DOI: 10.5061/dryad.ksn02v729
title: DNA methylation and phenotypic data from: Environmentally induced phenotypic plasticity and DNA methylation changes in a wild potato growing in two contrasting Andean experimental gardens

description: DNA methylation can be environmentally modulated and play a role in
 phenotypic plasticity. To understand the role of environmentally induced
 epigenetic variation and its dynamics in natural populations and
 ecosystems, it is relevant to place studies in a real-world context. Our
 experimental model is the wild potato Solanum kurtzianum, a close relative
 of the cultivated potato S. tuberosum. It was evaluated in its natural
 habitat, an arid Andean region in Argentina characterised by spatial and
 temporal environmental fluctuations. The dynamics of phenotypic and
 epigenetic variability (with Methyl Sensitive Amplified Polymorphism
 markers, MSAP) was assayed in three genotypes across three growing
 seasons. These genotypes were cultivated permanently and also reciprocally
 transplanted between experimental gardens (EG) differing in ca. 1000 m of
 altitude. In two seasons, the genotypes presented differential methylation
 patterns associated to the EG. In the reciprocal transplants, a rapid
 epigenomic remodelling occurred according to the growing season.
 Phenotypic plasticity, both spatial (between EGs within season) and
 temporal (between seasons), was detected. The epigenetic and phenotypic
 variability were positively correlated. The lack of an evident mitotic
 epigenetic memory would be a common response to short-term environmental
 fluctuations. Thus, the environmentally induced phenotypic and epigenetic
 variation could contribute to populations persistence through time. These
 results have implications for understanding the great ecological diversity
 of wild potatoes.
description: The spreadsheet has four sheets. The first titled MSAP_epiallele
 presents raw MSAP data with methylation patterns (0,1,2 and 3) for three
 MSAP markers. The second one titled MSAP_epiloci presents the binary data
 of the MSAP markers. The third and fourth show all the phenotypic features
 measured in leaves and tubers.
 <i><b>Experimental gardens and reciprocal transplants
 approach</b></i> We evaluated
 morphological, biochemical, and phenological phenotypes and analysed the
 dynamics of DNA methylation patterns in clones of three <i>S.
 kurtzianum</i> genotypes. These clones were cultivated during three
 successive years, both permanently and as reciprocally transplanted, in
 two Andean Experimental Gardens (EGs), located at 1141 and 2113 m a.s.l.
 in the VNR. To establish the EGs, and according to the number of available
 tubers, one genotype from each of the three populations collected in the
 VNR and studied by Marfil and Masuelli (2014) -1228M, QH5 and 2166M- were
 selected and labelled as G-1, G-2 and G-3, respectively. Twelve tubers
 (clones) per genotype were cultivated in pots at the campus of Facultad de
 Ciencias Agrarias, UNCuyo (33° 00′ 24´´ S, 68° 52′ 19´´ W, 940 m a.s.l.)
 from November 2013 to January 2014. When the pot-grown plants presented
 three fully expanded leaves, they were transplanted into 10 L pots with a
 mixture of autoclaved local soil and organic compost (3:1), to prevent
 seed contamination. These pots were used to establish the field experiment
 in January 2014, in two EGs located in the VNR. One EG (hereinafter called
 "EG-1100") was located at 1141 m a.s.l. (S32° 34' 39.57” S,
 68° 56' 45.65” W), and the other (hereinafter called
 "EG-2100") at 2113 m a.s.l. (32° 35' 06.47'' S,
 69° 05' 56.85'' W). Half of the clones of each genotype
 were placed in each EG following a completely random design (Ibañez et al.
 2017). Overall, six clones × three genotypes × two EG were evaluated.
 Plants were weekly irrigated with KSC II PHYT-actyl fertiliser (Timac
 Agro, USA). In September 2014, tubers formed during the growing season
 (November 2013- April-May 2014) were collected and conserved in the
 laboratory at 4º C until dormancy breakdown. The sprouted tubers were used
 as starting material for the next growing season. The cultivation
 conditions were repeated with the modifications detailed below. The
 cultivation conditions in 2015, 2016, and 2017 were modified by
 incorporating <i>reciprocal transplants</i> between EGs (Fig.
