ORIGINAL RESEARCH
published: 03 December 2020
doi: 10.3389/fpls.2020.607161
Simultaneous CRISPR/Cas9 Editing
of Three PPO Genes Reduces Fruit
Flesh Browning in Solanum
melongena L.
Alex Maioli 1† , Silvia Gianoglio 2† , Andrea Moglia 1*, Alberto Acquadro 1 , Danila Valentino 1 ,
Anna Maria Milani 1 , Jaime Prohens 3 , Diego Orzaez 2 , Antonio Granell 2 , Sergio Lanteri 1
and Cinzia Comino 1
1
DISAFA, Plant Genetics and Breeding, University of Torino, Grugliasco, Italy, 2 Crop Biotechnology Department, Instituto de
Biología Molecular y Celular de Plantas (IBMCP), CSIC-UPV, Valencia, Spain, 3 Instituto de Conservación y Mejora de la
Agrodiversidad Valenciana, Universitat Politècnica de València, Valencia, Spain
Edited by:
Amalia Barone,
University of Naples Federico II, Italy
Reviewed by:
Yoshihito Shinozaki,
Tokyo University of Agriculture and
Technology, Japan
Abdelali Hannoufa,
Agriculture and Agri-Food Canada
(AAFC), Canada
Marina Tucci,
National Research Council (CNR), Italy
*Correspondence:
Andrea Moglia
[email protected]
† These
authors have contributed
equally to this work
Specialty section:
This article was submitted to
Plant Metabolism and Chemodiversity,
a section of the journal
Frontiers in Plant Science
Received: 16 September 2020
Accepted: 06 November 2020
Published: 03 December 2020
Citation:
Maioli A, Gianoglio S, Moglia A,
Acquadro A, Valentino D, Milani AM,
Prohens J, Orzaez D, Granell A,
Lanteri S and Comino C (2020)
Simultaneous CRISPR/Cas9 Editing of
Three PPO Genes Reduces Fruit Flesh
Browning in Solanum melongena L.
Front. Plant Sci. 11:607161.
doi: 10.3389/fpls.2020.607161
Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes
the browning of the eggplant berry flesh after cutting. This has a negative impact on
fruit quality for both industrial transformation and fresh consumption. Ten PPO genes
(named SmelPPO1-10) were identified in eggplant thanks to the recent availability of
a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was
applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6),
which showed high transcript levels in the fruit after cutting. An optimized transformation
protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed
to a conserved region shared by the three PPO genes. The successful editing of
the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was
confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep
sequencing of amplicons of the potential off-target loci identified in silico proved the
absence of detectable non-specific mutations. The induced mutations were stably
inherited in the T1 and T2 progeny and were associated with a reduced PPO activity
and browning of the berry flesh after cutting. Our results provide the first example of
the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and
open the way to the development of eggplant genotypes with low flesh browning which
maintain a high polyphenol content in the berries.
Keywords: gene editing, CRISPR/Cas 9, eggplant, polyphenol oxydase, knock-out
INTRODUCTION
The polyphenol oxidases (PPOs) are a group of enzymes catalyzing the oxidation of phenolic
compounds into highly reactive quinones (Prohens et al., 2007; Mishra et al., 2013; Plazas et al.,
2013; García-Fortea et al., 2020). The physiological role of PPOs in plants has not been fully clarified
yet, but a defense role against pathogens and pests has been postulated because of their increased
localized activity in response to cutting and wounding. The relationship between PPO expression
or activation and pathogen infections was proved in tomato by either silencing (Thipyapong et al.,
2004) or over-expressing PPO genes (Li and Steffens, 2002). PPOs oxidize polyphenols to toxic
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Maioli et al.
CRISPR Cas9 in Eggplant
quinones which bind to amino acids in the insect gut, exerting an
anti-feeding role. Previous studies have associated PPO activity
with resistance to various types of insects (Mahanil et al., 2008).
In recent years, PPOs have been largely investigated for their
involvement in the browning process, a color reaction caused
by the oxidation of phenolic compounds during postharvest
processing and storage. Enzymatic browning is a two-step
reaction, consisting of the oxidation of a monophenol to a odiphenol (cresolase/monophenolase activity), which is further
oxidized to yield a o-quinone (catecholase/diphenolase activity).
O-quinones can then undergo condensation or polymerization
reactions, producing the dark pigments melanins. Fruit cutting
causes cellular disruption and damages membrane integrity,
allowing the PPOs sequestered in the plastid to come into contact
with the hydroxycinnamic acid derivatives, which are their
substrates. Extensive browning of cut fruit and vegetable surface
compromises food quality and usually impairs the properties
of the product, representing a major economic problem both
for the food industry (e.g., the industrial manipulation and
preservation of these products) and for consumers (in the case
of fresh and ready-to-eat fresh cut fruit and vegetables). Since
PPO activity is influenced by factors such as pH, temperature
and oxygen, the browning process is limited in the food industry
through the use of chemical and/or physical agents, with a
negative impact on nutritional and organoleptic properties.
Browning negatively affects the commercial value of many
key agricultural productions, including potato, lettuce, cereals,
banana, cucumber, grape and eggplant (Taranto et al., 2017).
Eggplant (Solanum melongena L.) berries are characterized by
a remarkable content in phenolic compounds, represented
mainly by chlorogenic acid (5-O-caffeoylquinic acid).
Chlorogenic acid plays important therapeutic roles due to
its antioxidant, antibacterial, hepatoprotective, cardioprotective,
anti-inflammatory and anti-microbial properties (Naveed et al.,
2018). In eggplant, a correlation between the concentration of
phenolics (mainly chlorogenic acid) and browning has been
detected in the fruit flesh, although additional morphological and
physiological factors may be involved in browning phenomena
(Kaushik et al., 2017). Furthermore, in commercial varieties,
the selection for berries with a reduced degree of browning in
the flesh has resulted in the indirect selection of accessions with
lower concentrations of phenolics (Prohens et al., 2007).
