Multiplex polymerase chain reaction (PCR) primers and method for identifying genotypes of green-shell laying hens

A green-shell laying hen, genotyping technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of close band distance and unclear individual separation, and achieve clear edges, Improve identification accuracy and clear separation effect

Inactive Publication Date: 2018-07-24
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the currently reported patented method of using molecular markers to identify the genotype of green-shelled eggs can identify the genotype of green-shelled eggs, some methods require secondary amp

Method used

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  • Multiplex polymerase chain reaction (PCR) primers and method for identifying genotypes of green-shell laying hens
  • Multiplex polymerase chain reaction (PCR) primers and method for identifying genotypes of green-shell laying hens

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Design and sequencing verification of multiple PCR primer combinations for identifying the genotype of green-shell layer hens

[0022] 1. Primer Design

[0023] According to the published sequences of SLCO1B3 (Genebank Accession: JN020139.1) and EAV-HP (GenBankaccession number: JF837512), design three primers F, R1, R2 (sequences such as Seq ID No: 1, Seq ID No: 2 and Seq ID No: 3), this set of primers is designed for researching whether there is a partial sequence of EAV-HP inserted in the 5' flanking region of chicken SLCO1B3. Wherein, F and R2 are fragments of the SLCO1B3 gene of chicken normal genome, and R1 is a fragment in the insertion sequence of EAV-HP.

[0024] If the individual does not carry the insert fragment, F and R2 are paired, and only a single target band with a fragment length of 397bp is amplified, indicating that the individual does not lay green eggs (genotype is NN / NN). If the individual carries the insert fragment, F and R1 are pair...

Embodiment 2

[0043] Example 2: Accuracy of large group detection and identification of genotypes of green-shell hens and non-green-shell hens

[0044] 1.1 Detection material

[0045] Table 1 list of individuals to be tested

[0046] Variety

male

mother

Wenshang Reed Chicken with Green Shell

30

28

Specialized strains of green-shell layer hens

13

Langya Chicken

12

48

Wenshang Reed Chicken

12

48

Lu Chicken

8

39

[0047] All 238 individuals to be tested were randomly sampled, and about 1 ml of blood was collected from the subwing vein, anticoagulated with ACD, and stored at -20°C for future use.

[0048] 1.2 Genomic DNA extraction

[0049] With embodiment 1.

[0050] 1.3 Genotype detection of individuals to be tested

[0051] Primer adopts F, R1 and R2 primer combination, with embodiment 1.

[0052] The multiplex PCR amplification system and PCR program are the same as in Example 1.

[0053] PCR produ...

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Abstract

The invention discloses multiplex polymerase chain reaction (PCR) primers and a method for identifying the genotypes of green-shell laying hens. Three primers F, R1 and R2 are firstly designed, wherein F and R2 are fragments in a SLCO1B3 gene of a chicken normal genome. If an individual does not carry an insert fragment, the F and the R2 are paired, a single purpose band of 397 bp is only amplified, and no green-shell egg is produced. The R1 is a fragment of an EAV-HP insertion sequence; if the individual carries the insert fragment, the F and the R1 are paired, a single purpose band of 223 bpis only amplified, and green-shell eggs are produced. If one chromosome of the individual carries the insert fragment and the other one chromosome does not carry the insert fragment, two purpose bands of 223 bp and 397 bp are amplified, and green-shell eggs are still produced. Compared with other identification methods, the primer combination enables the two bands in heterozygote to be more clearly separated, and is beneficial to increment of the genotype identification accuracy of the individual to be tested.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multiplex PCR primer and an identification method for identifying the genotype of green-shell laying hens. Background technique [0002] Egg shell color is an important egg trait and economic trait. The depth of egg shell color directly affects the sales of eggs, thereby affecting the economic benefits of farmers. Green-eggs are widely welcomed because of their color in line with the current needs of people in pursuit of health. In my country, only a few local chicken breeds such as Dongxiang green-shell hens and Changshun green-shell hens produce green-shell eggs, which have become valuable genetic resources. A number of domestic breeding companies have also introduced green-shell egg genes to carry out crossbreeding and cultivate specialized strains of green-shell layer hens for commercial production. However, in actual production, since heterozygous hens can also lay green egg...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2537/143
Inventor 周艳曹顶国雷秋霞韩海霞李福伟高金波刘玮刘杰李玉华
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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