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DNA Barcoding of spider mites (Prostigmata:Tetranychidae) on major crop plants of Kerala
Spider mites belonging to the family Tetranychidae are economically important pests of various agricultural and horticultural crops. Recently, spider mites emerged as serious pests of vegetables, banana and rice in Kerala. Though identification of spider mites is primarily based on external morpholo...
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| Format: | Ph.D Thesis |
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Vellanikkara
Centre for Plant Biotechnology and Molecular Biology, College of Horticulture
2017
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| Subjects: |
| LEADER | 06711nam a22001817a 4500 | ||
|---|---|---|---|
| 082 | |a 660.6 |b ARU/DN | ||
| 100 | |a Arunima V | ||
| 245 | |a DNA Barcoding of spider mites (Prostigmata:Tetranychidae) on major crop plants of Kerala | ||
| 260 | |a Vellanikkara |b Centre for Plant Biotechnology and Molecular Biology, College of Horticulture |c 2017 | ||
| 300 | |a 59p. | ||
| 502 | |a MSc | ||
| 520 | 3 | |a Spider mites belonging to the family Tetranychidae are economically important pests of various agricultural and horticultural crops. Recently, spider mites emerged as serious pests of vegetables, banana and rice in Kerala. Though identification of spider mites is primarily based on external morphology, the existence of cryptic species makes precise identification difficult. In this context, the present study entitled “DNA barcoding of spider mite (Prostigmata: Tetranychidae) was conducted in Centre for Plant Biotechnology and Molecular Biology (CPBMB) and Department of Agricultural Entomology, College of Horticulture, Vellanikkara with an objective to generate DNA barcodes for different species of spider mites on major crops of Kerala and to find out genetic variability among them. A purposive survey was conducted in four districts of Kerala viz., Thrissur, Palakkad, Ernakulam and Malappuram during the period of February-May and November- December 2016 and February-May 2017. Spider mites associated with ten different crops viz., cowpea, brinjal, okra, ashgourd, pumpkin, banana, papaya, Adenium, Dahlia and tapioca from 11 different localities were collected. The specimens were maintained in acarology laboratory as sixteen separate isolines assigning unique accession number. Permanent slides were prepared separately for adult female and male mites from each isoline for morphological characterization. The chaetotaxy of hysterosoma and legs as well as empodial characters of female were used for generic level identification. The shape of aedeagal knob in male was the characteristic feature utilised for species level identification. The study revealed that the spider mites collected from different crops belonged to three species of a single genus Tetranychus namely, T. truncatus, T. okinawanus and T. udaipurensis. The host range of T. truncatus includes tapioca, banana, pumpkin, Dahlia and cowpea T. okinawanus was recorded on papaya, ashgourd, Adenium, cowpea and brinjal, while T. udaipurensis was found only on okra, banana and tapioca. For molecular characterisation and identification, DNA was isolated from all the sixteen accessions using the modified CTAB method. The DNA was assessed for quality and quantity with NanoDrop spectrophotometer (ND-1000). The absorbance ratio A260/A280 for all accessions was in the range of 1.8-2.0 and concentration was obtained in the range 106-359 ng/μL indicating good quality. The COI loci was amplified with UBC6 (forward) and RCOI (reverse) primers while ITS2 loci was amplified with the primer ITS2 KAU (forward) and R ITS2 KAU (reverse primer). The reaction mixture proposed by Li et al. (2010) was used in study and for ITS2 loci the annealing temperature was standardised by gradient PCR (Polymerase Chain Reaction). The amplification was confirmed by two per cent Agarose Gel Electrophoresis (AGE) and the amplicon size obtained for ITS2 and COI were in the range of 600-700 bp and 800-900 bp, respectively. The PCR products were sequenced by outsourcing at AgriGenome Lab. Pvt. Ltd. Kochi. The forward and reverse sequences obtained after sequencing were merged with the CAP3 sequence assembler to obtain contigs. Fourteen out of sixteen sequences formed contigs. Using MEGA 7 software the sequences were analysed for the presence of stop codons. Stop codons were removed with BioEdit software. Nucleotide BLAST (BLASTn) was done for all the accessions and query coverage (%) and identity (%) were obtained in the range of 97-99 for most accessions. The BLASTn result was in consensus with the morphology based species determination. The nine COI sequences and five ITS2 sequences were aligned with Clustal W of MEGA 7 and barcode gaps were identified for both the loci. The sequences were aligned in MEGA 7 using Kimura 2 parameter (K2P) and pairwise distances between the sequences were analysed which showed that intraspecific divergences were always less than one, however accessions PmMk0927 showed maximum divergence within T. truncatus (0.812- 0.849) and accession BrTv0356 showed maximum divergence within T. okinawanus (0.835). Divergence of T. truncatus sequences from T. okinawanus (ITS2) and T. udaipurensis (COI) were found to be 0.113 and 0.140- 0.142 respectively. The distance summary of sequences when computed with the sequence analysis tool provided by BOLD (Barcode of Life Data systems) showed a sequence divergence within species in the range of 0.00 – 0.31 per cent for ITS2 sequences representing T. truncatus and T. okinawanus and 0.96 – 2.88 per cent for COI sequences representing T. truncatus and T. udaipurensis. A phylogenetic tree was constructed with the fourteen sequences in MEGA 7 using the neighbour joining method and it was seen that the COI and ITS2 sequences branched out into two monophyletic groups. The accessions PmMK0927 (Pumpkin, Manjakunnu) and BrTv0356 (Brinjal, Tavannur) formed out group for COI and ITS2 clusters respectively, as these sequences formed shorter contigs. The sequences were submitted to BOLD (Barcode of Life Data Systems) for generation of barcodes and also to GenBank (NCBI - National centre for Biotechnology Information). The study brings to light the occurrence of T. truncatus and T. okinawanus as the predominant species of spider mites on crops of Kerala. The study recorded four new hosts for T. okinawanus, a species reported only recently from India, indicating its potential to widen the host range. This is suggestive that this alien species has potential to turn invasive. Three new hosts were also recorded from Kerala for T. truncatus and T. udaipurensis. The pairwise distance analysis, distance summary analysis and phylogenetic tree are indicative that T. truncatus is closely related to T. okinawanus at ITS2 loci and to T. udaipurensis at COI loci. Present study confirms that ITS2 and COI loci of spider mite DNA provides species level resolution and thus can be used as reliable tool to differentiate closely related species of spider mites. | |
| 650 | |a Agriculture | ||
| 650 | |a Plant Biotechnology and Molecular biology | ||
| 700 | |a Haseena Bhaskar (Guide) | ||
| 942 | |2 ddc |c TH | ||
| 999 | |c 160035 |d 160035 | ||
| 952 | |0 0 |1 0 |4 0 |6 660_600000000000000_ARUDN |7 1 |8 REF |9 158608 |a KAUCLV |b KAUCLV |c THESES |d 2018-04-13 |o 660.6 ARU/DN |p 174233 |r 2018-04-13 |w 2018-04-13 |y TH | ||