1162777

research-article2023
BBI0010.1177/11779322231162777Bioinformatics and Biology InsightsKhan

Identification of Conserved and Novel MicroRNAs with Bioinformatics and Biology Insights
Volume 17: 1–12

their Targets in Garden Pea (Pisum Sativum L.) Leaves © The Author(s) 2023
Article reuse guidelines:

by High-Throughput Sequencing sagepub.com/journals-permissions

DOI: 10.1177/11779322231162777
https://doi.org/10.1177/11779322231162777

Qurshid Hasan Khan

Department of Plant Sciences, University of Hyderabad, Hyderabad, Telangana, India.

ABSTRACT: MicroRNAs (miRNAs) are single-stranded, endogenous, non-coding RNAs of 20–24 nucleotides that play a significant role in
post-transcriptional gene regulation. Various conserved and novel miRNAs have been characterized, especially from the plant species whose
genomes were well-characterized; however, information on miRNA in economically important plants such as pea (Pisum sativum L.) is limited.
In this study, I have identified conserved and novel miRNA in garden pea plant leaves samples along with their targets by analyzing the next
generation sequencing (NGS) data. The raw data obtained from NGS were processed and 1.38 million high-quality non-redundant reads were
retained for analysis, this tremendous quantity of reads indicates a large and diverse small RNA population in pea leaves. After analyzing the
deep sequencing data, 255 conserved and 11 novel miRNAs were identified in the garden pea leaves sample. Utilizing psRNATarget tool, the
miRNA targets of conserved and novel miRNA were predicted. Further, the functional annotation of the miRNA targets were performed using
blast2Go software and the target gene products were predicted. The miRNA target gene products along with GO_ID (Gene Ontology Identifier)
were categorized into biological processes, cellular components, and molecular functions. The information obtained from this study will provide
genomic resources that will help in understanding miRNA-mediated post-transcriptional gene regulation in garden peas.

Keywords: Conserved miRNA, novel miRNA, miRNA targets, small RNA, post-transcriptional gene regulation

RECEIVED: October 13, 2022. ACCEPTED: February 18, 2023.
Type: Original Research Article
Funding: The author(s) disclosed receipt of the following financial support for the
research, authorship, and/or publication of this article: This work was supported by BIRAC
SRISTI Appreciation Grant.
Declaration Of Conflicting Interests: The author(s) declared the following
potential conflicts of interest with respect to the research, authorship, and/or publication of
this article: The author declares that he has no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
article.
CORRESPONDING AUTHOR: Qurshid Hasan Khan, Department of Plant Sciences,
University of Hyderabad, Gachibowli, Hyderabad 500046, Telangana, India. Email:
hasanqurshid@uohyd.ac.in

Introduction translational repression or target mRNA degradation.1 In

The garden pea is an economically important cool-season crop,
which is grown across the globe. It is a rich source of protein,
fiber, starch, phytochemical substances, and trace elements.
Besides human consumption, pea has been grown as forage
crops for cattle as well as a cover crop to minimize soil erosion
(http://dpd.gov.in/Pea.pdf ).
Due to the increase in population and consumer awareness
about the consumption of healthy plant-based proteins, the
demand for peas has been increasing in developed and devel-
oping countries. In terms of production, the pea is the fourth
most important legume crop grown throughout the world.
From 2022 to 2027, the pea market is expected to register a
compound annual growth rate of 4.3%. (https://www.mor-
dorintelligence.com/industry-reports/peas-market)
This protein-rich pulse crop is widely grown in China,
India, the USA, France, Egypt, the UK, Pakistan, Algeria, Peru,
and Turkey. India is the second largest producer of green peas
in the world with a total production of 4.8 million tons (https://
worldmapper.org/maps/green-peas-production/).
In plant species, microRNAs (miRNAs) and small interfer-
ing RNAs (siRNAs) are two important types of small regula-
tory RNAs. These two types of RNA differ in their function
and biogenesis. In plants and animals, these small regulatory
RNAs are highly conserved and regulate gene expression.
miRNAs are 20–24 nucleotides long non-coding RNAs
that play a significant role in post-transcription gene regula-
tion. It complements the target mRNAs and causes
plants, RNA polymerase II transcribes miRNA genes into a
primary transcript (pri-miRNAs). Ribonuclease III-like Dicer
(DCL1) enzyme trims pri-miRNAs and produces miRNA
precursors (pre-miRNAs) that had a stem-loop (hairpin)
structure. Eventually, a short double-stranded RNA (dsRNA)
is produced by the second cleavage of DCL1 on the stem-loop
region of the hairpin. In the dsRNA, one strand acts as a mature
miRNA. Subsequently, the processed miRNA incorporates
with the RNA-induced silencing complex (RISC) and directs
the RISC to the target complementary mRNA for transla-
tional inhibition or degradation.2,3 Plant miRNA involves in
various biological processes such as signaling,4 development,5
and stress responses,6-11 and organ morphogenesis.12
Formerly, miRNAs were identified using computational
and hybridization-based experiments. As the computational-
based method was entirely dependent on the availability of the
EST or genome survey sequences in the database. If miRNA
was expressed at a very low level, then it was difficult to detect
miRNA using the hybridization-based method. On the con-
trary, using high-throughput next-generation sequencing
(NGS) technologies like Illumina Solexa, Roche 454, and ABI
SOLi, it is possible to differentiate lowly expressed miRNAs
from well-characterized or poorly characterized plant spe-
cies.13-18 Recent evidence indicates that plant miRNAs and
siRNAs play a role in biotic and abiotic stress responses
The biotic and abiotic stress has been relegating the produc-
tion of a garden pea. To minimize the damage caused by biotic

