Start Codon Targeted (SCoT) markers provide new insights into the genetic diversity analysis and characterization of Tunisian Citrus species

Biochemical Systematics and Ecology 61 (2015) 390e398

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Start Codon Targeted (SCoT) markers provide new insights into the genetic diversity analysis and characterization of Tunisian Citrus species Aymen Mahjbi, Ghada Baraket, Amel Oueslati, Amel Salhi-Hannachi* Laboratory of Molecular Genetics, Immunology & Biotechnology, Faculty of Sciences, University Tunis El Manar, Campus Universitaire, 2092 El Manar, Tunis, Tunisia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 March 2015 Received in revised form 9 July 2015 Accepted 11 July 2015 Available online xxx

Start Codon Targeted markers were used to establish phylogenetic relationship among seven species from Citrus L. genus. Twelve SCoT primers were used for their ability to reveal polymorphism of the targeted codon of initiation. A total of 132 amplicons were generated and 93.9% of them were polymorphic. The polymorphism information content of 0.884 and the resolving power of 75.22 illustrate the efficiency of the tested SCoT primers in highlighting polymorphism. The average Nei's (1973) gene diversity (0.376), the Schannon's index (0.548) and the Gst parameter (0.346) describe an important polymorphism at the interspecies level in Citrus genus. Analysis of molecular variance suggested significant genetic differences within species. In fact, 84% of variance occurs within the species, whereas 16% of the variation was recorded among the species of Citrus. The limited gene flow (Nm ¼ 0.941) was recognized as a major factor to explain the partition of the observed diversity. The principal coordinates analyses, Neighbor Joining and the Bayesian clustering approach based on the SCoT markers also confirm the discrimination of the species of Citrus. Our results confirm the relevance and suggest the effectiveness of the SCoT markers for assessing genetic diversity, characterization and identification of the species of Citrus. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Citrus L. Inter and intra-specific genetic diversity Molecular polymorphism Bayesian clustering approach SCoT

1. Introduction Citrus L. (Family: Rutaceae) is native to South-East Asia (Webber, 1967). The most important species are: citron (Citrus medica L.), lemon [Citrus limon (L.) Burm. F.], lime [Citrus aurantifolia (Christm.) Swing.], mandarin (Citrus reticulata Blanco), sour orange (Citrus aurantium L.), orange [Citrus sinensis (L.) Osb.] and grapefruit (Citrus paradisi Macf.). The differences between species are a matter of morphological and pomological characters such as size, shape, color and flavor of the fruit (Bonnassieux, 1988). In fact, two major classifications exist for the genus Citrus L.: Tanaka (1977) understands 162 species, whereas Swingle and Reece (1967) distinguish only 16 from it. Recently, Mabberley (1997) proposed a new classification of Citrus and suggests the presence of three ancestral species, namely Citrus maxima (Burm.) Merr. (pummelo), Citrus reticulata Blanco (mandarin) and Citrus medica L. (citron) and the hybrids of Citrus were obtained. The majority of Citrus is diploîd, only some natural polyploîd were identified (Fortunella hindsii. Tahiti lime) or artificially produced (Roose, 1988). Tunisia is

* Corresponding author. E-mail address: [email protected] (A. Salhi-Hannachi). http://dx.doi.org/10.1016/j.bse.2015.07.017 0305-1978/© 2015 Elsevier Ltd. All rights reserved.

