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Screening of SNP (Single Nucleotide Polymorphism) related to sheep wool traits and application

A sheep wool, sheep technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of small scope, high accuracy, high-throughput screening methods are not widely developed, etc. The effect of speeding up the breeding process, reducing the degree of dependence and reducing the cost of conventional breeding

Active Publication Date: 2016-08-24
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitation of detection cost, high-throughput screening methods have not been widely developed
At present, the mainstream SNP screening technology is still based on traditional technologies such as PCR-SSCP, PCR-RFLP, and HRM. The common feature is that a specific small region of the genome, or even a single SNP site, is the detection target. The range is small but the accuracy is high.

Method used

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  • Screening of SNP (Single Nucleotide Polymorphism) related to sheep wool traits and application
  • Screening of SNP (Single Nucleotide Polymorphism) related to sheep wool traits and application
  • Screening of SNP (Single Nucleotide Polymorphism) related to sheep wool traits and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Skin tissue DNA extraction

[0018] (1) Fully shred 0.3 g sheep skin tissue sample with surgical scissors, place it in a 1.5 mL centrifuge tube, and add 500 μL tissue DNA extraction solution.

[0019] (2) Add RNase solution to the above centrifuge tube to a final concentration of 20 μg / mL, mix thoroughly, and digest in a 37°C water bath for 1 hour.

[0020] (3) Add proteinase K to the above centrifuge tube to a final concentration of 150 μg / mL, mix well, and digest in a water bath shaker at 55°C for 12-24 hours.

[0021] (4) Add an equal volume of Tris saturated phenol into the centrifuge tube, and slowly invert up and down until fully mixed. Centrifuge at 1200 rpm for 10 min at 4°C, then transfer the supernatant to a new centrifuge tube, and repeat step (4) once.

[0022] (5) Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), mix well, and centrifuge at 1200 rpm for 10 min at 4°C.

[0023] (6) Transfer the supernatant to a new centrifuge...

Embodiment 2

[0028] Example 2 Genome PCR amplification

[0029] At present, there is no genome sequence information of sheep KAP13.1, and considering that the KAP family genes are single-exon genes, PCR-SSCP primers were designed using the predicted sequence of sheep KAP13-1-like mRNA (XM_004002776.3) as a template, primer information See the table below.

[0030] Table 1. 3 pairs of KAP13.1 gene-specific primer sequences, annealing temperature and target fragment length

[0031]

[0032] Add to PCR tube:

[0033]

[0034]

[0035] Setting of PCR amplification temperature:

[0036] Pre-denaturation at 95°C for 3 minutes

[0037] 35 cycles: Denaturation at 94°C, 30sec

[0038] See Table 1 for annealing temperature, 30sec

[0039] Extend 72℃, 30sec

[0040] Extend 72℃, 10min

[0041] Cool down to 12°C for storage

[0042] The results of PCR amplification were detected by 2% agarose gel electrophoresis. Electrophoresis detection showed that the KAP13.1 gene 2 primer PCR prod...

Embodiment 3

[0043] Embodiment 3 PCR product SSCP analysis

[0044] (1) Clean the glass plate with detergent, rinse with distilled water repeatedly and dry it for later use.

[0045] (2) Put the glass plate into the plastic frame, seal it with 2.5% agarose gel, prepare 12% polyacrylamide gel according to the standard formula, mix it well and pour it into the glass plate, insert the tooth comb and let it rest at room temperature for more than 30 minutes to make the gel The glue is fully solidified.

[0046] (3) After confirming that the gel is fully solidified, carefully pull out the tooth comb, wash the sampling hole with distilled water, dry it with absorbent paper, put the glass plate on the electrophoresis tank, add an appropriate amount of 1×TBE electrophoresis solution, and confirm that there is no leakage. Afterwards, 300V, 60mA pre-electrophoresis for about 10min.

[0047] (4) Take 2 μL of PCR product, add 8 μL of denaturing buffer, centrifuge briefly and then denature at 98°C for...

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Abstract

The invention discloses screening of SNP (Single Nucleotide Polymorphism) related to sheep wool traits and an application and belongs to the technical field of livestock marker assisted selection seedling. The sheep wool traits are analyzed and predicted with the SNP markers, and the total DNA of different sheep individuals is subjected to PCR (polymerase chain reaction) amplification with primers shown in SEQ ID NO: 2 and 3 or primers shown in SEQ ID NO: 4 and 5; products of the PCR amplification are subjected to SNP detection, bases in the 336th position, the 513rd position and the 572nd position from the 5' end are determined to be C or T, and the wool tensile length of the individuals containing CC and CT genes formed through mutation in the 336th position is remarkably increased; the wool yield and tensile length of the N haplotype homozygous type NN or heterozygous type NM individuals formed by TT in the 513rd position and the 572nd position are remarkably larger than those of the M haplotype homozygous type MM individuals. The SNP markers can be adopted as diagnostic markers for early breeding of superfine fine-wool sheep and can shorten the variety breeding period, reduce the degree of dependence on progeny testing, reduce the conventional breeding cost and accelerate the breeding process.

Description

technical field [0001] The invention belongs to the technical field of marker assisted selection (MAS) breeding of livestock, and specifically relates to the screening and application of the functional SNP of the functional gene keratin-associated protein 13.1 (keratin-associated protein 13.1, KAP13.1) gene for sheep wool traits . Background technique [0002] Wool is one of the main products of sheep farming. The quality of wool is the determinant of its economic value. The improvement of wool performance is an important goal of sheep breeding. In addition to being affected by factors such as environment and nutrition, the traits of wool are mainly determined by genetic factors. Keratin (KRT) and Keratin-associated protein (KAP), the main structural proteins of hair fiber, are the major genes that affect hair fiber diameter (WFD), hair fiber diameter coefficient of variation (CVFD) and hair length ( SL) and other traits. So far, studies have found that mammals contain a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张立春孙福亮张明新金海国朴庆林曹阳于永生
Owner JILIN ACAD OF AGRI SCI
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