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2 Bioinformatics and Biology Insights 
and abiotic stress, plants regulate gene expression at transcrip-
tional, post-transcriptional, and post-translational levels. A
thorough understanding of the gene expression level at post-
transcriptional will help in the development of new strategies
that will enhance plant stress tolerance.
To understand the transcriptional response in garden pea
plant, transcriptome analysis of pea tissues and organs were
fully investigated. To gain insights into garden pea plant post-
transcriptional and post-translational modification related to
stress, there is a need to study miRNAs in various organs and
tissues of pea plants under different conditions. As miRNAs
can control various protein-coding genes associated with
related gene families or genes that are involved in the same
pathway, it is interesting to study miRNA-mediated post-tran-
scriptional gene regulation in pea plants.19
The current literature lacks research studies on miRNA
related to pea plants. Therefore, in this study, to gain robust
information about the entire repertoire of miRNA present in
garden peas, a small RNA library was prepared from young
garden pea leaves tissues, followed by deep sequencing and bio-
informatic analyses.
Material and Methods
Sample preparation and extraction of total RNA
In this study, I have selected P. sativum (var. Arkel), which was
easily available in the local market of India. The growth of this
variety was vigorous, and the plant can grow up to 45 cm
(http://agropedia.iitk.ac.in/content/vegetable-pea-varieties).
Mature seeds of P. sativum (var. Arkel) were surface sterilized in
70% ethanol for 2 min and rinsed two times in sterile distilled
water. The seeds were soaked in water overnight. Then, the
seeds were sown in pots containing humus-rich soil. The plants
were allowed to grow for a month in a net house. The plants
were covered with plastic bags with holes. The young leaves
were collected and immediately chilled in liquid nitrogen. The
samples were stored at −80 C until RNA isolation. Utilizing
the Trizol reagent, as per the manufacturer’s instructions, total
RNA was extracted from the leaves.
Using Nanodrop Spectrophotometer, the RNA concentra-
tion and purity were estimated. A good quality RNA with a
very low amount of protein contamination would have A260/
A280 ratio greater than 1.8. Similarly, if the isolated RNA has a
very less amount of polysaccharides contamination, then A260/
A280 will be greater than 1.8. The total RNA extracted in this
study has a concentration of 352.8 ng/ul, A260/A280 ratio of
2.19, A260/A230 ratio of 2.22 indicating a good quality RNA.20,21
Small RNA library preparation and deep
sequencing
Small RNA sequencing (sRNA) library was prepared with
TruSeq Small RNA Sample Preparation protocol (Illumina,
San Diego, California, USA). 1000 ng of total RNA was
used as starting material. 3' adapters were ligated to the
specific 3’OH group of micro RNAs followed by ligation of
5' adapter.
Illumina Universal and Index Adapters utilized in this study
were as follows:
Universal Adapter:
5'-AATGATACGGCGACCACCGAGATCTACACGTTCA-
GAGTTCTACAGTCCGA-3'
Index Adapter:
5 ' - CA AG CAG A AG ACG G CATACG AG AT [ I N D EX ]
GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'
Reverse transcription of adapter-ligated fragments was per-
formed using Superscript III reverse transcriptase (Invitrogen).
The cDNA generated from the reverse transcription was
enriched and barcoded by PCR amplification (15 cycles).
Utilizing Polyacrylamide gel, the amplified library was size
selected in the range of 140-160 bp followed by overnight gel
elution and precipitation in the presence of glycogen, 3M
sodium acetate (Sigma, Saint Louis, Missouri, USA), and
absolute ethanol. Pellet was re-suspended in nuclease-free
water (Invitrogen, Whitefield, Bangalore). The workflow of
TruSeq Small RNA sample preparation is shown in Figure 1.
Qubit fluorometer (Thermo Fisher Scientific, MA, USA)
was used to quantify the Illumina-compatible sequencing
library. The Qubit concentration of the small RNA library was
13.4 (ng/ul). Agilent 2100 Bioanalyzer was used to analyze the
fragment size distribution. The fragment size of the Illumina-
Compatible sequencing library ranges between 130 and 180
bp. Since the combined adapter size was approximately 120 bp,
the effective user-defined insert size ranges between 10-60 bp.
The raw NGS data of P. sativum young leaves sample was
deposited in National Center for Biotechnology Information
(NCBI) Sequence Read Archive (SRA) database with acces-
sion PRJNA882352.
Data analysis of small RNA transcriptome
After completion of the NGS run, the raw data were analyzed
using UEA small RNA Workbench (Version 4.6; http://srna-
workbench.cmp.uea.ac.uk).22,23 In Oracle VM VirtualBox, a
virtual machine (VM) was created, and Ubuntu OS was
installed on it. The adaptor sequences and low-quality reads
were filtered and the sequences between 16 to 40 nucleotides
were retained for further analysis. Then, using Rfam database,
the reads that match with other ncRNAs such as rRNA,
tRNA, snRNA, and snoRNAs were removed.24 Glycine max
genome was used as a reference genome. Using Bowtie soft-
ware, the small RNA was aligned to the reference genome.25-27
The mapped and unmapped reads were separated using
SAMtools. Using the CD-HIT program, a read count was
generated.28-30 The mature sequences of miRNA were down-
loaded from miRbase (https://www.mirbase.org/), a local
Khan
Figure 1. Overview of small RNA sample preparation.
miRNA database was constructed, then a homology search
was performed using the ncbi-blast-2.13.0 + program to
identify the conserved miRNA with E-value of 0.001 and
non-gap alignment.31-35 Subsequently, the unaligned reads
were used for predicting novel miRNAs using the MIREAP
program. A total of 29 novel miRNAs were predicted using
the software; however, only 11 novel miRNAs have proper
precursor secondary structure and MFEI values of > 0.70. The
secondary structure of the precursors was predicted using the
RNAfold web server.17,27,28,36-42 The MFEI values were calcu-
lated utilizing the following formula:
( MFE / length of RNA sequence) × 100
MFEI =
%GC content
The targets for conserved and novel miRNA were predicted
using the psRNATarget tool.43,44 The steps involved in this
study are shown in Figure 2.
Results
Analysis of small RNA transcriptome of P. sativum
As limited EST sequence information was present in the
National Center for Biotechnology Information Expressed
Sequence Tags database, it is highly impossible to identify
P. sativum miRNAs merely based on computational analy-
sis. Next-generation high-throughput sequencing is a
robust tool for the de novo identification of miRNA present
in P. sativum.
By utilizing high-throughput Illumina sequencing in this
study, a total of 12,713,919 raw reads were generated from the
small RNA library of garden pea leaves tissue. After filtering
3
the low-quality reads, adapters, and redundant sequences along
with rRNA (2,106), tRNA (424), mRNA (75,037), snRNA
(1,024), and snoRNA (999), a total of 1,184,724 unique reads
with lengths ranging from 16 to 40 nucleotides were retained
for further analysis. It was observed that in P. sativum young
leaves tissue small RNA library, the size of the small RNA was
unevenly distributed as shown in Figure 3.
Identification of conserved miRNAs
To identify conserved miRNAs in P. sativum, Glycine max ref-
erence genome was downloaded from EnsemblPlants (https://
plants.ensembl.org/).The unique sequences ⩾16 bp and ⩽40 bp
length were considered for further analysis. Using Bowtie 1.3.1,
the reads were aligned to the Glycine max reference genome.
Then, the aligned reads were separated. The reads were checked
for rRNA, tRNA, snRNA, and snoRNA contamination using
the Rfam database. Finally, the filtered reads were used for con-
served miRNA prediction. Using the CD-HIT program, the
reads were made unique and the read count profile was
generated.
Using the NCBI-blast-2.13.0 + program, a homology
search of the unique miRNA reads was performed against
matured miRNA sequences retrieved from miRbase-22. A
total of 254 miRNA belonging to 56 different families were
identified. miRNA family analysis revealed MIR-156 family
was the most abundant, followed by MIR-166, MIR-159, etc.
All the identified conserved miRNAs in P. sativum were shown
in Table 1.
The rest of the information related to the conserved miR-
NAs such as length, reference miRNA, read count, percent
4 Bioinformatics and Biology Insights 
Figure 2. Steps involved in analyses of P. sativum next-generation sequencing data.
Figure 3. Small RNA length and reads distribution detected in P. sativum
young leaves tissue using Illumina Next Generation Sequencing.
identity, alignment length, mismatches, and E value were
attached as supplementary data S1.
Identification of novel miRNA
After the identification of conserved miRNA, the unaligned
reads were used to predict novel miRNA using MIREAP soft-
ware. The software integrates small RNA depth and position
with the miRNA biogenesis model for detecting miRNAs
from NGS small RNA libraries. A total of 11 novel miRNAs
were identified, and the read count of these miRNA candidates
varies from 20 to 5. psa-m0029-3p miRNA has the highest
number of read counts. The MFEI value of all the novel
miRNA precursors ranges from 0.72 to 1.40 ruling out the
possibility of other types of RNAs like mRNAs (0.62–0.66),
tRNAs (0.64), and rRNAs (0.59).45 The novel miRNAs
detected in P. sativum young leaf sample were shown in
Table 2. The red color sequences in the precursor sequence
represent mature miRNA. The rest of the information related
to the novel miRNAs such as strand, length, read count, and
MFEI were attached as supplementary data S2.
The secondary structure of the novel miRNA precursor
sequences was predicted using RNAfold WebServer (http://
rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.
cgi).46,47 Figure 4 shows novel psa-m0023-3p miRNA and
psa-m0028-5p generated from RNAfold WebServer.
Predicting target for conserved and novel miRNA
of P. sativum
To understand the functions of the identified P. sativum miR-
NAs, potential targets were predicted using psRNATarget tool
(https://www.zhaolab.org/psRNATarget/). miRNAs with copy
number ⩾5 were considered for target prediction. Pea mRNA
and EST sequences were downloaded from NCBI and used as
targets in psRNATarget tool with default parameters.
 Khan

