Euphytica (2017)213:44
DOI 10.1007/s10681-017-1839-y
Associating chemical analysis to molecular markers
for the valorization of Citrus aurantium leaves: a useful
starting point for marker-assisted selection
Myriam Lamine . Fatma Zohra Rahali . Ghaith Hamdaoui . Sawsen Selmi . Ahmed Mliki .
Mahmoud Gargouri
Received: 26 July 2016 / Accepted: 12 January 2017
 Springer Science+Business Media Dordrecht 2017
Abstract Variation in metabolite composition and
content is often observed in citrus, however, it is
poorly understood to what extent this variation has a
genetic basis. C. aurantium genotypes originating
from Tunisia were evaluated to detect genomic (SSR
markers) and chemotypic polymorphisms and to
discover possible associations between them. A total
of fifteen highly polymorphic SSR markers were
selected to screen the genetic variability of the most
widespread sour orange genotypes. Targeted sec-
ondary metabolite profiling analysis generated
twenty-one compounds differentially accumulated in
the leaves of sour orange genotypes. PCA analysis
revealed that genomic and chemotypic data generated
similar pattern of clustering, highlighting the intra-
specific variability in C. aurantium species. Both data
were integrated, leading to the identification of
associated SSR alleles with secondary metabolites.
Based on results, a relatively high correlation
(r = 0.381; p \ 0.0001) between chemotypic patterns
and genetic markers was identified. Associations
M. Lamine (&)
A. Mliki
M. Gargouri
Laboratory of Plant Molecular Physiology, Biotechnology
Center of Borj-Cedria, BP 901, 2050 Hammam-Lif,
Tunisia
e-mail: myriam_lamine@yahoo.fr
F. Z. Rahali
G. Hamdaoui
S. Selmi
Laboratory of Medicinal and Aromatic Plants,
Biotechnology Center of Borj-Cedria, BP 901,
2050 Hammam-Lif, Tunisia
between traits of interest for phenolic compounds
and genetic markers were tested using statistical
methods including three linear model approaches.
These results consolidate the presence of a chemical
fingerprint that may be suitable for assessing identity
and quality of a particular genotype which will be very
useful for citrus breeding programs.
Keywords Antioxidant activity
C. aurantium
leaves
Marker-metabolite association
Secondary
metabolites
SSR
Introduction
Phenolics are the most abundant secondary metabo-
lites of plants and are beneficial components in a daily
human diet (Ignat et al. 2011). These phytochemicals
exhibit a broad range of biological activities (antiox-
idant, anti-inflammatory and antimicrobial) (Tripoli
et al. 2007) and are potential agents for prevention and
treatment of many diseases (Szajdek and Borowska
2008).
Citrus is one of larges species among plant; it
consists of 40 species distributed in all continents
(Mehl et al. 2014). Citrus is believed to possess
bioactivities such as antioxidant (Tripoli et al. 2007),
anti-inflammatory (Menichini et al. 2011), antimicro-
bial (Jing et al. 2014), and is suggested to be
responsible for the prevention of cancer and
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44 Page 2 of 14 Euphytica (2017)213:44

