*3.5.1. Role of long noncoding RNAs in Solanaceae*

The lncRNAs are defined as a non-protein-coding functional RNAs of more than 200 bp in length with regulatory function and principally transcribed by RNA polymerase II. The identification of lncRNA in plants and especially in Solanaceae is still at infancy as compared with the human/animal genome. The application of high-throughput NGS technologies toward identification and the characterizations of lncRNAs are being reported. Recently, by analyzing around 200 *A. thaliana* transcriptome data sets, about 6480 lncRNAs were identified in the intergenic regions of the genome [98]. Further, 439 lncRNAs were identified in maize [99], and in a more comprehensive way by integrating all available data sets for maize transcriptome, high confidence 1704 lncRNAs were identified [100]. However, a systemic study on lncRNAs in Solanaceae has not been done except some few reports. In pepper, a total of 5976 long intergenic ncRNAs (lincRNAs), 222 intronic overlapping lncRNAs, and 329 bidirectional overlapping lncRNAs were identified from RNA-seq data of unopened flower buds [44]. Recently, a genome-wide identification of lncRNAs in tomato was reported [101]. The study identified a total of about 3679 lncRNAs from wild-type AC tomato and mutant ripening fruit (*rin*). The analysis further reported that out of 3530 and 3679 lncRNAs identified in wild-type and *rin* mutant tomatoes, only 23 and 126 lncRNAs were transcribed specifically in wild-type and *rin* mutant tomatoes, respectively. Most of the lncRNAs are derived from intergenic regions. It was also found that 490 lncRNAs were upregulated in ripening mutant fruits, while 187 lncRNAs were downregulated, suggesting the involvement of lncRNAs in the regulation of fruit ripening. However, the function of lncRNAs has not been fully under‐ stood and studied. In a more conclusive study, the role of lncRNAs known as *COOLAIR* (coolassisted intronic noncoding RNA) and *COLDAIR* (cold-assisted intronic noncoding RNA) during vernalization was investigated. These lncRNAs are involved in the epigenetic silencing of *FLC* gene that subsequently promotes flowering [102]. The identification and the charac‐ terization of novel lncRNAs have enormous potential to open new windows for crop im‐ provement. Therefore, databases of lncRNAs named as PLncDB (plant long noncoding RNA database) [103] and PNRD (plant ncRNA Database) [104] have been developed which provide information about the functions and role of lncRNAs in plants.

#### *3.5.2. Role of miRNAs in regulation of gene expression*

MicroRNAs (miRNAs) are approximately 21 nucleotides long in length, and they are a class of noncoding RNAs that play an important role in regulating gene expression in plants [105– 107]. Plant miRNAs mostly exert their effects by cleavage of target mRNA with full comple‐ mentarity, and their target sites are mostly found in coding regions thus altering the gene expression [105–107]. Recent studies have shown that plant miRNAs also repress translation via a slicer-independent mechanism and, therefore, mediates the expression of the genes posttranscriptionally [108, 109].

There are mainly two major approaches for identifying miRNAs in plants: (1) experimental and (2) bioinformatic approaches. An experimental approach includes forward genetics, direct cloning, and next-generation high-throughput sequencing. High-throughput sequencing technology showed significant progress in small RNA identification and has become com‐ monly available and affordable tool nowadays. A large number of miRNAs have been identified by means of high-throughput sequencing and available in online database (http:// www.mirbase.org, accessed June 21, 2014), which currently holds 35,828 mature miRNA products from 223 species. The majority of miRNAs identified so far have been obtained from only a few model plant species, such as *A. thaliana*, *Oryza sativa*, *Glycine max*, and *Medicago truncatula*. Despite the largest family in the plant kingdom, the annotated miRNAs are still very limited in Solanaceae [110–113]. It is necessary to understand the function of miRNAs in Solanaceae. The study of the miRNAs in pepper has been reported based on identification using an *in silico* approach [114]. However, there is a need to employ high-throughput sequencing approaches on the pepper to discover miRNAs. Recently high-throughput sequencing technologies have been employed to identify miRNAs in pepper from ten different tissues such as leaf, stem, root, flower, and six developmental stages of fruits. Based on a bioinformatics pipeline, the researchers successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. Moreover, their miRNA targets were also predicted com‐ putationally, many of which were experimentally validated using 5′ rapid amplification of cDNA ends (RACE) analysis. Among them, one of the confirmed novel targets of miR-396 was a domain-rearranged *methyltransferase*, the major *de novo* methylation enzyme responsible for RNA-directed DNA methylation in plants. These studies carried out using NGS technologies provide a basis for understanding the functional roles of miRNAs in pepper that can be explored for the crop improvement [115].

