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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 between traits of interest for phenolic compounds
content is often observed in citrus, however, it is and genetic markers were tested using statistical
poorly understood to what extent this variation has a methods including three linear model approaches.
genetic basis. C. aurantium genotypes originating These results consolidate the presence of a chemical
from Tunisia were evaluated to detect genomic (SSR fingerprint that may be suitable for assessing identity
markers) and chemotypic polymorphisms and to and quality of a particular genotype which will be very
discover possible associations between them. A total useful for citrus breeding programs.
of fifteen highly polymorphic SSR markers were
selected to screen the genetic variability of the most Keywords Antioxidant activity  C. aurantium
widespread sour orange genotypes. Targeted sec- leaves  Marker-metabolite association  Secondary
ondary metabolite profiling analysis generated metabolites  SSR
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- Introduction
specific variability in C. aurantium species. Both data
were integrated, leading to the identification of Phenolics are the most abundant secondary metabo-
associated SSR alleles with secondary metabolites. lites of plants and are beneficial components in a daily
Based on results, a relatively high correlation human diet (Ignat et al. 2011). These phytochemicals
(r = 0.381; p \ 0.0001) between chemotypic patterns exhibit a broad range of biological activities (antiox-
and genetic markers was identified. Associations idant, anti-inflammatory and antimicrobial) (Tripoli
et al. 2007) and are potential agents for prevention and
treatment of many diseases (Szajdek and Borowska
M. Lamine (&)  A. Mliki  M. Gargouri 2008).
Laboratory of Plant Molecular Physiology, Biotechnology
Citrus is one of larges species among plant; it
Center of Borj-Cedria, BP 901, 2050 Hammam-Lif,
Tunisia consists of 40 species distributed in all continents
e-mail: (Mehl et al. 2014). Citrus is believed to possess
bioactivities such as antioxidant (Tripoli et al. 2007),
F. Z. Rahali  G. Hamdaoui  S. Selmi
anti-inflammatory (Menichini et al. 2011), antimicro-
Laboratory of Medicinal and Aromatic Plants,
Biotechnology Center of Borj-Cedria, BP 901, bial (Jing et al. 2014), and is suggested to be
2050 Hammam-Lif, Tunisia responsible for the prevention of cancer and

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.

