agronomy

Review
A Review of Omics Technologies and Bioinformatics to
Accelerate Improvement of Papaya Traits
Rabiatul-Adawiah Zainal-Abidin 1 , Insyirah-Hannah Ruhaizat-Ooi 2 and Sarahani Harun 2, *

1 Biotechnology and Nanotechnology Research Centre, Malaysian Agricultural Research and Development
Institute (MARDI), Serdang 43400, Malaysia; [email protected]
2 Centre for Bioinformatics Research, Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia,
Bangi 43600, Malaysia; [email protected]
* Correspondence: [email protected]

Abstract: Papaya (Carica papaya) is an economically important fruit crop that is mostly planted in
tropical and subtropical regions. Major diseases of papaya, such as the papaya dieback disease
(PDD), papaya ringspot virus (PRSV) disease, and papaya sticky disease (PSD), have caused large
yield and economic losses in papaya-producing countries worldwide. Postharvest losses have
also contributed to the decline in papaya production. Hence, there is an urgent need to secure
the production of papaya for a growing world population. Integration of omics resources in crop
breeding is anticipated in order to facilitate better-designed crops in the breeding programme. In
papaya research, the application of omics and bioinformatics approaches are gradually increased





 and are underway. Hence, this review focuses on addressing omics technologies and bioinformatics
that are used in papaya research. To date, four traits of the papaya have been studied using omics
Citation: Zainal-Abidin, R.-A.;
Ruhaizat-Ooi, I.-H.; Harun, S. A
and bioinformatics approaches, which include its ripening process, abiotic stress, disease resistance,
Review of Omics Technologies and and fruit quality (i.e., sweetness, fruit shape, and fruit size). This review also highlights the potential
Bioinformatics to Accelerate of genetics and genomics data, as well as the systems biology approach that can be applied in a
Improvement of Papaya Traits. papaya-breeding programme in the near future.
Agronomy 2021, 11, 1356.
https://doi.org/10.3390/ Keywords: bioinformatics; comparative genomics; molecular markers; next-generation sequencing;
agronomy11071356 omics; papaya

Academic Editors: Roxana Yockteng,

Andrés J. Cortés and María
Ángeles Castillejo 1. Introduction
Papaya (Carica papaya) is one of the tropical fruits that is widely grown in tropical and
Received: 30 May 2021
Accepted: 17 June 2021
subtropical countries such as Australia, Brazil, Malaysia, Thailand, and South America. In
Published: 1 July 2021
worldwide fruit production, the papaya is ranked in third place after mango and pineapple
fruit production [1]. The papaya has a high nutritional content and medicinal value, which
Publisher’s Note: MDPI stays neutral
make it widely planted and globally popular. For instance, the fruit is rich in vitamin A,
with regard to jurisdictional claims in
vitamin C, folate, potassium, and niacin, and its leaves, stems, and roots are suitable for
published maps and institutional affil- alternative medicine [2].
iations. Papaya has nine pairs of chromosomes and is a diploid. The papaya, which has a
genome size of 372 Mb, belongs to the Brassicales order and Caricaceae family. Among
the well-known papaya cultivars in Hawaii are Solo, Sunrise, SunUp, and Rainbow [3],
while Eksotika and Sekaki are well-known cultivars in Malaysia [3]. To date, six papaya
Copyright: © 2021 by the authors.
cultivars, namely Eksotika, Eksotika II, Sekaki, Three Pillars, Frangi, and Viorica have been
Licensee MDPI, Basel, Switzerland.
registered with the Department of Agriculture, Malaysia [4]. Correspondingly, Japan also
This article is an open access article
produces papaya cultivars, namely Ishigaki Sango and Ishigaki Wondrous [5]; in India, the
distributed under the terms and well-known papaya cultivars are Co1 and Co2 [3].
conditions of the Creative Commons As a perennial herb, the breeding cycle of the papaya takes about seven to nine months
Attribution (CC BY) license (https:// until a ripe fruit is produced. The breeding cycle of the papaya is divided into three stages
creativecommons.org/licenses/by/ that are represented by seed germination, flowering, and fruit setting to fruit harvesting
4.0/). (Figure 1) [6,7]. The papaya seed germinates within two to four weeks after sowing [8].

Agronomy 2021, 11, 1356. https://doi.org/10.3390/agronomy11071356 https://www.mdpi.com/journal/agronomy

Agronomy 2021, 11, 1356 2 of 17

Then, after being transplanted, its vegetative state starts to grow into single-stemmed trees,
each bearing a rosette of large, deeply lobed leaves at the apex about two to three months
after transplantation [6–8]. The papaya plant produces its flowers at the age of three to
six months, and produces ripe fruits at seven to nine months [6,7].

