In 2016, the formation of the Republican DNA Bank Section “DNA Bank of Rare and Endangered Animal and Plant Species” commenced.
The Section is replenished with new samples jointly with the specialists of V.F. Kuprevich Institute of Experimental Botany and Brest State A.S. Pushkin University.
The priority samples of this Section are the biological material samples of plants listed in the Red Book of the Republic of Belarus and with the status I-IV of national nature protection categories. Alongside the classical methods of plant taxonomy, species verification is performed using a DNA barcoding technique.
DNA barcoding technology is an essential tool for the inventory of rare and endangered plants, allowing screening of flora diversity without removing samples from their natural habitat. To date, 67 samples of rare plant species collected in Belarus have been deposited with the DNA Bank. For their 46 representatives, DNA barcoding has been performed using a four-marker sequencing panel: nuclear ITS2 and 3 chloroplast – rbcL, psbA-trnH and matK. The two-locus genotyping scheme by ITS2 and rbcL markers was used as a basis, since their resolving power is quite high when used together.
Genotyping algorithm includes the steps as follows:
• DNA isolation (material sampling of three separate plants is required for reliable species identification)
• DNA quality assessment using the PCR control reaction with 26S rRNA (“housekeeping” gene) primers
• Amplification of two marker sequences — ITS2 and rbcL for each plant
• Sequencing of the obtained sequences (in the forward direction, and if required, in reverse)
• Analysis of the results obtained in the NCBI Database
For a more complete (comprehensive) assessment of species identification, chloroplast psbA-trnH and matK markers can be involved in work.
Currently, work on the establishment of a prototype Regional Reference DNA Barcodes Library as an independent structural unit of the Database of the Republican DNA Bank of a Human, Animals, Plants and Microorganisms (Belarus) is underway.