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CN-122012742-A - Transposon insertion molecular marker related to beach wool strand bending proportion and application thereof

CN122012742ACN 122012742 ACN122012742 ACN 122012742ACN-122012742-A

Abstract

The application relates to the technical field of animal molecular breeding and genetic engineering, and provides a transposon insertion molecular marker related to the bending proportion of beach wool strands and application thereof. The application identifies 2917bp transposon insertion extremely obviously related to wool bending proportion on the chromosome 13 of the beach sheep for the first time and establishes a corresponding PCR-gel electrophoresis typing method, and the molecular marker has extremely strong relevance with phenotype, so that early and accurate prediction of the beach wool strand bending proportion can be realized. The marker is used for auxiliary selection, so that the breeding period can be obviously shortened, the breeding cost can be reduced, the genetic improvement of the quality of the beach sheep fur can be accelerated, and the method has great economic and social benefits.

Inventors

  • WANG JIANKUI
  • DENG XUEMEI
  • YAO JIAJIE
  • MA QING
  • LIU JUEYING
  • WANG YIXIAN
  • ZHAO ZHENGWEI
  • MA LINA

Assignees

  • 中国农业大学
  • 宁夏农林科学院动物科学研究所(宁夏草畜工程技术研究中心)

Dates

Publication Date
20260512
Application Date
20260319

Claims (10)

  1. 1. A transposon insertion molecular marker associated with the bending proportion of beach wool strands, characterized in that the nucleotide sequence of the transposon insertion molecular marker is SEQ ID no.
  2. 2. A method for identifying the bending ratio of beach wool strands using the transposon insertion molecular markers of claim 1.
  3. 3. The method according to claim 2, wherein the method comprises the step of amplifying the sample of the beach sheep DNA to be detected by using a primer group consisting of a primer F1, a primer R1 and a primer R2, wherein the nucleotide sequence of the primer F1 is SEQ ID NO.4, the nucleotide sequence of the primer R1 is SEQ ID NO.5 and the nucleotide sequence of the primer R2 is SEQ ID NO.6.
  4. 4. The method of claim 2, further comprising detecting the PCR amplification products by agarose gel electrophoresis, determining the genotype of the body of the beach sheep DNA sample based on the band size and determining the beach sheep strand bending ratio thereof: only 528bp bands appear as non-inserted homozygotes; Two bands of 528bp and 288bp appear as heterozygotes at the same time; Only 288bp bands appear as insert homozygotes; the bending ratio of the hairs of the DNA sample body inserted into the homozygous beach sheep is higher than that of the DNA sample body inserted into the heterozygote beach sheep, and the bending ratio of the hairs of the DNA sample body inserted into the heterozygote beach sheep is higher than that of the DNA sample body not inserted into the homozygous beach sheep.
  5. 5. The method according to claim 2-4, wherein the PCR reaction system in the PCR amplification is 20. Mu.L, and the PCR reaction system comprises 2X Taq PCR MasterMix 10.0.0. Mu.L, 50-100 ng template DNA, 0.4. Mu.M each of primer F1, primer R1 and primer R2, and double distilled water to make up to 20. Mu.L.
  6. 6. The method according to any one of claims 2 to 4, wherein the PCR reaction is performed by pre-denaturing at 95℃for 5 minutes, then performing 35 cycles each comprising 95℃for 30 seconds, 60℃for 30 seconds, 72℃for 30 seconds, and finally extending at 72℃for 5 minutes.
  7. 7. Use of a transposon insertion molecular marker according to any one of claims 2-6 in the breeding of sheep, for breeding sheep individuals with a high wool curvature ratio, wherein the genotype is selected for the breeding of sheep individuals inserted with homozygotes or heterozygotes.
  8. 8. The kit for identifying the bending ratio of the beach and wool strands is characterized by comprising a primer F1, a primer R1 and a primer R2, wherein the nucleotide sequence of the primer F1 is SEQ ID NO., the nucleotide sequence of the primer R1 is SEQ ID NO., and the nucleotide sequence of the primer R2 is SEQ ID NO.
  9. 9. The kit of claim 8, comprising one or more components selected from DNA polymerase, magnesium ions, dntps, buffers.
  10. 10. The kit of claim 8 or 9, further comprising a DNA extraction reagent.