Shetty et al. (2011) identified six genes encoding PPOs in
eggplant and, on the basis of both protein sequence similarity
and organ-specific patterns of expression, they proposed the
distinction of eggplant PPOs in two clades: A and B, with clade
A encompassing genes expressed mostly in roots, while clade B
genes are involved in defense mechanisms. This categorization
was further extended to the rest of Solanaceae PPOs (Taranto
et al., 2017).
The development of new technologies to disable genes coding
for PPOs represents the most promising strategy to avoid
undesired browning in plant-derived products, as it would allow
to positively select genotypes enriched in beneficial phenolic
compounds, while reducing the need for physical and chemical
treatments in the food industry. The positive impact on the
storability of these foods, in addition, would help reduce waste.
Frontiers in Plant Science | www.frontiersin.org
Several examples are available on the adoption of RNA
silencing strategies to down-regulate PPO genes in order to
reduce the enzymatic browning in potato tubers (Bachem et al.,
1994; Coetzer et al., 2001; Rommens et al., 2006; Llorente
et al., 2011; Chi et al., 2014). By using artificial micro-RNAs
(amiRNAs) all StuPPO genes have been silenced individually or
in combination, identifying StuPPO2 as the main contributor to
PPO activity (Chi et al., 2014). A few notable examples exist
of commercially available genetically modified plants in which
PPOs have been silenced, such as the Arctic Apple R and the
Innate R potato. The emergent CRISPR/Cas9 technology has
proved extremely efficient in gene editing and is expected to
play a key role in crop breeding. This technology makes it
possible to induce point mutations in one or multiple target
sequences simultaneously, as well as to introduce new genetic
variants through homology directed recombination (HDR), or
to modulate transcription and chromatin structure at selected
target loci (Doudna and Charpentier, 2014). While this technique
has been successfully applied to some Solanaceae species, such as
tomato and potato, including the knock out of the StuPPO2 gene
in the potato tetraploid cultivar Desiree (González et al., 2020),
no examples of genome editing in eggplant have been reported in
literature so far (Van Eck, 2018).
In this study, thanks to the recent availability of a high
quality, annotated and anchored eggplant genome sequence
(https://solgenomics.net/organism/Solanum_melongena/
genome; Barchi et al., 2019), we report the homologybased characterization, functional domain identification
and phylogenetic analysis, of 10 PPO (SmelPPO1-PPO10) genes
in eggplant. On the basis of their expression in the fruit after
cutting, SmelPPO4, SmelPPO5, and SmelPPO6 were selected for
the generation of knock-out mutants using the CRISPR/Cas9
technology. Regenerated T0 , T1, and T2 lines were screened for
induced mutations in the target genes as well as in potential
off-target loci. In addition, PPO activity and the degree of
browning in the flesh of eggplant berries were analyzed in our
knock-out T1 and T2 edited lines.
MATERIALS AND METHODS
Mining of PPO in the Eggplant Genome
and Phylogenetic Analysis
The six eggplant PPO aminoacidic sequences previously reported
(Shetty et al., 2011) were used for a BlastP search of the
eggplant proteome (https://solgenomics.net/organism/Solanum_
melongena/genome) with an E-value threshold of 1 e−5 . The
polypeptide sequences of eggplant PPOs, together with those
of six tomato and nine potato PPOs (Data Sheet 1), were used
for a multiple alignment (Clustal Omega; https://www.ebi.ac.
uk/Tools/msa/clustalo/). A phylogenetic analysis was performed
with the MEGA X software. An unrooted phylogenetic tree
was generated, applying the Neighbor-Joining (NJ) algorithm.
The statistical significance of individual nodes was assessed by
bootstrap analysis with 1,000 replicates, and the evolutionary
distances were calculated using the p-distance method with
default parameters.
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CRISPR Cas9 in Eggplant
qPCR Analysis
pH 7) supplemented with 50 mg l−1 rifampicin and 50 mg l−1
kanamycin, and incubated overnight at 28◦ C. From this, a second
culture was set up in TY liquid medium (tryptone 5 g l−1 , yeast
extract 3 g l−1 , MgSO4 .7H2 O 0.5 g l−1 , pH 5.8) supplemented
with 200 µM acetosyringone and incubated overnight in the
dark at 28◦ C. Before transformation, the optical density of
the culture at 600 nm (OD600 ) was measured and the bacterial
culture was diluted to a final OD600 of 0.10–0.15 in TY medium
supplemented with 200 µM acetosyringone. Explants of about
5 mm in length were cut from the cotyledons of in vitro
germinated ‘Black Beauty’ seeds, dipped in the bacterial culture
for a minimum of 10 min, blotted dry on filter paper and
transferred for 48 h on a co-culture medium (MS basal salt
mixture 4.5 g l−1 , MES 0.5 g l−1 , sucrose 30 g l−1 , phytoagar 10 g
l−1 , Gamborg vitamin mixture 1 ml l−1 , trans-zeatin 2 mg l−1 ,
IAA 0.1 mg l−1 , acetosyringone 200 µM, pH 5.8), in the dark. For
organogenesis and shoot induction, a common basal induction
medium was used, as previously described (Muktadir et al., 2016)
(MS basal salt mixture 4.5 g l−1 , MES 0.5 g l−1 , sucrose 30 g
l−1 , phytoagar 10 g l−1 , Gamborg vitamin mixture 1 ml l−1 ,
trans-zeatin 2 mg l−1 , IAA 0.1 mg l−1 , kanamycin 30 mg l−1 ,
carbenicillin 400 mg l−1 , pH 5.8), with three different conditions:
without further additives, with supplementation of ascorbic acid
5 mg l−1 and citric acid 5 mg l−1 , and with supplementation of
polyvinylpyrrolidone (PVP40) 200 mg l−1 . Furthermore, for each
medium composition, two conditions were tested during the first
3 days of induction: no incubation, or 3 days of incubation in the
dark, after which explants were grown in the same conditions as
the untreated group (16:8 light:dark cycle, 24◦ C). Elongation and
rooting were performed on the same media for all conditions and
explants were moved to a fresh medium every 2–3 weeks. Both
media were previously described (Muktadir et al., 2016) and were
not supplemented with antioxidants, as no oxidative damage
was observed from this stage onwards. The elongation medium
was supplemented with kanamycin 30 mg l−1 and carbenicillin
400 mg l−1 , but did not contain any hormone. Kanamycin was
removed from the rooting medium to avoid inhibitory effects on
root development, and 0.2 mg l−1 indolebutyric acid were added.
