Approbation of the MS-ISSR method for assessing the DNA methylation profiles of Norway spruce
A.B. Pekedova, Калько Г. В. · Proceedings of the Saint Petersburg Forestry Research Institute · 2021
The article is devoted to the approbation of the MS-ISSR (methylation-sensitive inter simple sequence repeats) method for determining variations in the DNA methylation profiles of Norway spruce trees growing at 5 km from the metallurgical enterprise LLC Orion-Spetsplav-Gatchina, located in the city of Gatchina and in background planting of the Leningrad region.Earlier, the content of the metals Mn, Zn, Cu, Fe, Pb, Cr, Ni, Cd and sulfur was determined in the needles of the spruce trees used to test the method.The aim of the work was to determine the possibility of using the MS-ISSR method to assess the genetic stability of Norway spruce in the zone of anthropogenic emissions, namely, the stability of DNA methylation profiles.We used DNA isolated from fir needles by a modified method with CTAB.DNA restriction was performed using isoschizomer restriction enzymes Msp I or Hpa II (SibEnzyme, Russia) under conditions recommended by the enzyme manufacturer.The efficiency of using seven ISSR primers was evaluated.The amplification results were detected by electrophoresis in 1.5 % agarose gel in the presence of ethidium bromide.The four primers M 2, M 4, M 9 and M 14 have been shown to be useful in detecting changes in DNA methylation profiles.Primer M 9 showed the highest efficiency in detecting DNA methylation -75 %, primers M 2 and M 4 had an efficiency of 50 %, primer M 14 -25 %.It was concluded that the use of the MS-ISSR method is promising for the analysis of DNA methylation profiles of Norway spruce.