in the intergenic regions of the genome [98]. Further, 439 lncRNAs were identified in maize [99], and in a more comprehensive way by integrating all available data sets for maize transcriptome, high confidence 1704 lncRNAs were identified [100]. However, a systemic study on lncRNAs in Solanaceae has not been done except some few reports. In pepper, a total of 5976 long intergenic ncRNAs (lincRNAs), 222 intronic overlapping lncRNAs, and 329 bidirectional overlapping lncRNAs were identified from RNA-seq data of unopened flower buds [44]. Recently, a genome-wide identification of lncRNAs in tomato was reported [101]. The study identified a total of about 3679 lncRNAs from wild-type AC tomato and mutant ripening fruit (*rin*). The analysis further reported that out of 3530 and 3679 lncRNAs identified in wild-type and *rin* mutant tomatoes, only 23 and 126 lncRNAs were transcribed specifically in wild-type and *rin* mutant tomatoes, respectively. Most of the lncRNAs are derived from intergenic regions. It was also found that 490 lncRNAs were upregulated in ripening mutant fruits, while 187 lncRNAs were downregulated, suggesting the involvement of lncRNAs in the regulation of fruit ripening. However, the function of lncRNAs has not been fully under‐ stood and studied. In a more conclusive study, the role of lncRNAs known as *COOLAIR* (coolassisted intronic noncoding RNA) and *COLDAIR* (cold-assisted intronic noncoding RNA) during vernalization was investigated. These lncRNAs are involved in the epigenetic silencing of *FLC* gene that subsequently promotes flowering [102]. The identification and the charac‐ terization of novel lncRNAs have enormous potential to open new windows for crop im‐ provement. Therefore, databases of lncRNAs named as PLncDB (plant long noncoding RNA database) [103] and PNRD (plant ncRNA Database) [104] have been developed which provide

MicroRNAs (miRNAs) are approximately 21 nucleotides long in length, and they are a class of noncoding RNAs that play an important role in regulating gene expression in plants [105– 107]. Plant miRNAs mostly exert their effects by cleavage of target mRNA with full comple‐ mentarity, and their target sites are mostly found in coding regions thus altering the gene expression [105–107]. Recent studies have shown that plant miRNAs also repress translation via a slicer-independent mechanism and, therefore, mediates the expression of the genes

There are mainly two major approaches for identifying miRNAs in plants: (1) experimental and (2) bioinformatic approaches. An experimental approach includes forward genetics, direct cloning, and next-generation high-throughput sequencing. High-throughput sequencing technology showed significant progress in small RNA identification and has become com‐ monly available and affordable tool nowadays. A large number of miRNAs have been identified by means of high-throughput sequencing and available in online database (http:// www.mirbase.org, accessed June 21, 2014), which currently holds 35,828 mature miRNA products from 223 species. The majority of miRNAs identified so far have been obtained from only a few model plant species, such as *A. thaliana*, *Oryza sativa*, *Glycine max*, and *Medicago truncatula*. Despite the largest family in the plant kingdom, the annotated miRNAs are still

information about the functions and role of lncRNAs in plants.

*3.5.2. Role of miRNAs in regulation of gene expression*

262 Next Generation Sequencing - Advances, Applications and Challenges

posttranscriptionally [108, 109].

Kim et al. [114] identified miRNAs and their target genes by analyzing expressed sequence tag (EST) data from five different species of Solanaceae, wherein they revealed the presence of at least 11 miRNAs and 54 target genes in pepper (*C. annuum* L.) and 22 miRNAs with 221 target genes in potato (*S. tuberosum* L.). Apart from this, they identified a total of 12 miRNAs with 417 target genes in tomato, 46 miRNAs with 60 target genes in tobacco (*Nicotiana tabacum* L.), and 7 miRNAs with 28 target genes in *Nicotiana benthamiana*. Further, the identified miRNAs with their target genes were submitted to the SolmiRNA database, (http://gene‐ pool.kribb.re.kr/SolmiRNA). They showed the presence of both conserved and specific miRNAs, which may play crucial roles in the growth and development of Solanaceae plants. In addition, 12 miRNAs were randomly selected from a differentially expressed conserved miRNA family and subjected to qRT-PCR validation. Of these, the expression level of ntamiR167d was highly enriched in the leaf tissue, whereas the expression level of nta-miR319a and nta-miR160c were specifically found in stem and root tissues, respectively. The target prediction showed that most of the targets genes were those which codes for transcription factors involved in cellular and metabolic processes [116]. Similar study was performed where deep sequencing of leaf, stem, and root, and four early developmental stages of tubers were performed [117]. The study revealed a total of 89 conserved miRNAs belonging to 33 families and 147 novel miRNAs with 112 candidate potato-specific miRNAs. Digital expression profiling based on TPM (transcripts per million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue-specific expression (leaf, stem, and root), while a few demonstrated tuber-specific expressions. Further, targets were predicted for the identified conserved and potato-specific miRNAs. The predicted targets of four conserved miRNAs are as follows, *ARF16* (*auxin response factor* 16) for miR160, NAM (*no apical meristem*) for miR164, RAP1 (relative to *Apetala2* 1) for miR172, and HAM (*hairy meristem*) for miR171. Later they were experimentally validated using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). The list of databases for miRNA identifi‐ cation is presented as Table 2.


