CN114350816B - Molecular marker related to cashmere fiber diameter and variation coefficient of ARUM cashmere goat, specific primer pair and application - Google Patents
Molecular marker related to cashmere fiber diameter and variation coefficient of ARUM cashmere goat, specific primer pair and application Download PDFInfo
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Abstract
The invention relates to the technical field of animal molecular markers, in particular to a molecular marker and a specific primer pair related to the diameter and the variation coefficient of the cashmere fiber of a Jiangnan cashmere goat, wherein the molecular marker related to the diameter and the variation coefficient of the fiber diameter of the cashmere fiber of the Jiangnan cashmere goat is positioned on a KRAP 27-1 gene of a 1 st chromosome of the Jiangnan cashmere goat, the KRAP 27-1 gene is positioned at 3968645bp-3969193bp of a 1 st chromosome nucleotide sequence, the molecular marker is positioned at 3968799bp of the 1 st chromosome nucleotide sequence, and mutation bases are A or G. The molecular marker can realize early seed selection of the cashmere fiber diameter and fiber diameter variation coefficient character good sheep, and can be selected and remained after birth, thereby reducing the breeding cost, accelerating the breeding process of high-quality superfine cashmere goats and providing theoretical basis and guidance for genetic improvement of Jiang Nan cashmere goats.
Description
Technical Field
The invention relates to the technical field of animal molecular markers, in particular to a molecular marker related to the fiber diameter and the variation coefficient character of ARUM cashmere goat cashmere and a specific primer pair thereof, wherein the variation coefficient refers to the fiber diameter variation coefficient, the application of the specific primer pair in preparing a reagent or a kit for detecting the molecular marker related to the fiber diameter and the fiber diameter variation coefficient character of ARUM cashmere in vitro, and the application of the molecular marker in the selection of the fiber diameter and the fiber diameter variation coefficient character of ARUM cashmere goat cashmere.
Background
Along with the increasing living standard of people, more and more people pay attention to not only the style and comfort of clothes, but also the ecology and health care. The cashmere product integrating gorgeous, noble, light, thin, soft, comfortable and health care is certainly favored by more people.
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 price of cashmere is affected by the luster and fineness of the cashmere, and the finer the diameter of the fiber, the higher the price in the same-color system cashmere. Age, sex and feeding management can all influence the diameter of cashmere fibers. But in addition to these factors, inheritance is the root cause of the fineness of cashmere. 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 and popularization of new varieties of the down producing goats become an important way for the people to stand on the local resources to become lean and rich. 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 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. In the hair cortex, the hair keratin intermediate filaments are embedded in an interfilament matrix consisting of the hair keratin-related protein (KRTAP), which is cross-linked by its extensive disulfide bonds with the abundant cysteine residues in hair keratin, and is essential for the formation of a stiff and resistant hair shaft. KRAP 13, KRAP 15, KRAP 24, and KRAP 27 belong to the HS family of high sulfophurs (HS;. Ltoreq.30 mol% cysteine). Since HS protein is expressed first in hair, most scholars have focused on HS protein. Studies have shown that amino acid changes may affect the loss or absence of phosphorylation sites of proteins during post-translational modification, resulting in changes in the net charge of the protein.
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 KRTAP27-1 gene polymorphism of the ARUM 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 related to the diameter and the variation coefficient of cashmere fibers of ARUM cashmere goats, a specific primer pair and application, and discloses a molecular marker related to the characteristics of the diameter and the variation coefficient of the fiber diameters of cashmere fibers for the first time.
One of the technical schemes of the invention is realized by the following measures: a molecular marker (SNP 19 locus of the invention) related to the fiber diameter and fiber diameter variation coefficient characteristics of ARUM Nannochlori goat is positioned on KRAP 27-1 gene of ARUM Nannochlori goat chromosome 1, the KRAP 27-1 gene is positioned at 3968645bp-3969193bp of the nucleotide sequence of ARUM Nannochlori goat chromosome 1, the molecular marker is positioned at 3968799bp of the nucleotide sequence of ARUM Nannochlori, and the mutation base is A or G.