Fully developed plantlets were then moved to soil and gradually
acclimated to ex vitro conditions.
To identify PPO genes involved in the browning phenotype their
corresponding mRNA levels were analyzed in the flesh of fruits of
the “Black Beauty” variety harvested at the commercial ripening
stage (Mennella et al., 2012) after cutting them transversally with
a sharp knife. One gram of frozen fruit flesh was ground in
liquid nitrogen to a fine powder and RNA was extracted using the
“Spectrum plant total RNA kit” (Sigma-Aldrich, St. Louis, USA).
RNA was extracted in three biological replicates from commercial
grade ripe fruit 1 cm-wide slices exposed to air for 0 min (t0 ) and
30 min (t30 ).
cDNA was synthesized from 1 µg of RNA using a High
Capacity RNA-to-cDNA kit (Applied Biosystems, Foster
City, USA) as directed by the manufacturer. Using the
Primer 3 software (http://bioinfo.ut.ee/primer3), primers
targeting the ten identified eggplant PPO genes were designed
(Supplementary Table 1). PCR reactions were carried out in
three biological replicates using the StepOnePlus Real-Time
PCR System (Applied Biosystems). The following PCR program
was used: 95◦ C/10 min, followed by 40 cycles of 95◦ C/15 s
and 60◦ C/1 min. Data were quantified using the 2−11Ct
method based on Ct values of actin and elongation factor as
housekeeping genes. Values are expressed as relative mRNA
abundance at 30 min after cutting compared to time 0 (just
after cutting).
Target Identification, DNA Construct
Cloning, and Off-Target Search
Sequences of the wound-induced SmelPPO4, SmelPPO5, and
SmelPPO6 genes were aligned to find conserved regions, and
BlastX and Prosite were used to annotate functional domains.
A gRNA (ATGAATGGAAAGCAATCGGA) was designed to
target a conserved region of these three genes and assembled
into a CRISPR/Cas9 construct carrying the hCas9 and the
nptII gene for kanamycin resistance, using the GoldenBraid
(GB) assembly system and following GB software-directed
procedures (https://gbcloning.upv.es/). An additional guanine
was added at the 5’end in order to improve expression under
the U6-26 RNA PolIII promoter (Cong and Zhang, 2015).
The hCas9 expression is driven by the CaMV 35S promoter,
while the gRNA is placed under the control of the AtU6-26
RNA PolIII promoter. Putative off-target sites were identified
with the CasOT software (http://casot.cbi.pku.edu.cn/), using
the eggplant genome as reference. Four off-targets (OT1-OT4)
were selected based on the number and position of mismatches
(Supplementary Table 2); the corresponding loci (a 1 kb region
around the putative off-target site) were inspected, to determine
whether they corresponded to functional genes, and considered
for sequencing analyses.
Target and Off-Target Sequencing
Genomic DNA was extracted using a CTAB protocol (Doyle
and Doyle, 1987) from leaves sampled when plantlets were
transferred from in vitro growth conditions to soil. The
presence of the transgene was assessed by amplifying the hCas9
(Supplementary Table 3) gene by using qPCR (in three technical
replicates) according to the protocol described in the previous
paragraph. DNA was also extracted from T1 and T2 progeny
plants (Data Sheet 3).
Mutation frequencies at the target and off-target sites
were evaluated according to an adapted version of the 16S
Metagenomic Sequencing Library preparation protocol provided
by Illumina (16S Sample Preparation Guide). Amplifications
were carried out using the KAPA HiFi HotStart ReadyMix PCR
Kit (Kapa Biosystems, Boston, MA). Dual indexing was done
using the Nextera XT system (Illumina, San Diego, CA) using
Genetic Transformation of Plants
The final pCambia vector Tnos:nptII:Pnos-U6-26:gRNA:scaffoldP35S:hCas9:Tnos was transformed into LBA4404 Agrobacterium
tumefaciens strain. A pre-culture was set up in a modified
MGL liquid medium (tryptone 5 g l−1 , yeast extract 2.5 g l−1 ,
NaCl 0.1 g l−1 , mannitol 5 g l−1 , glutamic acid 1.15 g l−1 ,
KH2 PO4 0.25 g l−1 , MgSO4 .7H2 O 100 g l−1 , biotin 1 mg l−1 ,
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CRISPR Cas9 in Eggplant
RESULTS AND DISCUSSION
16 i5 indexes (S502-S522) and 24 i7 indexes (N701-N729),
enabling the multiplexing of 333 individual libraries. Due to their
high sequence identity, a differential amplification of SmelPPO4
and SmelPPO6 was obtained with a first specific PCR, using
primers designed on flanking non conserved regions, while a
second amplification was performed with non-specific primers
carrying Illumina adapter sequences (Supplementary Table 3).
Amplifications of SmelPPO5, OT1, OT2, OT3, OT4 were done
directly using primers modified with Illumina adapter sequences
(Supplementary Table 3). Products were diluted 1:50 and used
as templates to add dual Nextera XT indexes (Data Sheet 2).
Finally, indexed amplicons were purified using AmpureBeads
(0,7X) and quantified using Qubit 2.0 (Life Technologies,
Carlsbad, CA, USA), based on the Qubit dsDNA HS Assay
(Life Technologies, Carlsbad, CA, USA). All samples were
diluted to 4 nM and pooled in a single tube. Sequencing
was performed with an Illumina MiSeq sequencer (Illumina
Inc., San Diego, CA) and 150 bp paired-end reads were
generated. From reads generated by WGS sequencing, adapters
were removed and reads that were <50 nucleotides long
were discarded using Trimmomatic v0.39 (Bolger et al., 2014).