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considered one of the main Citrus producing countries in the Mediterranean basin (GIFruits, 2010) in which Citrus is a popular fruit crop of economic importance. In terms of production this sub-sector allows the supply of the local and international markets with fresh fruit for a period going six months a year. Citrus exports almost consist exclusively of Maltaise oranges. The Citrus resources are characterized by heterogeneity and diversity within and between major varietal, they represent a great potential for selection and breeding. It is thus imperative to use this potential for the conservation, improvement for better management and exploitation of these resources. In this contest, the development of molecular tools for the identification of local cultivars and the analysis of the genetic diversity of a national germplasm is necessary to develop varieties adapted to various environments and preserve our resources. Several molecular technics were used to generate nuclear molecular to et al., 2001; Barkley et al., 2006; Abkenar et al., 2007; Handaji et al., 2012; Garcia-Lor et al., 2013). The markers (Bre approach of chloroplast microsatellite has been demonstrated also for the evaluation of cpDNA in the Citrus L. genus (Deng et al., 2007). Recently, a novel and simple molecular technique termed Start Codon Targeted polymorphism (SCoT) has been demonstrated by Collard and Mackill (2009) and used to detect molecular markers in supplement of the popular markers RAPD and ISSR. This technique uses single 18-mer primers in single primer polymerase chain reaction (PCR) targeting different regions of the conserved region flanking the ATG start codon. Indeed, due to the simultaneous binding of the primer on both DNA strands, the sequence between the two binding sites is amplified. This technique has proven effective and reproducible markers and gave a high polymorphism correlated with traits of biological interest (Collard and Mackill, 2009). The goals of the present study, is to investigate the potential of the SCoT markers to reveal molecular polymorphism, to identify and investigate the genetic diversity of Tunisian Citrus germplasm represented by species of economical importance.

2. Materials and methods 2.1. Plant material and DNA extraction The plant material consists of seven Tunisian species of Citrus L. genus, including 54 cultivars and each variety is represented by two trees. Citrus aurantium L. and Citrus paradisi L. species including 10 cultivars originated from the Interprofessional Group Fruit (GIF) located in the region of Cap Bon. Citrus sinensis L., Citrus clementina L., Citrus reticulata L., Citrus  tangelo L. (Hybrid: Tangerine  pomelo) and Citrus limon L. species including 44 cultivars originating from the technical center of Citrus situated in Zaouiet Jdidi (Cap Bon) (Table 1). The extraction of total genomic DNA was performed by the automat of extraction (QIAGEN kit) from lyophilized young leaves (20 mg). The quality of the extracted DNA was checked on a 1% agarose gel. The concentration of the DNAs obtained was estimated using a QubitR fluorometer (Molecular Probes™ invitrogen detection technologies).

2.2. Oligonucleotides and PCR assays Twelve SCoT primers were used (Collard and Mackill, 2009) to amplify regions that frame ATG start codon in Citrus cultivars (Table 2). The solution of 25 mL contained 2.5 mL of Taq polymerase buffer (10X), 20 mM of dNTP, 25 mM of MgCl2, 10 mM of primers, 0.5 U of Taq DNA polymerase, 25e30 ng of DNA and 25 ml of H2O MiliQ, QSP. PCR reactions were performed in the PTC-100 thermal cycler (Programmable Thermal Controller). The amplification reaction begins with a phase of denaturation at 94  C for 3 min followed by 35 cycles (denaturation at 94  C for 1 min, specific appropriate melting temperature for each primer (Tm,  C) for 1 min, elongation at 72  C for 2 min) with a final extension at 72  C for 5 min.

2.3. Data analysis The size of the amplified fragments was estimated on 1.5% agarose gel. The amplicons were coded linearly as the following: the number 1 (present) or 0 (absent). Pairwise genetic dissimilarities of genotypes using Jaccard coefficient (1908) were calculated using DARwin version 5.0.158 (Perrier and Jacquemoud-Collet, 2006). The dissimilarity coefficients were used to perform principal coordinate analyses (PCoA) and construct Neighbor Joining trees (Saitou and Nei, 1987) with a bootstrapping value of 10,000 replications using DARwin version 5.0.158. Molecular variance (AMOVA) analysis was also carried out on SCoT dataset using GenAlEx 6.51 (Peakall and Smouse, 2006). The AMOVA components were used as an estimation of molecular diversity at the hierarchical level among and within the species of Citrus. The parameters of the genetic diversity such as the average Nei's (1973) gene diversity (H), the Schannon's index (I) and the effective number of alleles (Ne) (Maruyama and Kimura, 1980) were calculated by the POPGENE software version 1.32 (Yeh et al., 2000). The total genetic diversity (Ht) and the mean genetic diversity within species (Hs) were calculated using POPGENE software version 1.32 (Yeh et al., 2000). The Nei's Gst (Nei, 1977) and Nm parameters (Nei, 1987), a measurement of the inter-specific diversity and the gene flow were calculated. The Shannon's index (I) was calculated for each locus (Lewontin, 1972). The polymorphism information content (PIC) was estimated using the formula of Smith et al. (1997). The resolving power of primers (Rp) was calculated based on the formula of Gilbert et al. (1999).

