Developmental Gene Regulation Research Articles

Genetic cold cases: lessons from solving complex congenital limb disorders.

Congenital genetic disorders affecting limb morphology in humans and other mammals are particularly well described, due to both their rather high frequencies of occurrence and the ease of their detection when expressed as severe forms. In most cases, their molecular and cellular etiology remained unknown long after their initial description, often for several decades, and sometimes close to a century. Over the past 20 yr, however, experimental and conceptual advances in our understanding of gene regulation, in particular over large genomic distances, have allowed these cold cases to be reopened and, eventually, for some of them to be solved. These investigations led not only to the isolation of the culprit genes and mechanisms, but also to the understanding of the often complex regulatory processes that are disturbed in such mutant genetic configurations. Here, we present several cases in which dormant regulatory mutations have been retrieved from the archives, starting from a historical perspective up to their molecular explanations. While some cases remain open, waiting for new tools and/or concepts to bring their investigations to an end, the solutions to others have contributed to our understanding of particular features often found in the regulation of developmental genes and hence can be used as benchmarks to address the impact of noncoding variants in the future.

RRS1 shapes robust root system to enhance drought resistance in rice.

A strong root system facilitates the absorption of water and nutrients from the soil, to improve the growth of crops. However, to date, there are still very few root development regulatory genes that can be used in crop breeding for agriculture. In this study, we cloned a negative regulator gene of root development, Robust Root System 1 (RRS1), which encodes an R2R3-type MYB family transcription factor. RRS1 knockout plants showed enhanced root growth, including longer root length, longer lateral root length, and larger lateral root density. RRS1 represses root development by directly activating the expression of OsIAA3 which is involved in the auxin signaling pathway. A natural variation in the coding region of RRS1 changes the transcriptional activity of its protein. RRS1T allele, originating from wild rice, possibly increases root length by means of weakening regulation of OsIAA3. Knockout of RRS1 enhances drought resistance by promoting water absorption and improving water use efficiency. This study provides a new gene resource for improving root systems and cultivating drought-resistant rice varieties with important values in agricultural applications.

Evolution of Spatial and Temporal cis-Regulatory Divergence in Sticklebacks.

Cis-regulatory changes are thought to play a major role in adaptation. Threespine sticklebacks have repeatedly colonized freshwater habitats in the Northern Hemisphere, where they have evolved a suite of phenotypes that distinguish them from marine populations, including changes in physiology, behavior, and morphology. To understand the role of gene regulatory evolution in adaptive divergence, here we investigate cis-regulatory changes in gene expression between marine and freshwater ecotypes through allele-specific expression (ASE) in F1 hybrids. Surveying seven ecologically relevant tissues, including three sampled across two developmental stages, we identified cis-regulatory divergence affecting a third of genes, nearly half of which were tissue-specific. Next, we compared allele-specific expression in dental tissues at two timepoints to characterize cis-regulatory changes during development between marine and freshwater fish. Applying a genome-wide test for selection on cis-regulatory changes, we find evidence for lineage-specific selection on several processes between ecotypes, including the Wnt signaling pathway in dental tissues. Finally, we show that genes with ASE, particularly those that are tissue-specific, are strongly enriched in genomic regions of repeated marine-freshwater divergence, supporting an important role for these cis-regulatory differences in parallel adaptive evolution of sticklebacks to freshwater habitats. Altogether, our results provide insight into the cis-regulatory landscape of divergence between stickleback ecotypes across tissues and during development, and support a fundamental role for tissue-specific cis-regulatory changes in rapid adaptation to new environments.

Rapid evolution of insecticide resistance in the Colorado potato beetle, Leptinotarsa decemlineata.

Despite considerable research, efforts to manage insecticide resistance continue to fail. The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), epitomizes this problem, as it has repeatedly and rapidly evolved resistance to>50 insecticides. The patterns of resistance evolution are intriguing, as they defy models where resistance evolves from rare mutations. Here, we synthesize recent research on insecticide resistance in CPB showing that polygenic resistance drawn from standing genetic diversity explains genomic patterns of insecticide resistance evolution. However, rapid gene regulatory evolution suggests that other mechanisms might also facilitate adaptive change. We explore the hypothesis that sublethal stress from insecticide exposure could alter heritable epigenetic modifications, and discuss the range of experimental approaches needed to fully understand insecticide resistance evolution in this super pest.

