BACKGROUND: DNA methylation can change rapidly to regulate the expression of stress-responsive genes. Previous studies have shown that there are significant differences in the cold resistance of winter rapeseed (Brassica rapa L.) after being domesticated in different selection environments; however, little is known about the epigenetic regulatory mechanisms of its cold resistance formation.
METHODS: Four winter rapeseed materials (\'CT-2360\', \'MXW-1\', \'2018-FJT\', and \'DT-7\') domesticated in different environments were selected to analyze the DNA methylation level and pattern changes under low temperature using methylation-sensitive amplified polymorphism technology with 60 primer pairs.
RESULTS: A total of 18 pairs of primers with good polymorphism were screened, and 1426 clear bands were amplified, with 594 methylation sites, accounting for 41.65% of the total amplified bands. The total methylation ratios of the four materials were reduced after low-temperature treatment, in which the DNA methylation level of \'CT-2360\' was higher than that of the other three materials; the analysis of methylation patterns revealed that the degree of demethylation was higher than that of methylation in \'MXW-1\', \'2018-FJT\', and \'DT-7\', which were 22.99%, 19.77%, and 24.35%, respectively, and that the methylation events in \'CT-2360\' were predominantly dominant at 22.95%. Fifty-three polymorphic methylated DNA fragments were randomly selected and further analyzed, and twenty-nine of the cloned fragments were homologous to genes with known functions. The candidate genes VQ22 and LOC103871127 verified the existence of different expressive patterns before and after low-temperature treatment.
CONCLUSIONS: Our work implies the critical role of DNA methylation in the formation of cold resistance in winter rapeseed. These results provide a comprehensive insight into the adaptation epigenetic regulatory mechanism of Brassica rapa L. to low temperature, and the identified differentially methylated genes can also be used as important genetic resources for the multilateral breeding of winter-resistant varieties.

Background  The medial sural artery perforator (MSAP) flap was described by Cavadas et al in 2001. The aim of this study was to analyze the flap characteristics in the regional population and was planned as a cadaveric dissection study. Methods  Thirty-three legs of fresh cadavers were studied for perforator characteristics, length, and origin of pedicle and skin paddle thickness. Observations were documented and analyzed. Results  Seventeen right legs (51.5%) and sixteen left legs (48.5%) were studied. Twenty-five pedicles originated from popliteal artery (86.2%) and four (13.8%) from the common sural trunk. No perforators were seen in four legs. The mean number of perforators is 2 (0-6). The mean distance of perforator from midpoint of popliteal fossa was 10.7 cm (8-13 cm) and from posterior midline it was 3.2 cm. The mean size of the perforator was 1.1 ± 0.8 mm (0.8-1.5 mm). The mean pedicle length was 9.3 ± 1.3 cm. The mean flap thickness was 4.3 ± 0.7 mm (3.0-5.5 mm). There was no correlation for flap or perforator characteristics with side of leg. Conclusion  This study concludes that MSAP is a good flap in terms of perforator characteristics, pedicle length, and flap thickness, when a medium sized thin flap with long pedicle is needed. The location of perforator on calf varies in different population. Being a perforator flap, anatomical variability is common and should be thought of while choosing this flap.

In the present work, physio-biochemical and DNA methylation analysis were conducted in wheat (Triticum aestivum L.) cultivars \"Bolani\" (drought-tolerant) and \"Sistan\" (drought-sensitive) during drought treatments: well-watered (at 90% field capacity (FC)), mild stress (at 50% FC, and severe stress (at 25% FC). During severe stress, O2•- and H2O2 content in cultivar Sistan showed significant increase (by 1.3 and 2.5-fold, respectively) relative to cultivar Bolani. In Bolani, the increased levels of radical scavenging activity (by 32%), glycine betaine (GB) (by 11.44%), proline (4-fold), abscisic acid (by 63.76%), and more stability of relative water content (RWC) (2-fold) were observed against drought-induced oxidative stress. Methylation level significantly decreased from 70.26% to 60.64% in Bolani and from 69.06% to 59.85% in Sistan during stress, and higher decreased tendency was related to CG and CHG in Bolani but CG in Sistan under severe stress. Methylation patterns showed that the highest polymorphism in Bolani was mainly as CG. As the intensity of stress increased, the enhanced physio-biochemical responses of Bolani cultivar were accompanied by a more decrease in the number of unchanged bands. According to heat map analysis, the highest difference (84.38%) in methylation patterns was observed between control and severe stress. Multivariate analysis using principal component analysis (PCA) showed a cultivar-specific methylation during stress and that methylation changes between cultivars are much higher than that of within a cultivar. Higher methylation to demethylation in Bolani (30.06 vs. 22.12%) compared to that of cultivar Sistan (23.21 vs. 30.15%) indicated more demethylation did not induce tolerance responses in Sistan. Sequencing differentially methylated fragments along with qRT-PCR analysis showed the efficient role of various DNA fragments, including demethylated fragments such as phosphoenol pyruvate carboxylase (PEPC), beta-glucosidase (BGlu), glycosyltransferase (GT), glutathione S-transferase (GST) and lysine demethylase (LSD) genes and methylated fragments like ubiquitin E2 enzyme genes in the development of drought tolerance. These results suggested the specific roles of DNA methylation in development of drought tolerance in wheat landrace.