degenerative diseases (Jing et al. 2013). Those bioac- This information would help in the identification of
tivities of citrus are due to the present of bioactive genetic associations between the metabolic and/or
compound such as phenolics, flavonoids, essential oil, phenotypes (Schauer et al. 2008). To date, several
and vitamins (Ejaz et al. 2006). attempts have been performed to find correlative
Citrus aurantium is a wide spread tree in Cap Bon relationships between genetic and metabolic diversity
region in Tunisia (NorthEast) especially as a root- in various organisms (Laurentin et al. 2008). It is still
stock for other Citrus. In folk medicine, C. aurantium unclear whether possible correlations between each
is used for the treatment of sunstroke, gastrointestinal metabolite and the polymorphic pattern found at each
disturbances; it is also known to be a relaxant (Arias chromosomal location could exist to explain natural
and Ramon-Laca 2005). There are few reports men- variation. Considering the above, the novelty of the
tioning that C. aurantium leaves have nutritional present study consists on providing further knowledge
value, unique flavor and medicinal properties (Mo- regarding the chemical composition of sour orange
hamed et al. 2005). Interestingly, koca et al. (2003) leaves as well as the establishment of a possible
reported that orange leaf tissue contained significantly relationship between genetic and metabolic profile. To
more antioxidant flavonoids than fruit tissue. How- perform these analyses, we developed a robust proce-
ever, few investigations on secondary metabolite dure to explore the relationship between metabolic and
content in citrus leaves and their health benefits are genomic diversity based on correlation approach. As a
available. result, we present the first correlation network between
Commonly, DNA genetic markers have success- targeted secondary metabolite and SSR markers
fully established the fundamentals of citrus fruit among C. aurantium. Such integration analysis is
phylogeny and particularly SSRs have been shown required for identifying quality traits molecular mark-
to be highly useful for phylogenetic studies, assess- ers desirable for selection of appropriate parents for
ment of genetic variability and genome mapping future breeding programs of cultivated citrus.
(Snoussi et al. 2012; Lamine and Mliki 2015).
The evaluation of the genetic diversity and chem-
ical factors within and among populations is crucial Materials and methods
for understanding the adaptive ability of a species and
for the development of conservation programs (Mahar Plant materials
et al. 2011). Also, the genetic background might play a
pivotal role in the biosynthesis of secondary metabo- Leaves from eleven cultivars (Table 1) were collected
lites highlighting the necessity to investigate the in late January 2014 from adult (1015 years old)
correlation between them using advanced statistical cultivated healthy trees, uniform in size and growth
tools in managing germplasm collections, including
characterizing and establishing systematic relation-
Table 1 Sample information of 11 sour orange (C. aurantium)
ships (Zanor et al. 2009). Independent studies of either genotypes surveyed for SSR and HPLC fingerprinting
genetic diversity (Polat et al. 2012; Lamine and Mliki
Genotypes Origin Longitude (E) Latitude (S)
2015) or chemical profile of citrus (Tripoli et al. 2007;
Karimi et al. 2012; Xi et al. 2014; Zhang et al. 2014) OTD002 Beni khalled 10.59 36.64
have been conducted in recent years, but, to our OTD3 Beni khalled 10.59 36.64
knowledge, none of them addressed the influence of OTD4 Beni khalled 10.59 36.64
genetic variability on metabolic composition. So, we F15 Chrifet 10.62 36.79
hypothesize that combined analyses of metabolic F34 Chrifet 10.62 36.79
fingerprinting and molecular markers might be effec- H11 Chrifet 10.62 36.79
tive methods to reveal both chemical and genetic F40 Chrifet 10.62 36.79
diversities of citrus (Luro et al. 2012). Therefore, a Allch B3 Dar allouch 10.75 36.86
comprehensive exploration of the correlations W3 Menzel bouzelfa 10.58 36.68
between metabolic profile and genomic diversity Gafsi B2 Route sidi alaya 10.59 36.64
might provide information on both the broad and B2 Route sidi alaya 10.59 36.64
specific relationships between metabolo-genotypes.