Description

Transposon insertion molecular marker related to beach wool strand bending proportion and application thereof Technical Field The application relates to the technical field of animal molecular breeding and genetic engineering, and in particular provides a transposon insertion molecular marker related to the bending proportion of beach wool strands and application thereof. Background The beach sheep is a unique sheep variety for fur in China, and the core germplasm characteristics of the beach sheep are represented by a unique 'two Mao Qiupi' phenotype (indexes such as bending proportion, bending number and the like of wool strands are included in GB/T2033-2008 national standard) of the lambs at 30-40 days old. At present, the breeding of the Tan sheep mainly depends on traditional phenotype selection, namely, the breeding is carried out according to the quality of the fur of the adult sheep. The method has the inherent defects of long period (the sheep needs to be kept for adults), high cost, low efficiency, easy influence of environmental factors on accuracy and the like, and severely restricts the improvement process of the sheep germplasm resources. With the development of molecular biology, molecular Marker Assisted Selection (MAS) provides a new solution for animal breeding. Structural Variation (SV), particularly transposon insertion, is an important type of genetic variation in the genome that can be involved in the formation of important economic traits by affecting genetic structure or expression regulation. If the transposon inserted molecular marker closely linked with the bending proportion of the beach wool strands can be discovered, and a rapid and low-cost detection method is established, accurate breeding can be realized in the young or even embryo period of the beach sheep, the generation interval is greatly shortened, and the genetic progress is accelerated. At present, no report related to the relation of transposon insertion in specific region of the Tan sheep chromosome 13 and the wool bending proportion is found. Therefore, the development of the transposon insertion molecular marker related to the bending proportion of the beach wool strands has important significance for realizing molecular breeding of beach sheep and improving the industrial benefit of fur. Disclosure of Invention In one aspect, the present application provides a transposon insertion molecular marker related to the bending proportion of beach wool strands, the transposon insertion molecular marker having the nucleotide sequence of SEQ ID no. In another aspect, the present application provides a method for identifying the bending ratio of beach wool strands using the above transposon insertion molecular markers. Further, the method comprises the step of amplifying the to-be-detected sheep DNA sample by using a primer group consisting of the primer F1, the primer R1 and the primer R2. The nucleotide sequence of the primer F1 is SEQ ID NO.4, the nucleotide sequence of the primer R1 is SEQ ID NO.5, and the nucleotide sequence of the primer R2 is SEQ ID NO.6. The sheep DNA sample may be extracted from the tissues and organs of sheep, including but not limited to skin, blood, hair follicle, excrement, etc., and the extraction method may be selected from methods known in the art or commercial kits. Further, the method comprises detecting PCR amplification products by agarose gel electrophoresis, determining the genotype of the sheep DNA sample body according to the size of the band and determining the bending ratio of the sheep DNA sample body: only 528bp bands appear as non-inserted homozygotes; Two bands of 528bp and 288bp appear as heterozygotes at the same time; Only 288bp bands appear as insert homozygotes; the bending ratio of the hairs of the DNA sample body inserted into the homozygous beach sheep is higher than that of the DNA sample body inserted into the heterozygote beach sheep, and the bending ratio of the hairs of the DNA sample body inserted into the heterozygote beach sheep is higher than that of the DNA sample body not inserted into the homozygous beach sheep. Further, the PCR reaction system in the PCR amplification is 20 mu L, and comprises 2X Taq PCR MasterMix 10.0.0 mu L, 50-100 ng template DNA, 0.4 mu M each of the primer F1, the primer R1 and the primer R2, and double distilled water to make up to 20 mu L. Further, the PCR reaction procedure in the PCR amplification was 95℃for 5 minutes, followed by 35 cycles each including 95℃for 30 seconds, 60℃for 30 seconds, 72℃for 30 seconds, and finally 72℃for 5 minutes. On the other hand, the application provides the application of the method or the molecular marker in the breeding of the sheep, wherein the application is used for breeding sheep individuals with high wool bending ratio, and the genotype is selected from the sheep individuals inserted with homozygotes or heterozygotes for breeding. In another aspect, the application provides a kit for identifyin