The second technical scheme of the invention is realized by the following measures: a specific primer pair of molecular markers related to the characteristics of fiber diameter and fiber diameter variation coefficient of the cashmere of the Jiangnan cashmere goat, comprising an upstream primer and a downstream primer;
upstream primer F (shown as SEQ ID No. 1 of the sequence Listing): 5'-agtggcgtatcctacagaactcact-3';
downstream primer R (shown as SEQ ID No. 2 of the sequence Listing): 5'-cgctactcaggctcctcctg-3'.
The third technical scheme of the invention is realized by the following measures: the second technical proposal is the application of the specific primer pair in preparing a reagent or a kit for detecting in vitro molecular markers related to the fiber diameter and the fiber diameter variation coefficient property of the ARUM cashmere goat.
The fourth technical scheme of the invention is realized by the following measures: the application of the molecular marker in the selection of the fiber diameter and the fiber diameter variation coefficient characteristics of the ARUM cashmere goat.
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 PCR reaction is carried out for 5 minutes at 72 ℃, the temperature is kept at 4 ℃ after the PCR reaction is finished, 1% agarose gel electrophoresis is used for detecting the PCR product, the size of the product is determined to be correct, and the AMPure XP magnetic beads are used for purifying and recycling the PCR product;
upstream primer pool: 5'-AGTGGCGTATCCTACAGAACTCACT-3';
downstream primer pool: 5'-CGCTACTCAGGCTCCTCCTG-3';
third, performing a second round of PCR reaction by taking the first round of PCR products as templates to obtain a library with molecular tags in sequence;
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 based on the sequencing results.
For the cashmere fiber diameter property, in genotypes corresponding to the molecular markers (SNP 19 loci), the Jiangnan cashmere goats of the genotypes (GG or GA) containing G genes belong to dominant individuals with finer cashmere fiber diameter property; for the characteristic of the diameter variation coefficient of the cashmere fiber, in the genotypes corresponding to the molecular markers (SNP 19 loci), the AA genotype ARUM cashmere goat belongs to a dominant individual with smaller characteristic of the diameter variation coefficient of the cashmere fiber.
In the invention, multiple PCR (two rounds of PCR) and high-throughput sequencing are utilized to identify mutation sites of the KRAP 27-1 gene exon region of the ARUM Nannochlori goat; 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 research on germplasm characteristics of local cashmere goats in China and resource protection and utilization.
The invention discloses a method for applying a 3968799bp locus of a No. 1 chromosome nucleotide sequence to selection of the fiber diameter and the fiber diameter variation coefficient property of the ARUM cashmere goat for the first time, wherein a 3968799bp locus of the No. 1 chromosome nucleotide sequence is used as a molecular marker related to the fiber diameter and the fiber diameter variation coefficient property of the ARUM cashmere goat for the first time; when the molecular markers related to the fiber diameter and the fiber diameter variation coefficient characteristics of the ARUM cashmere goat are adopted to select the fiber diameter and the fiber diameter variation coefficient characteristics of the ARUM cashmere goat, the molecular marker detection layer is used for screening, 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 high-quality sheep with excellent properties of fiber diameter and fiber diameter variation coefficient of the ARUM cashmere can be realized, and the sheep can be selected and remained after birth, so that the breeding cost is reduced, the breeding process of the high-quality superfine cashmere goats is accelerated, and theoretical basis and guidance are provided for the genetic improvement of ARUM cashmere goats.
Drawings
FIG. 1 is an electrophoresis detection chart of genomic DNA of ARUM Nannochlori goat.
FIG. 2 shows the result of KRAP 27-1 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 19 locus) related to the fiber diameter and fiber diameter variation coefficient characteristics of the cashmere of the Jiangnan cashmere goat is positioned on the KRAP 27-1 gene of a chromosome 1 of the Jiangnan cashmere goat, the KRAP 27-1 gene is positioned at 3968645bp-3969193bp of the nucleotide sequence of the chromosome 1, the molecular marker is positioned at 3968799bp of the nucleotide sequence of the chromosome 1, and the mutation base is A or G.