Figure 1. The breeding cycle of papaya. DAG denotes day after germination.
Papaya is cultivated in fertile and well-drained soils with a pH of 5.5 to 6.5 [8,9].
Nutrients for the papaya are essential in order to increase the productivity and quality of
papaya production. Nitrogen (N) and phosphorus (P) are essential nutrients during the
early growth of the papaya in order to ensure an optimum growth of foliage, trunks, and
roots, as well as induce higher productivity [10]. Potassium (K) is needed at the papaya
fruiting stage because it is essential for improving the fruit quality (i.e., sweetness and
pulp thickness) [11,12]. Hence, these agronomic aspects are contributed to ensure that
breeding of papaya is successful. The breeding of papaya has produced more papaya
cultivars with desired agronomic traits such as high yield, fruit shape, fruit size, and
sweetness. However, the papaya is easily exposed to pathogens and being infected by
diseases, (i.e., the papaya dieback disease (PDD), papaya ringspot virus (PRSV) disease,
and papaya sticky disease (PSD)), which have resulted in a decline in papaya production.
In addition, high temperature and water stress in papaya plantation are being affected
by climate change, which is a challenge to the papaya industry, especially for its pro-
duction yield [13–15]. The high temperature (i.e., above 35 ◦ C) causes flowers to drop
and sex changes in female and hermaphrodite flowers [13]. Although high temperature
(i.e., 28 ◦ C) promotes the fast growth of papaya, low pollen viability and early maturation
result in imperfect quality fruits and a low yield [13]. Interestingly, high temperature
coupled with a higher moisture content produces higher total soluble solids (TSS) in pa-
paya [13]. A sufficient amount of water is essential to papaya because it determines the
fruit size and fruit quality [15]. For instance, in dry conditions, the fruits are smaller with a
hard texture when ripe.
One major way to overcome the constraints due to climate change and papaya dis-
eases is by breeding for new and improved papaya that has been enhanced with desired
traits, such as resistance to disease, resistance to abiotic stress, delayed ripening, and
sweetness. Recent trends in crop improvements have shown the integration of omics
approaches (i.e., genomics, transcriptomics, proteomics, and metabolomics) and bioinfor-
matics in breeding programmes [16–19]. The use of omics and bioinformatic approaches in
crop breeding helps to obtain a holistic understanding of the genetic and genomic bases
of the crop, as well as to understand the molecular interaction among genes, proteins,
and metabolites, especially regarding complex traits. Thus, the integration of omics and
bioinformatic approaches in crop-breeding programmes is anticipated to facilitate the
development of climate-resilient crop varieties and efficient germplasm screening, as well
as to accelerate the rate of the genetic gain in a crop [18,19].
To date, papaya-breeding programmes have focused on improving yield and quality,
resistance to abiotic stress condition, resistance to disease, as well as delayed ripening.
Remarkable success has been achieved in the genetic engineering of papaya: the first
transgenic papaya cultivar SunUp developed resistance to papaya ring spot virus (PRSV)
disease [20]. In 2008, the first papaya genome, from the cultivar SunUp, was sequenced
using whole-genome shotgun Sanger sequencing [21]. The papaya cultivar SunUp is a
transgenic papaya and was the first papaya genome sequenced [21]. The SunUp genome
Agronomy 2021, 11, 1356 3 of 17

sequence was annotated, and yielded 27,793 protein-coding transcripts. To date, SunUp has
been used as a reference genome for various comparative genomics analyses of the papaya
genome. Previous efforts have exploited the genome sequences, molecular markers, and
physical and genetic maps for improvement of papaya traits [22–25]. The domestication
and genetics of the papaya have also been discussed [26]; while Dhekney et al. [8] and
Palei et al. [27] have summarised the use of biotechnology tools and the progress of research
that has been conducted on papaya, they have not covered all the omics and bioinformatic
approaches that have been used in the improvement of papaya traits.
Hence, this review paper aims to examine the recent progress of the improvement
of papaya traits using the omics and bioinformatic approaches, and their application in
breeding for new and improved papaya cultivars (Figure 2). Each omics approach is shown
in Figure 2, outlining how most of the generated molecular or omics data are analysed
and visualised using bioinformatics approaches. The integration of systems biology with
the analysed omics data analysis will cater to the identification of molecular markers, as
well as candidate genes, proteins, and metabolites, for application in papaya-breeding
programmes. In this review paper, the future perspective of using omics approaches in
improving the desired papaya traits are also discussed.

Figure 2. A summary of the omics approaches that are used in the improvement of papaya traits. Four traits have been
studied using omics and bioinformatic approaches, including ripening, abiotic stress, disease resistance, and fruit quality.

2. The Role of Bioinformatics in Analysing Omics Data

Bioinformatics is an interdisciplinary field of various research backgrounds that com-
prises biology, computer science, mathematics, and statistics in order to extract, analyse,
integrate, and visualise the biological data that are generated from omics platform tech-
nologies [28]. Several bioinformatics tools and biological databases have been developed
and are accessible to other researchers in similar fields [19,28]. The development of useful
bioinformatics tools is also important for crop breeders in order to inform them of the target
selection of traits effortlessly [19,28]. The continuous effort in building and managing the
Agronomy 2021, 11, 1356 4 of 17

integrated databases would contribute to the translational research in which multiomics

data analysis has become an integral part of systems biology.