The function and evolution of a genetic switch controlling sexually dimorphic eye differentiation in honeybees

Animals develop sex-specific morphological structures that are diverse between organisms. However, understanding the developmental and evolutionary mechanisms governing these traits is still limited and largely restricted to DM domain genes, which are conserved, sex-specific developmental regulators identified in genetic models. Here, we report a sex-specific developmental regulator gene, glubschauge (glu) that selectively regulates sexually dimorphic eye differentiation in honeybees. We found that the sex determination gene feminizer (fem) controls sex-specific splicing of glu transcripts, establishing a genetic switch in which Glu proteins with a zinc finger (ZnF) domain are only expressed in females. We showed that female coding sequence was essential and sufficient for partial feminization. Comparative sequence and functional studies revealed that the evolutionary origination of the genetic switch was followed by the mutational origin of the essential ZnF domain. Our results demonstrate that glu is a newly evolved sex-specific genetic switch for region-specific regulation of a dimorphic character.

The Role of IRX Homeobox Genes in Hematopoietic Progenitors and Leukemia.

IRX genes are members of the TALE homeobox gene class and encode six related transcription factors (IRX1-IRX6) controlling development and cell differentiation of several tissues in humans. Classification of TALE homeobox gene expression patterns for the hematopoietic compartment, termed TALE-code, has revealed exclusive IRX1 activity in pro-B-cells and megakaryocyte erythroid progenitors (MEPs), highlighting its specific contribution to developmental processes at these early stages of hematopoietic lineage differentiation. Moreover, aberrant expression of IRX homeobox genes IRX1, IRX2, IRX3 and IRX5 has been detected in hematopoietic malignancies, including B-cell precursor acute lymphoblastic leukemia (BCP-ALL), T-cell ALL, and some subtypes of acute myeloid leukemia (AML). Expression analyses of patient samples and experimental studies using cell lines and mouse models have revealed oncogenic functions in cell differentiation arrest and upstream and downstream genes, thus, revealing normal and aberrant regulatory networks. These studies have shown how IRX genes play key roles in the development of both normal blood and immune cells, and hematopoietic malignancies. Understanding their biology serves to illuminate developmental gene regulation in the hematopoietic compartment, and may improve diagnostic classification of leukemias in the clinic and reveal new therapeutic targets and strategies.

Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells

The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimulation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to developmental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.

Diversity of σ66-Specific Promoters Contributes to Regulation of Developmental Gene Expression in Chlamydia trachomatis.

Promoter recognition by the RNA polymerase (RNAP) holoenzyme is a key step in gene regulation. In Chlamydia trachomatis, a medically important obligate intracellular bacterium, σ66 allows the RNAP to initiate promoter-specific transcription throughout the chlamydial developmental cycle. Here, we investigated the intrinsic properties of σ66-specific promoters with emphasis on their role in the developmental gene expression of C. trachomatis. First, we examined whether promoters that contain a 5'-T(-15)G(-14)-3' (TG) motif upstream from the -10 element appear more often than others in genes that are preferentially expressed during the early, middle, or late stages of the C. trachomatis developmental cycle. We then determined the critical genetic elements that are required for transcription initiation in vitro. We also assessed the activity of promoters in the presence of Scc4, which can directly interact with σ66RNAP. Finally, we evaluated the promoter-specific dynamics during C. trachomatis infection using a reporter assay. These results reveal that the TG motif is an important determinant in certain early or late promoters. The TG promoters that have the -35 element are recognized by σ66RNAP and Scc4 differently from those lacking the -35 element. Based on these properties, the σ66-specific promoters can fall into three classes. Architectural diversity, behavioral plasticity, and the specific interplays between promoters and the σ66RNAP likely contribute to developmental gene transcription in C. trachomatis. IMPORTANCE Meticulous promoter elucidation is required to understand the foundations of transcription initiation. However, knowledge of promoter-specific transcription remains limited in C. trachomatis. This work underscores the structural and functional plasticity of σ66-specific promoters that are regulated by σ66RNAP, as well as their importance in the developmental gene regulation of C. trachomatis.

Designing rice panicle architecture via developmental regulatory genes.