123
Euphytica (2017)213:44
vigor, grown under the same pedoclimatic conditions
and located in the main production orange areas in
Tunisia. The samples were stored in dry ice after
collection, transferred to the laboratory and immedi-
ately grinded into liquid nitrogen and kept at 80 C
until analyzed. Three biological replicates were col-
lected for each cultivar.
Determination of total phenolic and flavonoid
contents
The grounded citrus leaf material (3 g) of each sample
was separately extracted by shaking with 30 ml of
pure methanol for 30 min. The extracts were then kept
for 24 h at 4 C, filtered through a Whatman filter
paper (No. 4), dried under vacuum and stored at 4 C
until their analysis as described by (Mau et al. 2001).
Total phenolic and flavonoid contents were deter-
mined according to (Dewanto et al. 2002). Total
phenolic content was assayed by adding 0.125 ml of
the FolinCiocalteu reagent and 0.5 ml of deionized
water was mixed well and immediately, the absor-
bance was measured at 510 nm using UVVis
spectrophotometer.
Identification of phenolic compounds
For HPLC analysis, 500 ll of BHT (Butylated
Hydroxytoluene) (1 mg ml-1), as internal standard,
was added to the methanolic extract. The phenolic
compound analysis was carried out using an Agilent
Technologies 1100 series liquid chromatograph (RP-
HPLC) coupled with a UVVis multi-wavelength
detector and equipped with a 250 9 4.6 mm, 4 lm
Hypersil ODS C18 reversed phase column kept at
ambient temperature. The mobile phase consisted of
aceto-nitrile (solvent A) and water with 0.2% sul-
phuric acid (solvent B). The flow rate was of 0.5 ml/
min. The gradient program was as follows: 15%
A/85% B 012 min, 40% A/60% B 1214 min, 60%
A/40% B 1418 min, 80% A/20% B 1820 min, 90%
A/10% B 2024 min, and 100% A 2428 min. The
injection volume was of 20 ll, and peaks were
monitored at 280 nm. Samples were filtered through
a 0.45 lm membrane filter before injection. Phenolic
compounds were identified according to their reten-
tion time as well as by spiking the sample with
standards. Analyses were performed in triplicate.
Page 3 of 14 44
DPPH assay for antioxidant capacity
The antioxidant ability of the citrus methanolic extract
was assessed using the stable free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH) (Hatano et al.
1988). The antiradical activity was expressed as IC50
(concentration required to cause a 50% DPPH inhibi-
tion: lg/ml). The inhibition percentage I% of radical-
scavenging activity was calculated as DPPH scaveng-
ing effect (%) = [(A0-A1)/A0] 9 100; where A0 is
the absorbance of the control and AS is the absorbance
of the sample after 30 min of incubation. All tests
were run in triplicate and the results expressed as
mean standard deviation (SD). BHT in the
1.0100 lM range was chosen as a standard
antioxidant.
DNA isolation, PCRs and electrophoresis
Leaf genomic DNA isolation and PCR amplifications
were carried out as previously described by Lamine and
Mliki (2015). Leaf samples were ground with liquid
nitrogen in separate 2 ml eppendorf tubes. Extraction
buffer [100 mM TrisHcl, 50 mM EDTA, 500 mM
NaCl, SDS (20%) and 140 mM B-mercaptoethanol]
was added, vortexed and incubated at 65 C for 30 min.
After that, potassium acetate (5 M) was added in each
tube and the mixture was incubated on ice for 30 min.
Subsequently, all tubes were centrifuged at 12000 rpm
for 30 min. Then, isopropanol was added to the aqueous
layer, the DNA was pelleted by centrifugating for
20 min and washed with 80% ethanol. The DNA was
dissolved in TE buffer and quantified before a dilution
step to obtain uniform concentration. Fifteen primer
pairs (http://www.plantbiology.ucr.edu/faculty/SSR-
Marker-Tables-Germplasm-v5.pdf) were used in this
study and it has been selected among available public
SSR markers of citrus. PCR was performed in a final
volume of 15 ll. Each PCR reaction consisted of
1.5 mM of MgCl2, 2.5 mM of dNTPs, 0.2 lM of each
primer and 1U of DNA Taq polymerase with 50 ng of
DNA. Cycling conditions were: 94 C for 5 min as an
initial denaturation step before entering 40 cycles each
composed of 30 s at 94 C, 30 s at annealing temper-
ature, 1 min at 72 C and a final extension step of 4 min
at 72 C. Capillary electrophoresis was carried out and
alleles were sized based on a DNA size standard
(600 bp, Vivantis).
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44 Page 4 of 14
Statistical analysis
For genetic analysis, the allele numbers and frequen-
cies, the expected (He) and observed (Ho) heterozy-
gosity were calculated using GenAlEx v. 6.4 (Peakall
and Smouse 2006). In order to determine the informa-
tiveness of the microsatellites, the PIC (Polymorphic
Index Content) values were calculated using the
CERVUS 3.0.3 software (Marshall et al. 1998). For
metabolite data, statistical analysis was performed
using SPSSv21.0 software and significant differences
among the different samples were determined by one-
way ANOVA using Duncans multiple-range test at a
significance level of 5% (p B 0.05). Coefficients of
variation (CV%) were determined as indicators of
variability. In order to explain the potentiality of these
variables (chemical and SSR) and to depict relation-
ships among individuals, principal component analysis
(PCA) was applied using PAST software (Hammer
et al. 2001). To check for possible correlations between
genetic and chemical distances among cultivars, Mantel
test was performed using XLSTAT-Pro 7.5.3 software.
Furthermore, associations between molecular markers
(as independent variables) and metabolite data (as
dependent variables) were performed by a multiple
regression analysis (MRA) using stepwise method of TF levels fluctuated between 4.47 and 89.8 mg EC/g
linear regression analysis option of SPSS version DW, W3 had the lowest levels, while GafsiB2 had the
21. The robustness of this MRA model is in providing
an adequate setting for association tests (Lourenco et al.
2011). The analysis was based on the following model:
Y = a ? b1m1 ? b2m2 ? ? bjmj ? ? bnmn ?
d ? e, which related to the variation in the dependent
variable (Y cultivar means for a quantitative trait) to a
linear function of the set of independent variables mj,
