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New specific primers for amplification of the Internal Transcribed Spacer region in Clitellata (Annelida)

Artikel i vetenskaplig tidskrift
Författare Christer Erséus
Yingkui Liu
Publicerad i Ecology and Evolution
Volym 7
Nummer/häfte 23
Sidor 10421-10439
ISSN 2045-7758
Publiceringsår 2017
Publicerad vid Institutionen för biologi och miljövetenskap
Sidor 10421-10439
Språk en
Länkar https://doi.org/10.1002/ece3.3212
Ämnesord Hirudinida, Internal Transcribed Spacer region, Oligochaeta, polymerase chain reactions, primers
Ämneskategorier Biologisk systematik, Biokemi och molekylärbiologi

Sammanfattning

Nuclear molecular evidence, for example, the rapidly evolving Internal Transcribed Spacer region (ITS), integrated with maternally inherited (mitochondrial) COI barcodes, has provided new insights into the diversity of clitellate annelids. PCR amplification and sequencing of ITS, however, are often hampered by poor specificity of primers used. Therefore, new clitellate-specific primers for amplifying the whole ITS region (ITS: 29F/1084R) and a part of it (ITS2: 606F/1082R) were developed on the basis of a collection of previously published ITS sequences with flanking rDNA coding regions. The specificity of these and other ITS primers used for clitellates were then tested in silico by evaluating their mismatches with all assembled and annotated sequences (STD, version r127) from EMBL, and the new primers were also tested in vitro for a taxonomically broad sample of clitellate species (71 specimens representing 11 families). The in silico analyses showed that the newly designed primers have a better performance than the universal ones when amplifying clitellate ITS sequences. In vitro PCR and sequencing using the new primers were successful, in particular, for the 606F/1082R pair, which worked well for 65 of the 71 specimens. Thus, using this pair for amplifying the ITS2 will facilitate further molecular systematic investigation of various clitellates. The other pair (29F/1084R), will be a useful complement to existing ITS primers, when amplifying ITS as a whole.

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