Table 1. Conserved miRNAs identified in P. sativum.

miRNA Name Sequence (5'-3') miRNA Name Sequence (5'-3')

Family Family

MIR156 psa-miR156a UUGACAGAAGAGAGUGAGCACAUUUUGGGGAGGAGUCAUA MIR397 psa-miR397a UCAUUGAGUGCAGCGUUGAUG

psa-miR156b-3p GCUCACCCUCUAUCUGUCACCAAUCCCUCCUCGCCCACCA psa-miR397-3p AUUGAAUGUAGCGUUGAUGAAAUU

psa-miR156 ACCACGUUCCCGUGGUUGACAGAAGAUAGAGAGCAC MIR398 psa-miR398c UGUGUUCUCAGGUCGCCCCUG

psa-miR156 g-3p CGCUCUCUAGACUUCUGUCAUCC psa-miR398b-3p UUGUGUUCUCAGGUCACCCCU

psa-miR156f UUGACAGAAGAGAGAGAGCACAGAUGAGUGCUCUCCU psa-miR398a-3p UGUGUUCUCAGGUCACCCCUU

psa-miR156i-3p UGCUCACUUCUCUUUCUGUCAUC psa-miR398a-5p GGAGUGAAACUGAGAACACAAG

psa-miR156q UUGACAGAAGAGAGUAAGCACU psa-miR398b GUGUUCUCAGGUCGCCCCUGC

psa-miR156d-3p GCUCUCUAUGCUUCUGUCAUCA psa-miR399c-3p UGCCAAAGGAGAGUUGCCCUG

psa-miR156c-3p GCUUACCCUCUAUCUGUCACC MIR399 psa-miR399o UGCCAAAGGAGAGCUGCCCUG

psa-miR156e-3p GCUCACUGCUCUCUCUGUCAAU psa-miR399q UGCCAAAGGAGAGCUGCUCUU

psa-miR156b UGACAGAAGAGAGUGAGCAU psa-miR399e-5p GGGCUUCUCUUUCUUGGCAGG

psa-miR156b-3p GGGUGACAGAUAGAAAGUGAGCAC MIR408 psa-miR408-3p UGCACUGCCUCUUCCCUGUUUU