representing SSR markers. The bj terms are the partial
regression coefficients that specify the empirical rela-
tionships between Y and mj, d represents between
accession residuals, which is left after regression, and
e is the random error of Y that includes environmental
variation (Virk et al. 1996). To select independent
variables for the regression equation, F values with
0.045 and 0.099 probabilities were used to enter and
remove, respectively (Afifi and Clark 1999). Selected
markers were further tested independently with linear
curve fitting using linear models for confirming the
significance of b-statistics for each band identified by
MRA. b can be defined as the standardized regression
coefficient = BxSx/Sy, where B is the regression coef-
ficient or slope and Sx and Sy are the standard deviations
123
Euphytica (2017)213:44
of independent (x) and dependent (y) variables (Afifi
and Clark 1999; Kar et al. 2008). Students t test was
performed to test significance between mean trait
estimates of genotypes where specific markers are
present or absent. Markers showing significant regres-
sion values were considered as associated with the trait
under consideration. The criteria for correlation deter-
mination were correlation values of C0.65 or B-0.65
and p-values B 0.05.
Results and discussion
Total phenolic and total flavonoid content
For all sour orange genotypes, the TP levels ranged
from 64.2 to 288.5 mg GAE/g DW, with the W3
having the lowest content, and GafsiB2 with the
highest content. Its noteworthy to mention that all the
tested genotypes had high TP levels, with more than
50 mg GAE/g FW (Fig. 1a). Our results farther
exceeded those obtained in flowers (4.83 mg GAE/g
DW; Karimi et al. 2012), in peels (31.62 mg GAE/g
DW; Lagha Benamrouche and Madani 2013) and in
seeds (1.35 mg GAE/g DW; Moulehi et al. 2012). The
highest. GafsiB2 and F15 had significantly higher TF
contents than the other genotypes (Fig. 1b). In fact,
these levels were higher than those reported in the and
zest (0.33 mg EC/g DW; Jabri karoui and Marzouk
2013), flowers (4.11 mg rutin equivalent/g DW;
Karimi et al. 2012) and seeds (1.7 mg EC/g DW;
Moulehi et al. 2012) of C. aurantium.
These findings demonstrate that the high levels of
polyphenols obtained from sour orange leaves extract
far exceeded the levels obtained different other sour
orange organs analyzed and suggest that the leaves are
rich in phenolic compounds. Thus we may consider
them as important source of substantial secondary
metabolites. Indeed, various studies have shown that
they can be valued as natural antioxidants (Koca et al.
2003; Lamport et al. 2012).
Antioxidant activities of leaves extract from C.
aurantium genotypes
In order to assess whether the phenolic and flavonoid
contents positively correlated to the antioxidant
Euphytica (2017)213:44
Fig. 1 a Total Phenolic Content (mg GAE/g DW); b Total
Flavonoid Content (mg EQ/g DW) and c Scavenging of the 1,1-
diphenyl-2-picrylhydrazyl Radical (DPPH, IC50 lg/ml) of sour
orange leaf extracts
activity, DPPH analysis was achieved for the different
genotypes. The radical scavenging ability was high
with IC50 values ranging 581600 lg/ml. Further-
more, allchB3 leaves extract had the highest antirad-
ical activity while F15 displayed the lowest activity
(Fig. 1c).
The relationship between the total polyphenol
content and antioxidant activity is very controversial.
While some authors support the existence of a strong
correlation between phenolic content and antioxidant
activity (Ouchemoukhe et al. 2012; Benmeddour et al.
2013), others contradict (Anagnostopoulou et al. 2006;
Kamran et al. 2009). Correlation analysis was used to
Page 5 of 14 44
investigate the relationship between the phenolic
content of sour orange leaves extract and their
antioxidant capacity. The correlation graphs are
depicted in Fig. 1a, b. Usually, extracts or fractions
with a high radical scavenging activity showed a high
phenolic content as well, but correlations could not be
found among them (Fig. 1a) and was failed to
demonstrate by linear regression analysis.
Besides, there was no correlation between total
flavonoids and radical scavenging activity, too
(Fig. 2). This lack of relationship is in agreement
with other works (Anagnostopoulou et al. 2006;
Nickavar et al. 2007). Furthermore, the extracts are
very complex mixtures of many different compounds
with distinct activities (Mensor et al. 2001; Hou et al.
2003). In fact, according to Huang et al. (2005), the
FolinCiocalteu reagent is nonspecific for phenolic
compounds since it measures sample reducing capac-
ity through electronic transfer-based antioxidant
capacity, and thus it can be reduced by many non-
phenolic compounds such as vitamin C. Since syn-
thetic antioxidants have side effects, the search for a
natural source of antioxidants has attracted attention
recently. The study proved that the citrus leaves and
specifically those of sour orange are rich in antioxi-
dants. Therefore, they have the potential to be used as
an alternative to the synthetic antioxidants. This result
was supported by Koca et al. (2003), who mentioned
that both young and old leaves displayed the highest
antioxidant potential among other tissue types in three
citrus species. Collectively, as mentioned in previous
studies (Lamport et al. 2012), we might consider that
citrus leaves are an important source of secondary
metabolites.
Contents of individual phenolic compounds
A total of 21 phenolic compounds were identified from
the tested sour orange leaves, including eleven phe-
nolic acids and ten flavonoids (Table 2). A large
variation patterns of phenolic acids components and
contents were also observed for the different sour
orange genotypes studied (Table 2). The gallic acid,
syringic acid and tannic acid have been identified as
the major phenolic compounds in the sour orange
leaves (65.44, 61.27 and 58.78 lg/g DW, respec-
tively). AllchB3 had the highest gallic (20.25 lg/g
DW), syringic (58.12 lg/g DW) and tannic acid
(21.89 lg/g DW) content, while these compounds
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44 Page 6 of 14 Euphytica (2017)213:44