**Table 2.** List of databases for miRNA identification

#### *3.5.3. miRNAs in plant growth and development*

To investigate the role of miRNAs in ovary and fruit development of tomatoes, transgenic plants were generated by overexpressing MIR167. The transgenic plants showed a reduction in leaf size and internode length as well as shortened petals, stamens, and styles. The RNA-Seq analysis identified many genes with altered expression patterns in tomato. Of these, *SpARF6* and *SpARF8* genes involved in flower maturation in *Arabidopsis* have been found to be significantly down regulated [129]. In a separate study, it was found that transgenic tomato plants harboring AtMIR156b (*A. thaliana* miRNA 156b family) precursor resulted in abnormal flower and fruit morphology; in addition, the fruits were characterized by the growth of extra carpels and ectopic structures [130]. Moreover, these transgenic lines also displayed increased the expression of genes, which are involved in maintenance of meristem and formation of new organs such as *LeT6/TKN2* (a KNOX-like class I gene) and *GOBLET* (a NAM/CUC-like gene). Overall, these observations suggest that the miR156 is involved in the maintenance of the meristematic activity of ovary tissues and participates in the normal fleshy fruit development. Several miRNAs have been identified in the fruit tissue. However, no miRNA has been experimentally validated to be involved in fruit ripening. Recently, *SlymiR157* and *Sly‐ miR156* have been shown to regulate ripening and softening of tomato fruits. SlymiR157 governs the expression of key ripening gene *LeSPL-CNR* by miRNA-induced mRNA degra‐ dation and by translational repression. Furthermore, qRT-PCR profiling of key ripeningrelated genes reveals that the SlymiR157-target LeSPL-CNR may also affect the expression of *LeMADS-RIN*, *LeHB1*, *SlAP2a*, and *SlTAGL1* [131]. Table 3 contains the list of databases for miRNA target gene prediction.

**Database Description Link Reference**

To investigate the role of miRNAs in ovary and fruit development of tomatoes, transgenic plants were generated by overexpressing MIR167. The transgenic plants showed a reduction in leaf size and internode length as well as shortened petals, stamens, and styles. The RNA-Seq analysis identified many genes with altered expression patterns in tomato. Of these, *SpARF6* and *SpARF8* genes involved in flower maturation in *Arabidopsis* have been found to be significantly down regulated [129]. In a separate study, it was found that transgenic tomato plants harboring AtMIR156b (*A. thaliana* miRNA 156b family) precursor resulted in abnormal flower and fruit morphology; in addition, the fruits were characterized by the growth of extra carpels and ectopic structures [130]. Moreover, these transgenic lines also displayed increased the expression of genes, which are involved in maintenance of meristem and formation of new organs such as *LeT6/TKN2* (a KNOX-like class I gene) and *GOBLET* (a NAM/CUC-like gene). Overall, these observations suggest that the miR156 is involved in the maintenance of the meristematic activity of ovary tissues and participates in the normal fleshy fruit development.

http://www.mirbase.org/ [118–122]

http://deepbase.sysu.edu.cn/ [123]

http://www.microrna.org/microrna/home.do [124]

http://diana.cslab.ece.ntua.gr/mirgen/ [125]

http://bioinformatics.cau.edu.cn/PMRD/ [128]

http://mirnamap.mbc.nctu.edu.tw/ [126, 127]

miRBase Database of published miRNA

deepBase A platform for annotating and

sequencing data

miRNAMap miRNAMap Genomic maps of

PMRD Plant miRNA database with large

browser, etc.