Processed reads were analyzed for CRISPR/Cas9 editing events
with CRISPResso2 [http://crispresso2.pinellolab.org (Clement
et al., 2019)] (Supplementary Table 4). Sequences can be can be
downloaded at https://www.crispr-plants.unito.it/eggplant.
SmelPPO Identification and Phylogenetic
Analysis
In addition to the six sequences previously reported (Shetty et al.,
2011), four new loci in the eggplant genome were found to
encode polyphenol oxidases and named SmelPPO7-10 (Table 1).
Coding sequences retain extensive structural similarities both
within S. melongena and with homologs in tomato and potato.
The CDS of PPOs range in size from 1,686 to 2,466 bp; all
genes except SmelPPO3 and SmelPPO4 are on the negative strand
and, like the PPO genes of tomato and potato, eggplant PPOs
do not possess introns. In all the three Solanum species, PPO
genes cluster on chromosome 8 (Figure 1A), with the exception
of one orthologous gene (SmelPPO10 in eggplant, StuPPO9 in
potato, and SlPPOG in tomato), mapping on chromosome 2. This
suggests that PPO genes evolved from tandem duplications and
further supports the notion that the structure of this gene family,
and possibly its functional specializations, are conserved across
Solanaceae species.
The PPO encoded proteins range in size from 562 to 822 aa
(Table 1). All polypeptides possess the same functional domains,
namely the central tyrosinase and PPO1_DWL domains, and
a C-terminal domain of unknown function (DUF_B2219),
characterized by the KFDV conserved motif. In accordance with
previous reports (Taranto et al., 2017), we confirmed that in the
Solanaceae family two main clusters can be distinguished among
PPO proteins (Figure 1B), which correspond to a functional
separation between PPOs that are preferentially expressed in
roots (tomato SlPPO A-D, potato StuPPO2 and StuPPO4 and
eggplant class A proteins, i.e., SmelPPO1-3) and PPOs whose
expression is associated to defense responses (tomato SlPPO
E and F, potato StuPPO1 and eggplant class B proteins, i.e.
SmelPPO4-6). Among the newly identified proteins, SmelPPO7
clusters with class A proteins, SmelPPO8 with StuPPO5 and
SmelPPO9 with StuPPO8. Finally, SmelPPO10 clusters with
StuPPO9 and SlPPOG.
PPO Activity Assay
Fruits of the wild type and edited lines (T1 and T2 ) were collected
in eight biological replicates at the commercial ripening stage
(Mennella et al., 2012). Flesh slices about 1 cm thick, cut at the
midpoint between the blossom and stem ends, were exposed
to air for 30 min (t30 ) before pictures were taken. After the
exposition, all fresh tissues were immediately frozen in liquid
nitrogen and stored at −80◦ C for PPO activity measurement
of the eight biological replicates. PPO activity analysis was
performed according to previously described protocols (Bellés
et al., 2006; Plazas et al., 2013) with minor modifications: 1 g
of fresh frozen peel tissue was taken and ground in a mortar
with liquid nitrogen and 50 mg of polyvinylpolypyrrolidone
before being resuspended in 4 ml 0.1 M sodium phosphate
′
buffer pH 6. Samples were sonicated in a water bath for 10
′
◦
◦
at 20 C, centrifuged at 12,000 rpm for 15 at 4 C and the
supernatant was collected. Protein concentration was evaluated
using Bradford’s dye (Sigma Aldrich) binding assay using
bovine serum albumin (Sigma Aldrich) as a standard (Bradford,
1976). PPO activity was measured colorimetrically at room
temperature using a spectrophotometer (Beckman Coulter, Brea,
CA, USA) to follow the emerging enzymatic reaction. For
sample analysis, 145 µl sodium phosphate buffer (0.1 M, pH
6, RT), 15 µl chlorogenic acid (Sigma Aldrich, 35.5 mg ml−1 ),
and 40 µl of protein extract were mixed and absorbance
(415 nm) measured every 10 s for 25 min. A negative control
without protein extract was even analyzed. One unit of enzyme
activity was defined as the increase in 0.1 absorbance unit
per minute per milligram of fresh weight (Kaushik et al.,
2017).
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Transcriptional Profiling in Response to
Wounding
Oxidative browning in eggplant is influenced by multiple factors,
including total phenolic content, PPO expression and also the
way in which the plant integrates environmental stimuli to elicit
defense responses (Mishra et al., 2013; Plazas et al., 2013; Docimo
et al., 2016). The differential spatial and temporal expression
patterns of PPOs in planta reflect the functional diversity among
the PPO gene members. In eggplant, the expression of SmelPPO16 genes was higher in young tissues and declined during plant
development in mature and reproductive organs (Shetty et al.,
2011). In fruits, PPO expression was mainly concentrated in
the exocarp and in the areas surrounding the seeds in the
mesocarp (Shetty et al., 2011). PPO expression is mainly induced
by herbivores or by mechanical damage, such as cutting.
The promoters of group B genes (Shetty et al., 2011) are
characterized by the presence of several responsive elements
for wounding stress and defense response (Thipyapong et al.,
1997). The structural similarity of eggplant class B PPO
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CRISPR Cas9 in Eggplant
TABLE 1 | Characteristics of PPO encoding genes and of PPO proteins.