psa-miR156c UUGUCAGAAGAGAGUGAGCAC psa-miR408 UUGCACUGCCUCUUCCCUGG

psa-miR156e UUGACAGAGGAGAGUGAGCAC psa-miR408-5p UCAUCCAUGCUCAGCCUGUUCCCU

psa-miR156j UUUAAUCGGAAAAUGGUUGACAGAAGAGAGUGAGCAC psa-miR408 AUGCACUGCCUCUUCCCUGGCACCGAUAGACUUGAAC

psa-miR156ad UGACAGAAGAAAGUGAGCAC MIR414 psa-miR414 GAGGUGGAGAUAUGGACGAGGAUGAUGAGGAAGAGGAGG

psa-miR156b-3p GCUCUCUAAACUUCUGUCAUCC psa-miR414 CAUCUUCAUCUUCAUCAUCGUCGU

psa-miR156d-3p GCUCACUUUCUUUCUGUCACC MIR437 psa-miR437 UGUGCGAGUUAUAGAAGUUUGACUU

psa-miR156 CAACUACUCAUAUCAUCGUCUGACAGAAGAGAGUGAGCAC MIR4376 psa-miR4376-5p UACGCAGGAGAGAUGAUGCCG

psa-miR156f-3p GCUCACCCUCUAUCCGUCACC MIR4416 psa-miR4416c CAGGUGAGAGAAACACGUAUC

psa-miR156 g-5p UUGACAGAAGAUAGAGGGCAC MIR474 psa-miR474b UGAACCCACCCAAACCCAACAACC

psa-miR156k UGACAGAAGAGAGGGAGCAC MIR5037 psa-miR5037c AACCCUCAAAGGCUUCCACGG

psa-miR156e UGACAGAAGAGAGCGAGCAC MIR5067 psa-miR5049c CGUCCCAAAAUAAUUGUCAUAUUU

psa-miR156a CGACAGAAGAGAGUGAGCAC MIR5079 psa-miR5079b AAUUUGGAUAUGUUAUUUUGGGAUC

(Continued)
5
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Table 1. (Continued)
miRNA Name Sequence (5'-3') miRNA Name Sequence (5'-3')
Family Family

MIR159 psa-miR159a UUUGGAUUGAAGGGAGCUCUAUCGUUCCCGGGCCUUGUAC MIR5268 psa-miR5268b UAUCUUCAUUCCACUCUGCUCCAU

psa-miR319d-3p CUUGGACUGAAGGGAGCUCCCUA MIR5272 psa-miR5272e ACAGAAUUGAUUUAUGUUUGGAU

psa-miR159d NAGAGCUGCUUAGCUAUGGAUCCC MIR5291 psa-miR5291c UUUGAUGAAUAGAUUGGAUGGAU

psa-miR319d GCAUGGACUGAAGGGGAGCUCCUUC MIR530 psa-miR530 UGCAUUUGCACCUGCACUUUCCUCCGCACCCA

psa-miR159c UUUGGAUUGAAGGGAGCUUUUUUAC MIR5303 psa-miR5303f UUUUUGGAGAAUCUGACAGGACGU

psa-miR159c-5p UUUGGAUCGAAGGGAGCUCUA MIR535 psa-miR535 UUGACAAAGAUAGAGAGCAC

psa-miR159b UUUGGAUUGAAGGGAGCUCUCCUAAGAUGAGUGCUCUCCU MIR5770 psa-miR5770b UUAGGACUAUGGUUUGGACAAUUACCAGGUCCAGACAUA

psa-miR159 UUUGGACUGAAGGGAGCUCUA MIR6024 psa-miR6024-3p GAAAACAACUCUUGAUAAAACAUG

psa-miR159 UUUGGGUUGAAGGGAGCUCUA MIR7540 psa-miR7540b GUAACACAUCACUUAUCAUAUCCU

psa-miR159b UUUGGAUUGAAGAGAGCUCUA MIR818 psa-miR1130b-3p UUGCUUAUAAUAUGGGACGGAGGG

psa-miR319 g UUGGACUGAAGGGAGCUCCUUC psa-miR1436 UACUCCCUCCGUCCCAUAAUGAGU

psa-miR319a-5p GAGCUUCCUUCAGUCCACUC psa-miR5568 g-3p AAACGUCUUAGAAUUUGGAACAGA

psa-miR159b UUUGGAUUGAAGUGAGCUCU psa-miR5568 g-5p UAAAUUAUAAGAUGUUUUGGACAU

psa-miR159c AUUGGAUUGAAGGGAGCUUUA MIR831 psa-miR831-5p UUACUCAUCUCCUUGUAUCUCUU

psa-miR159f AUUGGAUUGAAGGGAUCUCUA MIR838 psa-miR838-3p CGAAGAGUUGUGCAAGAAGAGGAAGAAAGUCUGAUU

psa-miR159i-3p UUUGGAGUGAAGGGAGCUCUA MIR84 psa-miR845b-5p CCUGCUCUGAUACCAAUUGAAAUU