Table 2 Contents (lg/g DW) of targeted secondary metabolites (flavonoids and phenolic acids) in sour orange leaves
C. aurantium Allch B3 Gafsi B2 F34 B2 OTD002 OTD4

Phenolic acids 148.77 5.32 79.71 0.21 15.32 6.04 2.04 0.09 0.31 0.032 4.58 0.1
Tannic acid 21.89 1.01 9.18 0.72 _ _ _ _
Gallic acid 20.25 0.53 7.43 0.16 _ _ _ _
Resorcinol 14.07 0.53 8.8 0.26 _ _ _ _
Methyl,3,4,5- 5.05 0.59 4.34 0.63 _ _ _ _
dihydroxyxybenzoate
Caffeic acid 22.21 0.74 10.98 0.06 _ _ _ _
Gentisic acid 2.55 0.43 2.73 0.25 _ _ _ _
Vanillic acid 4.63 0.59 24.79 1.00 _ _ _ _
Syringic acid 58.1 1.34 2.42 0.13 _ _ _ _
A-2, hydroxyphenyl acetic _ 9.04 0.255 _ _ _ _
Ferulic acid _ _ 15.32 6.04 0.98 0.08 3.13 0.33 4.58 0.18
Carnosic acid _ _ 1.06 0.06 7.18 0.06
Flavonoids 14.46 2.32 10.67 0.01 122.05 3.1 19.4 2.3 26.02 4.06 32.33 136
Catechin 6.48 0.32 5.95 0.10 _ _ _ _
Epicatechin 7.98 0.50 4.72 0.71 _ _ _ _
Rutin _ _ 19.91 5.32 _ _
Hesperetin _ _ 7.24 2.58 _ _ _
Quercetin _ _ 23.78 3.83 2.22 0.02 6.67 0.17 0.35 0.06
Naringin _ _ 35.20 8.66 8.50 0.63 15.6 0.22 28.93 0.99
Flavone _ _ 11.75 3.53 1.80 0.02 0.76 0.03 0.14 0.015
Naringenine-7-o-glucoside _ _ 21.52 5.11 4.76 0.20 2.99 0.19 2.67 0.21
5,7dihydroxyflavone _ _ _ 1.49 0.03 _ _
Flavonose _ _ 2.65 0.49 0.63 0.14 _ 0.24 0.007
Total 163.23 3.56 90.38 0.02 137.37 2.14 21.44 0.3 26.33 1.22 36.91 0.04
C. aurantium W3 OTD3 H11 F15 F40

Phenolic acids 16.58 0.22 40.45 0.47 17.9 2.35 10.1 2.6 6.42 0.02
Tannic acid 2.13 0.62 15.41 0.23 3.25 2.18 4.53 2.55 2.39 0.01
Gallic acid 3.82 0.26 19.17 0.18 11.33 3.03 2.15 1.27 1.29 0.10
Resorcinol _ 3.83 0.18 2.15 0.19 0.54 0.29 _
Methyl,3,4,5- _ _ 0.13 0.06 0.71 0.36 0.51 0.001
dihydroxyxybenzoate
Caffeic acid 1.26 0.05 0.38 0.05 _ 1.02 0.13 _
Gentisic acid _ _ _ 0.12 0.38 _
Vanillic acid 1.91 0.15 1.66 0.05 0.86 0.01 0.71 0.10 0.16 0.01
Syringic acid _ _ _ _ 0.73 0.07
A-2, hydroxyphenyl acetic 7.14 0.02 _ 0.18 0.09 0.32 0.002 1.29 0.004
Ferulic acid _ _ _ _ _
Carnosic acid 0.32 0.12 _ _ _ 0.05 0.008
Flavonoids 8.55 0.25 57.27 3.84 6.36 0.56 6.8 0.02 4.5 0.2
Catechin 0.26 0.05 24.55 0.76 4.65 0.55 3.9 2.05 1.62 0.08
Epicatechin 3.66 0.07 32.72 2.48 1.71 0.30 2.9 1.63 1.29 0.01
Rutin _ _ _ _ 0.4 0.005
Hesperetin 4.63 0.47 _ _ _ _

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Euphytica (2017)213:44 Page 7 of 14 44

Table 2 continued
C. aurantium W3 OTD3 H11 F15 F40

Quercetin _ _ _ _ _
Naringin _ _ _ _ 0.34 0.020
Flavone _ _ _ _ _
Naringenine-7-o-glucoside _ _ _ _ 0.06 0.002
5,7dihydroxyflavone _ _ _ _ 0.04 0.004
Flavonose _ _ _ _ 0.75 0.02
Total 25.13 0.8 97.72 1.03 24.26 1.34 16.9 1.25 10.92 0.36

were not detectable in some other genotypes (F34, B2, (a)

2.5 y = -0.0027x + 1.5652
OTD002, OTD4). Several studies supported the exis-

DPPH (IC 50, g/mL)