Locus
Gene name
Chr
Chromosome location
ORF
length
(bp)
Strand
Size
(aa)
Protein domains
Pfam domains
SMEL_008g312510.1.01
Smel_PPO1
8
97,412,508: 97,414,307
1,800
-
600
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312500.1.01
Smel_PPO2
8
97,401,279: 97,403,066
1,788
-
596
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312430.1.01
Smel_PPO3
8
97,284,426: 97,286,198
1,773
+
591
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312420.1.01
Smel_PPO4
8
97,238,764: 97,239,741
1,734
+
578
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g311990.1.01
Smel_PPO5
8
96,314,480: 96,316,243
1,764
-
588
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312010.1.01
Smel_PPO6
8
96,395,550: 96,397,448
1,899
-
633
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312490.1.01
Smel_PPO7
8
97,397,374: 97,399,167
1,794
-
598
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312460.1.01
Smel_PPO8
8
97,349,335: 97,351,020
1,686
-
562
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_008g312520.1.01
Smel_PPO9
8
97,429,811: 97,432,277
2,466
-
822
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SMEL_000g064350.1.01
Smel_PPO10
2
982,270: 984,463
2,193
-
731
PPO1_DWL–
DUF_B2219–Tyrosinase
Pfam 12142–Pfam 1243Pfam 00264
SmelPPO1-9 cluster on chromosome 8, while PPO10, which was initially located on an unanchored scaffold, is probably located on chromosome 2. All PPOs share the same functional
domains (PPO1_DWL and Tyrosinase, and a conserved domain of unknown function, DUF_B2219).
being mostly inefficient or highly dependent on the genotype,
no examples of genome editing in this species has been reported
in literature so far. After the first report of the Agrobacteriummediated transformation of eggplant (Guri and Sink, 1988),
several examples of genetic transformation have been proposed
using seedling explants like the hypocotyl, epicotyl, and node
segments and cotyledon segments, leaf disks or roots (Rotino
et al., 2014; Saini and Kaushik, 2019; García-Fortea et al., 2020).
In many plant species, the browning of tissues, which
leads to toxicity and necrosis, is one of the major causes
of unsuccessful in vitro organogenesis and regeneration from
explants. Browning is associated with the oxidation of phenolics,
whose release is caused by cutting and manipulating explants
and calli. This problem is particularly relevant in eggplant, whose
tissues are rich in phenolic compounds. Among strategies to
avoid browning, the most common include the supplementation
of culture media with antioxidant or adsorbent compounds
(Abdelwahd et al., 2008; Menin et al., 2013).
We tested different strategies to reduce browning during
eggplant tissue culture, including the addition of citric and
ascorbic acid and PVP supplementation, and we found out
that PVP supplementation exerts a positive effect on shoot
regeneration. Among a total of 15 rooted shoots, 10 derived from
the PVP-supplemented medium, four from not supplemented
medium, and only 1 from the medium supplemented with
ascorbic and citric acids. No differences were found in the
phenotype of regenerants from different culture conditions.
However, in spite of their notably higher number, the emergence
of shoots on the PVP-supplemented medium was slower.
genes (SmelPPO4-5-6) to wound-induced tomato SlPPOF might
suggest an analogous pattern of gene regulation (Thipyapong
et al., 1997).
In our study we analyzed the transcript levels of PPO genes
in the flesh of full-ripe eggplant berries of the “Black Beauty”
variety 30 min after cutting (Figure 2). A strong increase in gene
transcription in the flesh was observed for all PPOs, and especially
for SmelPPO1 (7.45X), SmelPPO4 (3.03X), SmelPPO6 (4.00X),
SmelPPO8 (3.59X), and SmelPPO10 (4.01X). The simultaneous
activation of both A and B classes of PPO genes was already
observed in the eggplant cultivars AM086 (Docimo et al.,
2016) and Arka Shirish (Shetty et al., 2011). Based on this
transcriptional profile, we hypothesized that the design of an
appropriate editing strategy directed at reducing detrimental
oxidative browning in fruit tissues might require simultaneous
suppression of several members of this multigene family. In
our experiments we targeted class B PPO genes (SmelPPO4,
SmelPPO5, and SmelPPO6) through a CRISPR/Cas9 editing
strategy. Due to their extremely high level of similarity, it was
possible to design a unique gRNA against the tyrosinase domain
of all class B genes.
Plant Regeneration
The development of new genome editing technologies in plant
breeding has fostered a growing interest for in vitro culture and
regeneration protocols, which represent a major bottleneck in
the application of these techniques in many plant species of
agricultural and industrial interest. Due to the difficulties often
encountered in eggplant regeneration, with available protocols
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FIGURE 1 | (A) Relative position and organization of PPO1-9 genes on chromosome 8 of Solanum melongena. All eggplant PPOs except SmelPPO10 are located on
chromosome 8. (B) Phylogenetic analysis of PPO proteins. The neighbor-joining trees was constructed by aligning the PPO protein sequences contained in
Data Sheet 1. Clade A (orange) and B (yellow) proteins. The number at each node represents the bootstrap percentage value from 1,000 replicates. Smel, Solanum
melongena; Sl, Solanum lycopersicum; Stu, Solanum tuberosum.
targeting both SmelPPO4 and SmelPPO5, as well as SmelPPO6
(Figure 3A).
After transformation of the CRISPR/Cas9 constructs in
eggplant cotyledons and regeneration, 12 eggplant T0 individuals
(T0_ 1-T0_ 12) were analyzed (Data Sheet 3). The qPCR analysis
using Cas9 gene-specific primers revealed genomic integration
of the construct in nine T0 plants, while T0_ 1, T0_ 6 and T0_ 10
did not possess the transgene. In order to detect mutations
in SmelPPO4-5-6, we employed targeted deep sequencing of
genomic DNA, which allowed us to comprehensively assess the
editing efficiency and the types of mutations (Data Sheet 3).
Among the nine transformed plants, the Illumina amplicon
sequencing revealed that simultaneous editing of SmelPPO4,
SmelPPO5, and SmelPPO6 genes occurred in 2 lines (T0_ 3 and
T0_ 4) (Figure 3B). For the remaining lines, T0_ 5 and T0_ 12
showed editing only at the SmelPPO5 locus, while T0_ 10 at
the SmelPPO4 locus (Data Sheet 3). In most transformants,
SmelPPO5 appears edited to a higher extent than SmelPPO4
and SmelPPO6, with the exceptions of T0_ 3, T0_ 4 and T0_ 10.
Notably, in T0_ 5 SmelPPO5 reached an editing efficiency of 50%
while the other two loci displayed negligible levels of mutation.