DNA methylation plays a critical role in the modulation of gene expression. The role of DNA methylation in sex determination was investigated in spinach. The differentiated cytosine CpG methylation profiles of CCGG motifs were assessed with methylation sensitivity amplification polymorphism (MSAP) in spinach. Among 442 DNA fragments from four plants, 134 methylated fragments were found. Relative proportions of methylation sites were 28.8% in male plants and 31.8% in female plants. At the same time, cytosine methylation levels were higher in females than in males in CCGG motifs of genomes in the spinach. These findings suggest that methylation of CG islands is involved in sex determination and differentiation in spinach.

Whether conifers can withstand environmental changes especially temperature fluctuations has been controversial. Epigenetic analysis may provide new perspectives for solving the issue. Pseudotaxus chienii is an endangered gymnosperm species endemic to China. In this study, we have examined the genetic and epigenetic variations in its natural populations aiming to disentangle the synergistic effects of climate and soil on its population (epi)genetic differentiation by using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MSAP) techniques. We identified 23 AFLP and 26, 7, and 5 MSAP outliers in P. chienii. Twenty-one of the putative adaptive AFLP loci were found associated with climate and/or soil variables including precipitation, temperature, K, Fe, Zn, and Cu, whereas 21, 7, and 4 MSAP outliers were significantly related to precipitation of wettest month (Bio13), precipitation driest of month (Bio14), percent tree cover (PTC), and soil Fe, Mn, and Cu compositions. Total precipitation and precipitation in the driest seasons were the most influential factors for genetic and epigenetic variation, respectively. In addition, a high full-methylation level and a strong correlation between genetic and epigenetic variation were detected in P. chienii. Climate is found of greater importance than soil in shaping adaptive (epi)genetic differentiation, and the synergistic effects of climate and climate-soil variables were also observed. The identified climate and soil variables should be considered when applying ex situ conservation.

BACKGROUND: Sphagneticola trilobata (L.) Pruski is a prevalent and widely distributed invasive plant in South China. To investigate the molecular mechanisms underlying its rapid adaptation, we employed DNA methylation-sensitive amplified polymorphism (MSAP) and simple sequence repeat (SSR) analysis to study 60 S. trilobata individuals collected from Fuzhou (FZ), Haikou (HK), Jinghong (JH) and Guangzhou (GZ).
RESULTS: In this study, we computed the Shannon diversity index (I) of SSR and MSAP as 0.354 and 0.303, respectively. The UPGMA phylogenetic tree and PCoA analyses showed that MSAP had a better discriminatory power to distinguish populations from different regions. Notably, the GZ population was found to be the most distinct from the other three populations. Moreover, Mantel analysis revealed a significantly higher correlation between epigenetic distance and geographic distance as compared to genetic distance and geographic distance. Consequently, the correlation between epigenetic distance and geographic distance observed to be markedly stronger than that between genetic distance and geographical distance on Mantel analysis.
CONCLUSIONS: The S. trilobata populations in various regions displayed a high of complementary genetic and epigenetic diversity, which was a key feature contributing to their rapid invasion. Interestingly, the correlation between epigenetics and geographical distance was significantly stronger than that observed for genetics and geographical distance. These findings indicated that the epigenetic mechanism of S. trilobar exhibited high plasticity, leading to significant differences in methylation pattern across different populations.

Forest species in the course of their evolution have experienced several environmental challenges, which since historic times include anthropogenic pollution. The effects of pollution on the genetic and epigenetic diversity in black pine (Pinus nigra) forests were investigated in the Amyntaio - Ptolemais - Kozani Basin, which has been for decades the largest lignite mining and burning center of Greece, with a total installed generating capacity of about 4.5 GW, operating for more than 70 years and resulting in large amounts of primary air pollutant emissions, mainly SO2, NOx and PM10. P. nigra, a biomarker for air pollution and a keystone species of affected natural ecosystems, was examined in terms of phenology (cone and seed parameters), genetics (283 AFLP loci) and epigenetics (606 MSAP epiloci), using two populations (exposed to pollution and control) of the current (mature trees) and future (embryos) stand. It was found that cone, seed, as well as genetic diversity parameters, did not show statistically significant differences between the exposed population and the control. Nevertheless, statistically significant differences were detected at the population epigenetic level. Moreover, there was a further differentiation regarding the intergenerational comparison: while the epigenetic diversity does not substantially change in the two generations assessed in the control population, epigenetic diversity is significantly higher in the embryo population compared to the parental stand in the exposed population. This study sheds a light to genome dynamics in a forest tree population exposed to long term atmospheric pollution burden and stresses the importance of assessing both genetics and epigenetics in biomonitoring applications.