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Table 1 List of the considered Tunisian Citrus cultivars. Cultivar name

Group

Code

Origin

Species namea

Mandarinier Fortune (I)

Mandarin

MF

Ctrus reticulata Blanco

Mandarinier Encore (I)

Mandarin

ME

Mandarinier Ortanique (I)

Mandarin

MOT

Mandarinier Nova (I)

Mandarin

MN

Tangelo Minneola (I)

hybrid (Tangerine  pomelo)

TM

mentinier MA3 (I) Cle

Clementine

CMA

mentinier Hernandina (I) Cle

Clementine

CHA

mentinier Marisol (I) Cle

Clementine

CML

mentinier Cassar (L) Cle

Clementine

CCA

Washington Navel (L)

Navel orange

WN

Navelina (I)

Navel orange

NA

Newhall (I)

Sweet orange

NWL

Lane Late (I)

Sweet orange

LAL

Navel Late (I)

Navel orange

NAT

Maltaise 1/2 Sanguine (L)

Blood orange

MDS

Maltaise Barlerin (L)

Blood orange

MB

Valencia Late (L)

Valencia orange

VL

Orange Tarroco (I)

Sweet orange

OT

reza (I) Citronnier Santa Te

Lemon

CZ

Citronnier Interdonato (I)

Lemon

CI

Citronnier Monachello (I)

Lemon

CM

minello (I) Citronnier Fe

Lemon

CFM

Bigaradier (I) Bigaradier Maroc (I) Pomelo Star Ruby (I)

Sour orange Sour orange Grapefruit

BG BGM PSR

Pomelo Marsh (I) Pomelo Ruby (I)

Grapefruit Grapefruit

PM PR

The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) The Technical Center of Citrus (Zaouiet Jdidi) Interprofessional Group Fruit (GIF) Interprofessional Group Fruit (GIF) The Technical Center of Citrus (Zaouiet Jdidi) Interprofessional Group Fruit (GIF) Interprofessional Group Fruit (GIF)

Ctrus reticulata Blanco Ctrus reticulata Blanco Ctrus reticulata Blanco Citrus  tangelo L. Citrus clementina L. Citrus clementina L. Citrus clementina L. Citrus clementina L. Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus sinensis (L.) Osbeck Citrus limon (L.) Burm.f. Citrus limon (L.) Burm.f. Citrus limon (L.) Burm.f. Citrus limon (L.) Burm.f. Citrus aurantium L. Citrus aurantium L. Citrus paradisi Macf. Citrus paradisi Macf. Citrus paradisi Macf.

I: introduced variety; L: local variety; a: species name according to Swingle system (1967).

2.4. The Bayesian clustering approach The STRUCTURE (Pritchard et al., 2000; Falush et al., 2003) software was run using a model with admixture and correlated allele frequencies, with 10 independent replicate running for each K value (number of genetic clusters) ranging from 1 to 10. Ten runs of the structure were performed with 100,000 steps of burning followed by 100,000 Monte Carlo Markov Chain (MCMC) repetitions. The run with the maximum likelihood was used to assign the most probable number of clusters, which was validated with an ad hoc statistic based on the second order rate of change in the log probability of data between successive K values (Evanno et al., 2005). To find optimal alignments of independent runs, the CLUMPP version 1.1 software program (Jakobsson and Rosenberg, 2007) was used with greedy algorithms, 10,000 random input orders and 10,000 repeats, to calculate the average pairwise similarity (H0 ) of runs.