R = 0.0568

Antioxidant activity
tence of gallic acid as major compound in different 2
P = 0.486
citrus species (Moulehi et al. 2012). 1.5
Among the flavonoids, naringin (94.26 lg/g of
1
DW) was the most abundant compound, followed by
0.5
epicatechin (55.5 lg/g of DW) and catechin
(51.12 lg/g of DW) (Table 2). F34 and OTD4 had 0
0 100 200 300 400
the highest content of naringin (35.2 and 28.93 lg/g Total polyphenol content
DW, respectively).OTD3 had higher level of epicat- mg GAE/g DW

echin (32.72 28.93 lg/g DW) and catechin (24.55 (b)

2.5 y = 0.0079x + 0.766
28.93 lg/g DW) compared to other genotypes. These R = 0.0561
DPPH (IC 50, g/mL)
Antioxidant activity

results are in accordance with previous works that 2 P = 0.480

reported similar findings regarding the abundance of
naringin in different citrus organs (Karimi et al. 2012;
Moulehi et al. 2012; Jabri karoui and Marzouk 2013).
In fact, flavanones are the typical polyphenols of citrus
species (Khan et al. 2014).
We found that the similar variation patterns of the
polyphenols components and contents were observed
in the different genotypes studied (Table 2), which is
similar to previous results (Zhang et al. 2014). The
conclusion that polyphenols are genetically controlled
was further confirmed by the present result (Fig. 2).
Usefulness of target secondary metabolite
profiling for intra-specific variability of C.
aurantium
Sour orange genotypes were subjected to clustering
analysis using principal component analysis (PCA), in
order to differentiate them based on their phenolic
profile. The first two axes of PCA plot accounting for
66.21%. In the PCA score plot, 11 genotypes were
separated into four groups (Fig. 3).
Group I, represented by the genotypes allchB3 and
GafsiB2, was characterized by a significant amount
1.5
1
0.5
0
0 20 40 60 80 100
Flavonoids content
mg EQ/g DW
Fig. 2 Correlation of antioxidant activity (DPPH method) of
sour orange leaves methanolic tissues extracts with a polyphenol
content and b flavonoids content
of phenolic acids particularly syringic, vanillic, caf-
feic, tannic and gallic acids. The second group (II),
formed by the genotypes OTD3, H11 and F15, was
characterized by, practically, equal amounts of
flavonoids and phenolic acids. Catechin and epicate-
chin were the major flavonoid components of this
group, while, gallic and tannic acid were the predom-
inant phenolic acids in this group. A high accumula-
tion of the naringenin-7-O-glucoside, rutin and
naringin, quercetin and ferulic acid was spotted in
genotypes OTD 002, OTD 4, W3, B2 and F40
within the third group (III). F34 (Group IV), being
separated from the other groups, was characterized by
very high levels of naringin, quercetin, naringenine7-

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44 Page 8 of 14
Fig. 3 Principal
component analysis (PCA)
of sour orange genotypes
based on metabolite profiles
O-glucoside, rutin and ferulic acid. Our results
revealed that the quantitative and qualitative diversity
of secondary metabolites may be useful to determine
the intra-specific variability in the studied sour orange.
Genetic polymorphism and allelic diversity based
on SSR markers
To establish the authenticity of the tested sour orange
genotypes, we carried out a genetic profiling analysis
using 15 pairs of SSR markers. The set of primers were
selected for amplification based on their high poly-
morphic and unambiguous profiles. The statistical
analysis of SSR loci is reported in Table 3. A total of
37 alleles were identified in the analyzed cultivars.
Specifically, the number of alleles per locus ranged
from 2 (TAA27, GT03, CAG01, cAGG9, CT21,
CT02, TAA41, CT19 and CCT01) to 4 (TAA15),
with an average of 2.46 alleles per locus. The
amplification of more than one band per genotype by
some SSR primers may be due to residual heterozy-
gosity (Yates et al. 2012). Thus, the observed
heterozygosity was calculated for each individual
marker as a measure of marker diversity. The
percentage of heterozygotes per marker detected in
123
Euphytica (2017)213:44
our citrus sample ranged from 0 (TAA27, GT03) to
100% for the marker ATC09. The mean observed
heterozygosity for all markers was 52%. The esti-
mated He in citrus germplasm of this study (0.397)
(Table 3) was in the same range of the level of
polymorphism reported for local sour orange cultivars
in Tunisia (Snoussi et al. 2012; Lamine and Mliki
2015). Most of the SSR loci, except TAA27 and GT03,
showed higher values for Ho than He (Table 3),
indicating an excess number of heterozygotes. This
may be due to the presence of over dominant selection
(where heterozygous individuals have higher survival
or fitness due to heterozygous advantage than homozy-
gous individuals). We found that the PIC assayed
within loci ranged from 0.2 (CT02) to 0.890 (TAA1)
and most are around the 0.5 threshold value, indicating
the high discriminating capacity of the selected
markers (Table 3). Moreover, the mean value of the
PIC obtained in this study was 0.489, indicating that
the generated SSR-data is informative and adequate to
discriminate among most accessions included in the
study (Amar et al. 2011). Altogether, our data
indicated that the SSR marker set employed was
effective in elucidating intraspecific variability of C.
aurantium. Furthermore, the principal component
Euphytica (2017)213:44 Page 9 of 14 44