Rice panicle architecture displays remarkable diversity in branch number, branch length, and grain arrangement; however, much remains unknown about how such diversity in patterns is generated. Although several genes related to panicle branch number and panicle length have been identified, how panicle branch number and panicle length are coordinately regulated is unclear. Here, we show that panicle length and panicle branch number are independently regulated by the genes Prl5/OsGA20ox4, Pbl6/APO1, and Gn1a/OsCKX2. We produced near-isogenic lines (NILs) in the Koshihikari genetic background harboring the elite alleles for Prl5, regulating panicle rachis length; Pbl6, regulating primary branch length; and Gn1a, regulating panicle branching in various combinations. A pyramiding line carrying Prl5, Pbl6, and Gn1a showed increased panicle length and branching without any trade-off relationship between branch length or number. We successfully produced various arrangement patterns of grains by changing the combination of alleles at these three loci. Improvement of panicle architecture raised yield without associated negative effects on yield-related traits except for panicle number. Three-dimensional (3D) analyses by X-ray computed tomography (CT) of panicles revealed that differences in panicle architecture affect grain filling. Importantly, we determined that Prl5 improves grain filling without affecting grain number.

Synergistic interaction of two bHLH transcription factors positively regulates artemisinin biosynthetic pathway in Artemisia annua L.

The wonder drug artemisinin, a sesquiterpene lactone endoperoxide from Artemisia annua is the million-dollar molecule required to curb the deadliest disease, Malaria. One of the major challenges even today is to increase the concentration of artemisinin within plants. The transcription factors are important regulators of plant secondary metabolites and have the potential to regulate key steps or the whole biosynthetic pathway. In this study, we have identified and characterized two bHLH transcription factors (Aa6119 and Aa7162) from A. annua. Both the transcription factors turned out to be transcriptionally active and nuclear-localised typical bHLH proteins. In our study, we found that Aa6119 specifically binds to the E-box element present on the promoter of artemisinin biosynthetic gene, AMORPHA-4,11-DIENE SYNTHASE (ADS). The protein-DNA interaction confirmed by Yeast One-hybrid assay was specific as Aa6119 was unable to bind to the mutated E-boxes of ADS. Further, Aa6119 interacted physically with Aa7162, which was confirmed in vitro by Yeast Two-hybrid assay and in vivo by Bimolecular Fluorescent complementation assay. Our quantitative expression studies have confirmed that Aa6119 and Aa7162 act synergistically in the regulation of artemisinin biosynthetic and trichome developmental genes. The higher accumulation of artemisinin content in the transient co-transformed transgenic plants than in the individual over-expression transgenic plants has further validated that Aa6119 and Aa7162 act positively and synergistically to regulate artemisinin accumulation.

A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10-another important ENS developmental regulator gene-GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.

Mechanisms of Male Reproductive Toxicity of Polybrominated Diphenyl Ethers.

Polybrominated diphenyl ethers (PBDE) are a group of flame retardants used in a variety of artificial materials. Despite being phased out in most industrial countries, they remain in the environment and human tissues due to their persistence, lipophilicity, and bioaccumulation. Populational and experimental studies demonstrate the male reproductive toxicity of PBDEs including increased incidence of genital malformations (hypospadias and cryptorchidism), altered weight of testes and other reproductive tissues, altered testes histology and transcriptome, decreased sperm production and sperm quality, altered epigenetic regulation of developmental genes in spermatozoa, and altered secretion of reproductive hormones. A broad range of mechanistic hypotheses of PBDE reproductive toxicity has been suggested. Among these hypotheses, oxidative stress, the disruption of estrogenic signaling, and mitochondria disruption are affected by PBDE concentrations much higher than concentrations found in human tissues, making them unlikely links between exposures and adverse reproductive outcomes in the general population. Robust evidence suggests that at environmentally relevant doses, PBDEs and their metabolites may affect male reproductive health via mechanisms including AR antagonism and the disruption of a complex network of metabolic signaling.

Dynamic regulation of gene expression and morphogenesis in the zebrafish embryo test after exposure to all-trans retinoic acid

The zebrafish embryotoxicity test (ZET) is widely used in developmental toxicology. The analysis of gene expression regulation in ZET after chemical exposure provides mechanistic information about the effects of chemicals on morphogenesis in the test. The gene expression response magnitude has been shown to change with exposure duration. The objective of this work is to study the effect of the exposure duration on the magnitude of gene expression changes in the all-trans retinoic acid (ATRA) signaling pathway in the ZET. Retinoic acid regulation is a key driver of morphogenesis and is therefore employed here as an indicator for the regulation of developmental genes. A teratogenic concentration of 7.5 nM of ATRA was given at 3 hrs post fertilization (hpf) for a range of exposure durations until 120 hrs of development. The expression of a selection of genes related to ATRA signaling and downstream developmental genes was determined. The highest magnitudes of gene expression regulation were observed after 2–24 hrs exposure with an optimal response after 4 hrs. Longer exposures showed a decrease in the gene expression response, although continued exposure to 120 hpf caused malformations and lethality. This study shows that assessment of gene expression regulation at early time points after the onset of exposure in the ZET may be optimal for the prediction of developmental toxicity. We believe these results could help optimize sensitivity in future studies with ZET.