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Table 2 Characteristics of the tested SCoT primers. Code primers

Sequences (50 -30 )

%GC

SCoT2 SCOT5 SCoT8 SCoT11 SCoT13 SCoT16 SCoT19 SCoT22 SCoT26 SCoT28 SCoT29 SCoT32

CAACAATGGCTACCACCC CAACAATGGCTACCACGA CAACAATGGCTACCACGT AAGCAATGGCTACCACCA ACGACATGGCGACCATCG ACCATGGCTACCACCGAC ACCATGGCTACCACCGGC AACCATGGCTACCACCAC ACCATGGCTACCACCGTC CCATGGCTACCACCGCCA CCATGGCTACCACCGGCC CCATGGCTACCACCGCAC

56 50 50 50 61 56 67 56 61 67 72 67

3. Results 3.1. Molecular polymorphism The analysis of SCoT patterns shows that among the 12 primers used, 10 led to reproducible and easily interpretable amplifications. A total of 132 bands was generated from the 54 Citrus cultivars studied, including 124 polymorphic bands (93.9%) with an average of 12.4 bands per primer. The size of these bands ranged from 240 bp for the SCoT13 to 3800 bp for SCoT11 primer. The number of bands per primer ranged from 4 for the SCoT19 primer to 22 for SCoT13 primer with an average of 13.2 bands per primer. The greatest number of SCoT markers was recorded by SCoT13 primer (20 bands), which shows that it is most effective in the detection of polymorphism. While, the SCoT19 primer seems to be the less informative ones since it generates the lower number of polymorphic bands (3 bands). It is worthy to note that SCoT2, SCoT22, SCoT26 and SCoT32 primers provided 100% of polymorphic bands. While SCoT19 primer provided 75% of polymorphic bands (Table 3). The polymorphism information content (PIC) ranged from 0.616 (SCoT19) primer to 0.944 (SCoT13) primer with an average of 0.884 which shows a high content of polymorphism and the primers used are very discriminating (Table 3). The resolving power (Rp) ranged from 1.45 for SCoT19 primer to 11.53 for SCoT13 primer with an average of 7.52. The collective value of resolving power (75.22) highlights the reproducibility of the loci tested (Table 3). The total number of polymorphic bands for each species of Citrus genus ranged from 41 bands for the Citrus  tangelo L. to 114 bands for Citrus sinensis L. (Table 4). A percentage of polymorphic bands of 86.3% reflected the highest rate of polymorphism among Citrus sinensis L. Whereas, the Citrus  tangelo L. showed the lowest rate of polymorphism with a percentage of polymorphic bands of 31% (Table 4). The Citrus limon L. provided the highest level of polymorphism compared to the other two species Citrus clementina L. and Citrus reticulata L. which are represented by the same number of cultivars.

3.2. Genetic diversity The average Nei's (1973) gene diversity (H) ranged from 0.128 for Citrus  tangelo L. to 0.346 for Citrus sinensis L. with an overall genetic diversity for the seven species of 0.376. The Shannon's index (I) ranged from 0.187 for Citrus  tangelo L. to 0.505 for Citrus sinensis L., with a total value of 0.548 (Table 4). Therefore, this index shows a significant genetic diversity at the interspecies level. The effective number of alleles (Ne) ranged from 1.219 for Citrus  tangelo L. to 1.613 for Citrus sinensis L. Table 3 Summary of polymorphism generated by SCoT primers. Primer

Size range (bp)

TB

PB

PPB

PIC

Rp

SCoT2 SCoT8 SCoT11 SCoT13 SCoT16 SCoT19 SCoT22 SCoT26 SCoT29 SCoT32 Total Mean

310e2100 530e2400 506e3800 240e2250 380e3000 570e3000 360e2590 450e3600 290e2450 300e2340 e e

12 14 11 22 14 4 11 12 14 18 132 13.2

12 12 10 20 13 3 11 12 13 18 124 12.4

100 85.71 90.9 90.9 92.8 75 100 100 92.85 100 93.94 e

0.913 0.912 0.889 0.944 0.919 0.616 0.903 0.909 0.899 0.939 e 0.884

7.469 9.703 7.814 11.536 7.02 1.452 5.37 7.562 7.888 9.408 75.222 7.522

TB: total number of bands, PB: number of polymorphic bands, PPB: percentage of polymorphic bands, Rp: resolving power, PIC: polymorphism information content.