Euphytica (2017)213:44 Page 3 of 14 44

vigor, grown under the same pedoclimatic conditions DPPH assay for antioxidant capacity
and located in the main production orange areas in
Tunisia. The samples were stored in dry ice after The antioxidant ability of the citrus methanolic extract
collection, transferred to the laboratory and immedi- was assessed using the stable free radical 2,2-
ately grinded into liquid nitrogen and kept at 80 C diphenyl-1-picrylhydrazyl (DPPH) (Hatano et al.
until analyzed. Three biological replicates were col- 1988). The antiradical activity was expressed as IC50
lected for each cultivar. (concentration required to cause a 50% DPPH inhibi-
tion: lg/ml). The inhibition percentage I% of radical-
Determination of total phenolic and flavonoid scavenging activity was calculated as DPPH scaveng-
contents ing effect (%) = [(A0-A1)/A0] 9 100; where A0 is
the absorbance of the control and AS is the absorbance
The grounded citrus leaf material (3 g) of each sample of the sample after 30 min of incubation. All tests
was separately extracted by shaking with 30 ml of were run in triplicate and the results expressed as
pure methanol for 30 min. The extracts were then kept mean standard deviation (SD). BHT in the
for 24 h at 4 C, filtered through a Whatman filter 1.0100 lM range was chosen as a standard
paper (No. 4), dried under vacuum and stored at 4 C antioxidant.
until their analysis as described by (Mau et al. 2001).
Total phenolic and flavonoid contents were deter- DNA isolation, PCRs and electrophoresis
mined according to (Dewanto et al. 2002). Total
phenolic content was assayed by adding 0.125 ml of Leaf genomic DNA isolation and PCR amplifications
the FolinCiocalteu reagent and 0.5 ml of deionized were carried out as previously described by Lamine and
water was mixed well and immediately, the absor- Mliki (2015). Leaf samples were ground with liquid
bance was measured at 510 nm using UVVis nitrogen in separate 2 ml eppendorf tubes. Extraction
spectrophotometer. buffer [100 mM TrisHcl, 50 mM EDTA, 500 mM
NaCl, SDS (20%) and 140 mM B-mercaptoethanol]
Identification of phenolic compounds was added, vortexed and incubated at 65 C for 30 min.
After that, potassium acetate (5 M) was added in each
For HPLC analysis, 500 ll of BHT (Butylated tube and the mixture was incubated on ice for 30 min.
Hydroxytoluene) (1 mg ml-1), as internal standard, Subsequently, all tubes were centrifuged at 12000 rpm
was added to the methanolic extract. The phenolic for 30 min. Then, isopropanol was added to the aqueous
compound analysis was carried out using an Agilent layer, the DNA was pelleted by centrifugating for
Technologies 1100 series liquid chromatograph (RP- 20 min and washed with 80% ethanol. The DNA was
HPLC) coupled with a UVVis multi-wavelength dissolved in TE buffer and quantified before a dilution
detector and equipped with a 250 9 4.6 mm, 4 lm step to obtain uniform concentration. Fifteen primer
Hypersil ODS C18 reversed phase column kept at pairs (
ambient temperature. The mobile phase consisted of Marker-Tables-Germplasm-v5.pdf) were used in this
aceto-nitrile (solvent A) and water with 0.2% sul- study and it has been selected among available public
phuric acid (solvent B). The flow rate was of 0.5 ml/ SSR markers of citrus. PCR was performed in a final
min. The gradient program was as follows: 15% volume of 15 ll. Each PCR reaction consisted of
A/85% B 012 min, 40% A/60% B 1214 min, 60% 1.5 mM of MgCl2, 2.5 mM of dNTPs, 0.2 lM of each
A/40% B 1418 min, 80% A/20% B 1820 min, 90% primer and 1U of DNA Taq polymerase with 50 ng of
A/10% B 2024 min, and 100% A 2428 min. The DNA. Cycling conditions were: 94 C for 5 min as an
injection volume was of 20 ll, and peaks were initial denaturation step before entering 40 cycles each
monitored at 280 nm. Samples were filtered through composed of 30 s at 94 C, 30 s at annealing temper-
a 0.45 lm membrane filter before injection. Phenolic ature, 1 min at 72 C and a final extension step of 4 min
compounds were identified according to their reten- at 72 C. Capillary electrophoresis was carried out and
tion time as well as by spiking the sample with alleles were sized based on a DNA size standard
standards. Analyses were performed in triplicate. (600 bp, Vivantis).