Dark treatments are known to increase adventitious shoot
formation in cotyledon, leaf and hypocotyl explants in a number
of species, including eggplant (Muktadir et al., 2016). However,
we did not observe differences between shoots which underwent
the 3-days dark treatment and those which did not. For all
regenerating conditions, shoots apt for rooting were recovered
in as short as 6 weeks (Supplementary Figure 1).
Screening for Mutations in the T0
Generation
CRISPR/Cas9 induced mutagenesis can be employed to induce
the simultaneous knockout of multiple targets within a gene
family (Karunarathna et al., 2020; Sashidhar et al., 2020).
Targeting a conserved gene family poses some challenges
regarding the design of the gRNAs as well as the screening of
edited genotypes and off-target effects. This can be particularly
problematic for PPOs, since different members of this gene
family, including the ones implicated in defense response, possess
distinct activation patterns and specialized metabolic functions.
By identifying a conserved region of SmelPPO4 and SmelPPO5,
corresponding to the tyrosinase domain, we designed a gRNA
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FIGURE 2 | Transcriptional levels of 10 PPO-encoding genes in the Black Beauty variety 30 min after fruit cutting. The values are expressed as relative mRNA
abundance at 30 min after cutting compared to time 0 (just after cutting). Eggplant actin and elongation factor genes were used as the reference genes. Data are
means of three biological replicates ± SE. Different letters associated with the set of means indicate a significant difference based on Tukey b test (P ≤ 0.05).
tomato (Pan et al., 2016; Nonaka et al., 2017). Chimerism suggests
that gene editing occurred after the emergence of differentiated
tissues, leading to a heterogeneous mutation pattern within
the same plant. Transgene expression might be influenced by
the chromatin status at its insertion locus and, of course, by
the choice of promoters. In Arabidopsis, where mutants are
obtained through floral dipping and where the expression of
Cas9 in the germline is crucial to fix edited alleles, the use
of egg cell-specific promoters for Cas9 expression allowed to
efficiently obtain non-mosaic T1 mutants for multiple target
genes (Wang et al., 2015). In plants regenerated through somatic
organogenesis, the use of egg-cell and embryo-specific promoters
might also help retrieving T1 generations with higher levels
of homozygous or biallelic mutations (Zheng et al., 2020).
Since no previous reports of gene editing dynamics existed for
eggplant, and because we predicted the regeneration process
to be the limiting factor (both in terms of efficiency and time
consumption), we prioritized the use of standard gene editing
constructs to maximize Cas9 expression and establish a baseline
protocol. Based on this, other variants (e.g., tissue- or speciesspecific promoters) can be successively factored in to fine-tune
the editing outcome.
Previous observations showed that small indels are the
predominant mutations introduced in plants by gene editing
and that the breakpoint introduced by Cas9 is placed at 3
nucleotides upstream of the PAM (Bortesi et al., 2016; Pan
et al., 2016; Andersson et al., 2017). In plants, insertion of
one nucleotide or deletion of 1–10 nucleotides are the most
common mutations (Pan et al., 2016). The most common
Preferential editing of one member of a family sharing the same
gRNA recognition sequence might depend on the transcriptional
status of the target sequences. Although after wounding the
expression of SmelPPO5 is induced at lower levels than the ones
of SmelPPO4 and SmelPPO6, its transcript abundance seems to be
generally higher, as suggested by its Ct values. The transcriptional
accessibility of this locus might also reflect on its availability for
the Cas9 endonuclease. It is interesting to point out that, in T0_ 10,
SmelPPO4 was edited with an efficiency of over 70% although the
transgene was not integrated, which highlights that it is possible
to retrieve non-transgenic plants derived from edited cells in
which the editing machinery presumably acted in a “transient”
fashion.
T0_ 3 showed the greatest editing efficiency for all three
loci, i.e., 76% for SmelPPO4, 74% for SmelPPO5 and 60%
for SmelPPO6. In T0_ 4, a high editing efficiency was also
detected, i.e., 76% for SmelPPO4, 20% for SmelPPO5 and 21%
for SmelPPO6 (Data Sheet 3 and Figure 3B), although these
values are lower than those of T0_ 3. The number of plants
edited at all loci was low (22%) and editing efficiencies were
also significantly below the ones observed in tomato and potato,
presumably as a consequence of low levels of expression of Cas9
and gRNAs (Pan et al., 2016). T0_ 3 and T0_ 4 had chimeric
mutations (with at least 3 different alleles) in all targeted loci
and retained a proportion of the wild type allele. The wild type
copy of the target gene in chimeric plants could thus continue
to mutate either in T0 or in the following generations if the
Cas9 transgene does not segregate. The predominance of this
chimeric status in the T0 resembles the pattern described in
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FIGURE 3 | (A) Alignment of SmelPPO4, SmelPPO5 SmelPPO6 with selected gRNA. (B) Genotyping of targeted gene mutations induced by CRISPR/Cas9 in the T0
generation. Quantification of Illumina reads edited at the target locus in T0 _3 and T0 _4. For each line, the percentage of reads carrying mutated (blue) as well as not
mutated (red) target sequence is reported together with the pattern and frequency of targeted gene mutations.
mutations in our T0 eggplant plantlets were represented by
a single nucleotide insertion (+G; T0_ 3-SmelPPO4) and by
a deletion of one (T0_ 4-SmelPPO4/6; T0_ 5-SmelPPO5), two
(T0_ 3-SmelPPO5), three (T0_ 3-SmelPPO6), or four (T0_ 4SmelPPO5; T0_ 10-SmelPPO4; T0_ 12-SmelPPO5) nucleotides
(Data Sheet 3).
(Fu et al., 2013). The risk of off-target effects has been reported as
comparable to that of somaclonal variation deriving from plant
tissue culture itself (Ma et al., 2015). In order to reduce offtarget effects, a strategy based on Cas9/sgRNA ribonucleoprotein
complexes has been proposed (Hahn and Nekrasov, 2019).