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Table 4 Parameters of genetic diversity calculated for Citrus species. Species

PB

Ne

H

I

PPB%

Citrus reticulata L. Hybrid: C.  tangelo L. (Tangerine  pomelo) Citrus clementina L. Citrus sinensis L. Citrus limon L. Citrus paradisi L. Citrus aurantium L. Overall

87 41 94 114 96 82 56

1.456 1.219 1.473 1.613 1.468 1.420 1.340 1.667

0.256 0.128 0.268 0.346 0.270 0.239 0.182 0.376

0.375 0.187 0.395 0.505 0.401 0.352 0.262 0.548

65.9 31 71.2 86.3 72.7 62.1 42.4 93.9

PB: number of polymorphic bands, Ne: effective number of alleles, H: average Nei's (1973) gene diversity, I: Shannon's information index, PPB: percentage of polymorphic bands.

with an average of 1.667 for the all samples. These results reflected the high level of polymorphism found in the Citrus sinensis L., followed by Citrus limon L. and Citrus clementina L. species. Contrastingly, Citrus  tangelo L. shows the low level of polymorphism. The average of intra-specific genetic diversity (Hs) is 0.241. The total genetic diversity (Ht) of all the species is 0.370. The overall genetic differentiation of the all species (Gst) is 0.346. This result shows a significant genetic diversity among species and supported the results of Shannon's information measure that there was a relatively level of genetic differentiation within the species of Citrus. The total value of gene flow (Nm) is 0.941. AMOVA analysis revealed 16% and 84% of variance among and within groups of the species of Citrus, respectively. This result suggests that genetic variance was high detected within the species of Citrus and low among these groups. These results confirm the Gst value obtained. 3.3. Phylogenetic relationship The coefficient of genetic dissimilarity of Jaccard ranged from 0.159 to 0.689 with an average of 0.492. The low genetic distance was estimated between the two ‘Newhall 1’ [NWL1] and ‘Navelina 2’ [NA2] of Citrus sinensis L., reflecting their genetic similarity. The highest distance was recorded between Citrus sinensis L. and Citrus clementina L. species. This result mentinier Cassar 2’ [CCA2] reflects the high genetic divergence between ‘Maltaise Barlerin 2’ [MB2] of Citrus sinensis L and ‘Cle of Citrus clementina L. The Neighbor Joining phylogenetic tree (Fig. 1) generated underlines the presence of three major clusters. The first one labeled (I) which supported by bootstrap values ranging from 66% to 92% includes Citrus aurantium L., [‘Pomelo Marsh’ [PM], ‘Pomelo Ruby’ [PR] cultivars of Citrus paradisi L. ‘Maltaise ½ Sanguine’ [MDS], ‘Washington Navel’ [WN], minello’ [CFM] of Citrus limon L.]. ‘Maltaise Barlerin’ [MB], ‘Valencia Late’ [VL] cultivars of Citrus sinensis L. and ‘Citronnier Fe The second major cluster noted (II) which supported by bootstrap values varied from 54% to 94% includes three species: Citrus reticulata L., Citrus  tangelo L. and Citrus clementina L. [‘Newhall’ [NWL], ‘Navelina’ [NA], ‘Lane Late’ [LAL] and ‘Orange Tarroco 1’ [OT1] cultivars of Citrus sinensis L. and ‘Pomelo Star Ruby’ [PSR] of Citrus paradisi L.]. The third major cluster labeled (III) which supported by bootstrap values ranged from 66% to 92% includes two species: Citrus limon L. and Citrus sinensis L. reza’ [CZ]] and [‘Navel Late’ [NAT] and [‘Citronnier Interdonato’ [CI], ‘Citronnier Monachello’ [CM] and ‘Citronnier Santa Te ‘Orange Tarroco 2’ [OT2] of Citrus sinensis L.]. The results demonstrate the effectiveness and usefulness of SCoT markers to detect polymorphism and discriminate the species of Citrus. Dissimilarity between Citrus limon L. and the other species and genetic convergence between Citrus clementina L., Citrus reticulata L. and Citrus  tangelo L. species were noted. In addition, Citrus aurantium L. and Citrus paradisi L. species are genetically close. 3.4. Principal coordinate analyses The principal coordinate analyses (PCoA) (Fig. 2) shows that the first two axes account for 24.3% of the total variability. The Scatter plot show the divergence of Citrus reticulata L., Citrus  tangelo L. and Citrus clementina L. from the other species by SCoT markers that define the PC1 and PC2 axes. Similarly, the genetic convergence was also recorded between Citrus aurantium L. and Citrus paradisi L. [‘Pomelo Marsh’ [PM] and ‘Pomelo Ruby’ [PR]]. In addition, the main coordinated PC2 separates the twenty orange cultivars of Citrus paradisi L., Citrus limon L. and the ‘Mandarinier Ortanique1’ [MOT1] of Citrus reticulata L. into two distinct groups. We also noted a remarkable intra-specific genetic diversity obtained within Citrus limon L., Citrus paradisi L. and Citrus sinensis L. which is in agreement with the result obtained by the Neighbor Joining phylogenetic tree (Fig. 1). 3.5. Genetic structure analysis To obtain a more detailed result of the genetic structure of cultivated Citrus genotypes in Tunisia, the Bayesian clustering analysis, implemented in the program STRUCTURE (Pritchard et al., 2000), was used. STRUCTURE results which are based on the admixture model and correlation of allele frequencies showed that the maximum likelihood described by the logarithm of the probability (ln Pr (XjK)), and used to determine the number of clusters K possible is achieved for K ¼ 4. The ad hoc method developed by Evanno et al. (2005) showed a possible representation of the genetic structure of cultivated Citrus for the model