**Table 2.** List of databases for miRNA identification

*3.5.3. miRNAs in plant growth and development*

miRandamicroRNA.org

DIANA-mirGen

2.0

sequences and their annotation

Database for predicted microRNA targets, target downregulation scores and experimentally observed expression patterns

Database of miRNA genomic information and regulation

miRNA genes and their target genes in human, mouse, rat, and other metazoan genomes

information of plant microRNAs data, consisting of microRNA sequence and their target genes, secondary dimension structure, expression profiling, genome

discovering small and long ncRNAs (microRNAs, siRNAs, and piRNAs) from next generation

264 Next Generation Sequencing - Advances, Applications and Challenges



**Table 3.** List of databases for miRNA target gene prediction

#### *3.5.4. miRNAs in biotic stress*

miRNAs have been identified in many plants with their diverse regulatory roles in biotic stresses. miRNA sequencing was used to investigate the miRNA expression difference between the tomatoes treated with and without *Phytophthora infestans*. Using high-throughput sequencing technologies, they could identify a total of 207 known miRNAs and 67 novel miRNAs. In addition to this, a total of 70 miRNAs were differentially regulated in the plants treated with *P. infestans*; of these, 50 were downregulated and 20 were upregulated. Also, a total of 73 target genes were identified for 28 differentially expressed miRNAs by using psRNATarget analysis [157].

The fungus *Fusarium oxysporum* f. sp. *lycopersici* causes vascular wilt disease in tomato. A comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars were performed to explore the role of miRNAs in tomato defense against *F. oxyspo‐ rum*. *SlmiR482f* and *SlmiR5300* were repressed during infection of Motelle with *F. oxysporum*. Four predicted mRNA targets, two each of slmiR482f and slmiR5300, displayed increased expression in resistant Motelle. This was further confirmed by co-expression analysis in *N. benthamiana*. Silencing of the targets in the resistant Motelle cultivar compromised the resist‐ ance to *F. oxysporum* and confirmed the role of these genes in fungal resistance [158].

#### *3.5.5. miRNAs in abiotic stress*

**Database Description Link References**

A Plant Small RNA Target Analysis Server http://plantgrn.noble.org/

TAPIR Target prediction for plant microRNAs http://bioinformatics.psb.ugent.be/

http://rnalogo.mbc.nctu.edu.tw/ [145]

http://mirgator.kobic.re.kr/ [146–148]

http://mirnamap.mbc.nctu.edu.tw/ [112]

http://mirdb.org/miRDB/ [149]

[150]

[151]

[152]

[153]

[155]

[156]

http://bibiserv.techfak.unibielefeld.de/rnahybrid/

http://rhesus.amu.edu.pl/mirnest/

http://www.comgen.pl/mirex2/ [154]

http://pmted.agrinome.org/

http://pcsb.ahau.edu.cn:8080/

psRNATarget/

copy/browse.php

by\_mirna.jsp

webtools/tapir/

PASmiR/

miRNAs have been identified in many plants with their diverse regulatory roles in biotic stresses. miRNA sequencing was used to investigate the miRNA expression difference between the tomatoes treated with and without *Phytophthora infestans*. Using high-throughput sequencing technologies, they could identify a total of 207 known miRNAs and 67 novel miRNAs. In addition to this, a total of 70 miRNAs were differentially regulated in the plants treated with *P. infestans*; of these, 50 were downregulated and 20 were upregulated. Also, a total of 73 target genes were identified for 28 differentially expressed miRNAs by using

representation of the patterns in an aligned RNA sequences with a consensus structure

RNALogo Database with novel graphical

miRGator Database with microRNA diversity,

266 Next Generation Sequencing - Advances, Applications and Challenges

relationships

miRNAMap miRNAMap Genomic maps of miRNA

miRDB Webserver for miRNA target prediction and functional annotation

RNA hybrid This tool is primarily meant as a means for microRNA target prediction

miRNEST miRNEST is an integrative collection of

PMTED Plant MicroRNA Target Expression Database

PASmiR A database for miRNA molecular

*3.5.4. miRNAs in biotic stress*

psRNATarget analysis [157].

**Table 3.** List of databases for miRNA target gene prediction

MIREX A platform for comparative exploration of

plant pri-miRNA expression data

regulation in plant abiotic stress

miRU, psRNAtarget expression profiles, and target

genes and their target genes in human, mouse, rat, and other metazoan genomes

animal, plant and virus microRNA data

Abiotic stress (such as salt, drought, and heat) is becoming a major constraint to crop produc‐ tion due to the climate change. miRNAs have been found to play a significant role in tolerance to these stresses. For example, in tomato, transgenic lines were generated by the overexpression of miR169 family member: Sly-miR169c that displayed reduced stomatal opening, decreased transpiration rate, reduced water loss, and enhanced drought tolerance [159]. In eggplant, the high-throughput sequencing of salt tolerant species was performed and identified 98 con‐ served miRNAs from 37 families [160]. Some of them were found to be expressed under salt stress. These studies provide a better understanding about the regulation of gene expression under abiotic stresses for genetic improvement of crops.