MIR160 psa-miR160 g UGCCUGGCUCCCUGUAUGCCAUGGGGAGUUUGGCUGGGGC psa-miR845b UCAAUUGGUAUCAGAGCUUUGGCU

psa-miR162b-3p UCGAUAAACCUCUGCAUCCAGUUUUA MIR862 psa-miR5204 UGCUGGAAGGUUUUGUAGGAAC

MIR162 psa-miR162a UCGAUAAACCUCUGCAUCCUA MIR8643 psa-miR8643a AUUCGACUUCUCAAUAUGUAACUGC

psa-miR162b UCGAUAAGCCUCUGCAUCCAG MIR8781 psa-miR8781b UUAGGAACUUUGAGGAUCACCAAC

MIR164 psa-miR164b-5p CCCGUGGUGGAGAAGCAGGGCACGUGCA MIR1120 psa-miR1120a CUCCCUCCGUCUCAUAUUAUAAGC

MIR166 psa-miR166a UCGGACCAGGCUUCAUUCCCCUCAUGGAGGCAUACAUUUU MIR1122 psa-miR5281e CUUAUAAAUAAGGCCGGAGGGAGU

psa-miR166e-3p UUCUACAGUCCGACGAUCUCGGACCAGGCUUCAUUCCCC psa-miR1133 AUACAUAUACUCCCUCCGUCCCAU

psa-miR166c UCUCGGACCAGGCUUCAUUCCUCAAACGAGGAAAGGCUU MIR1507 psa-miR1507-3p AGAACUGGCGAUGCGGGCCUCAUUCCAUACAUCAUCUAA

psa-miR166b UCGGACCAGGCUUCAUUCCCGAUUGGGGUUUGAAGAAAAA psa-miR1507-5p UAGAGUUGUAUGGAAUGAAAGAU

psa-miR166u UCUCGGACCAGGCUUCAUUCUUUUC psa-miR1507a CCUCAUUCCAUAUAUCGUCUAA

(Continued)
Bioinformatics and Biology Insights 
 Khan
Table 1. (Continued)
miRNA Name Sequence (5'-3') miRNA Name Sequence (5'-3')
Family Family

psa-miR166a UCGGACCAGGCUUCAUUUCCCC MIR1511 psa-miR1511-5p UUAACAAGACUCUGAUACCACACU

psa-miR166e-3p UCGAACCAGGCUUCAUUCCCC MIR1514 psa-miR1514a-5p UUCAUUUCUAAAAUAGGCAUUG

psa-miR166i-3p UUCGGAUCAGGCUUCAUUCCCC MIR1520 psa-miR1520 m AAAUCAGGACAUGACAUGUGCGUC

psa-miR165b UCGGACCAGGCUUCAUACCCC MIR2119 psa-miR2119 UCAAAGGGAGGUGUGGAGUAG

psa-miR166d UCGGGCCAGGCUUCAUUCCCC MIR2593 psa-miR5287a CAUCCUCCGGUCACUAUUAUAAAC

psa-miR166k-3p GCGGACCAGGCUUCAAUCCCC psa-miR5287b ACACCCUCUGAUCACUAUUAUAAG

psa-miR166 m UCGGACCAGGCAUCAUUCCCC MIR3630 psa-miR3630-3p GAAAAUGAUGAUUUGUCGUUGGGAAUCUCUCUGAUGCAUA

psa-miR166p UUCGGACCAGGCUCCAUUCCCC MIR390 psa-miR390b AAGCUCAGGAGGGAUAGCGCC

psa-miR166b UCGGACCAGGCUUCAUUCCUAUUAUUA psa-miR390a-3p CGCUAUCCAUCCUGAGUUUC

psa-miR166q UCGGACCAGGCUUCAUUCCUUCAUGAAGCCGG MIR393 psa-miR393 CGGAAGGUCCAAAGGGAUCGCAUUGAUCU

psa-miR166k UCUCGGACCAGGCUUCGUUCC psa-miR393c-3p AUCAUGCCAUCCCUUUGGAUU

psa-miR166 m GCGGACCAGGCUUCAGUCCCC MIR394 psa-miR394b-5p UUUGGCAUUCUGUCCACCUCC

psa-miR166i UUGGACGAGGCUUCAUUCCCC MIR395 psa-miR395i CGAUGAAGUGUUUGGGGGAACUCU

psa-miR166 h-5p GGAAUGUUGUUUGGCUCGAGG psa-miR6300 GUCGUUGUAGUAUAGUGGUGAGUAUUCCCGCCUGUCACGC

psa-miR166c-5p GGAACGUUGUCUGGCUCGA NA psa-miR6173 GUAGUCCUAGCCGUAAACGAUGGAUACUAAGUGGCUGUGC

psa-miR166i UCGGACCAGGCUUCAUUCUC psa-miR894 GAUCAGAAGGUUGCGUGUUCGUUUCACGUCAGGUUCACCA

psa-miR166k CCGGACCAGGCUUCAUUCUUAA psa-miR5368 GUGCCAAGACAGACCAAGGGACAGUCUCAGGUAGACAGUU

psa-miR166j-5p UUUGAACCAAACAAACAAACUAUU psa-miR5232 CCUUGUCGCUUCGAUUCGUAUACAUGUCGCUCUCACCUUG

MIR167 psa-miR167d UGAAGCUGCCAGCAUGAUCUGGCGGCGAUGUGUCUCGGUC psa-miR6483 ACCUAAUCUUAUUGUAGAAAUUUUCAGGAUCAAGGAUUGU

psa-miR167d UGAAGCUGCCAGCAUGAUCUGACAGCUUUCUUGAACUG psa-miR1511 UACAGUCCGACGAUCAACCAGGCUCUGAUACCAUGA

psa-miR167f-3p AUCAGAUCAUGUGGCAGUUUCACC psa-miR2916 AAAGUUGGGGGCUCGAAGACGAUCAGAUACCGUCCUAGUC

psa-miR167b-3p AGGUCAUCUUGCAGCUUCAAUU psa-miR6478 CCGACCUUAGCUCAGUUGGUAGAGCGGAGGACUGUAGUGG

psa-miR167b-3p AGAUCAUGUCGGAGCUUCACCCGAACGGGUGAGUAACGCG psa-miR4995 UCAUAGGCAGUGGCUUGGUUAAGGGAAACCACCGGAGCCG

psa-miR167c-5p UGAAGCUGCCAGCAUGAUCUGCUGGUGUAUUCGGCGGCUC psa-miR5213-5p UACGUGUGUCUUCACCUCUGAAUU

psa-miR167a UCAGGUCAUCUUGCAGCUUCA psa-miR8175 CACGCGGGUAACCCGGGUUCGAUCCCCGGCAACGGCGCCA

psa-miR167i UGAAGCUGCCAGCAUGAUCUUAUCCCCAGUAGCUCAG psa-miR1527 AACCGGUUUUGUAGGGUUGAGUUAGGUCCAACCACAUU

(Continued)
7
 8
Table 1. (Continued)
miRNA Name Sequence (5'-3') miRNA Name Sequence (5'-3')
Family Family