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Fig. 1. Neighbor Joining tree based on Jaccard dissimilarity coefficient showing the genetic relationship among 54 cultivated Citrus using SCoT markers (Bootstrap analysis was performed with 10,000 replications [50]).

K ¼ 2 (DK ¼ 57.655). However, for model K ¼ 2, the values of DK are not enough to generate a resolution. Moreover, the calculation of the average pairwise similarity (H0 ) between the simulations obtained taken in pairs, carried out using CLUMPP (Jakobsson and Rosenberg, 2007) program showed that the highest values are obtained for K ¼ 2 (H0 ¼ 0.993), and K ¼ 4 (H0 ¼ 0.994). Thus, about these results, a preliminary analysis of the assignment obtained for each cultivar for the model K ¼ 2 probabilities allows us to distinguish on one hand the six species (Citrus reticulata L., Citrus  tangelo L., Citrus clementina L., minello’ [CFM]], Citrus sinensis L. (Except ‘Washington Citrus limon L. (Except the two trees from the cultivar ‘Citronnier Fe Navel 1’ [WN1], ‘Maltaise ½ Sanguine 2’ [MDS2], ‘Maltaise Barlerin 1’ [MB1], ‘Maltaise Barlerin 2’ [MB2], ‘Valencia Late 1’ [VL1] and ‘Valencia Late 2’ [VL2] cultivars) and on the other the both trees from the cultivar ‘Pomelo Star Ruby’ [PSR] of Citrus paradisi L.. Citrus aurantium L. and Citrus paradisi L. [‘Pomelo Marsh’ [PM] and ‘Pomelo Ruby’ [PR]] clustered together in the same group colored in red (in the web version). When, the model K ¼ 4, it classifies 54 Citrus genotypes into four genetically distinct clusters. A second analysis of each cultivar assignment probabilities for the model K ¼ 4 shows that cluster 1 is composed of three species; Citrus reticulata L. with the exception of ‘Mandarinier Ortanique 1’ [MOT1], Citrus  tangelo L., Citrus clementina L. and ‘Maltaise ½ Sanguine 1’ [MDS1] cultivar of Citrus sinensis L. The cluster 2 (yellow) (in the web version) includes five trees; ‘Navelina 2’ [NA2], ‘Newhall 1’ [NWL1], ‘Newhall 2’ [NWL2], ‘Lane Late 2’ [LAL2] and ‘Orange Tarroco 1’ [OT1] of Citrus sinensis L.. The third cluster (blue) (in the web version) includes ‘Orange Tarroco 2’ [OT2], ‘Lane Late 1’ [LAL1], ‘Navel Late 1’ [NAT1] and ‘Navel Late 2’ [NAT2] of Citrus sinensis L. and two trees of ‘Citronnier Monachello’ [CM], ‘Citronnier reza’ [CZ] of Citrus limon L.. Finally, cluster 4 consists of the cultivars ‘Washington Interdonato’ [CI] and ‘Citronnier Santa Te Navel 1’ [WN1], ‘Washington Navel 2’ [WN2], ‘Maltaise ½ Sanguine 2’ [MDS2], ‘Maltaise Barlerin 1’ [MB1], ‘Maltaise Barlerin 2’

