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Method for analyzing genetic diversity of Candidatus Liberibacter by utilizing SSR (simple sequence repeat) molecular marker primer system

A technology of citrus huanglongbing bacteria and genetic diversity, applied in the field of molecular biology, can solve the problems of inability to extract the nucleic acid of pure bacteria, no genetic diversity analysis method, citrus huanglongbing bacteria cannot be cultured in vitro, etc., and achieve good promotion and application value , Overcoming technical defects, the effect of fewer steps

Active Publication Date: 2014-03-26
嘉兴卓十生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Citrus Huanglongbing bacteria cannot be cultured in vitro at present, and the nucleic acid of pure bacteria cannot be extracted
There is no simple and effective method for genetic diversity analysis

Method used

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  • Method for analyzing genetic diversity of Candidatus Liberibacter by utilizing SSR (simple sequence repeat) molecular marker primer system
  • Method for analyzing genetic diversity of Candidatus Liberibacter by utilizing SSR (simple sequence repeat) molecular marker primer system
  • Method for analyzing genetic diversity of Candidatus Liberibacter by utilizing SSR (simple sequence repeat) molecular marker primer system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Screening of the Las-SSR molecular marker primer system of the present invention

[0039] 1. Using the Genome database in the Genebank database, two Asian species of citrus Huanglongbing, gb|CP001677.5 and gb|CP004005.1 were found ( Ca. L. asiaticus) genome sequence, the nucleotide lengths are 1.23 Mb and 1.27 Mb respectively.

[0040] The analysis software used in this example is the SSR information search software Tandem Repeats Finder (version 4.07b) online analysis system (http: / / tandem.bu.edu / trf / trf.html) and the primer synthesis software Primer Premier 5.0.

[0041] The experimental methods are conventional PCR, agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE).

[0042] 2. Use the SSR retrieval software Tandem Repeats Finder (version 4.07b) to find all the SSR site information of all 2-500 base repeat motifs in the two genomes, and obtain 36 SSR sites after screening. The screening criteria are: (1) If the matching rate (Perce...

Embodiment 2

[0049] Example 2 Acquisition of core primers for Las-SSR molecular markers and genetic polymorphism analysis of H. citri

[0050] Citrus leaf samples infected with citrus H. citrus L. were selected from six major citrus producing areas in Fujian, Guangdong, Guangxi, Jiangxi, Yunnan and Zhejiang, and each sample was photographed and recorded. Genomic DNA was extracted using OMEGA E.Z.N.A.TM Plant Genomic DNA Kit (HP plant DNA kit (200), D2485-02, OMEGA bio-tek), and 0.15 g of leaf midrib was cut and placed in a 2.0 mL centrifuge tube, and 500 mL of The extract CPL and 2 magnetic beads were crushed on the MP FastPrep-24 grinder. The quality of genomic DNA was detected by 1% (w / v) agarose gel electrophoresis.

[0051] Using the Asian species of Huanglongbing citri ( Ca.L. asiaticus) 16s rDNA specific primer OI1 / OI2c (OI1: 5'- GCGCGTATCCAATACGAGCGGCA-3'; OI2c: 5'- GCCTCGCGACTTCGCAACCCAT-3') was used to analyze the extracted samples, and 32 positive samples were screened out, ...

Embodiment 3

[0062] Example 3 The Asian species of citrus Huanglongbing in 6 provinces of China ( Ca. Preliminary analysis of strain diversity of L. asiaticus

[0063] 1. Analysis method

[0064] Method is with embodiment 2.

[0065] 2. Result analysis

[0066] The band information of all 32 available SSR primers (excluding Las-ssr28) from 32 strains in 6 provinces of China was collected, and different band types were indicated by different Arabic numerals.

[0067] The polymorphic site frequency (PPB), Nei's gene diversity index (h) and Shannon's information index (I) of SSR markers were respectively obtained by POPGENE1.32 software (as shown in Table 3 and Table 4), reflecting 32 strains genetic diversity.

[0068] Table 3 Among them, 32 pairs of polymorphic site frequencies (PPB) in 32 strains that can be marked by SSR

[0069]

[0070] Table 4 Nei's gene diversity index (h) and Shannon's information index (I) of 32 pairs of polymorphic sites that can be marked by SSR

[007...

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Abstract

The invention discloses a method for analyzing genetic diversity of a Candidatus Liberibacter by utilizing an SSR (simple sequence repeat) molecular marker primer system. The SSR molecular marker primer system comprises 33 pairs of Las-SSR available primers, 18 pairs of which are SSR core primers. The invention puts forward the SSR molecular marker based method for analyzing diversity of a Ca.L.asiaticus strain in combination with PAGE (polyacrylamide gelelectrophoresis). The method is simple and convenient, is high in analysis efficiency, and lays a molecular foundation for study of Candidatus Liberibacter molecular epidemiology.

Description

technical field [0001] The invention relates to the technical field of molecular biology. More specifically, it relates to a method for analyzing the genetic diversity of H. citri by using the SSR molecular marker primer system. Background technique [0002] Citrus huanglongbing is the most important disease endangering many citrus producing areas in the world and in my country. The disease was first discovered in Guangdong, my country, and has a history of nearly 100 years. At present, Huanglongbing is widely distributed in Asia and Africa, and it also occurs seriously in some major citrus producing areas in Oceania, South America and North America, and it has been reported that it has caused huge losses in more than 50 countries and regions. The understanding of the pathogen of Huanglongbing has gone through a long time. At present, it is generally believed that Huanglongbing is caused by Gram-negative bacteria that are limited to the phloem of the host plant and cannot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10G06F19/20C12R1/01
CPCC12Q1/6858C12Q2531/113C12Q2565/125
Inventor 许美容邓晓玲郑正李昕昱洪红霞朱青
Owner 嘉兴卓十生物科技有限公司
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