites were obtained in the KRAP 24-1 gene of ARUM Nannochlori goat, as shown in Table 2.
The result of the KRAP 24-1 gene mutation site heterozygous first generation sequencing is shown in FIG. 2, and the high-throughput typing result is consistent with the first generation sequencing result, which shows that the high-throughput sequencing result is reliable.
2.4 population genetic polymorphism analysis
The results of the 6 mutation site genotyping and the hypo-allele frequency analysis of the KRAP 24-1 gene of the down producing goat group are shown in Table 3.
As can be seen from the data in Table 3, SNP24 and SNP26 have minor allele frequencies of less than 0.03 in the ARUM Nannoch goat population.
2.5SNP Effect analysis
The correlation analysis of SNP and cashmere fiber diameter was performed using SAS 9.2 software as shown in table 4.
As can be seen from the data in table 4, SNP28 significantly affected the average fiber diameter (P < 0.05) of the down population of the jiang south down goats.
From this, it is clear that SNP28 significantly affects the cashmere fiber diameter. Thus, the SNP28 is related to the cashmere fiber diameter property, and the SNP28 can be used as a molecular marker for influencing the cashmere fiber diameter property of the Jiangnan cashmere goat.
As can be seen from the gene types in Table 4, in the genotype corresponding to the SNP28 locus, the Jiangnan cashmere goat containing TT or CT genes of T genes belongs to dominant individuals with finer cashmere fiber diameter properties.
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 descriptive statistics of fiber diameter traits of ARUM goat breeds
| Trais (character) | Mean value of | Standard deviation of | Range |
| (average fiber diameter) MFD/. Mu.m | 15.69 | 1.19 | 11.70 to 19.40 |
| (average fiber diameter Standard deviation) FDSD/. Mu.m | 3.26 | 0.25 | 2.40 to 4.00 |
| (average fiber diameter variation coefficient) CVFD/% | 20.86 | 1.44 | 16.90 to 25.20 |
TABLE 2 KRTAP24-1 Gene mutation site information Table
TABLE 3 essential characteristics of 6 mutations of the KRTAP24-1 gene
TABLE 4 analysis of the correlation between mutation sites and Cashmere fiber diameters
Note that: 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.
TABLE S1 information on multiplex PCR amplification primers
TABLE S2 first round PCR reaction System
| System composition | volume/μL |
| DNA template (10 ng/. Mu.l) | 2.0 |
| Upstream primer pool (10 mu M) | 1.0 |
| Downstream primer pool (10. Mu.M) | 1.0 |
| 2×PCR Ready Mix | 15 |
| ddH 2 O | 6 |
| Total volume of | 25 |
Table S3 first round procedure in PCR reactions
Table S4 second round PCR reaction System
| System components System composition | Volume/. Mu.L |
| First round PCR products | 2.0 |
| Universal P7 primer (containing molecular tag, 10. Mu.M) | 1.0 |
| General P5 primer (10. Mu.M) | 1.0 |
| 2×PCR Ready Mix | 15 |
| ddH 2 O | 11 |
| Total volume of | 30 |
Table S5 second round procedure in PCR reactions
Sequence listing
<110> Xinjiang livestock research institute and Erdos market science institute of agriculture and animal husbandry (Erdos division of inner Mongolia academy of agriculture and animal husbandry)
<120> molecular marker affecting fiber diameter properties of ARUM cashmere goat, specific primer pair and application thereof
<130>
<160>2
<170>PatentIn 3.5
<210> 1
<211>25
<212> mRNA
<213> artificial sequence
<400> 1
tggtgtgaca tagaccaaag agcta 25
<210> 2
<211>25
<212> mRNA
<213> artificial sequence
<400> 1
ctcttggata agcagttaag ttgcc 25
Claims (2)
1. The application of the molecular marker affecting the diameter of the cashmere fiber of the Jiangnan cashmere goat in the selection of the diameter property of the cashmere fiber of the Jiangnan cashmere goat is characterized in that the molecular marker affecting the diameter property of the cashmere fiber of the Jiangnan cashmere goat is positioned on the KRAP 24-1 gene of a chromosome 1 of the Jiangnan cashmere goat, the KRAP 24-1 gene is positioned at 4033765bp-4034298bp of a nucleotide sequence of the chromosome 1, the molecular marker is positioned at 4034201bp of the nucleotide sequence of the chromosome 1, and a mutant base is C or T; for the cashmere fiber diameter property, in the genotypes corresponding to the molecular markers, the Jiangnan cashmere goats of the CT genotypes belong to dominant individuals with smaller cashmere fiber diameter property, and the Jiangnan cashmere goats of the CC genotypes belong to individuals with larger cashmere fiber diameter property.
2. The use of molecular markers affecting the diameter of the cashmere fiber of Jiangnan cashmere goats according to claim 1 in the selection of the characteristics of the diameter of the cashmere fiber of Jiangnan cashmere goats, comprising the steps of: firstly, taking genomic DNA of a to-be-detected Jiangnan cashmere goat; the second step, taking genome DNA as a template, and carrying out first round of amplification by utilizing a specific primer pair, wherein a first round of PCR system is a reaction system of 25 mu l, and the first round of PCR system consists of the following components:
the first round of PCR amplification conditions were: pre-denaturation at 98 degrees for 3 min, followed by 8 cycles of 94 degrees for 30 seconds, 50 degrees for 30 seconds, 72 degrees for 30 seconds; immediately after 25 cycles, the conditions were 98 degree denaturation for 30 seconds, 66 degree annealing for 30 seconds, 72 degree extension for 30 seconds; finally, the extension is carried out at 72 ℃ for 5 minutes, after the PCR reaction is finished, the temperature is kept at 4 ℃,
upstream primer pool: 5'-tggtgtgacatagaccaaagagcta-3';
downstream primer pool: 5'-ctcttggataagcagttaagttgcc-3';
third, the first round of PCR products are used as templates to execute the second round of PCR reaction, the second round of PCR system is a reaction system of 30 mul, and the second round of PCR system consists of the following components:
the second round of PCR amplification conditions were: the method comprises the steps of pre-denaturing at 98 ℃ for 5 minutes, then performing 5 cycles under the conditions of denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 20 seconds, extension at 72 ℃ for 30 seconds, and finally extension at 72 ℃ for 5 minutes, preserving heat at 4 ℃ after the PCR reaction is finished, mixing all PCR products in equal amounts, sequencing, and genotyping according to the sequencing result.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009011602A1 (en) * | 2007-07-13 | 2009-01-22 | Ovita Limited | Ovine identification method |
| CN110373474A (en) * | 2019-07-03 | 2019-10-25 | 甘肃农业大学 | The relevant genetic marker of Gansu Province east Cashmere Goat diameter and its application |
| CN111549143A (en) * | 2020-02-21 | 2020-08-18 | 甘肃农业大学 | Genetic marker related to diameter of cashmere fiber of goat and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009011602A1 (en) * | 2007-07-13 | 2009-01-22 | Ovita Limited | Ovine identification method |
| CN110373474A (en) * | 2019-07-03 | 2019-10-25 | 甘肃农业大学 | The relevant genetic marker of Gansu Province east Cashmere Goat diameter and its application |
| CN111549143A (en) * | 2020-02-21 | 2020-08-18 | 甘肃农业大学 | Genetic marker related to diameter of cashmere fiber of goat and application thereof |
Non-Patent Citations (1)
| Title |
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| Variation in the Caprine KAP24-1 Gene Affects Cashmere Fibre Diameter;Jiqing Wang 等;Animals;第9卷(第15期);1-12 * |
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