Soil heavy metal pollution is one of the most challenging problems. Kenaf is an important natural fiber crop with strong heterosis and a higher tolerance to heavy metals. However, little is known about the molecular mechanisms of kenaf heavy metal tolerance, especially the mechanism of genomic DNA methylation regulating heterosis. In this study, kenaf cultivars CP085, CP089, and their hybrid F1 seedlings were subjected to 300 µM cadmium stress and found obvious heterosis of cadmium resistance in morphology and antioxidant enzyme activity of F1 hybrid seedlings. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that the total DNA methylation level under cadmium decreased by 16.9 % in F1 and increased by 14.0 % and 3.0 % in parents CP085 and CP089, respectively. The hypomethylation rate was highest (21.84 %), but hypermethylation was lowest (17.24 %) in F1 compared to parent cultivars. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between F1 and its parents under cadmium. Furthermore, 21 differentially methylated DNA fragments (DMFs) were analyzed. Especially, the expression of NPF2.7, NADP-ME, NAC71, TPP-D, LRR-RLKs, and DHX51 genes were changed due to cadmium stress and related to cytosine methylation regulation. Finally, the knocked-down of the differentially methylated gene NPF2.7 by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under cadmium stress. It is speculated that low DNA methylation levels can regulate gene expression that led to the heterosis of cadmium tolerance in kenaf.

Age information is often non-existent for most shark populations due to a lack of measurable physiological and morphological traits that can be used to estimate age. Recently, epigenetic clocks have been found to accurately estimate age for mammals, birds, and fish. However, since these clocks rely, among other things, on the availability of reference genomes, their application is hampered in non-traditional model organisms lacking such molecular resources. The technique known as Methyl-Sensitive Amplified Polymorphism (MSAP) has emerged as a valid alternative for studying DNA methylation biomarkers when reference genome information is missing, and large numbers of samples need to be processed. Accordingly, the MSAP technique was used in the present study to characterize global DNA methylation patterns in lemon sharks from three different age groups (juveniles, subadults, and adults). The obtained results reveal that, while MSAP analyses lack enough resolution as a standalone approach to infer age in these organisms, the global DNA methylation patterns observed using this technique displayed significant differences between age groups. Overall, these results confer that DNA methylation does change with age in sharks like what has been seen for other vertebrates and that MSAP could be useful as part of an epigenetics pipeline to infer the broad range of ages found in large samples sizes.

The genetic incompatibility of the seedlings which are used as rootstocks (stock-scion interactions) and the mechanical stress induced by grafting are two major factors responsible for the high intraclonal variations observed in tree crops which are propagated through bud grafting. Since stress-induced DNA methylation changes associated with heterografting is a major contributor of such variations in grafted tree crops, a proper assessment of this epigenetic phenomenon is inevitable to devise strategies for the development of more uniform planting materials with minimal intraclonal variations in the future. In order to evaluate and establish the effects of heterografting on the epigenome of plants, availability of ideal plant materials and a standard procedure for testing is very essential. Development of genetically uniform own-rooted seedlings through induction of cleavage polyembryony by a novel technique of half ovulo embryo culture is the first step. Grafting of buds from these genetically and epigenetically uniform plants to genetically divergent rootstock and identification of DNA methylation polymorphism among them forms the second part of the methodology for detecting epigenetic changes associated with grafting in tree crops. Methylation-sensitive amplification polymorphism technique (MSAP), a modified version of AFLP using a pair of methylation-sensitive and insensitive isoschizomers (such as HpaII and MspI), is an ideal methodology to assess DNA methylation polymorphisms on a genomic scale in such plants. Comparative analysis of two sets of restriction digestion products (EcoRI/HpaII and EcoRI/MspI) allows the identification of DNA methylation polymorphisms induced by grafting and will aid in the detection of differentially methylated regions (DMRs) among grafted plants. This chapter describes a detailed protocol for inducing multiple embryos of single zygotic origin and regeneration of seedlings in rubber tree (Hevea brasiliensis), grafting of buds from these genetically uniform own-rooted seedlings to divergent rootstocks, identification of epigenetic changes induced by grafting or stock-scion interactions through MSAP analysis, and locating the differentially methylated genomic region. The methodology described here could be applied to any tree species commercially propagated through grafting for detecting epigenetic changes putatively associated with intraclonal variability.