44 Page 4 of 14 Euphytica (2017)213:44

Statistical analysis of independent (x) and dependent (y) variables (Afifi

and Clark 1999; Kar et al. 2008). Students t test was
For genetic analysis, the allele numbers and frequen- performed to test significance between mean trait
cies, the expected (He) and observed (Ho) heterozy- estimates of genotypes where specific markers are
gosity were calculated using GenAlEx v. 6.4 (Peakall present or absent. Markers showing significant regres-
and Smouse 2006). In order to determine the informa- sion values were considered as associated with the trait
tiveness of the microsatellites, the PIC (Polymorphic under consideration. The criteria for correlation deter-
Index Content) values were calculated using the mination were correlation values of C0.65 or B-0.65
CERVUS 3.0.3 software (Marshall et al. 1998). For and p-values B 0.05.
metabolite data, statistical analysis was performed
using SPSSv21.0 software and significant differences
among the different samples were determined by one- Results and discussion
way ANOVA using Duncans multiple-range test at a
significance level of 5% (p B 0.05). Coefficients of Total phenolic and total flavonoid content
variation (CV%) were determined as indicators of
variability. In order to explain the potentiality of these For all sour orange genotypes, the TP levels ranged
variables (chemical and SSR) and to depict relation- from 64.2 to 288.5 mg GAE/g DW, with the W3
ships among individuals, principal component analysis having the lowest content, and GafsiB2 with the
(PCA) was applied using PAST software (Hammer highest content. Its noteworthy to mention that all the
et al. 2001). To check for possible correlations between tested genotypes had high TP levels, with more than
genetic and chemical distances among cultivars, Mantel 50 mg GAE/g FW (Fig. 1a). Our results farther
test was performed using XLSTAT-Pro 7.5.3 software. exceeded those obtained in flowers (4.83 mg GAE/g
Furthermore, associations between molecular markers DW; Karimi et al. 2012), in peels (31.62 mg GAE/g
(as independent variables) and metabolite data (as DW; Lagha Benamrouche and Madani 2013) and in
dependent variables) were performed by a multiple seeds (1.35 mg GAE/g DW; Moulehi et al. 2012). The
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 highest. GafsiB2 and F15 had significantly higher TF
an adequate setting for association tests (Lourenco et al. contents than the other genotypes (Fig. 1b). In fact,
2011). The analysis was based on the following model: these levels were higher than those reported in the and
Y = a ? b1m1 ? b2m2 ? ? bjmj ? ? bnmn ? zest (0.33 mg EC/g DW; Jabri karoui and Marzouk
d ? e, which related to the variation in the dependent 2013), flowers (4.11 mg rutin equivalent/g DW;
variable (Y cultivar means for a quantitative trait) to a Karimi et al. 2012) and seeds (1.7 mg EC/g DW;
linear function of the set of independent variables mj, Moulehi et al. 2012) of C. aurantium.
representing SSR markers. The bj terms are the partial These findings demonstrate that the high levels of
regression coefficients that specify the empirical rela- polyphenols obtained from sour orange leaves extract
tionships between Y and mj, d represents between far exceeded the levels obtained different other sour
accession residuals, which is left after regression, and orange organs analyzed and suggest that the leaves are
e is the random error of Y that includes environmental rich in phenolic compounds. Thus we may consider
variation (Virk et al. 1996). To select independent them as important source of substantial secondary
variables for the regression equation, F values with metabolites. Indeed, various studies have shown that
0.045 and 0.099 probabilities were used to enter and they can be valued as natural antioxidants (Koca et al.
remove, respectively (Afifi and Clark 1999). Selected 2003; Lamport et al. 2012).
markers were further tested independently with linear
curve fitting using linear models for confirming the Antioxidant activities of leaves extract from C.
significance of b-statistics for each band identified by aurantium genotypes
MRA. b can be defined as the standardized regression
coefficient = BxSx/Sy, where B is the regression coef- In order to assess whether the phenolic and flavonoid
ficient or slope and Sx and Sy are the standard deviations contents positively correlated to the antioxidant

Euphytica (2017)213:44 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
Fig. 1 a Total Phenolic Content (mg GAE/g DW); b Total studies (Lamport et al. 2012), we might consider that
Flavonoid Content (mg EQ/g DW) and c Scavenging of the 1,1- citrus leaves are an important source of secondary
diphenyl-2-picrylhydrazyl Radical (DPPH, IC50 lg/ml) of sour metabolites.
orange leaf extracts

Contents of individual phenolic compounds

activity, DPPH analysis was achieved for the different
genotypes. The radical scavenging ability was high A total of 21 phenolic compounds were identified from
with IC50 values ranging 581600 lg/ml. Further- the tested sour orange leaves, including eleven phe-
more, allchB3 leaves extract had the highest antirad- nolic acids and ten flavonoids (Table 2). A large
ical activity while F15 displayed the lowest activity variation patterns of phenolic acids components and
(Fig. 1c). contents were also observed for the different sour
The relationship between the total polyphenol orange genotypes studied (Table 2). The gallic acid,
content and antioxidant activity is very controversial. syringic acid and tannic acid have been identified as
While some authors support the existence of a strong the major phenolic compounds in the sour orange
correlation between phenolic content and antioxidant leaves (65.44, 61.27 and 58.78 lg/g DW, respec-
activity (Ouchemoukhe et al. 2012; Benmeddour et al. tively). AllchB3 had the highest gallic (20.25 lg/g
2013), others contradict (Anagnostopoulou et al. 2006; DW), syringic (58.12 lg/g DW) and tannic acid
Kamran et al. 2009). Correlation analysis was used to (21.89 lg/g DW) content, while these compounds