Example 2: specific primer pairs for amplifying the molecular markers related to the fiber diameter and fiber diameter variation coefficient characteristics of the ARUM cashmere goat cashmere of the embodiment 1, comprising an upstream primer and a downstream primer;
upstream primer F (shown as SEQ ID No. 1 of the sequence Listing): 5'-agtggcgtatcctacagaactcact-3';
downstream primer R (shown as SEQ ID No. 2 of the sequence Listing): 5'-cgctactcaggctcctcctg-3'.
Example 3: application of the specific primer pair described in example 2 in preparing a reagent or a kit for in vitro detection of molecular markers related to the fiber diameter and fiber diameter variation coefficient characteristics of ARUM cashmere goats.
Example 4: the application of the molecular marker in the selection of the fiber diameter and the fiber diameter variation coefficient character of cashmere of the embodiment 1 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 PCR reaction is carried out for 5 minutes at 72 ℃, the temperature is kept at 4 ℃ after the PCR reaction is finished, 1% agarose gel electrophoresis is used for detecting the PCR product, the size of the product is determined to be correct, and the AMPure XP magnetic beads are used for purifying and recycling the PCR product;
upstream primer pool: 5'-AGTGGCGTATCCTACAGAACTCACT-3';
downstream primer pool: 5'-CGCTACTCAGGCTCCTCCTG-3';
third, performing a second round of PCR reaction by taking the first round of PCR products as templates to obtain a library with molecular tags in sequence;
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 based on the sequencing results.
For the cashmere fiber diameter property, in genotypes corresponding to the molecular markers (SNP 19 loci), the Jiangnan cashmere goats of the genotypes (GG or GA) containing G genes belong to dominant individuals with finer cashmere fiber diameter property; for the characteristic of the diameter variation coefficient of the cashmere fiber, in the genotypes corresponding to the molecular markers (SNP 19 loci), the AA genotype ARUM cashmere goat belongs to a dominant individual with smaller characteristic of the diameter variation coefficient of the cashmere fiber.
The following is the experimental study of the molecular marker related to the fiber diameter and the fiber diameter variation coefficient 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 exon region of the KRAP 27-1 gene was selected based on 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 is 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 results and analysis
2.1 Cashmere fiber diameter Property descriptive statistics
The characteristics of average fiber diameter (MFD), standard deviation of fiber diameter (FDSD) and Coefficient of Variation of Fiber Diameter (CVFD) of the Jiangnan cashmere goats were respectively subjected to descriptive statistical analysis, and basic statistics are shown in Table 1.
As shown in Table 1, the average fiber diameter of the ARUM cashmere goat cashmere is 15.69 μm, the standard deviation of the average fiber diameter is 3.26, and the variation coefficient of the average fiber diameter is 20.86.
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, 7 mutation sites were obtained in the KRAP 27-1 gene of ARUM Nannochlori goat, as shown in Table 2.
The result of the KRTAP27-1 gene mutation site heterozygous first generation sequencing is shown in figure 2, and the high-throughput typing result is consistent with the first generation sequencing result, so that the high-throughput sequencing result is reliable.
2.4 population genetic polymorphism analysis
The results of the analysis of the frequency of 7 mutation sites genotyping and the hypo-allele of the KRAP 27-1 gene of the down producing goat are shown in Table 3.
As can be seen from Table 3, the minor allele frequencies of SNP16, SNP17 and SNP18 in the ARUM Nannochlori goat population are all less than 0.03; SNP17, SNP18. Wherein SNP18 is not typed in the ARUM Nannocher goat population.
2.5 SNP 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, SNP19 and SNP22 significantly affect the cashmere fiber diameter (P < 0.01), SNP21 significantly affects the cashmere fiber diameter (P < 0.05), SNP21 significantly affects the fiber diameter standard deviation (P < 0.05), and SNP19 significantly affects the fiber diameter variation coefficient (P < 0.05).
Therefore, the SNP19 has a very significant influence on the cashmere fiber diameter and a significant influence on the fiber diameter variation coefficient, so that the SNP19 is related to the cashmere fiber diameter property and the fiber diameter variation coefficient property, and the SNP19 can be used as a molecular marker related to the cashmere fiber diameter and the fiber diameter variation coefficient property of the Jiang Nannocheir goat.