psa-miR167d UGAAGCUGCCAGCAUGAUUUCU psa-miR5261 AAGAUUGUAGAUAGCUUUGGCUGU

psa-miR167b-3p GUGAGGCUGUCACAGCAUGACCU psa-miR5083 ACGAUUGUAAAAUUUCAGACUACAAUUAUCUGAUCAAA

psa-miR167b UGAAGCUGCCAGCAUGAUCUAAAAC psa-miR6485 AGAAAGACCUAGGAUGUAGAAGAUCAUAACAUGAGU

psa-miR167b-5p UAAAGCUGCUAGCAUGAUCUGC psa-miR5523 AUAUAACUAGUAAAUAUGUCCCUCCUCAUGUCAAUAU

psa-miR167j-3p AGAUCAUGUGGCAGUUUCAUU psa-miR6167 ACCCAGGUGGUAGCUUUGACCCAUUGG

MIR168 psa-miR168b-5p UCGCUUGGUGCAGGUCGGGAAUUCGACCCGUC psa-miR408 UGCACUGCCUCUUCCCUGGCUUUAUUU

psa-miR168 UCCCGCCUUGCAUCAACUGAAUU psa-miR5141 UAACGUCGGCUUAUCUGUCAGUCGCGUCGGGUCUGCCGC

psa-miR168a-3p CCCGCCUCGCAUCAACUGAAU psa-miR5211 CUGUCGCAGGAGUGAUGGGACC

MIR169 psa-miR169d UGAGCCAAGGAUGACUUGCCGG psa-miR5054 CGUGGGUUCGUUCCCCACGGUCGGCGCCA

psa-miR169s UUCUCCGGCAAGUCAUCCUUGGCU psa-miR4403 ACGGACACCGAACACGACACAGAG

psa-miR169e-3p GGCAGGUCAUCCUUCGGCUA psa-miR2614 UCGGUUCAACUCGUUAGGUUCAGU

psa-miR169 h UAGCCAAGGAUGGCUUGCCUGGC psa-miR2666 UGAAAGUGAGGCUAUCAAGGAUAUUGUU

psa-miR169j UGAGCCAGGAUGACUUGCCGG psa-miR2678 UGAAAUUGUUGCGAGUGUCUU

MIR171 psa-miR171p UUGAGCCGCGUCAAUAUCUUGUU psa-miR5248 UUUUUAGUUGGAAUGCAUUCAAGCAAGCGAGUCCUAC

psa-miR171e UUGAGCCGCGUCAAUAUCUCGA psa-miR845 GGCUCUGAUACCAAUUGUUGGUGC

psa-miR171k-3p UUGAGCCGCGCCAAUAUCACU psa-miR156r UUGACAGAAGAUAGAGAGCAUUUU

psa-miR171a UUGAGCCGUGCCAAUAUCACU psa-miR5299 AUUCAUUGGUAUUAUAAAGCGACU

psa-miR170-5p UAUUGGCCUGGUUCACUCAGA psa-miR5742 ACAUCAAUCGUCGUUGGAUCGAGU

psa-miR171c-5p AGAUAUUGGUGCGGUUCAAUC psa-miR5072 AUUCUGGGUUUCCCCAGCAGAGUCGCCA

psa-miR171d UGAUUGAGCCGUGCCAAUAUC psa-miR5538 UAACGGCAGCAAGUGAUUGAGUUCAGUAGUUCC

psa-miR171 g UUGAGCCGCGCCAAUAUCUCG psa-miR8030-3p AUUGAUUUGGUUUGAUUUGGUUU

psa-miR171l CGAUGUUGGUGAGGUUCAAUC psa-miR4233 AUAUGCGGGGAGUUGAUGUUGAUGAUCGUCAGU

psa-miR171q UUGAGCCGUGCCAAAAUCACA psa-miR5653 CGUAAACUCAACCCAACUCAACCC

psa-miR171b UGAUUGAGCCGCGUCAAUAUC psa-miR5658 ACUGACUAUGAUAAUGAUGAUGAUGACGAGG

psa-miR171e GGAUAUUGGUCCGGUUCAAUA psa-miR829-3p.1 AUAUCUCCAUCAUUUUGUAUCAGA

(Continued)
Bioinformatics and Biology Insights 
Table 1. (Continued) Khan
miRNA Name Sequence (5'-3') miRNA Name Sequence (5'-3')
Family Family

