Identifying Plant Parasitic Nematodes from Imported Plant Products Using Pyrosequencing Approach
Date Issued
2015
Date
2015
Author(s)
Liu, Hsiang-Chun
Abstract
Several plant parasitic nematodes are quarantine pests listed in the ‘Quarantine Requirements for The Importation of Plants or Plant Products into The Republic of China’. The plant parasitic nematodes intercepted in imported plants or plant products are usually only with limited number, and are short of identifiable characteristics resulting from non-female individuals or larval stage. With such difficulty in identification of potential nematode pests, additional cost may apply to consignee as the responsible agency may have to implement quarantine treatment. In this study, nematode samples are classified into genus level based on morphological characters, and furthermore into species level via pyrosequencing. Pyrosequencing method does not require fluorescent DNA sequence analyses, and sequence products with 200-300 bp can be readily read for data analysis, which enables rapid quantification of sequence variations, thus successful identification. In this study, universal primers rDNA2-M/rDNA1.58S were used to amplify partial 18S, ITS1 and partial 5.8S rDNA fragments (500-700 bp) of various nematodes, but amplicons are too long to be utilized for pyrosequencing. Therefore, three modified primers including Aph, Dit, and Xip, coupling with rDNA2-M, are used as pyrosequencing specific primers for Aphelenchoides spp., Ditylenchus spp., and Xiphinema spp., respectively, and approximately 200-300 bp partial 18S rDNA fragments were successfully amplified. These sequences are blasted on NCBI GenBank to obtain similar aligned sequences of quarantine nematode pests and other potential organisms, and these data were integrated as database. The sequences obtained from various nematode samples via pyrosequencing were also used to construct another database. Results of sequence alignment indicate that the resolution of pyrosequencing in plant parasitic nematodes seems limited. In near future, the resolution can be elevated by adding dNTPs according to predicted sequences during pyrosequencing, instead of adding in a random fashion. This study demonstrates the utility of pyrosequening technique especially for establishment of a rapid identification platform of quarantine purpose. Also, the expandable nature of the database allows pyrosequening technology even more suitable for quarantine pest identification and database development.
Subjects
identification
plant parasitic nematodes
pyrosequencing
quarantine
Type
thesis
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