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Fig. 2. Dispersion of 54 cultivated Citrus genotypes (Citrus L.) in the two-dimensional plane of the principal coordinates analyses (24.3% of the total variability).

minello’ [CFM] cultivar [MB2], ‘Valencia Late 1’ [VL1] and ‘Valencia Late 2’ [VL2] of Citrus sinensis L., both trees of ‘Citronnier Fe of Citrus limon L., Citrus paradisi L. [‘Pomelo Marsh’ [PM] and ‘Pomelo Ruby’ [PR]] and Citrus aurantium L. species. A total of 50/ 54 cultivars is clearly assigned with an assigned probability of more than 50% (model K ¼ 4). The four cultivars unassigned [‘Mandarinier Ortanique 1’ [MOT1], ‘Navelina 1’ [NA1], ‘Pomelo Star Ruby 1’ [PSR1] and ‘Pomelo Star Ruby 2’ [PSR2]] are considered as the result of a mixture between clusters. These results therefore tend to a net structure of cultivated species of Citrus L. genus. 4. Discussion A dominant molecular marker was used to evaluate molecular polymorphism and differentiate the species of Citrus. The results showed by this study indicate that a high genetic diversity that occurs among and within Tunisian cultivars of Citrus L. species. The size of amplicons ranges up to 3800 bp is higher than that obtained by Han et al. (2011), which ranged from 150 bp to 2100 bp. The level of polymorphism observed is higher than that obtained by Han et al. (2011) for some species of Citrus by seven SCoT primers (TB ¼ 74; PB ¼ 48 and PPB ¼ 64.9%). For grapes, the molecular analysis of 64 varieties by 17 SCoT primers shows that the average rate of polymorphism of 93.1%, which is close to that founded in our study (93.9%) (Guo et al., 2012). The high collective value of resolving power (Rp) and the average value of polymorphism information content (PIC) prove the usefulness of the tested primers. Our results show that the SCoT technique reveals a higher level of polymorphism than that shown by ISSR markers (Handaji et al., 2012) in mandarin (Citrus reticulata L.) species (PB ¼ 3.9, PPB ¼ 49%). Moreover, Gorji et al. (2011) and Luo et al. (2011) showed that the SCoT markers are more informative and reproducible that ISSR and RAPD markers to detect polymorphism in potato and mango, respectively. The occurrence of somatic mutations, introgression, polyploidy, the sexual mating system (Moore, 2001) may partly explain the detected diversity or natural hybridization that is often high in the Citrus L. genus (Garcia-Lor et al., 2012a). In fact, the two species Citrus sinensis L. and Citrus limon L. showed a significant intra-specific molecular polymorphism. Based on the structure analysis, the four unassigned cultivars of three species Citrus reticulata L., Citrus sinensis L. and Citrus paradisi L. (‘Mandarinier Ortanique 1’ [MOT1], ‘Navelina 1’ [NA1], ‘Pomelo Star Ruby 1’ [PSR1] and ‘Pomelo Star Ruby 2’ [PSR2]) are considered as a result of the contribution of the genomes of other species. Indeed, several hypotheses have been proposed for the origin of Citrus sinensis L.. According