As can be seen from the gene types in table 4, in the genotypes corresponding to the SNP19 loci, the Jiangnan cashmere goats containing the genotypes of the G genes (GG or GA) belong to dominant individuals with finer cashmere fiber diameter traits; for the cashmere fiber diameter variation coefficient character, in genotypes corresponding to SNP19 loci, AA genotype ARUM cashmere goats belong to dominant individuals with smaller cashmere fiber diameter variation coefficient character.
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)/μm | 3.26 | 0.25 | 2.40 to 4.00 |
| Average fiber diameter coefficient of variation) CVFD/% | 20.86 | 1.44 | 16.90 to 25.20 |
TABLE 2 KTAP 27-1 Gene mutation site information Table
TABLE 3 essential characteristics of 7 mutations of the KRTAP27-1 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 |
| ddH2O | 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 |
| ddH2O | 11 |
| Total volume of | 30 |
Table S5 second round procedure in PCR reactions
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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 related to diameter and variation coefficient of cashmere fiber of Jiangnan cashmere goat, specific primer pair and application
<130>
<160>2
<170>PatentIn 3.5
<210> 1
<211>25
<212> mRNA
<213> artificial sequence
<400> 1
agtggcgtat cctacagaac tcact 25
<210> 2
<211>20
<212> mRNA
<213> artificial sequence
<400> 1
cgctactcag gctcctcctg 20
Claims (2)
1. The application of a molecular marker in the selection of the fiber diameter and fiber diameter variation coefficient characteristics of the cashmere of the ARUM cashmere goat is characterized in that the molecular marker is positioned on the KRTAP27-1 gene of the ARUM cashmere goat chromosome 1, wherein the accession number of the chromosome 1 in the NCBI database is NC_030808.1, the KRTAP27-1 gene is positioned at 3968645bp-3969193bp of the nucleotide sequence of the chromosome 1, the molecular marker is positioned at 3968799bp of the nucleotide sequence of the chromosome 1, and the mutation base is A or G; for the diameter property of the cashmere fiber, in the genotypes corresponding to the molecular markers, the Jiangnan cashmere goats containing G genes GG or GA genotypes belong to dominant individuals with finer diameter property of the cashmere fiber; and for the characteristic of the diameter variation coefficient of the cashmere fiber, in genotypes corresponding to the molecular markers, the AA genotype ARUM cashmere goat belongs to a dominant individual with smaller characteristic of the diameter variation coefficient of the cashmere fiber.
2. The use of molecular markers according to claim 1 in selection of fiber diameter and fiber diameter coefficient of variation characteristics of ARUM cashmere goat, characterized by 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 PCR reaction is carried out for 5 minutes at 72 ℃, and the temperature is kept at 4 DEG after the completion of the PCR reaction
Upstream primer pool: 5'-agtggcgtat cctacagaac tcact-3';
downstream primer pool: 5'-cgctactcag gctcctcctg-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:
10 ng/. Mu.l DNA template 2. Mu.l
10 mu M Universal P7 primer 1. Mu.l
10 mu M Universal P5 primer 1. Mu.l
2×PCR Ready Mix15μl
ddH 2 O11μl
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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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| cuiling wu等.correlation analysis of four krtap gene polymorphisms and cashmere fiber diameters in two cashmere goat breeds.canadian journal of animal science.2022,第561-570页. * |
| Mengli Zhao等.Variation in the Caprine Keratin-Associated Protein 27-1 Gene is Associated with Cashmere Fiber Diameter.Genes (Basel).2020,第11卷(第8期),第1-11页. * |
| 车陇杰 ; 王继卿 ; Huitong Zhou ; 李涛 ; 赵孟丽 ; 胡江 ; 刘秀 ; 李少斌 ; 罗玉柱 ; .山羊KRTAP26-1基因鉴定及其遗传特征研究.基因组学与应用生物学.2018,(02),第239-246页. * |
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