psa-miR171b-3p CGAGCCGAAUCAAUAUCACUC psa-miR8599 GCAAAUGACUUGAGUUCUAACUUU

psa-miR171l-5p CGUGAUAUUGAUCCGGCUCAUC psa-miR8613 AUCGAUCAAACUGGAUGACUUGGU

MIR172 psa-miR172a AGAAUCUUGAUGAUGCUGCAUC psa-miR5174e- AAUAUGGAACGGAGGGAGUACUUG

3p.2

psa-miR172c-5p GUAGCAUCAUCAAGAUUCACU psa-miR5174e- CUAGUACUCCCUCUUUUCCAUAAU

5p.2

psa-miR172a AGAAUCCUGAUGAUGCUGCAG psa-miR5281b ACCUCCGUUCCUAUUUGUAAGAGA

psa-miR172c-5p GUGGCAUCAUCAAGAUUCAC psa-miR7782-3p AUCAAGGCUCUGAUACCAUGUUGU

psa-miR172b AGAAUCUUGAUAAUGCUGCAU psa-miR6104 AUACGGCAAAUCGAACAAAUAAAU

psa-miR172c GCAGCAUCAUCAAGAUUCAC psa-miR6113 CGAUGAACCUCAAGAACACGUUCC

psa-miR172d UUGUCAUGAGAAUCUUGAUGAUGCU psa-miR169t UGAGCCAGGAAUGACUUGCCGG

MIR1846 psa-miR1846d-5p UGGUCGCACCCCACCCAGCAGCCGGAUCU psa-miR8155 CGUAACCGGGCUCUGAUACCA

MIR1863 psa-miR1863b UAAAAGCUCUGAUACCAUGUUAAG psa-miR1171 AUGGAGUGGAAUGAAGUGGAGUGG

MIR2111 psa-miR2111a-5p UAAUCUGCAUCCUGAGGUUUA psa-miR3946 CUAACAGAGAAAGAGAAAAGAGCA

MIR396 psa-miR396 g-3p GUUCUACAGUCCGACGAUCUUCCACAGCUUUCUUGAACUU psa-miR9753 CUAGCAGCCCUAAGUACCAAAUUG

psa-miR396a-5p UUCCACAGCUUUCUUAAACUGA psa-miR2637 UAGUGAUCAGAGGAUGUAUUGAAU

psa-miR396a-3p GUUCAAUAAAGCUGUGGGAAG psa-miR1424 AACUGACAAUCAGCAUUAGUGUGC

psa-miR396 UUCCACAGCUUUCUUGAACUUAUUCAGGAGAGAGGACUG psa-miR1863a UUAGCUCUGAUACCAUGUAAGAUA

psa-miR396b-3p CGGUUCAAUAAAGCUGUGGGA psa-miR1873 ACUAGGCUCUGAUACCAUGUUGAA

psa-miR396j UUCCACAGCUAUCUUGAACUG psa-miR414 AGGCCACUGAAGCUGAGGACGAUGAUGAUGACGAUGUGG

psa-miR396 g-5p UUCCACAGCUUUAUUGAACUU psa-miR5808 AAAUUGUUUCUGAUCGUUGGCCUU

psa-miR396 g-5p UUCCACGGCUUUCUUGAACUU psa-miR7828 ACGAUGACAUGGACACCAAACUCC

psa-miR396a AUCCACAGCUUUCUUGAACUA psa-miR7804-3p AUCGAAUGAAGAUUUUAAAAGACU

psa-miR396b UUCCACAGCUUUCUUGAAUU psa-miR5385 UACCAAUCCCACCGCUUCUCUGGC

psa-miR396-3p UUCCACAGCUUUCUUGAGCUU psa-miR1128 AUACUACUCCCUCCAUCCCACGGA

psa-miR396d AAGAAAGCUGUGGGAGAAUAUGGC psa-miR8028-3p GGUGCAUAAUUAUAGUAUAAGGCU

psa-miR396k-3p GCUCAAGAAGGCUGUGGGAGA psa-miR8040-3p AUAUAAUUGUAAUAAUGAUCCGA

psa-miR396a UUCACAGCUUUCUUGAACUU psa-miR6197-5p AACUGUAAAGAAAUGUAGGACACC

9
10 Bioinformatics and Biology Insights 

Table 2. Potential novel miRNA identified in P. sativum.

Name Mature miRNA sequence (5'-3') Precursor sequence

psa-m0029-3p UCGGGGUUCUUGUAUCUUUGUCA AAAAUAUUUUUGAUAAAGAUUCAAGAUUACUGAUAUAAAUUAUA

UUGGUUGUUCACUUGUUCUGCUAUGUGCAUGCUAAAGAUUGUG
CAAAUACAAAUGAUCAAAAUUUAACAUGUUUGCCACUAAACUUU
UUUUGUGACUAAUUCGGGGUUCUUGUAUCUUUGUCAAAUUGU
CACA

psa-m0016-3p CAGUUUCAGCAGCAGCAUGGGCA AGUUGAGCAAGGCUGGCCGCGCUGCGAGCUGCCGCUGCUGCAC

GUUCAGCAGUUUCAGCAGCAGCAUGGGCAGCAGCCAACA

psa-m0004-3p AGCCAUGUACUUUGAUUGAGCC GGUAUUGCCGUGCCUCAAUCUGAAUACAUGACUAUUAUAACAA

AAUCAGCCAUGUACUUUGAUUGAGCCGCGUCAAUAU

psa-m0025-3p UAAGUUUGACGGAAAUUUAAA UUAAAAUAUAUUUUUAGUAUUGGACAAAUUUGAUUGUUAUAUUU

UAAAAAUAGAUUAAUUUAAUCCUUGUACAUUGAUAACUGUGGAU
AAAUAAGGAUUAAAUUCGUUUGGUUUUUAAAAUAUAAAGAUGCA
AUUAAUUAAUUUAAAAUUAUAUAAAUUGAAAUUAAGUUUGACGGA
AAUUUAAAAACAUUUUAU

psa-m0006-3p UUUUCGCGGGCAAAAGUUUGAUU UUUUUUGAAAGAUUGAUUUUUGUUGCGAACAAAAGUCGUUUAAGG

CGUUGGACCUUUAAACGAUCUUUUGAUUUUUUAAAAGAGAUAAUCG
UUAAGGCACCGGACCCUUUUGAACGAUCUCUUGAUUUUUGAAUGGA
GGGGAGAAUCGUUAAGGUGUUGGACCUUGAAUGGUUUCAAAUGACC
UUUUCGCGGGCAAAAGUUUGAUUUGUGAGUUGA

psa-m0020-3p AUUCAAAGACGAUCAUUACAUA AAGACGGUUAUUAUCUAAUAAUCGUCUUUGAUGCUGCAUCUGCUUGG

ACAUUCAAAGACGGUCAUUAGAUAAUAAUCGUCUUUGAAUGCUGCUU
GUUCAAAGACGGUUAUUAGAUAAUAACCGUCUUUGAAUGUUGCGUCU
GCAUCGACAUUCAAAGACGAUCAUUACAUAAUAACCGUCU