Developmental toxicity of remdesivir, an anti-COVID-19 drug, is implicated by in vitro assays using morphogenetic embryoid bodies of mouse and human pluripotent stem cells.

Remdesivir is an antiviral drug approved for the treatment of COVID-19, whose developmental toxicity remains unclear. More information about the safety of remdesivir is urgently needed for people of childbearing potential, who are affected by the ongoing pandemic. Morphogenetic embryoid bodies (MEBs) are three-dimensional (3D) aggregates of pluripotent stem cells that recapitulate embryonic body patterning in vitro, and have been used as effective embryo models to detect the developmental toxicity of chemical exposures specifically and sensitively. MEBs were generated from mouse P19C5 and human H9 pluripotent stem cells, and used to examine the effects of remdesivir. The morphological effects were assessed by analyzing the morphometric parameters of MEBs after exposure to varying concentrations of remdesivir. The molecular impact of remdesivir was evaluated by measuring the transcript levels of developmental regulator genes. The mouse MEB morphogenesis was impaired by remdesivir at 1-8μM. Remdesivir affected MEBs in a manner dependent on metabolic conversion, and its potency was higher than GS-441524 and GS-621763, presumptive anti-COVID-19 drugs that act similarly to remdesivir. The expressions of developmental regulator genes, particularly those involved in axial and somite patterning, were dysregulated by remdesivir. The early stage of MEB development was more vulnerable to remdesivir exposure than the later stage. The morphogenesis and gene expression profiles of human MEBs were also impaired by remdesivir at 1-8μM. Remdesivir impaired mouse and human MEBs at concentrations that are comparable to the therapeutic plasma levels in humans, urging further investigation into the potential impact of remdesivir on developing embryos.

Comprehensive analysis of gene regulatory dynamics, fitness landscape, and population evolution during sexual reproduction

The fitness landscape is a critical concept in biophysics, evolutionary biology, and genetics that depicts fitness in the genotype space and visualizes the relationship between genotype and fitness. However, the fitness landscape is challenging to characterize because the quantitative relationships between genotype and phenotype and their association to fitness has not been comprehensively well described. To address this challenge, we adopted gene regulatory networks to determine gene expression dynamics. We analyzed how phenotype and fitness are shaped by the genotype in two-gene networks. A two-by-two matrix provided the two-gene regulatory network in which a vector with two angle values (Θ) was introduced to characterize the genotype. Mapping from this angle vector to phenotypes allowed for the classification of steady-state expression patterns of genes into seven types. We then studied all possible fitness functions given by the Boolean output from the on/off expression of the two genes. The possible fitness landscapes were obtained as a function of the genetic parameters Θ. Finally, the evolution of the population distribution under sexual reproduction was investigated in the obtained landscape. We found that the distribution was restricted to a convex region within the landscape, resulting in the branching of population distribution, including the speciation process.

Proteogenomic analysis of cancer aneuploidy and normal tissues reveals divergent modes of gene regulation across cellular pathways.

How cells control gene expression is a fundamental question. The relative contribution of protein-level and RNA-level regulation to this process remains unclear. Here, we perform a proteogenomic analysis of tumors and untransformed cells containing somatic copy number alterations (SCNAs). By revealing how cells regulate RNA and protein abundances of genes with SCNAs, we provide insights into the rules of gene regulation. Protein complex genes have a strong protein-level regulation while non-complex genes have a strong RNA-level regulation. Notable exceptions are plasma membrane protein complex genes, which show a weak protein-level regulation and a stronger RNA-level regulation. Strikingly, we find a strong negative association between the degree of RNA-level and protein-level regulation across genes and cellular pathways. Moreover, genes participating in the same pathway show a similar degree of RNA- and protein-level regulation. Pathways including translation, splicing, RNA processing, and mitochondrial function show a stronger protein-level regulation while cell adhesion and migration pathways show a stronger RNA-level regulation. These results suggest that the evolution of gene regulation is shaped by functional constraints and that many cellular pathways tend to evolve one predominant mechanism of gene regulation at the protein level or at the RNA level.