Comparative Genomics Analysis of Carica papaya

The completion of the SunUp papaya reference genome sequence provides the oppor-
tunity for researchers to perform a comparative genomics analysis of the papaya genome.
Comparative genomics, which is also known as comparative genome-wide analysis, is
an area of study in which the structure and function of genomes from different species or
varieties are compared [29]. Using computational approaches, the comparison of genomes
and their contents among the cultivars or species enable researchers to identify sequence
features (i.e., genes and proteins) that are conserved among the species [30].
Such examples include the comparative genome-wide analysis of the papaya genome
in order to identify, annotate, and classify the genes into several gene and transcription-
factor families that are associated with abiotic stress [31], disease resistance [24,32], ripen-
ing [33,34], and flower development [35]. The identification and annotation of the selected
gene families in the papaya genome have also provided information on their gene struc-
ture, and phylogenetic tree relationships in relation to orthologs or paralogs from other
species. Consequently, the potential genes and transcription factors were validated to
further investigate their expression patterns under different conditions [31–35].
The papaya is susceptible to multiple pathogens, such as Erwinia mallotivora [36],
ringspot virus [37], and papaya meleira virus (PMeV) [38]. Hence, in order to under-
stand the basis of papaya resistance and susceptibility, the method is to perform a com-
parative genome-wide analysis of the papaya genome. The genome-wide analysis of
papaya sequences has revealed that the papaya has fewer (0.2%) disease-resistance genes
(i.e., nucleotide binding site (NBS)-containing R genes) than Arabidopsis thaliana (0.68%) and
O. sativa (1.38%), which makes conventional breeding for resistance difficult [24]. Similarly,
Praza-Echeverria et al. [32] investigated the potential of NPR1, a pathogenesis-related gene,
in papaya resistance against a pathogen by comparing the NPR1 in Arabidopsis and tomato
genome sequences. This could lead to the application of the identified gene in genetic
engineering for crop improvement.
The understanding of the papaya ripening process is important in order to reduce
the occurrence of postharvest losses in the papaya industry. To improve the ripening trait
in papaya, it is crucial to identify the genes that are responsible for the ripening process.
Coupled with bioinformatics analysis, Liu et al. [33] identified 14 potential SQUAMOSA
protein-binding protein-like (SPL) genes, whereas Xu et al. [34] investigated 18 potential
auxin/indole-3-acetic acid (Aux/IAA) genes in the papaya ripening process. The involve-
ment of SPL and Aux/IAA gene families in the papaya ripening process is not well known.
Hence, these efforts provide an opportunity to understand the roles of auxin-responsive
genes and SPL in the papaya ripening process.
Using the genome sequence of the papaya, the basic helix–loop–helix (bHLH) tran-
scription factors that were associated with abiotic stress were identified [31]. A total of 73
candidate genes from the bHLH family were detected using comparative genome-wide
analysis. The quantitative real time PCT (qRT-PCR) experiment also revealed the role of
candidate bHLHs that might be responsible for abiotic stress responses (i.e., salt, drought,
and cold stresses) in papaya. Using a similar approach, Liu et al. [35] identified 11 potential
genes in the auxin response factor (ARF) transcription factor family, and their role in
papaya flower development.
In summary, a comparative genomics analysis of papaya can be initiated by retrieving
the papaya genome sequence from public databases. To date, five databases can be used to
retrieve the papaya genome sequence; namely, the NCBI (https://ftp.ncbi.nlm.nih.gov/
genomes/all/annotation_releases/3649/100/GCF_000150535.2_Papaya1.0/ accessed on
22 June 2021) [39], PlantGDB [40], Phytozome [41], EnsemblPlants [42], and PLAZA [43].
HMMER [44] and BLAST [45] were developed to perform similarity and homolog sequence
analyses. InterProScan [46], Pfam [47], and SMART [48] tools were designed to identify
Agronomy 2021, 11, 1356 5 of 17

the gene family, motif sequence, and domain association, respectively. PLACE [49] and
PlantCARE [50] are tools that are used for the analysis of cis-regulatory elements and pro-
moter identification of the genes of interest. The Gene Structure Display Server (GSDS) [51]
annotates gene structures; i.e., exon, intron, 30 UTR, and 50 UTR. To construct a phylogenetic
tree, multiple sequence alignment must be performed using the gene sequences containing
the domain or motif of interest as input data. ClustalX [52] and MAFFT [53] are multiple
sequence alignment programmes that are used for aligning the gene sequences, and MEGA
is a tool that is used for constructing a phylogenetic tree [54].
Figure 3 summarises the workflow of the comparative genomics analysis of the papaya
gene families using bioinformatic approaches that are related to disease resistance, ripening,
flower development, and abiotic stress.

Figure 3. Workflow for comparative genomics analysis of the papaya gene families.

3. Application of Omics Technologies in Carica papaya

The suffix ‘omics’ can be described as the screening of the whole molecular data from
a living organism for as genes (genomics), mRNA (transcriptomics), proteins (proteomics),
and metabolites (metabolomics). As seen by the expanding number of publications over
the years, these omics methods, as well as systems and synthetic biology, are becoming
increasingly popular [55]. As compared to other model fruit crops such as the tomato
and grape, the multiomics study of papaya is still in its infancy. However, various omics
data of papaya have been generated from each omics platform, which can lead to the
understanding of the complex phenotypic variations that can facilitate papaya breeding
strategies in the improvement of papaya traits (Table 1) [56].

