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Application of COI gene in identification of luehdorfia chinensis

A Chinese tiger swallowtail and gene technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of being easily affected by the environment, poor accuracy, etc., and achieve the effect of shortening the identification time and accurate identification

Pending Publication Date: 2020-11-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The applicant found that in the research on the identification method of the genus S. chinensis in the prior art, there is no application and related analysis method of the barcode of the COI gene of S. chinensis to judge the COI gene as the gene barcode in the identification of S. chinensis. The existing methods for identifying butterfly species are mainly based on morphological classification, but the external morphology is highly plastic and easily affected by the environment, so the accuracy of identification is poor

Method used

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  • Application of COI gene in identification of luehdorfia chinensis
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  • Application of COI gene in identification of luehdorfia chinensis

Examples

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Effect test

Embodiment 1

[0072] Example 1 Design and Verification of Specific Primers for the COI Gene Fragment of Tiger Papilio chinensis

[0073] Sequencing quality analysis: The present invention designs 3 pairs of primers for the Chinese tiger swallowtail, and analyzes the sequencing quality and screens the available primer sequences through the following steps:

[0074] 1) Test sample:

[0075] The sample S1 (Luehdorfia chinensis 1) in the present invention was taken from Nanjing Animal Protection Association, Jiangsu Province in mid-April 2019. It is a living adult sample. Result accuracy.

[0076] 2) Use the following primers to amplify the target COI gene fragment of S. chinensis S1, wherein the PCR amplification primer pair (primer pair COI1, primer pair COI2, primer pair COI3) and PCR reaction system and program are as follows, primer The sequence is (F represents the upstream primer, R represents the downstream primer):

[0077] COI 1-F: (SEQ ID NO: 1);

[0078] COI 1-R: (SEQ ID NO:...

Embodiment 2

[0086] Example 2 Primer pair COI1 amplified gene barcode applicability analysis of the COI gene fragment of the Chinese tiger swallowtail butterfly

[0087] 1) Test sample: See Example 1) for the source of the Chinese tiger swallowtail sample S1 of the present invention.

[0088] 2) Sequence information:

[0089] Log in to the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ), download the existing COI sequences of Papilio species, and download the COI sequences of some outgroups, as follows Show:

[0090] Table 1 COI sequences downloaded from GenBank

[0091]

[0092] 3) Method:

[0093] (3.1) Use the DNeasy Blood&Tissue Kit (Qingke) kit to extract leg muscle DNA from the sample, and dilute it into a 50ng / μL working solution for later use. The extracted DNA sample should be stored at -20°C.

[0094] (3.2) Use the following primers to amplify the COI gene fragment of the order S. sinensis, wherein the PCR amplification primers and PCR r...

Embodiment 3

[0120] Example 3 Application of Primer to COI1 Amplified COI Gene Barcode of Tiger Papilio chinensis

[0121] S2 (Luehdorfia chinensis 2) and S3 (Luehdorfia chinensis 3) were identified by primer COI1. The sources of S. chinensis individual S2 and S3 are the same as that of S. chinensis S1 in embodiment 1-1). The gene sequence homology between the sequencing result and SEQ ID NO.3 is more than 98% (the gene sequence homology between S2 and SEQ ID NO.3 in the present invention is 99.69%, and S3 and SEQ ID NO.3 The homology of the gene sequence is 100%), so it can be determined that the tissue to be tested is the Chinese tiger swallowtail. The amplified sequences and genetic distances were calculated as follows. The average genetic distance between species was 0.0839, the average genetic distance within a species was 0.0030, and the average genetic distance between species was 28 times of the average genetic distance within a species. The minimum interspecific genetic distanc...

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Abstract

The invention discloses application of a COI gene in identification of luehdorfia chinensis, and provides a plurality of analysis methods compared with a traditional DNA bar code analysis method, andthe bar code screened by the method is higher in accuracy. The invention also discloses an analysis method for evaluating the applicability of luehdorfia chinensis COI gene as a gene bar code. By means of the analysis method, two proper COI gene bar codes are provided, rapid and accurate identification of different insect states (including adults, eggs, larvae and pupae) and incomplete specimens (including partial tissues of muscles, feet, wings and body walls) is facilitated, and the identification time is shortened. The analysis method has important reference significance for screening DNA barcodes, and has a great application prospect.

Description

technical field [0001] The invention belongs to the field of animal molecular biology, and specifically refers to the application of COI gene in identification of Chinese tiger swallowtail. Background technique [0002] There are 369 genera, 1222 species, and 1851 subspecies of butterflies in China, among which there are 5 species in the genus Tiger Swallowtail, namely Tiger Swallowtail, Chinese Tiger Swallowtail, Japanese Tiger Swallowtail, Zhou's Tiger Swallowtail, Boss's Tiger Swallowtail Among them, the Chinese tiger swallowtail is a rare butterfly unique to my country, and it is a second-level wild animal under national key protection. All species of the tiger swallowtail are included in the key protection scope of the State Forestry Administration. In recent years, due to the reduction of host plants caused by environmental damage, and the small number of breeding generations of tiger swallowtails each year, the number of wild tiger swallowtails has declined sharply, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/12C12N15/11
CPCC12Q1/6888C12Q1/6869C07K14/43563C12Q2531/113C12Q2537/165C12Q2565/125C12Q2535/122
Inventor 张克云肖振华窦振国包浩然蔡亚非张威高国富
Owner NANJING AGRICULTURAL UNIVERSITY
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