Table 3 Data summary for 15 microsatellite markers across separated into four main groups (Fig. 4). The first
the C. aurantium genotypes group (I) contains the genotypes allchB3, GafsiB2 and
Locus Size range (bp) N Ho He Pic F15. The second group (II) includes OTD3, OTD4,
OTD002, W3, B2 and H11 genotypes. Groups III and
TAA1 190202 3.000 0.600 0.464 0.890 IV each include one genotype (F34 and F40,
TAA27 130140 2.000 0.000 0.124 0.421 respectively).
GT 03 170180 2.000 0.000 0.124 0.465
CAG01 165170 2.000 0.733 0.491 0.461 Correlation analysis
ATC09 180220 3.000 1.000 0.598 0.492
CT21 170190 2.000 0.733 0.464 0.517 The relationship between genetics and chemistry is
CAGG9 200220 2.000 0.857 0.490 0.557 very controversial as some authors have reported that
TAA41 240260 2.000 0.450 0.350 0.435 genetic variability is reflected in the chemical profile
CAC15 140190 3.000 0.400 0.320 0.380 (Morone-Fortunato et al. 2010), while others describe
CAC23 150200 3.000 0.650 0.490 0.500 the opposite (Mendes et al. 2011).
TAA15 190200 4.000 0.750 0.660 0.665 The genotypes grouping generated on the basis of
CT02 155170 2.000 0.235 0.180 0.200 molecular data (SSR) was almost similar to that
AG14 110140 3.000 0.400 0.350 0.365 designed by metabolites (Figs. 3, 4). In addition, using
CT19 165180 2.000 0.485 0.461 0.385 Mantel test, a significant correlation (r = 0.381;
CCT01 220250 2.000 0.500 0.385 0.600 p \ 0.0001), was found between SSR genetic distance
Mean 2.467 0.520 0.397 0.489 and secondary metabolite-based matrixes.
N no. of different alleles, Ho observed heterozygosity, He
expected heterozygosity, PIC polymorphism information content Metabolite-metabolite correlation
analysis (PCA) revealed that 62.64% of variation was
explained by the first two axes was (axis138.77%, Correlation analysis is a statistical method used to
axis223.87%), where sour orange genotypes were evaluate the associations between variables and can be

Fig. 4 Principal
component analysis (PCA)
of sour orange genotypes
based on SSR markers

123
44 Page 10 of 14
Fig. 5 Correlation matrix of metabolites from C. aurantium
genotypes. Each square indicates r (Pearsons correlation
coefficient values for a pair of metabolites). The value of the
used to establish relationships between metabolites
belonging to a biological system (Kim et al. 2013).
Pearson correlation analysis and HCA of the acces-
sions were implemented to determine the relationships
among levels of the 21 metabolites in sour orange
(Fig. 5). Analytical results revealed the existence of
123
Euphytica (2017)213:44
correlation coefficient is represented by the intensity of the blue
(-1 \ r \ 0) or pink (0 \ r \ 1) color, as indicated on the color
scale
27 pairs of positive correlations and 18 pairs of
negative correlations. These analytical results
revealed strong correlations between metabolites
involved in closely related pathways and demon-
strated the robustness of the present experimental
system. The highest positive correlation was detected
Euphytica (2017)213:44 Page 11 of 14 44