Indeed, only through a whole genome resequencing of the edited
lines is it possible to exhaustively evaluate the presence of offtarget mutations induced by the selected sgRNAs. However,
other screening methods make it possible to rule out the
occurrence of undesired mutations at selected loci, which
is reliable particularly if they correspond to transcriptionally
active sequences.
Analysis of Off-Target Mutations
Only few occurrences of low-frequency off-target mutations
induced by CRISPR/Cas9 have been reported in plant species so
far (Feng et al., 2013; Peterson et al., 2016; Wolt et al., 2016; Hahn
and Nekrasov, 2019) contrary to what observed in human cells
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TABLE 2 | Quantification of Illumina reads edited at putative off-target loci in T0
generation.
Sample
Target
Reads
WT
OT1
17,926
549
OT2
17,855
1,354
7.58
OT3
27,671
10,758
38.88
T0 _3
T0 _4
Number of
mutated reads
% of
mutated
reads
3.06
OT4
26,977
803
2.98
OT1
24,196
747
3.09
OT2
21,205
922
4.35
OT3
30,086
3,203
10.65
OT4
34,541
822
2.38
OT1
20,621
591
2.87
OT2
13,231
476
3.60
OT3
20,282
1,648
8.13
OT4
274
8
2.92
For each individual and for each locus the total number of reads is reported, together
with the percentage of reads carrying the mutated off-target sequence. The percentage
of mutated sequences is reported.
One of the major risks related to targeting conserved regions
in a gene family is that putative off-targets are most likely other
members of the same family which, in the case of PPOs, are
also located in close proximity on the genome. This makes
it much more difficult to eliminate potentially undesired offtargets by breeding, than it is for non-linked loci. With respect
to our gRNA, four putative off-target sequences were identified:
one was an intergenic sequence, while three corresponded to
other members of the PPO family (SmelPPO2, SmelPPO3, and
SmelPPO7) (Supplementary Table 2).
In order to confirm that our selected T0 edited lines (T0_ 3
and T0_ 4) displayed mutations only in the SmelPPO4-5-6 loci,
we sequenced the candidate off-target loci by applying the
same Illumina Amplicon Sequencing Protocol we used for the
sequencing of target loci, and which allowed us to get a deep
insight into possible non-specific editing activity. The total
variation at putative off-target sites was compared between
edited and wild type plants (Table 2). We seldom observed
only base substitutions consistent with SNPs or sequencing
errors and, even considering those, no increase in total variation
was observed between wild type and mutants. Our analyses
thus demonstrated the lack of off-target effects, confirming the
specificity of Cas9-mediated PPO gene editing in eggplant. The
presence of mismatches in the seed region between our selected
sgRNA and the off-target SmelPPOs supports the specificity of
′
our results (Hahn and Nekrasov, 2019), since this 3 terminal
region of the target sequence is known to strongly affect
recognition by the Cas9 endonuclease.
FIGURE 4 | Genotyping of targeted gene mutations induced by CRISPR/Cas9
in the T1 and T2 generations. (A) Mutagenesis frequencies for all three
targeted loci in T1 and T2 progenies. (B) Zygosity of targeted gene mutations
in T1 and T2 populations.
heritability of CRISPR/Cas-induced mutations were initially
raised (Feng et al., 2013). However, in all other edited monocot
and dicot species, T1 generations with high mutation efficiencies
have been obtained, demonstrating the heritability of edited
alleles (Miao et al., 2013; Gao et al., 2015; Li et al., 2015; Svitashev
et al., 2015; Pan et al., 2016). In our case, 14 T1 plants of the
T0 _4 progeny were examined to investigate the transmission
pattern of CRISPR/Cas9-induced mutations. Out of 14 analyzed
individuals, 4 presented no detectable amplification of hCas9
and therefore it is reasonable to deduce that the transgene was
segregated (Data Sheet 3).
In order to detect the mutation efficiency and patterns at
different sites in SmelPPO4-5-6 genes, we employed targeted
deep sequencing. The average editing efficiency was 60%
for SmelPPO4, 52% for SmelPPO5, and 52% for SmelPPO6
(Figure 4A). Focusing on the SmelPPO4 locus, five were
heterozygous mutants, four chimeric, three homozygous and two
WT. At the SmelPPO5 locus, four were homozygous mutants,
eight chimeric and two WT. At the SmelPPO6 locus, three were
homozygous mutants, seven chimeric and four WT (Figure 4B;
Data Sheet 3).
The most common mutation at the SmelPPO4 locus was a
single nucleotide deletion, followed by a 4 nucleotide deletion.
At the SmelPPO5 locus different mutations were present:−2/1/-4/-3. The segregation pattern at the SmelPPO6 locus (which
Segregation of the Transgene and of
Mutated Alleles in the T1 and T2 Progeny
Due to the early finding that in Arabidopsis many somatic
mutations were not efficiently inherited, concerns about the
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FIGURE 5 | Genotyping of targeted gene mutations induced by CRISPR/Cas9 in the T2 _4_10_1 plant. The dashed lines represent nucleotide deletions. The reported
number represents the frequency and the number of reads carrying mutated (edited) target sequence.
FIGURE 6 | Phenotypical and biochemical changes associated with postcut browning. (A) Cut fruits of Wild Type, T1 _4_8, T1 _4_9 and T1 _4_10 showing post-cut
browning 30 min after cutting. (B) Polyphenoloxidase (PPO) activity in fruits of Wild Type, T1 _4_8, T1 _4_9 and T1 _4_10. Data are means of eight biological replicates ±
SD. Asterisks indicate a significant difference based on Tukey’s HSD test (P ≤ 0.05).
stably passed to the next generation in a Mendelian fashion.
As previously observed in other species (Pan et al., 2016),
the segregation patterns of the T1 chimera lines were less
predictable and a number of new mutants were obtained due to
the probable continued Cas9 activity. Interestingly, T2 _4_10_1
showed homozygous mutations for SmelPPO4 (-1/-1), SmelPPO5
(-4/-4) and SmelPPO6 (-4/-4) (Figure 5).