3.1. Genomics and Molecular Markers

Advancements in next-generation sequencing technology (NGS) have resulted in
the different types of sequencing platforms (i.e., Illumina, PacBio, and Oxford Nanopore
Technologies), which produce high-quality sequences, including longer sequence reads
and fewer sequence error rates [56]. In addition, NGS has offered an affordable cost,
facilitating the sequencing of the genomes of various plant species and cultivars [57]. For
instance, the whole-genome resequencing of the papaya cultivars Eksotika and Sekaki from
Malaysia [58], as well as the Sunset cultivar from Hawaii [59], have been carried out using
the Illumina platform. The whole genome resequencing of papaya cultivars has led to the
identification of a large number of variants that have been annotated into coding genes,
where the identified SNPs could be developed as molecular markers in the application of a
marker-assisted selection of papaya breeding.
The large volumes of data that have been generated using next-generation sequencing
technology have increased the efficiency of the development of molecular markers, such as
simple sequence repeat (SSR) and single nucleotide polymorphism (SNP). SSR and SNP are
types of molecular markers that are widely used to improve the agronomic traits of fruit
Agronomy 2021, 11, 1356 6 of 17

trees [5]. Molecular markers, such as restriction fragment length polymorphism (RFLP),
amplified fragment length polymorphism (AFLP), and random amplified polymorphic
DNA (RAPD), are often limited by low reproducibility, laborious techniques, and time-
consuming processes [60]; using SSR and SNP will greatly expedite the detection by using
a high-throughput genotyping platform.
Previous efforts have identified 116,43 SSRs in the SunUp genome sequence [61]. In
addition, 73 SSRs were found in 25 genes that were related to fruit ripening. A similar
study focused on the development of high polymorphic SSR from the whole-genome
resequencing of Sunrise Solo (Hawaiian cultivar) and RB2 (Australian cultivar) [62]. The
developed SSR markers can be used to differentiate the elite cultivars, and can be used in
papaya breeding selection.
Genomics variants such as SNP and insertion–deletion (InDel) are highly abundant in
plant genomes [63]. Previous efforts have discovered putative SNPs in the genome-wide
analysis of papaya genome resequencing [20,21,48]. Zainal-Abidin et al. [58] sequenced
the genome of the papaya cultivars Eksotika and Sekaki in order to identify and annotate
SNPs. The identified SNPs will be useful for genotyping to develop a papaya SNP panel.
Similarly, the whole-genome resequencing of papaya cultivars such as SunUp (transgenic)
and SunSet (nontransgenic) has identified SNPs, InDels, and structural variations [59].
These variants’ data have been used to perform a comparative genomics analysis between
transgenic and nontransgenic papaya. Recently, Bohry et al. [64] carried out genome-wide
identification of SNP and InDels between the two parental lines of UC10 hybrids, the
Formosa elite lines Sekati and JS-12, using the Illumina MiSeq (Illumina Inc., San Diego,
CA, USA) platform. Interestingly, the putative variants that were located in the ripening-
related genes (RRGs) were suggested to be validated through functional analysis and the
genotyping platform. Subsequently, these candidate SNPs that are associated with RRGs
can be applied in diversity and genetic-mapping studies, as well as for application in
marker-assisted selection (MAS) of papaya.
Recent efforts have been conducted to identify the structural variations (SVs) in the
genomes of 25 wild and 42 cultivated papaya [65]. The SVs (i.e., insertions, deletions,
inversions, transposable elements, and copy number variations) contain relatively long
DNA changes as compared to SNPs and InDels [63,65]. These SV data have been used to
unravel the effect of SVs on the papaya phenotype and its adaptation during the domesti-
cation process [65]. Detailed GO enrichment between the wild and cultivated papaya has
identified genes that are artificially selected during papaya domestication. Environmental
adaptability, sexual reproduction, and essential traits such as pistil development, embry-
onic development, flowering duration, crop yield, pedicel elongation, defence response,
and disease response are all influenced by these genes. This study would facilitate the un-
derstanding of the genes that are involved during the process of papaya domestication, and
provide potential SV data to develop molecular markers in papaya breeding programmes.
The establishment of a bioinformatics pipeline in discovering large genomic variants
(i.e., SNP, InDel) has made it possible to unravel the genomic variants in the papaya genome
from the various cultivars. The identified and annotated SNPs in disease-resistance and
ripening-related genes could be applied in the MAS of the papaya as a tool to aid the
selection process in papaya germplasm, as well as in the study of its genetic diversity.
The genetic map is useful for dissecting the genetic components of complex traits [66].
The first genetic map of papaya was developed between Sunrise Solo x Line UH536, which
comprised 61 RAPD markers and was distributed in 11 linkage groups (LG) [22]. Then,
Deputy et al. [67] developed the genetic map of Kapoho x SunUp that comprised 1498
AFLP in 12 LG; while Chen et al. [23] developed the genetic map of AU9 x SunUp, which
comprised 706 SSR in 12 LGs. Blas et al. identified 14 QTL controlling fruit size and shape
of papaya [68]. However, progress in the development of papaya QTL has been limited
by a lack of genetic and genomic information. Genome-wide identification of SNPs in the
papaya genome sequence has allowed the development of a high-density genetic map.
This approach has been applied for unravelling the fruit quality of papaya, which has been
Agronomy 2021, 11, 1356 7 of 17

developed in the F2 population from the RB2 x Sunrise Solo cross using the genotyping-
by-sequencing approach (GBS) [69]. The highlights include the candidate genes that are
related to the fruit quality traits, such as fruit size, fruit shape, sweetness, length, and
firmness. These QTL data that are associated with the genes, and which are related to the
fruit quality traits, can be used as candidates for gene exploration in the selection of SNPs
and InDels using bioinformatics analysis [64]. Notably, candidate genes with associated
SNP markers represent a valuable resource for the future of strategic selective breeding of
elite papaya cultivars.
Although the genome sequences of papaya cultivars have been determined, no work
has been carried out on the pangenome analysis of C. papaya. Pangenome analysis enables
us to capture the entire set of genes from papaya cultivars, as well as to overcome the
limitations of relying on a single reference genome [70,71]. The identification of SVs can
represent pangenome analysis that would enable us to capture the entire set of genes from
papaya cultivars, as well as to overcome the limitations of relying on a single reference
genome [70,71]. In addition, more new candidate genes (i.e., genes for disease resistance
and ripening) or gene pools can be identified from the wild germplasm and molecular
markers, and these can be developed to screen for resistant varieties in the field [70,71].