between quercetin and ferulic acid (r = 0.999; Metabolite-SSR correlation

p \ 0.0001), however, a strong negative correlation
between gallic acid and naringin (r = -0.802;
p \ 0.0001) is also observed. The results of the
CAH obtained from Pearson correlation analysis of
the 21 metabolites revealed several large groups of
metabolites. A group containing quercetin includes the
precursors of the flavonol biosynthetic pathway such
as naringenin. Flavanones such as hesperetin, naringin
and naringenin gathered in the same group. Correla-
tion analysis showed that caffeic, vanillic and gentisic
were strongly positively correlated. Tannic acid
exhibited the greatest number of correlation pairs. In
fact, these analytical results revealed strong correla-
tions between metabolites involved in closely related
pathways. Not surprising that some flavonoids such
catechin and epicatechin were found to be highly
correlated to phenolic acids (tannic acid and gallic
acid) since they derive from the same degradation
pathway of condensed tannins. This indicates that
correlations between metabolites of the same class are
conserved while those between the different classes of
metabolites are quite diverse. Therefore, metabolic
profiling of the phenolic compounds can be used to
track their metabolic links on one hand, and the
observed correlations between the concentrations of
metabolites in a biological sample, may be used to
gain additional information about the physiological
state of a given tissue on the other hand (Steuer et al.
2003).
The biosynthesis and accumulation of metabolites are
mainly controlled by enzymes and regulatory factors
which are encoded by genes in the genome. The SSR
marker analysis revealed a number of markers show-
ing statistically significant correlation with different
metabolite components, as identified through MRA.
Five SSR markers were correlated with 12 metabolites
showing a significant correlation (r [ 0.6 or r \ -0.6;
P \ 0.001; n = 11). Several SSR markers (Table 4)
showed a significant correlation with specific metabo-
lites in sour orange. Indeed, the strongest correlation
was that of cAGG9200 with rutin (r = 0.989;
b = -0898; P = 0.00). This marker (cAGG9200)
was also negatively correlated with flavone
(r = 0.761; b = -0.83), however, with tannic acid,
it was positively correlated (r = 0.71; b = 0.729)
indicating an inverse or negative correlation between
flavone/rutin and tannic acid. CT21190 was the most
important marker which correlates with high number
of metabolites (4 metabolites). The highest negative
correlation value (b = -0.988) was detected with
gentisic acid, followed by vanillic acid (b = -0.966);
caffeic acid (b = -0.939) and Methyl, 3,4,5-dihy-
droxy-benzoate (b = -0.648). In addition, CAG01165
was positively correlated with syringic acid (r = 0.71)
and negatively correlated with hydroxyphenyl-acetic
acid and hespertine (b = -0926 and b = -0944,
respectively). The marker GT03170 displayed a

Table 4 SSR markers associated with phenolic compounds in sour orange as revealed by MRA and coefficients
SSR markers (alleles) Phenolic compound R R2 Standardized beta coefficients T value P value

cAGG9200 Rutin 0.989 0.978 0.898 15.53 0.00

Flavone 0.761 0.579 0.832 3.27 0.02
Tannic acid 0.705 0.496 0.729 2.62 0.04
CT21190 Gentisic acid 0.981 0.961 0.988 15.61 0.00
Vanillic acid 0.966 0.933 0.966 11.48 0.00
Caffeic acid 0.939 0.882 0.939 8.62 0.00
Methyl,3,4,5-dihydroxyxybenzoate 0.648 0.42 0.648 2.93 0.02
CAG01165 Hesperetin 0.956 0.913 0.944 9.47 0.00
A-2, hydroxyphenyl acetic 0.942 0.884 0.926 8.03 0.00
Syringuc acid 0.710 0.513 0.033 0.141 0.03
TAA27130 Naringenin-7-o-b-D-glucoside 0.685 0.462 0.682 2.93 0.01
GT03170 Gallic acid 0.626 0.391 0.626 2.53 0.03

123
44 Page 12 of 14
positive correlation b (r = 0.63; b = 0.626) with
gallic acid, while TAA27130 was positively correlated
with a naringenin-7-O-glucoside (b = 0.682).
Some of the SSR markers were found to be
associated with more than one metabolite in MRA.
Such an association may arise due to pleiotropic
effect of the linked QTL on different traits (Culp et al.
1979). The described methods provide a reliable and
easy analysis for the identification of promising
cultivars at early stages of breeding programs (Virk
et al. 1996; Kar et al. 2008). The MRA approach is a
suitable tool for tree crops and a quick method for
establishing marker trait association avoiding the
need for mapping populations. The markers identi-
fied in this study can be used for MAS breeding
programs. The SSR markers identified to be associ-
ated with major secondary metabolites in this study
can be used for QTL and marker-assisted selection
(MAS) breeding programs (Kar et al. 2008; Khadivi-
Khub 2014).
The SSR-based fingerprinting can identify the
genotypes unequivocally, while the HPLC-based
metabolic fingerprinting is reliable for rapid analysis
of a large number of citrus materials, revealing the
potential differences in metabolites within sour orange
leaves. Correlation network approach used for
metabolites and SSR markers provided a basis for
deeper insight into metabolic pathways and the
regulation networks in sour orange. In fact, in breeding
programs, selection of high quality material is often a
time consuming process, and thus markers-assisted
selection could be of great help in the identification
and selection of elite genotypes mainly when genetic
and genomic information (linkage maps, quantitative
trait loci and genome database) are not available. As
far as we know, this is the first report that correlates
SSR markers with metabolic dataset in citrus leaves.
We believe that our approach might be reasonably
adopted in the future for the characterization and the
traceability purposes of citrus germplasm.
Acknowledgements This work was supported by Grants from
the Tunisian Ministry of Higher Education and Scientific
Research.
Compliance with ethical standards
Conflict of interest The authors declare no competing finan-
cial interest.
123
Euphytica (2017)213:44
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