psa-m0001-3p CCGUACACAUUGCUAGGAAUCAA UUGUCAGAGAUUAAUUCCUUUUGAAGUGUAGAGAUCACUAGUAAAGU

CUUCUCCGUACACAUUGCUAGGAAUCAAAUCCUUGACA

psa-m0012-5p AAUUGUGGACGAUUUUUCUAGAG CAAUUGAGUUAAUUGUGGACGAUUUUUCUAGAGUUUCAAUUUCUACAA

GUUAAUUCAUAUUGUUUCACAAGCUUCUCACAAUUGUUUGGCUAUAUAGA
AGGUUGAAAUUCUGUGGGAACUCUCACAAUUAUCUCAAUUGU

psa-m0013-3p CUAAAAUGGGUAUAUUUAAUUUU AGUGAUAUUUUAUUAAAAAAUUUAUACAUAUUCCAGAAGUAUAUCUUU

CAAACUAUGAAUCACAAUUCGGAAGAUAUAUUGCUAAAAUGGGUAUA
UUUAAUUUUGAAAAUACAU

psa-m0023-3p GCAUCUCACUCCUUUGUGCUCUC ACAGAAGAUAGAGAGCACAGAUGAUGAUAUGCACAUAUACAUGGAAC

AGGAAUUUAAGCAAUUGCAUCUCACUCCUUUGUGCUCUCUAAGCUUCUG

psa-m0028-5p UUGGAUGAAAAUCAGAUGACUC AUCAGAAUUGUUGGAUGAAAAUCAGAUGACUCAUUUUAUCUUGAU

GAAUGAAUCCGACAUAAAACAUGCAUCUGAGAUGUGAAUAGUCCG
AUUUAUUUUCAUCCAAUGGUAUCGAUU

In this study, a total of 12,838 corresponding potential tar-
get gene products of P. sativum miRNAs were detected (9,715
target gene products for conserved miRNA and 1,064 target
gene products for novel miRNA). Approximately 83% of the
conserved miRNA directly cleaves the target mRNA whereas
the rest of the conserved miRNA performs translational repres-
sion on the target mRNA. As 11 novel miRNAs were identi-
fied, some of them directly cleave the target mRNA and some
perform translational repression.
In the conserved miRNAs, psa-miR1527 (191) has the
highest number of potential targets, followed by psa-miR5261
(171) and psa-miR396 g-3p (165), whereas in novel miRNAs
psa-m0025-3p (161) has the higher number of targets, fol-
lowed by psa-m0028-5p (145) and psa-m0016-3p (121).
Functional annotation of the miRNA target was performed
using Blast2Go software. The GO (Gene Ontology) terms
were obtained. The miRNA target gene products along with
GO_ID were categorized into biological processes, cellular
components, and molecular functions.48,49 All the data related
to conserved and novel miRNA targets, miRNA target gene
products, and gene ontology functional categorization were
attached as Supplementary Data S3 and S4.
Discussion
Over the past few years, NGS or deep sequencing technologies
have been used in different molecular biology studies.15,50
Various studies have been conducted to identify conserved and
novel miRNAs in different plant species; however, miRNA and
their target remain unknown in an economically important
plant such as garden pea.
The small RNA size distribution pattern in P. sativum
young leaves shows that 63% of small RNA reads lie between
20 and 24 nucleotides and 24 nucleotides length small RNA
reads are dominant (41%) in the entire sRNA transcriptome. A
large number of sequencing reads of miRNA belonging to
MIR-156, MIR-159, and MIR-166 families support the deep
Khan
Figure 4. Secondary structure of novel psa-m0023-3p miRNA and
psa-m0028-5p miRNA.
sequencing data in other leguminous species such as Medicago
and peanut.51,52
Conversely, in wheat and rice plant species, MIR-169 fam-
ily sequencing reads are high in number and MIR-156 family
reads were very low in abundance indicating the presence of
species-specific expression profile of miRNA.53 All the detected
novel miRNAs had MFEI value > 72 and appropriate second-
ary structures.
The variation in sequencing reads numbers of different
members or isoforms of a miRNA family in P. sativum indicates
that the regulatory role of the isoform is dominant during the
particular stage of development. For instance, the MIR-172
family is likely to play a significant role in phase transformation
and floral organ development in various plant species, and the
reads number of psa-miR172a (64) belonging to the MIR-172
family was high compared to other isoforms of the family.54 The
comparative study of various miRNA isoforms present in a
miRNA family during a particular stage of development or at
11
specific conditions will provide valuable insights into the role-
played by the miRNA in growth and development.
The lack of funding was the major limitation of the study.
As leaf tissue contains a large repertoire of miRNAs;55,56 there-
fore, I have focused primarily on garden pea leaves in this study.
It would be interesting to study the distribution of the identi-
fied miRNA in roots, stems, and other important organs and
tissues of garden pea plants under various stress conditions to
understand the regulatory mechanism of miRNAs and to
develop strategies that will help in enhancing the production of
garden pea crops.
Conclusions
In conclusion, in this study, a small-RNA library was prepared
by high-throughput sequencing of P. sativum leaves to identify
the conserved and novel miRNAs along with their potential
targets. This study provides the first report on the identifica-
tion of conserved and novel miRNAs in P. sativum leaves by
deep sequencing. The valuable information obtained from this
study will provide a strong base for the researchers to select the
candidate miRNA or MIR family in P. sativum plants and
check the regulatory role-played by the miRNA in response to
biotic and abiotic stress, growth, and development. In addition,
this study will enlighten our understanding of miRNA-medi-
ated post-transcriptional gene regulation and could be helpful
in the annotation of the genome.
Acknowledgements
I am thankful to the Department of Plant Sciences and
School of Life Science, University of Hyderabad, for their
support and cooperation. I would like to express my deep-
est gratitude to Prof. Ch. Venkataramana, Department of
Plant Sciences, University of Hyderabad, and Dr. Satendra
Kumar Mangrauthia, Senior Scientist, IIRR, for their
support.
Author Contributions
Q.H.K. conceived, planned, and carried out the experiment, processed
the experimental data, performed the analysis and interpretation of
results, and prepared the manuscript.
Supplemental Material
Supplemental material for this article is available online.
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