Naslov
Nova metoda genske identifikacije vrsta u integriranoj zaštiti šuma
(New method of molecular identification of species in integral forest protection)

Autori
Franjević, Milivoj ; Hrašovec, Boris ; Blašković, Petra ; Višić, Hrvoje ; Franjević, Damjan

Izvornik
Naše šume (1840-1678) 17 (2019); 45-48

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Castanea sp. ; Dryocosmus kuriphilus ; parasitoid ; Torymus sinensis

Sažetak
For genetic identification of Torymus sinensis species total genomic DNA was extracted from 25mg of tissue with the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer protocol and stored at -20°C until the polymerase chain reaction (PCR) was conducted. Mitochondrial DNA fragment of COI coding gene, the so called Folmer region (Folmer et al 1994), was proposed as a universal DNA barcode region for animals (Hebert et al 2003). In this research we used the COI gene fragment primers LCO 1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and HCO 2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) in order to amplify Folmer region of COI gene and utilize it as a barcoding marker for genetic identification of our samples. The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. Sequence similarity searching is a method of searching sequence databases by using alignment to a query sequence. By statistically assessing how well database and query sequences match, one can infer homology and transfer information to the query sequence. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. We used NCBI’s blast optimized for megablast to find similar sequences in GenBank. Results retrieved using BLAST service showed a high sequence similarity between our sequences with 11 sequences of Torymus sinensis from GenBank from China and Italy. Query coverage (percent of the query sequence that overlaps the subject sequence) was 80%. E-value (see below) was 0.0 and identity score was 89% (percent similarity between the query and subject sequences over the length of the coverage area) corroborating that the sample from Zagreb (Croatia) is indeed T. sinensis species. The Expect value (E-value) is a parameter that describes the number of hits one can ‘expect’ to see by chance when searching a database of a particular size. It decreases exponentially as the Score (S) of the match increases. Essentially, the E value describes the random background noise. The lower the E- value, or the closer it is to zero, the more ‘significant’ the match is. E-value of zero for our sequences proves unambiguously that the T. sinensis parasitoid is present in chestnut gall wasps from Dotrščina. Successful genetic identification of T.sinensis from collected samples of chestnuts gall wasps indicates that biological control of this pest is established in park forest Dotrščina near Zagreb.

Izvorni jezik
Hrvatski

Znanstvena područja
Biologija, Šumarstvo

POVEZANOST RADA