It has been previously demonstrated how off-target effects
can be further exacerbated in the T2 progeny as compared to
T0 and T1 (Zhang et al., 2018). Targeted deep sequencing
at putative off-target loci once again demonstrated the
lack of significant mutated off-targets in our T2 progeny,
confirming the specificity of Cas9-mediated PPO gene editing
in eggplant (Data Sheet 3). We observed only base substitutions
consistent with SNPs or sequencing errors, with similar
frequencies to those observed in the T0 , which did not
represent an increase in total variation between wild type and
mutant lines.
was less mutated in T0 ) was less predictable and a number
of new mutations (-2/-3/-7) were found in the T1 lines. The
highest editing efficiency was highlighted for T1 _4_10: 85%
for SmelPPO4, 90% for SmelPPO5 and 90.7% for SmelPPO6
(Data Sheet 3).
To further investigate the genetic stability of the targeted
mutations we screened the T2 plants derived from selfing T1 _4_8,
T1 _4_9 and T1 _4_10 (Data Sheet 3). The presence of a transgene
in most of the analyzed T2 plants (19/21) suggested that more
than one copy of the transgene was inserted in those T0
regenerants, which explains that Cas9 can still be active in all
T2 plants.
Compared to the T1 generation, the mutagenesis frequency
(99% for SmelPPO4, 85% for SmelPPO5 and 85% for SmelPPO6)
as well as the overall proportion of homozygous, biallelic
and chimeric assets increased (Figure 4). As expected, all 7
T2 progeny of T1 _4_11 were homozygous at the SmelPPO4
locus, indicating that the mutations in the homozygotes were
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Enzymatic Browning and PPO Activity
Analysis in Eggplant Berries
detectable off-target effects on other members of the same
gene family.
Upon cutting, edited T1 and T2 eggplant fruits showed
a reduction of the typical brown coloration due to phenolic
oxidation. Through our approach it will be possible to develop
eggplant varieties that maintain their antioxidant and nutritional
properties during harvest and post-harvest procedures, without
reducing the content in phenolics, which are beneficial for
human health.
Phenolics provide a substrate to oxidative reactions catalyzed
by PPOs that, consuming oxygen and producing fungitoxic
quinones, play a role in making the medium unfavorable to the
further development of pathogens (Taranto et al., 2017); however,
contrasting results are reported in literature, as PPO silenced lines
of potato were found to acquire higher resistance to P. infestans
(Llorente et al., 2014). Our future studies will be thus focused in
assessing the relationship between PPO knock-out and pathogen
response in our mutant eggplant lines.
We hypothesized that the CRISPR/Cas-mediated knock out of
PPOs would result in a lowered enzymatic browning, due to
the reduced PPO activity. Selected T1 lines (T1 _4_8, T1 _4_9
and T1 _4_10) carrying mutations in SmelPPO4-5-6 genes were
subjected to phenotypic analysis of enzymatic browning and
PPO activity in berries. The lines were grown in a greenhouse
and no growth alteration or changes in berry size/weight were
observed during plant development when compared to wild type,
as previously observed in potato (Llorente et al., 2011). The
berries were cut and exposed to air for browning induction. After
30 min, the typical brown discoloration due to phenolic oxidation
was detected and it was clearly more evident in wild type plants
in comparison to edited lines (Figure 6A). The average PPO
activity of T1 _4_8, T1 _4_9 and T1 _4_10 lines was also found to
be reduced by 48, 61, and 52%, respectively, compared to the wild
type (Figure 6B). By comparing the T2 edited lines with wild type
a reduction of PPO activity as well as of browning discoloration
upon cutting was highlighted (Supplementary Figure 2).
Several studies applied RNA silencing technologies to downregulate the expression of PPO genes in potato tubers (Bachem
et al., 1994; Rommens et al., 2006; Llorente et al., 2011; Chi et al.,
2014). In this species, by using the amiRNA technology (Chi et al.,
2014), a reduction in PPO activity of 15–95% was obtained and
it was more marked when StuPPO1 to 4 were simultaneously
suppressed. Furthermore, in a more recent study in potato,
CRISPR/Cas mutants for the four alleles of the StuPPO2 gene
(which is considered the major contributor to the PPO protein
content) displayed a reduction up to 69 and 73% in the PPO
activity and enzymatic browning, respectively (González et al.,
2020)
We can hypothesize that the partial reduction of PPO activity
in eggplant, comparable to the one observed in potato mutants,
might be enhanced through the knockout not only of class B
PPOs (SmelPPO4-5-6), but even of class A PPOs (SmelPPO1 and
SmelPPO3). However, this approach could provoke downside
effects, due to the involvement of the PPO multigene family in
important cell functions (Jukanti and Bhatt, 2015).
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Materials, further inquiries can be
directed to the corresponding author/s.
AUTHOR CONTRIBUTIONS
AMo, SG, and CC: conceptualization. AMa, SG, AMo, and AA:
data curation. AMa, SG, AMo, AA, DV, and AMM: investigation.
AMo and CC: supervision. AMa, SG, and AMo: writing—original
draft preparation. SG, AMo, JP, DO, AG, SL, and CC: writing—
review and editing preparation. All authors contributed to the
article and approved the submitted version.
FUNDING
Research was financially supported by the project CRISPR/Cas9–
mediated gene knock-out in eggplant financed by Compagnia
San Paolo.
ACKNOWLEDGMENTS
CONCLUSIONS
We thank Prof. Luigi Bertolotti (University of Turin) for his
technical assistance.
We have established a successful protocol for gene editing
in eggplant, adding to the list of Solanaceae species for
which CRISPR/Cas9 represents an alluring option for the
introduction of specific traits through a biotechnological
approach. Our system, based on the use of one guide RNA
directed simultaneously at three members of the PPO gene
family, demonstrated to be specific for the target genes, without
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpls.2020.
607161/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Maioli, Gianoglio, Moglia, Acquadro, Valentino, Milani, Prohens,
Orzaez, Granell, Lanteri and Comino. This is an open-access article distributed
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