Dna extraction

DNA extraction , amplification, and sequencing

Total genomic DNA was extracted as described previously by Echt et al. (1992) with some modifications ( Kiselev et al. 2015). Part of the rbc L gene was amplified as described by Romanov et al. (2022b). The nuclear ribosomal internal transcribed spacer region (ITS1 + 5.8S + ITS2) was amplified by nested PCR in a T100 Thermal Cycler (Bio-Rad Laboratories, Inc., USA). The first PCR was performed with primers ITS-36F and ITS-R ( Hall et al. 2010) and the second PCR—with primers ChITS-1F ( Romanov et al. 2022b) and ITS-R. The PCR cycling profile for this region included denaturation at 95 °C for 3 min, followed by 44 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 7 min. The PCR products were purified using ExoSAP-IT PCR Product Cleanup Reagent (Affymetrix Inc., USA) and sequenced in both directions at the Instrumental Centre of Biotechnology and Gene Engineering of FSCEATB FEB RAS using an ABI 3500 genetic analyzer (Applied Biosystems, Maryland, USA) with a BigDye terminator v. 3.1 sequencing kit (Applied Biosystems, USA) and primers used for PCR. Sequences were assembled with the Staden Package v.1.4 ( Bonfield et al. 1995), and aligned manually in the SeaView program ( Galtier et al. 1996).

An isotype of Nitella heteroteles J. Groves & E.L. Stephens (1933: 277) {[ Madagascar], Imerina, Jul[y] 1880, J.M. Hildenbrandt 3525 (LE)} was used for barcoding. Its ITS sequence is similar to the species described here (see section “Results” below). This is why it was included in the consideration irrespective of clear dissimilarity in morphological traits. All efforts to obtain the rbc L gene sequence for N. heteroteles were unsuccessful. The newly obtained DNA sequences were deposited in GenBank (Supplementary Table 1, 2).