Integration of multi-omics data reveals cis-regulatory variants that are associated with phenotypic differentiation of eastern from western pigs

BackgroundThe genetic mechanisms that underlie phenotypic differentiation in breeding animals have important implications in evolutionary biology and agriculture. However, the contribution of cis-regulatory variants to pig phenotypes is poorly understood. Therefore, our aim was to elucidate the molecular mechanisms by which non-coding variants cause phenotypic differences in pigs by combining evolutionary biology analyses and functional genomics.ResultsWe obtained a high-resolution phased chromosome-scale reference genome with a contig N50 of 18.03 Mb for the Luchuan pig breed (a representative eastern breed) and profiled potential selective sweeps in eastern and western pigs by resequencing the genomes of 234 pigs. Multi-tissue transcriptome and chromatin accessibility analyses of these regions suggest that tissue-specific selection pressure is mediated by promoters and distal cis-regulatory elements. Promoter variants that are associated with increased expression of the lysozyme (LYZ) gene in the small intestine might enhance the immunity of the gastrointestinal tract and roughage tolerance in pigs. In skeletal muscle, an enhancer-modulating single-nucleotide polymorphism that is associated with up-regulation of the expression of the troponin C1, slow skeletal and cardiac type (TNNC1) gene might increase the proportion of slow muscle fibers and affect meat quality.ConclusionsOur work sheds light on the molecular mechanisms by which non-coding variants shape phenotypic differences in pigs and provides valuable resources and novel perspectives to dissect the role of gene regulatory evolution in animal domestication and breeding.

Ruminant-specific retrotransposons shape regulatory evolution of bovine immunity.

Cattle are an important livestock species, and mapping the genomic architecture of agriculturally relevant traits such as disease susceptibility is a major challenge in the bovine research community. Lineage-specific transposable elements (TEs) are increasingly recognized to contribute to gene regulatory evolution and variation, but this possibility has been largely unexplored in ruminant genomes. We conducted epigenomic profiling of the type II interferon (IFN) response in bovine cells and found thousands of ruminant-specific TEs including MER41_BT and Bov-A2 elements predicted to act as IFN-inducible enhancer elements. CRISPR knockout experiments in bovine cells established that critical immune factors including IFNAR2 and IL2RB are transcriptionally regulated by TE-derived enhancers. Finally, population genomic analysis of 38 individuals revealed that a subset of polymorphic TE insertions may function as enhancers in modern cattle. Our study reveals that lineage-specific TEs have shaped the evolution of ruminant IFN responses and potentially continue to contribute to immune gene regulatory differences across modern breeds and individuals. Together with previous work in human cells, our findings demonstrate that lineage-specific TEs have been independently co-opted to regulate IFN-inducible gene expression in multiple species, supporting TE co-option as a recurrent mechanism driving the evolution of IFN-inducible transcriptional networks.

Sex-biased and parental allele-specific gene regulation by KDM6A

BackgroundKDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer.MethodsKdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus. Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses.ResultsWe report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes.ConclusionsOur study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles.

Epigenomic insight of lingonberry and health-promoting traits during micropropagation

Epigenetic variation plays a role in developmental gene regulation and responses to the environment. An efficient interaction of zeatin-induced cytosine methylation and secondary compounds has been displayed for the first time in tissue-culture shoots/plants of lingonberry (Vaccinium vitis-idaea L.) cultivar Erntedank in vitro (NC1, in a liquid medium; NC2, on a semi-solid medium), ex vitro (NC3, node culture-derived plants; LC1, leaf culture-derived plants) and its cutting-propagated (ED) plants. Through methylation-sensitive amplification polymorphism (MSAP) assay, we observed highest methylated sites in leaf regenerants (LC1) from all primer combinations (108 bands), along with the highest secondary metabolites. The four types of tissue culture-derived shoots/plants (NC1, NC2, NC3, LC1) showed higher methylation bands than cutting propagated donor plants (ED) that exhibited 79 bands of methylation, which is comparatively low. Our study showed more methylation in micropropagated shoots/plants than those derived from ED plants. On the contrary, we observed higher secondary metabolites in ED plants but comparatively less in micropropagated shoots (NC1, NC2) and plants (NC3, LC1).