3.2. Transcriptomics
The transcriptome is defined as the whole set of transcripts in a cell and the quan-
tification of its specific developmental stage or physiological condition [72]. Hence, tran-
scriptome plays a role in estimating the expression of genes, as well as in deciphering
the regulation of genes in tissues and organs. Unravelling the transcriptome of tissues
and organs of the species that are of interest can be carried out using RNA sequencing
technology (RNA-seq), which has been shown to be highly reproducible and enables the
simultaneous study of expressed gene samples [73–79]. These RNA-seq features have
made RNA-seq experiments widely used in most transcriptome studies, including the
transcriptome analysis of papaya plants.
Previous studies on papaya transcriptomes have focused on the analysis of several
papaya traits, such as drought effects [73], fruit quality [74,75], sex determination [76,77],
and disease mechanisms [78,79] (Table 1). The molecular response of papaya plants can
be observed by analysing the tissue-specific differentially expressed genes (DEGs) using a
gene-enrichment analysis. Gene-enrichment analysis using AgriGO [80] or ShinyGO [81]
provides information on the biological processes that are regulated in response to the
desired traits in papaya.
A transcriptome study of the delayed sticky disease symptoms in papaya has revealed
the involvement of stress-responses genes in tolerance mechanisms at the pre-flowering
stage [79]. In addition, the authors found that the salicylic-activated genes (i.e., PR1, PR2,
PR5, WRKY) contributed to the delayed symptoms, while the activation of candidate
genes such as NPR1, UDP-glucuronosyltransferase (UGT), and ethylene limit salicylic acid
allowed the PSD symptoms to develop. The nutrient transporter gene family (i.e., nitrate,
ammonium, potassium, sodium, phosphate, and sulfate) was the upregulated gene in the
host during the infection, and had been shown to act as sensors for plant immunity [78,79].
Shen et al. [75] used RNA-seq technology to elucidate the fruit-colouration process in
the ripening condition of papaya. A total of 13 candidate genes, including beta-carotene hy-
droxylase (CHYB), carotene ε-monooxygenase (LUT1), violaxanthin de-epoxidase (VDE),
phytoene synthases (PSY1, PSY2), phytoene desaturases (PDS1, PDS2), zeta-carotene desat-
urae (ZDS), lycopene cyclases (CYCB, LCYB1, LCYB2, LCYE), and zeaxanthin epoxidase
(ZEP), were detected in the papaya fruit transcriptome, which showed that these genes
were involved in the carotenoid biosynthetic pathway.
A transcriptome study of the papaya male flower and male-to-hermaphrodite sex-
reversal flower demonstrated the involvement of 1756 differentially expressed genes in sex
determination [76]. Of these, four papaya homologous genes, including three PIN1 and one
PIN3, were found to be upregulated in the male-to-hermaphrodite sexual-reversal flowers.
Agronomy 2021, 11, 1356 8 of 17

In addition, the authors found the phytohormone-biosynthesis and signal-transduction

pathways in the male-to-hermaphrodite sex-reversal flowers. Similarly, Zhou et al. [77]
used RNA-seq technology to investigate the underlying mechanism in DNA methyla-
tion contribution to the male and female flowers. Dissecting the molecular mechanism
that regulates sex expression in the papaya provides valuable resources that facilitate an
understanding of sexual differentiation in the papaya.
In the papaya disease-resistance trait, the transcriptomes of SunUp and Sunset leaves
were sequenced to investigate the expression changes in both of the papaya cultivars, and
were compared to the regulated genes in the resistant and susceptible varieties [78]. This
comparative transcriptomic analysis found that there were few disease-resistance and
hormone-related genes in SunUp, indicating the PRSV resistance in SunUp transgenic
papaya. The finding of this study also provided evidence that genetically modified papaya
is not harmful.
Using transcriptome data, gene clusters can be observed by performing a gene coexpres-
sion network analysis (Figure 4). This analysis identifies gene–gene interaction, regulation
of biological processes, and molecular functions that occur within a cluster of genes. In
a transcriptome study of papaya leaves, sap, and roots that were under mild and severe
drought, several transcription factors (i.e., WRKY, MYB, bHLH) that were commonly linked
to abiotic stress conditions were identified [73]. These transcription factors can be potential
regulators in the leaves and roots of papaya plants under drought-stress conditions.

Figure 4. Schematic representation of the papaya RNA-seq analysis using a bioinformatics approach. The potential genes
and transcription factor can be validated using qRT-PCR detection, which can be applied in precision breeding through
gene editing or marker-assisted selection (MAS).

