Morfoloogilisel vs. molekulaarsel meetodil mullas elavate lestade (Acari) identifitseerimine

Abstract

DNA meta-triipkoodistamine on kiiresti arenev meetod, kus huvipakkuvate organismide määramine DNA triipkoodide põhjal pakub alternatiivi määramisele morfoloogiliste tunnuste põhjal. Antud magistritöö eesmärk on välja selgitada, kas mullaproovidest eraldatud DNA (PacBio ja Illumina sekveneerimistehnoloogiad) abil lestade koosluste määramine kattub omavahel ja morfoloogilisel teel saadud määrangute tulemustega. Kõik kolm kasutatud meetodit andsid erinevad tulemused. Enim leiti eri lestade taksoneid morfoloogilise määramise põhjal (vähemalt 103 unikaalset taksonit), OTU’sid leiti PacBio’ga 62 ja Illumina’ga 63. Liikide kattuvuste osakaal varieerub 2,3% kuni 4,6% vahel (keskmiselt 3,8%). DNA kaudu väheste lestade tuvastamine võib olla põhjustatud DNA eraldamiseks kasutatud palju väiksematest mullakogustest (2 g ja 0,2 g). Lisaks, lestade molekulaarne signaal võib olla nõrgenenud, kuna mullaproovidest eraldatud DNA amplifitseeriti universaalsete COI praimeritega. Samuti ei esinenud eri sekveneerimismeetoditel saadud taksonite määrangute vahel olulist kattuvust (18,6%). Antud tööst järeldub, et lestakoosluste uurimiseks toimis kõige paremini morfoloogiline määramine (tuvastati kõige rohkem taksoneid), kuid DNA meetodi eelisteks on võimalus tuvastada liike, mida Tullgreni aparaati kasutades ei õnnestu mullast eraldada, lisaks lestadele muid loomataksoneid samaaegselt. Kasutades eri meetodeid kombineerituna saaks antud proovi lestafaunast parima ülevaate.

Identifying soil-dwelling mites (Acari): morphological vs. molecular methods DNA metabarcoding is a rapidly developing method in which the identification of organisms of interest based on DNA barcodes offers an alternative to identification based on morphological features. The aim of this master’s thesis is to determine whether the identification of mite communities based on DNA extracted from soil samples (using PacBio and Illumina sequencing technologies) coincides with each other and with the result obtained using morphological identification. All three methods gave different results. The highest number of distinct mite taxa were found by morphological identification (at least 103), while using PacBio and Illumina 62 and 63 OTUs were obtained respectively. The percentage of common species to both methods varies between 2.3% and 4.6% (with an average of 3.8%). The detection of fewer mites through DNA may be attributed to a smaller amount of soil in samples used for DNA extraction (2 g and 0.2 g) and potential dilution of molecular signals due to amplification of DNA from soil samples using universal COI primers. There was no significant overlap (18.6%) between the taxonomic identifications obtained by the results of different sequencing methods. This study concludes that morphological identification worked best for studying mite communities (the most taxa were identified), however the advantages of the DNA methods include the ability to identify species that can’t be extracted from soil using Tullgren funnel as well as simultaneously detecting other animal taxa besides mites. The most comprehensive overview of the mite fauna from a sample can be obtained by combining different methods.