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 _ _ _ _
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
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 _ _ _ _

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

44 Page 8 of 14 Euphytica (2017)213:44

Fig. 3 Principal
component analysis (PCA)
of sour orange genotypes
based on metabolite profiles

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

44 Page 10 of 14 Euphytica (2017)213:44

Fig. 5 Correlation matrix of metabolites from C. aurantium correlation coefficient is represented by the intensity of the blue
genotypes. Each square indicates r (Pearsons correlation (-1 \ r \ 0) or pink (0 \ r \ 1) color, as indicated on the color
coefficient values for a pair of metabolites). The value of the scale

used to establish relationships between metabolites 27 pairs of positive correlations and 18 pairs of
belonging to a biological system (Kim et al. 2013). negative correlations. These analytical results
Pearson correlation analysis and HCA of the acces- revealed strong correlations between metabolites
sions were implemented to determine the relationships involved in closely related pathways and demon-
among levels of the 21 metabolites in sour orange strated the robustness of the present experimental
(Fig. 5). Analytical results revealed the existence of 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; The biosynthesis and accumulation of metabolites are
p \ 0.0001) is also observed. The results of the mainly controlled by enzymes and regulatory factors
CAH obtained from Pearson correlation analysis of which are encoded by genes in the genome. The SSR
the 21 metabolites revealed several large groups of marker analysis revealed a number of markers show-
metabolites. A group containing quercetin includes the ing statistically significant correlation with different
precursors of the flavonol biosynthetic pathway such metabolite components, as identified through MRA.
as naringenin. Flavanones such as hesperetin, naringin Five SSR markers were correlated with 12 metabolites
and naringenin gathered in the same group. Correla- showing a significant correlation (r [ 0.6 or r \ -0.6;
tion analysis showed that caffeic, vanillic and gentisic P \ 0.001; n = 11). Several SSR markers (Table 4)
were strongly positively correlated. Tannic acid showed a significant correlation with specific metabo-
exhibited the greatest number of correlation pairs. In lites in sour orange. Indeed, the strongest correlation
fact, these analytical results revealed strong correla- was that of cAGG9200 with rutin (r = 0.989;
tions between metabolites involved in closely related b = -0898; P = 0.00). This marker (cAGG9200)
pathways. Not surprising that some flavonoids such was also negatively correlated with flavone
catechin and epicatechin were found to be highly (r = 0.761; b = -0.83), however, with tannic acid,
correlated to phenolic acids (tannic acid and gallic it was positively correlated (r = 0.71; b = 0.729)
acid) since they derive from the same degradation indicating an inverse or negative correlation between
pathway of condensed tannins. This indicates that flavone/rutin and tannic acid. CT21190 was the most
correlations between metabolites of the same class are important marker which correlates with high number
conserved while those between the different classes of of metabolites (4 metabolites). The highest negative
metabolites are quite diverse. Therefore, metabolic correlation value (b = -0.988) was detected with
profiling of the phenolic compounds can be used to gentisic acid, followed by vanillic acid (b = -0.966);
track their metabolic links on one hand, and the caffeic acid (b = -0.939) and Methyl, 3,4,5-dihy-
observed correlations between the concentrations of droxy-benzoate (b = -0.648). In addition, CAG01165
metabolites in a biological sample, may be used to was positively correlated with syringic acid (r = 0.71)
gain additional information about the physiological and negatively correlated with hydroxyphenyl-acetic
state of a given tissue on the other hand (Steuer et al. acid and hespertine (b = -0926 and b = -0944,
2003). 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

44 Page 12 of 14 Euphytica (2017)213:44

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