3.3. Proteomics
The two-dimensional differential gel electrophoresis (2D-DIGE), isobaric-tags for rela-
tive and absolute quantification (iTRAQ), and matrix-assisted laser desorption/ionisation
time-of-flight mass spectrometry (MALDI-TOF/TOF MS) platforms have been widely used
to estimate the expressed and abundance of proteins in plants, animals, and humans [82].
In papaya, the use of a proteomics platform has focused on the ripening process [83,84]
and disease mechanism [85,86] (Table 1).
The use of a proteomics platform to study the underlying mechanism in papaya
fruit ripening was first reported in 2011. Nogueira et al. [83] performed a comparative
proteomic analysis of climacteric and preclimacteric papaya cultivars using the 2D-DIGE
platform. Several proteins that were closely related to metabolic changes (i.e., cell wall,
ethylene biosynthesis, climacteric respiratory burst, stress response, and chromoplast
differentiation) in the ripening papaya were found, suggesting that these candidate proteins
might be involved in the fruit-ripening process. The 2D-DIGE protein analysis, which was
performed in order to identify the differentially expressed proteins during papaya ripening,
also suggested the role of 1-methylcyclopropene (1-MCP) in affecting the fruit-ripening
Agronomy 2021, 11, 1356 9 of 17

process [83]. In addition, several expressed proteins were associated with sugar and cell-
wall metabolism, signalling, defence and stress responses, folding and protein stability, and
ion channels. Proteins related to pectinesterases, SODs, and the dienelactone hydrolase
family deserve further attention, as these proteins might be involved in the ripening
processes [84]. In a proteomic study that was applied to different ripening stages in papaya,
differential accumulated proteins (DAPs) were quantified using HPLC fractionation and
LC-MS/MS [85]. Interestingly, unsaturated fatty acids (i.e., methyl palmitoleate and methyl
alpha-octadecatrienoic) were increased during the ripening, indicating that they might be
associated with volatile formation in papaya fruit.
Rodrigues et al. [86] used 2-DE and DIGE to identify and distinguish proteins that
had accumulated during sticky disease infection, which was caused by the papaya meleira
virus (PeMV). A proteomics study of the compatible reaction of C. papaya cv. Eksotika in
response to E. mallotivora attack was carried out using an iTRAQ mass spectrometry analy-
sis, and showed differentially expressed proteins that were related to metabolic processes,
defence response, and response to stress [87]. Similarly, iTRAQ mass spectrometry was
used to quantify the effector proteins in E. mallotivora, and suggested the type III secretion
system (T3SS) as an important protein that contributed to the bacterial pathogenicity and
virulance [88]. A high-throughput proteomic study using an LC-MS/MS-based label-free
proteomics approach was used to assess the protein expression between PMeV-infected
preflowering C. papaya and control plants [89]. It was suggested that the increased modula-
tion of photosynthesis, the 26S proteasome, and cell-wall remodelling-associated proteins
were involved in the initiation of papaya plant immunity.
In general, identifying differentially expressed proteins provides valuable resources
for selecting essential proteins in improving C. papaya agronomic traits (i.e., disease re-
sistance and ripening). To encourage reproducibility of proteomics data, the MS-based
proteomics data can be uploaded to a public database such as the ProteomeXchange Consor-
tium [90]. This effort adds additional value of the data in efforts towards the improvement
of papaya traits.

3.4. Metabolomics
Metabolomics is the study of small molecules such as metabolites, substrates, and
metabolic production in an organ, tissue, or cell [5]. Metabolomics has been used to identify
and measure differentially expressed metabolites and to gain an insight into biochemical
composition under different environmental conditions [91]. Papaya is rich in secondary
metabolites that serve as a source of nutrients for human health. High-performance liquid
chromatography or gas chromatography tandem mass spectrometry (LC/GC-MS) and
liquid chromatography-quadrupole time-of-flight (LC-QTOF) have been widely used in
studies of plant metabolomics [92]. Determining the standard chemical compounds of
metabolites and their MS spectrum data is essential in a metabolomic-profiling analysis.
Previous studies performed a comparative analysis of metabolites among papaya
cultivars [93,94] (Table 1). The identification of metabolites among these papaya cultivars is
important in order to identify the cultivar with the essential metabolites that are associated
with quality traits (i.e., sweetness and ripening) that subsequently are to be applied in
papaya breeding programmes. For example, carotenoids, tocopherols, and glucosinolates
are highly abundant in papaya, and all of them are sources of antioxidants [95,96]. The
identification of unique metabolites in a papaya cultivar has highlighted the importance of
the chemical marker in authenticating papaya-based food products [94].
Chilling injury is a part of the constraint in maintaining the freshness of papaya,
especially for exported papaya. A study by Wu et al. [97] identified different metabolite
profiles in the papaya peel at a temperature of 4 ◦ C. The metabolites were associated with
aroma traits, such as organic acid, amino acids, hexanal, carbonic acid, pentadecyl propyl
ester, and methyl geranate, in papaya peel [97]. The elucidation of the metabolite profile
that involves chilling stress at 4 ◦ C can be applied to regulate the storage temperature of
the papaya to prevent chilling injury, and to extend its storage period.
Agronomy 2021, 11, 1356 10 of 17

