*3.4.1. DNA methylation*

DNA methylation refers to the covalent addition of methyl group to the cytosine base at position 5 by the action of DNA methyl transferases. In mammals, cytosine methylation occurs mostly at CG sites and rarely at non-CG sites, while in plants, cytosine methylation can occur in both CG and non-CG contexts. Non-CG methylation involves both symmetrical and asymmetrical sites, CHG and CHH, respectively (H = A, T, or C). Much of our knowledge with respect to DNA methylation is based on the studies performed on model plant *Arabidopsis thaliana*. DNA methylation in plants is being catalyzed principally by three different enzymes. The maintenance of symmetrical CG methylation during DNA replication is carried out by *Methyltransferase1* (*MET1*) (homolog of animal DNA methyltransferase DNMT1), while CHG methylation is catalyzed by the plant-specific *chromomethylase 3* (CMT3) and asymmetric CHH methylation is mediated by *domains rearranged methyltransferase 2* (*DRM2*) (similar to the mammalian *DNMT3* family) activity, which works through RNA-directed DNA methylation (RdDM) pathway [83, 84].

The first ever single-base resolution methylomes of tomato fruits were established, which revealed that fruit epigenome is not static, and the changes occur continuously during different stages of fruit development. The whole genome bisulfite sequencing was employed to study four different stages of fruit development. This study identified 52,095 differentially methy‐ lated regions of the 90% of the genome covered in this analysis in wild-type tomato fruits. Comparative analysis of fruits from two nonripening mutants of tomato viz ripening-inhibitor (rin) and Colorless nonripening (Cnr) demonstrated the changes in the methylation patterns in the wild type and the mutants [85]. The *Cnr* mutation in tomato restricts normal ripening process in tomato resulted in a colorless fruits develop a colorless pericarp [86]. Silencing of the *SlCMT3* gene in tomato resulted in the increased expression of *LeSPL-CNR* that encodes for SBP-box transcription factor, which was located in the *Cnr* locus that ultimately triggers *Cnr* fruits to ripen normally. These studies revealed that the induced ripening of *Cnr* fruits is associated with a reduction of methylation at CHG sites of the *LeSPL-CNR* promoter, while a decrease of DNA methylation in differentially methylated regions associated with the *LeMADS-RIN* binding sites [87, 88].

#### *3.4.2. Histone modifications*

The interaction between DNA and proteins has a crucial role in the regulation of gene expression. Chromatin immunoprecipitation (ChIP) can be employed to study such interac‐ tions. These interactions can be explored using a technique called ChIP, microarray platforms (ChIP-on-chip or ChIP-chip) [89, 90]. More recently, NGS-based techniques are being used for studying histone modifications where ChIP-Seq combines ChIP with massively parallel direct sequencing. ChIP-enriched DNA is sequenced directly, using the Solexa/Illumina platform, and the readings were mapped to the reference genome. Histone modification phenomenon includes methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and ADPribosylation. These modifications bring changes directly and cause structural changes to the chromatin or indirectly through the mediator proteins. All histone modifications are reversible and provide versatile ways for regulating gene expression during plant development and their responses to environmental stimuli. The study found that the reversible acetylation and deacetylation of specific *Lys* residues on core histone N-terminal tails catalyzed by histone acetyltransferases (HDA) and histone deacetylases (HDAC), respectively [91, 92]. The action of both enzymes regulates biological processes like transcriptional regulation. It was found that generally, hyperacetylated histones are associated with gene activation, whereas hypoa‐ cetylated histones were involved in gene inactivation. ChIP-seq was employed to identify the targets of *ASR1* starting out with the purification of *ASR1*, by using the high-quality anti-*ASR1* antibody. ChIP-seq data generated through this helped in identifying the genes encoding aquaporins and those associated with the cell wall; these genes were associated with drought stress response [93]. There are several studies reported where ChIP-seq along with ChIP-chip methods were used to search genomes for locations associated with binding of several transcription factors (TFs) such as *RIN* and *fruitful* homologs (FUL1/FUL2) [85, 94, 95]. The investigation of genome-wide targets for the main regulators of fruit ripening viz. *RIN*, *FUL1*, and *FUL2* by combining RNA-Seq with ChIP-chip assay identified a total of 292, 860, and 878 target ripening-associated genes in tomato [85, 95]. Therefore, a combination of ChIPseq and RNA-Seq with ChIP-chip are imperative tools nowadays and can be employed for better understanding of transcriptional networks underlying tomato development.