 1). In each season, clones were divided into two groups. Three clones per
 genotype were cultivated repeatedly in the same EG as in the previous
 season (permanent clones). For example, clones of G-1, G-2 and G3
 cultivated in EG-1100 in 2014 were cultivated again in EG-1100 in 2015.
 The other three clones were transplanted (transplanted clones) into the
 other EG (<i>reciprocal transplants</i>). For
 example,<i> </i>clones of G-1, G2 and G3 cultivated in EG-1100
 in 2014 were transplanted into EG-2100 in 2015, whereas those grown in
 EG-2100 in 2014 were transplanted into EG-1100 in 2015 (Fig. 1). In
 summary, in 2015, 2016 and 2017, three biological replicates of each of
 the three genotypes and condition (permanent or transplanted) were
 cultivated in each EG, that is,<i> </i>three permanently grown
 clones and three transplanted clones of each of the three genotypes were
 cultivated in each EG, totalling 18 plants per EG .
 <i><b>MSAP analysis</b></i>
 MSAP markers were used to estimate epigenetic variability
 according to Cara et al. (2013). Three selective primer combinations
 without fluorescence were assayed:
 <i>Eco</i>RI+AGC⁄<i>Hpa</i>II-<i>Msp</i>I+ATC, <i>Eco</i>RI+AGA⁄<i>Hpa</i>II-<i>Msp</i>I+ATC and <i>Eco</i>RI+ACA⁄<i>Hpa</i>II-<i>Msp</i>I+ATC. The amplification products were separated by electrophoresis on a 6% denaturing polyacrylamide gel, silver stained, and scanned for manual scoring. To calculate the error rate, a subset of 12 samples were analysed in duplicate for each season, using the same extracted DNA. From the <i>Eco</i>RI/<i>Hpa</i>II and <i>Eco</i>RI/<i>Msp</i>I raw data, each locus could present multistate information (MSAP_epiallele). To construct binary epigenetic matrices, each locus in the multistate matrix was split into three subloci (or subepiloci; MSAP_epiloci) following the codification ‘Mixed Scoring 2’ described by Schulz et al. (2013): fragments present in both the <i>Hpa</i>II and <i>Msp</i>I lanes were codified as Type I, fragments present in the <i>Hpa</i>II lane but absent in <i>Msp</i>I lane were codified as Type II, and fragments present in the <i>Msp</i>I lane but absent in <i>Hpa</i>II lane were codified as Type III. Then, two types of methylation states were considered: non-methylated (Type I) and methylated (types II +III)<i>. </i>In the methylated state, both amplification patterns (II + III) were clustered in one category according to the findings of Fulneček and Kovařík (2014), who indicated that the hemi-methylated state (fragment presence in the <i>Hpa</i>II lane) could not be determined with accuracy. The absence of fragments on both lanes was not used for calculating the methylation state because it could be the result of genetic changes. <i><b>Phenotypic characterisation</b></i> All variables described below were measured for all the plants cultivated in 2015, 2016 and 2017 at both EGs.<i><b> </b></i>The 6<sup>th</sup> and 7<sup>th</sup> fully expanded leaves were removed from plants in anthesis, wrapped with aluminium foil and conserved on ice. Once in the laboratory, leaves were photographed and the total leaf area, terminal leaflet area and rachis length were measured with AxioVison software (Carl Zeiss MicroImaging GmbH, Jena, Deutschland); leaflet number was also registered. Leaflet thickness was estimated through the area/weight ratio in two 2 cm<sup>2</sup> leaflet discs dried at 40°C until reaching a constant weight. Photoprotective pigments as ultraviolet absorbing compounds (UVAC) and anthocyanins were determined in the leaflet discs according to Ibañez et al. (2017). At harvest, the total tuber number and total and mean tuber weight were recorded for each plant, except in the 2017 season in which data from EG-2100 had to be excluded due to the harmful action of stray animals on both substrate and tubers. Harvested tubers were stored at 4ºC and periodically examined for sprouting (dormancy breakdown). Sprouted tubers were maintained at room temperature in darkness, and the sprouting percentage and sprout length were recorded every two weeks for 60 days.