Apart from the fruit, studies of metabolite profiling in other parts of the papaya, in-
cluding its leaves and seeds, have been conducted to detect the metabolites’ phytomedicinal
properties [98,99]. Papaya leaves are rich in metabolites (i.e., phenolics, flavonoid, saponins,
and tocopherol) that have potential antimicrobial, anticancer, antioxidant, and pesticide
properties [100]. Hence, papaya leaves have been widely used in the pharmacological
industry for drug development.
Metabolomics that has been combined with other high-throughput omics technologies,
such as transcriptomics and proteomics, is referred to as integrated metabolomics, which is
sometimes used in studies that are aimed at understanding metabolism as the phenotype of
genome function [92]. However, little is known on the integration between metabolomics
and transcriptomics to understand the mechanism during the fruit-ripening process. It has
been reported that metabolites (i.e., flavonoids, terpenoids, organic acids, phenolic acids, and
alkaloids) are closely related to the ripening disorder of fruits [101–103]. The elucidation of these
potential metabolites during papaya-ripening processes will provide valuable information for
developing a strategy for postharvest storage and improving the fruit quality.
Table 1. A summary of the recent omics and bioinformatics approaches that are used in the improvement of papaya traits.

Type of Omics
Traits/Conditions Descriptions Approach Reference
Platform
Whole-genome sequences of papaya cultivar
Whole-genome shotgun
Genomics - SunUp. Development of first papaya [2]
Sanger sequencing
reference genome sequences.
Whole-genome resequencing of papaya
Whole-genome
cultivars Eksotika and Sekaki to identify
resequencing using
putative SNPs. The identified SNPs between
- Illumina HiSeq2000 [58]
Eksotika and Sekaki located in genes of
(Illumina Inc., CA, USA)
interest could be suggested for validation
and bioinformatic analysis
using a genotyping platform.
Whole-genome resequencing of papaya
cultivar SunUp (transgenic) and Sunset
Whole-genome
(nontransgenic) to identify SNPs and InDels,
resequencing using
and used in comparing transgenic and
- Illumina HiSeq2000 [59]
nontransgenic papaya. The identified SNPs
(Illumina Inc., CA, USA)
and InDels that were located in high-impact
and bioinformatic analysis
genes could be applied in marker-assisted
PRSV disease-resistance breeding in papaya.
Whole-genome resequencing of wild-type Whole-genome
and cultivated papaya to detect structural resequencing using
- variations in papaya, and used in Illumina HiSeq2500 [65]
understanding the process of papaya (Illumina Inc., CA, USA)
domestication. and bioinformatic analysis
Gene-based SSR marker development Bioinformatics and
Ripening [61]
focusing on genes related to fruit ripening. genotyping
Whole-genome
resequencing using
Illumina HiSeq4000
Polymorphic SSR marker development for
(Illumina Inc., Foster City, [62]
marker-assisted breeding in papaya.
CA, USA),
bioinformatics, and
genotyping
Genome-wide identification of SNPs and
InDels using whole-genome resequencing of
two papaya cultivars, namely Sekati and Whole-genome
JS-12. The SNPs that were located in RRGs are resequencing using
[64]
potential SNPs to be converted in PCR Illumina Miseq (Illumina
markers, and could be applied in papaya Inc., Foster City, CA, USA)
genetic mapping and diversity studies, as
well as marker-assisted selection.
Agronomy 2021, 11, 1356 11 of 17

Table 1. Cont.

Type of Omics
Traits/Conditions Descriptions Approach Reference
Platform
Genome-wide analysis of basic
Comparative genomics
Abiotic helix–loop–helix (bHLH) transcription factors.
and quantitative real-time [31]
stress Candidate bHLH genes that might be
PCR (qRT-PCR)
responsible for abiotic stress.
Genome-wide analysis of NBS resistance gene
Comparative genomics
family. Candidate resistance (R) genes
Disease resistance and quantitative real-time [24]
potentially responsible for disease-resistance
PCR (qRT-PCR)
mechanism.
Genome-wide analysis of NPR1 family.
Comparative genomics
Candidate pathogenesis-related genes that
Disease resistance and quantitative real-time [32]
might be responsible for a disease-resistance
PCR (qRT-PCR)
mechanism.
Genome-wide analysis of SQUAMOSA
Comparative genomics
promoter binding protein-like gene family in [33]
Ripening and quantitative real-time
papaya. Candidate ripening- and
PCR (qRT-PCR)
development-related genes.
Genome-wide analysis of Aux/IAA gene Comparative genomics
Ripening family. Candidate ripening-related genes in and quantitative real-time [34]
papaya. PCR (qRT-PCR)
Genome-wide analysis of auxin response
factor (ARF) family genes related to flower Comparative genomics
Flower
and fruit development in papaya. and Quantitative real-time [35]
development
Candidate genes related to flower and fruit PCR (qRT-PCR)
development.
Transcriptome sequencing
using Illumina NextSeq500
Coexpression network analysis to identify
(Illumina Inc., Foster City,
Transcriptomics Drought tolerance genes and transcription factors related to [73]
CA, USA)
abiotic stress.
and coexpression network
analysis
Identification of potential regulatory genes Transcriptome sequencing
Ripening
during papaya ripening underlying 1-MCP using Hiseq Xten (Illumina [74]
mechanism
treatment. Inc., Foster City, CA, USA)
Transcriptome sequencing
Identification of potential TF regulating the
Fruit colouration using [75]
carotenoid biosynthetic pathway.
Illumina HiSeq2500
(Illumina Inc., Foster City,
CA, USA)
Differential expressed genes in sex Transcriptome sequencing
Sex determination determination of papaya, in using [76]
male-to-hermaphrodite and male flowers. Illumina HiSeq2500
(Illumina Inc., Foster City,
CA, USA)
Transcriptome sequencing
using
Identification of disease-resistance genes in
Disease resistance Illumina HiSeq2500 [78]
PRSV-resistant and susceptible cultivars.
(Illumina Inc., Foster City,
CA, USA)
Transcriptome sequencing
Identification of stress-response genes and
using Illumina HiSeq2000
Disease resistance nutrient upregulated genes in tolerance [79]
(Illumina Inc., Foster City,
mechanism of papaya sticky disease.
CA, USA)
Comparative proteomic analysis of
Ripening
Proteomics climacteric and preclimacteric papaya 2-DGE and LC-MS/MS [83]
mechanism
cultivars.
Agronomy 2021, 11, 1356 12 of 17

Table 1. Cont.