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Moore (2001), sweet orange should be a direct inter-specific hybrid between a pomelo (Citrus maxima L.) and mandarin (Citrus reticulata L.). In addition, Barkley et al. (2006) reported that sweet orange inherited the majority of its genetic makeup from mandarin and only a small proportion from pummelo. While, Roose et al. (2009) and Garcia-Lor et al. (2012a) suggest that Citrus sinensis L. resulted from a backcross 1 (BC1) [(C. maxima  C. reticulata)  C. reticulata]. On the other hand, Jena et al. (2009) based on cpDNA data have also elucidated the involvement of Citrus reticulata L. in the origin of sweet orange (Citrus sinensis L.), although some workers suggest that Citrus maxima L. (grapefruit) is the maternal parent of sweet orange mentinier Cassar 2’ [CCA2] very near (Penjor et al., 2013). According to the NJ phylogenetic tree, the local variety ‘Cle genetically to the orangbe e trees ‘Lane Late 1’ [LAL1] and ‘Navelina 1’ [NA1] appeared to be different from other introduced varieties of clementine through the absence of some SCoT markers. This result confirmed the hypothesis that the clementine is a hybrid between the mandarin and sweet orange (Ollitrault et al., 2012). While, the two trees ‘Tangelo Minneola’ [TM] (tangerine  grapefruit) of Citrus  tangelo L. are genetically close to the two species of clementine and mandarin. The bayesienne analysis clustered together Citrus reticulata L., Citrus clementina L. and Citrus  tangelo L species and supports their hybrid status resulting from Citrus reticulata L. and Citrus paradisi L. species (Garcia-Lor et al., 2012a, b). Similarly, we note the presence of a specific marker of 240 bp by SCoT13 primer that characterizes the cultivar ‘Bigaradier Maroc 2’ [BGM2] compared to other varieties of Citrus aurantium L. and other species, confirming that this cultivar derived from natural hybridization. The cultivars of Citrus reticulata L. are genetically very close together and in the same group, a similar result was found by Barkley et al. (2006) who studied the group of mandarins and closest varieties by SSR markers, this could be explained by apomixis (polyembryony) which has been demonstrated by Froelicher et al. (2011). According to the result obtained by the NJ phylogenetic tree and principal coordinate analyses (PCoA), Citrus reticulata L. is genetically close to Citrus clementina L. and Citrus  tangelo L. (hybrid: Tangerine  pomelo) species, which was confirmed by the structure analysis, which puts into consideration the hypothesis that mandarin is the ancestral species for both clementine and tangelo Garciaminello’ [CFM] of Citrus limon L. Lor et al. (2012a). Structure analysis revealed that the two trees of the cultivar ‘Citronnier Fe are genetically divergent from other cultivars of Citrus and are grouped in the same cluster with Citrus paradisi L. and Citrus aurantium L. species, this could be explained that Citrus limon L. is a secondary species derived from natural hybridization between Citrus aurantium L. and Citrus aurantifolia L. species, which has been shown by other studies (Barrett and Rhodes, 1976; Herrero et al., 1996). In addition, Barkley et al. (2006) and Uzun et al. (2014) suggested that genetic makeups of grapefruits and sour oranges were derived from pummelo and mandarin. Moreover, Ramadugu et al. (2013) suggest that sour oranges were found to be of hybrid origin, with pummelo and mandarin parentage. The limited gene flow (Nm ¼ 0.941) was recognized. This is due to the facultative apomixis characterizing most Citrus (Froelicher et al., 2011). The analysis of molecular variance (AMOVA) showed that the genetic diversity is higher at the intra-specific level. Similarly, it was recorded both in the inter-specific level, which has been demonstrated by the overall Schannon's index (I ¼ 0.548), bayesian and multivariate analyses has been discriminated Citrus  tangelo L. and Citrus aurantium L. species, Citrus clementina L. and Citrus aurantium L. species, Citrus limon L. and Citrus clementina L. species and between Citrus paradisi L. and Citrus clementina L. species. A similar result was found by Uzun et al. (2009) studying Citrus L genus and related genera by SRAP markers. 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