A method for identifying the parentage relationship of single-parent buffaloes and its primers and kit

A technology of paternity relationship and identification sheet, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of group inbreeding, population degradation, etc., and achieve high accuracy, low sample requirements, and reliable test results Effect

Inactive Publication Date: 2012-02-22
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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Problems solved by technology

Errors caused by pedigree information errors will be magnified step by step throughout the breeding process, and it is very likely to cause large-scale inbreeding of the population, resulting in population degradation, resulting in irreparable losses in the breeding process
However, there is no report on the method of identifying the paternity relationship of single parents of Chinese buffaloes using microsatellite markers

Method used

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  • A method for identifying the parentage relationship of single-parent buffaloes and its primers and kit
  • A method for identifying the parentage relationship of single-parent buffaloes and its primers and kit
  • A method for identifying the parentage relationship of single-parent buffaloes and its primers and kit

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Embodiment 1

[0051] Example 1. Screening of microsatellite markers and mitochondrial primers for paternity identification of Chinese buffaloes

[0052] 1. Primer design

[0053] Referring to GenBank, after repeated screening tests, polymorphic-rich microsatellite primers were screened from the common bovine microsatellite markers recommended by the Roslin Institute in the United Kingdom. The sequences of the screened microsatellite primers are shown in Table 1.

[0054] The primers were designed according to the D-Loop region of common bovine mtDNA, and the internal primers were designed for the convenience of sequencing. The primer sequences are shown in Table 2. The primers were synthesized by TaKaRabao Bioengineering (Dalian) Co., Ltd. The length of the amplified fragment is expected to be about 900bp.

[0055] Table 1 Selected STR loci 8 microsatellite loci and their primer sequences

[0056]

[0057] The sequence of the primers in the D-Loop region of mtDNA selected in Table 2

...

Embodiment 2

[0107] Embodiment 2, carry out paternity identification to Chinese buffalo with microsatellite marker

[0108] The method for identifying the paternity relationship of single-parent buffalo in this embodiment comprises the following steps: screen polymorphic-rich microsatellite primers from common bovine microsatellite markers; Under the guidance of microsatellite primers, the blood samples of a cow (m), a Chinese buffalo known to be the offspring c1 of the cow and another suspected c2 of the cow's offspring were collected from a farmer's house in Xichang, Sichuan, China Carry out PCR amplification; conduct 8% polyacrylamide gel electrophoresis detection on microsatellite PCR amplification fragments; judge the genotype of each individual according to the electrophoresis detection results, and calculate the uniparental parentage index PI value and parent-child relative probability value according to the genotype W value, according to the W value to judge whether the parental re...

Embodiment 3

[0127] Example 3 Using microsatellite markers and mitochondrial genetic markers to carry out paternity identification on Chinese buffaloes

[0128] The microsatellite marker paternity testing method is the same as in Example 2.

[0129] The paternity identification of Chinese buffalo by mitochondrial genetic markers includes the following steps: design primers according to the D-Loop region of common bovine mtDNA, and the primer sequences are shown in Table 2; A farmer in Xichang, Sichuan, China collected blood samples from a cow, a buffalo known to be the offspring of the cow, and another buffalo suspected to be the offspring of the cow, and performed PCR amplification in a 50 μL reaction system, and the PCR amplification fragment of mitochondria Agarose gel purification was carried out, and the purified PCR products were sequenced by the dideoxy termination method, and the sequencing results were analyzed and judged by DNAman software.

[0130] 1. PCR amplification

[0131...

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Abstract

The invention discloses a method for identifying the parentage relationship of single-parent buffaloes, primers and a kit thereof, and belongs to the technical field of genetic engineering. The present invention screens out 8 microsatellite primers with rich polymorphisms, performs PCR amplification with the buffalo genome DNA to be tested as a template, performs polyacrylamide gel electrophoresis detection on the PCR amplified fragments, and judges each individual's Genotype: Calculate the single-parent parentage index PI value and parent-child relative probability value W value according to the genotype, and judge whether the parentage relationship exists according to the W value. In order to further improve the stability, accuracy and sensitivity of the identification, the present invention also designs primers according to the D-Loop region of common bovine mtDNA to carry out mitochondrial genetic identification. The invention combines two methods of microsatellite and mitochondrial genetic markers to identify from different angles, and the identification results are consistent, which improves the stability, accuracy and sensitivity of paternity identification, and makes the identification range wider and the sample requirements more precise. Low.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method for paternity identification of Chinese buffalo using microsatellite and mitochondrial genetic marker technology, special primers and a kit. Background technique [0002] In modern animal production and breeding, it is necessary to use the phenotypic information of various relatives to determine accurate genetic pedigrees and understand the breeding value of male animals through correct pedigrees, so as to determine individual selection and avoid individual inbreeding. With the development of large-scale joint breeding and the wide application of BLUP technology, the influence of pedigree information on breeding is increasing. Errors caused by pedigree information errors will be magnified step by step throughout the breeding process, and it is very likely to cause large-scale inbreeding of the population, resulting in population degradation, resulting in irr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 徐亚欧孙蕊毛亮
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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