Type of Omics
Traits/Conditions Descriptions Approach Reference
Platform
Ripening Differentially expressed proteins during 2-DGE and QTRAP hybrid
[84]
mechanism papaya ripening. tandem mass spectrometer
Ripening Differentially accumulated proteins (DAPs)
HPLC and LC-MS/MS [85]
mechanism during papaya ripening.
Identification of differentially expressed
proteins in healthy and PMev disease leaf
MALDI-TOF-MS/MS and
Disease samples in the Golden cultivar.
DIGE/LC-IonTrap- [86]
mechanism Metabolism-related proteins were
MS/MS
downregulated, and stress-responsive
proteins were upregulated.
Differentially expressed proteins of
Disease
compatible reaction between Eksotika papaya iTRAQ mass spectrometry [87]
mechanism
and E. mallotivora
Disease Protein expression between PMeV-infected LC-MS/MS-based
[88]
mechanism preflowering C. papaya and control plants label-free proteomics
Fruit Comparative analysis of metabolite profiling
Metabolomics GC-MS [93]
ripening between Eksotika and Sekaki cultivars.
Profiling analysis of bioactive and volatile
Fruit
compounds in two papaya cultivars, namely HPLC-ESI-MS/MS [94]
ripening
Sel-42 and Tainung.
Comparative profiling of carotenoids and
Fruit
volatile in yellow and red flashed between Sui HPLC-ApCI-MS [95]
ripening
huang and Sui hong cultivars.
Transcriptome sequencing
using Illumina Hiseq Xten
Identification of genes and metabolites
Fruit (Illumina Inc., Foster City,
regulating fruit ripening and softening in [103]
ripening CA, USA) and
papaya cultivar Suiyou-2.
metabolomics profiling
using HPLC-ESI-MS/MS
Elucidating of primary metabolites and
Chilling
volatile changes in papaya peel in response to GC-MS /MS [97]
injury
chilling stress.
Bioactive UPLC-ESI-MS
Metabolite profiling in papaya leaves. [98–100]
properties and GC-MS/MS

4. Future Perspective
The progress of the improvement of papaya traits has been limited due to the lack
of genetic and genomic information on papaya. The outcome of recent omics studies of
papaya plants suggests that there is potential for using these valuable genetic and genomic
resources as a breeding tool to improve the desired traits in papaya. Incorporating omics
data in papaya breeding programmes with a focus on abiotic stress, disease resistance, de-
layed ripening, and sweetness offers a promising strategy for developing high-quality traits
in papaya cultivars without compromising their yields or agronomic traits. A previous
study in Mexico conducted a network analysis of the interaction between viruses in papaya
orchards. This viral metagenomics study, which was coupled with a network analysis,
could contribute to the understanding of the host–pathogen interactions, which would
cater to the management strategies against PRSV and non-PRSV symptoms in papaya [104].
The potential of computational approaches in understanding these biological systems has
been employed in crop improvement. The computational models that were constructed
integrate genome and phenome information, which led to new experimental strategies in
improving crop production [105].
In papaya, pangenome analysis has not received much attention. Using whole-genome
resequencing of papaya cultivars from diverse germplasms enables researchers to perform
pangenome analyses, which would facilitate the identification of core and variable genes in
Agronomy 2021, 11, 1356 13 of 17

various papaya genomes. This effort has been performed in soy bean [106] and B. rapa [107].
A further area of interest is to screen for favourable alleles of diverse resistance genes
sourced from the wild germplasm of papaya.
Another direction in papaya-trait improvement is employing the genome-editing ap-
proach. Functional analysis of candidate genes in the papaya–pathogen system can be per-
formed using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-
associated protein 9(Cas9) system. The CRISPR-Cas9 system was successfully applied in
banana [108], apple [109], and kiwifruit [110].

5. Conclusions
The use of omics data plays a role as an advanced breeding tool that will enable the
faster and more accurate selection of key consumer-driven traits. Integration of various
high-throughput omics platforms may accelerate the research on papaya crop improvement.
In addition, the application of computational approaches is key in revealing and filling data
gaps, which will be valuable in the designing of new experimentation and measurement
strategies that would result in enhanced papaya quality, as well as the ability of it to be
sustained under various environmental conditions.
Author Contributions: R.-A.Z.-A. and S.H. conceived the idea; R.-A.Z.-A. wrote the original draft;
S.H. and I.-H.R.-O. reviewed and edited the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was funded by the Government of Malaysia under the 12th Malaysia Plan
Development through Malaysian Agricultural Research and Development Institute (MARDI), grant
number (PRB502).
Conflicts of Interest: The authors declare no conflict of interest.

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