Effects of Temperature Treatments on Cytosine-Methylation Profiles of Diploid and Autotetraploid Plants of the Alpine Species Ranunculus kuepferi (Ranunculaceae)

Abstract

The exposure to environmental stress can trigger epigenetic variation, which may have several evolutionary consequences. Polyploidy seems to affect the DNA methylation profiles. Nevertheless, it abides unclear whether temperature stress can induce methylations changes in different cytotypes and to what extent a treatment shift is translated to an epigenetic response. A suitable model system for studying these questions is Ranunculus kuepferi, an alpine perennial herb. Diploid and autotetraploid individuals of R. kuepferi were exposed to cold (+7°C day/+2°C night; frost treatment −1°C cold shocks for 3 nights per week) and warm (+15°day/+10°C night) conditions in climate growth chambers for two consecutive flowering periods and shifted from one condition to the other after the first flowering period. Methylation-sensitive amplified fragment-length polymorphism markers were applied for both years, to track down possible alterations induced by the stress treatments. Patterns of methylation suggested that cytotypes differed significantly in their profiles, independent from year of treatment. Likewise, the treatment shift had an impact on both cytotypes, resulting in significantly less epiloci, regardless the shift's direction. The AMOVAs revealed higher variation within than among treatments in diploids. In tetraploids, internally-methylated loci had a higher variation among than within treatments, as a response to temperature's change in both directions, and support the hypothesis of temperature stress affecting the epigenetic variation. Results suggest that the temperature-sensitivity of DNA methylation patterns shows a highly dynamic phenotypic plasticity in R. kuepferi, as both cytotypes responded to temperature shifts. Furthermore, ploidy level, even without effects of hybridization, has an important effect on epigenetic background variation, which may be correlated with the DNA methylation dynamics during cold acclimation.

Phenotypic Responses, Reproduction Mode and Epigenetic Patterns under Temperature Treatments in the Alpine Plant Species Ranunculus kuepferi (Ranunculaceae)

“…The current experimental design was favored for the purpose of investigating the temperature preferences of the two cytotypes implied by Schinkel et al [ 47 ] and Kirchheimer et al [ 63 ], with cold temperature treatments simulating the harsh high alpine temperature conditions of the tetraploid cytotype’s typical habitats. The treatment of Klatt et al [ 41 ] was kept until 2016 and rotated in 2017 before the beginning of the present study [ 69 ]. All measures were taken from 2017 under the conditions specified in Table 1 .…”

“…Regarding fragment scoring, the resulted electropherograms from the ABI Prism 3700/3730 (Applied Biosystems, Waltham, MA, USA) capillary sequencer went through the following scoring pipeline: Peakscanner v.2 (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA), RawGeno 2.0-1 [ 73 ], and MSAP_calc script [ 74 ]. A detailed wet lab and fragment scoring methodology can be found in Methods S1 (Supplementary Materials) , and the DNA methylation study results on the species Ranunculus kuepferi are presented elsewhere [ 69 ].…”

“…A molecular dating revealed that the tetraploid cytotype originated only 10–80 thousand years ago [ 66 ], probably via multiple and recurrent polyploidization events [ 61 , 67 ]. Epigenetic studies using methylation-sensitive amplified fragment-length polymorphisms (MS-AFLPs) on the species suggested differential profiles in the cytotypes and a connection to abiotic environmental conditions of the epigenetic variation in natural populations and experimental treatments [ 68 , 69 ]. This epigenetic variation was further correlated with an elevation in natural populations [ 68 ] and showed higher persistence under cold treatment in experimental conditions [ 69 ].…”

“…Epigenetic studies using methylation-sensitive amplified fragment-length polymorphisms (MS-AFLPs) on the species suggested differential profiles in the cytotypes and a connection to abiotic environmental conditions of the epigenetic variation in natural populations and experimental treatments [ 68 , 69 ]. This epigenetic variation was further correlated with an elevation in natural populations [ 68 ] and showed higher persistence under cold treatment in experimental conditions [ 69 ]. Thus, we assume a putative epigenetic background of the niche shift of tetraploids in the Alps, which helps to understand the geographical parthenogenesis scenario for the species.…”

“…By assessing methylation variation data of the same year of treatment [ 69 ], we focused on possible correlations of phenotypic patterns with MS-AFLP profiles. We investigated whether the methylation profiles of vegetative parts differentiate for phenotypic traits according to ploidy and treatment conditions.…”