#### **3.5. Noncoding RNA (ncRNAs) in crop improvement**

changes in the alteration of DNA sequences but are triggered by chemical modifications on the DNA (cytosine methylation) or on histone modifications (e.g., acetylation, methylation) bringing about modulation of chromatin structure and function [83]. In recent years, small RNAs have been emerged as key players in controlling epigenetic changes throughout the

DNA methylation refers to the covalent addition of methyl group to the cytosine base at position 5 by the action of DNA methyl transferases. In mammals, cytosine methylation occurs mostly at CG sites and rarely at non-CG sites, while in plants, cytosine methylation can occur in both CG and non-CG contexts. Non-CG methylation involves both symmetrical and asymmetrical sites, CHG and CHH, respectively (H = A, T, or C). Much of our knowledge with respect to DNA methylation is based on the studies performed on model plant *Arabidopsis thaliana*. DNA methylation in plants is being catalyzed principally by three different enzymes. The maintenance of symmetrical CG methylation during DNA replication is carried out by *Methyltransferase1* (*MET1*) (homolog of animal DNA methyltransferase DNMT1), while CHG methylation is catalyzed by the plant-specific *chromomethylase 3* (CMT3) and asymmetric CHH methylation is mediated by *domains rearranged methyltransferase 2* (*DRM2*) (similar to the mammalian *DNMT3* family) activity, which works through RNA-directed DNA methylation

The first ever single-base resolution methylomes of tomato fruits were established, which revealed that fruit epigenome is not static, and the changes occur continuously during different stages of fruit development. The whole genome bisulfite sequencing was employed to study four different stages of fruit development. This study identified 52,095 differentially methy‐ lated regions of the 90% of the genome covered in this analysis in wild-type tomato fruits. Comparative analysis of fruits from two nonripening mutants of tomato viz ripening-inhibitor (rin) and Colorless nonripening (Cnr) demonstrated the changes in the methylation patterns in the wild type and the mutants [85]. The *Cnr* mutation in tomato restricts normal ripening process in tomato resulted in a colorless fruits develop a colorless pericarp [86]. Silencing of the *SlCMT3* gene in tomato resulted in the increased expression of *LeSPL-CNR* that encodes for SBP-box transcription factor, which was located in the *Cnr* locus that ultimately triggers *Cnr* fruits to ripen normally. These studies revealed that the induced ripening of *Cnr* fruits is associated with a reduction of methylation at CHG sites of the *LeSPL-CNR* promoter, while a decrease of DNA methylation in differentially methylated regions associated with the

The interaction between DNA and proteins has a crucial role in the regulation of gene expression. Chromatin immunoprecipitation (ChIP) can be employed to study such interac‐ tions. These interactions can be explored using a technique called ChIP, microarray platforms (ChIP-on-chip or ChIP-chip) [89, 90]. More recently, NGS-based techniques are being used for studying histone modifications where ChIP-Seq combines ChIP with massively parallel direct

plant genome.

*3.4.1. DNA methylation*

260 Next Generation Sequencing - Advances, Applications and Challenges

(RdDM) pathway [83, 84].

*LeMADS-RIN* binding sites [87, 88].

*3.4.2. Histone modifications*

Recent advances in next-generation genome and transcriptome sequencing with thorough bioinformatics and computational analysis laid to the discovery of numerous RNA types. The ncRNAs are one of the great examples of such techniques. The ncRNAs has emerged as a key product of eukaryotic transcriptionary machinery with a critical role in the regulatory mechanism. The ncRNAs are being classified as housekeeping ncRNAs and regulatory ncRNAs [96]. The rRNAs, tRNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) are under the "housekeeping" ncRNAs, whereas the "regulato‐ ry" ncRNAs are known as small ncRNAs (such as miRNAs and siRNAs) and long noncoding RNA (lncRNAs) [96, 97].
