DNA barcoding of spider mites (Prostigmata: Tetranychidae) in vegetable crops
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Date
2015
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short DNA sequences from a standardized region of the genome. It helps in precise identification of species that can be applicable as a diagnostic tool for quarantine and other pest management activities. DNA barcoding is based on the variation on the sequences identified in genomic regions, which can distinguish individuals of a species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e. the ‘barcode’ from a standard region of the genome, a specific gene region either from mitochondria or nuclear genome. The barcode sequence from the unknown specimen could be compared with a library of reference barcode sequences derived from individuals of known identity.
The family Tetranychidae or spider mites include the most injurious plant-feeding mites. Some infest a wide range of host plant whereas some others are highly specific. The family includes about 12,000 species of phytophagous mites which can rapidly disperse to exploit new feeding sites, damage agricultural and horticultural crops causing severe economic losses. Precise identification up to species level is difficult since all species look similar. So molecular methods are increasingly applied in tetranychid mite taxonomy to establish species identity and DNA barcoding is the best option for this.
The study entitled “DNA barcoding of spider mites (Prostigmata :
Tetranychidae) in vegetables” was done at Centre for Plant Biotechnology and Molecular Biology and Department of Agricultural Entomology, College of Horticulture, Vellanikkara. The objective of this study was to generate DNA barcodes for different species of spider mites in vegetable crops and to study the intra and inter species genetic relationship. For this, spider mites were collected from different locations viz; Anthikad, Alathore, Elenad, KVK, Thrissur and Vellanikkara and from different vegetable crops viz; Amaranthus, brinjal, cowpea, cucumber, dolichos bean, okra and ridge gourd. After collection, spider mites were reared in the laboratory to get an isoline from which few males and females were used for microscopic slide preparation. Acarologists used slides to identify the spider mite species based on morphological taxonomic keys.
Total genomic DNA isolated using modified CTAB method (Rogers and Benedict, 1994) was subjected to PCR assay using markers for two different loci ITS2 (second internal transcribed spacer) and COI (mitochondrial cytochrome c oxidase subunit I) which yielded bands of 620 and 868bp respectively. The obtained bands were eluted and subjected for sequencing. Nucleotide divergence among the sequences was calculated using Barcode of Life Data (BOLD) tool ‘Distance summary’. It is desirable for barcodes to show very low sequence divergence within species.
Multiple sequence alignment using clustalW of MEGA6 software was performed for all the sequences and phylogenetic analysis has discriminated three different species of Tetranychidae family: Tetranychus truncatus, T.macferlanei and T.okinawanus. Barcoding gap, a position in the sequence at which unique nucleotide is present in all the members of a Tetranychus species was also assessed. Morphological and molecular analysis results were correlated with each other and results matched. Tetranychus okinawanus found on cucumber during the study is first time getting reported from India.
The study had shown that both ITS2 (99.25%) and COI (98.45%) sequences efficiently classified the spider mite species. However, ITS2 was found to be an efficient tool which gave species level resolution in spider mites. This can be used as supporting marker for COI to barcode spider mites species. In future, these primers can be used to barcode other Tetranychus species also and sequence analysis can also be done using ITS1 locus and its efficiency can be measured for DNA barcoding of Tetranychus species.
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