Selection of nuclear and plastid DNA markers for comparison of Lithuanian populations of Phalaris arundinacea

Abstract

Reed canarygrass (Phalaris arundinacea L.) belongs to family Poaceae, subfamily Pooideae, tribe Poeae. This species is spread all over the northern hemisphere. It is common perennial herb of Lithuania. Phalaris arundinacea is valuable plant due to adaptation to grow in both dry and moisture areas, also it is economically important species used as an animal feed, ornamental plant, biofuel, also applied for bioremediation purposes. Recently, P. arundinacea has been studied at the molecular level, aiming to determine how invasive populations of this plant have spread along North America. Till now there is a lack of information about reed canarygrass sequences of chloroplast and nuclear DNA. It is important to get more information for understand such processes as plant evolution and ecology, as well as to find relation with a lot of questions posting topic – anthropogenic effects on natural ecosystems. DNA was extracted using modified CTAB method. Extraction of pure DNA is an important prerequisite for efficient sequencing of chloroplast genome. It is also important to find out the best conditions for polymerase chain reactions. The purpose of the study was to select various chloroplast and ribosomal DNA markers finding out the best reaction mix, temperature, cycles for further investigation steps getting information about genome regions of populations of Phalaris arundinacea growing along riverbanks in Lithuania. In our study nuclear ribosomal markers ITS4, ITS5, ITS7 also chloroplast markers atpB, rbcL, trnH (GUG), psbA, petA, psbJ, atpI, atpH, F71, R1516, R1661 were tested. Obtained DNA fragments were visualized on 1.5 % agarose gel, cut out and cleaned with columns produced by manufactory. Finally, according to elaborated protocol, one primer pair (ITS4, ITS5) out of two nrDNA primer pairs and three (petA-psbJ, atpI-atpH, F71-R1661) out of six cpDNA primers have generated clear and readable fragments for sequencing.