1. bookVolume 28 (2020): Issue 1 (June 2020)
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2353-3978
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30 Jul 2013
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access type Open Access

Genetic Characterization of Tunisian Lime Genotypes Using Pomological Traits

Published Online: 30 Jun 2020
Volume & Issue: Volume 28 (2020) - Issue 1 (June 2020)
Page range: 65 - 76
Received: 01 Oct 2019
Accepted: 01 Mar 2020
Journal Details
License
Format
Journal
eISSN
2353-3978
First Published
30 Jul 2013
Publication timeframe
2 times per year
Languages
English
Abstract

Citrus genus includes a wide number of species that have been long cultivated and well adapted in Tunisia. It is represented by small number of plantations and considered as underutilized in Tunisia. Our goal was to genetically characterize Tunisian lime genotypes to obtain data useful for gene conservation and breeding purposes. The survey of genotypes was conducted in the Cap Bon region, where citrus cultivation is the most spread. Sixteen quantitative and 19 qualitative parameters were evaluated. The observed accessions belonged to three different species: Citrus limetta, Citrus latifolia (limes Byrsa), and Citrus limettioides (limes of Palestine) according to Tanaka classification. Principal component analysis confirmed these classifications. Four-cell analysis (FCA) was used to determine the most threatened genotypes. Quantitative traits were evaluated and allowed the discrimination between genotypes. Many quantitative traits of fruit and juice were highly positively and significantly correlated. Phenotypic diversity was determined using Shannon–Wiener diversity index (H’). The highest value of diversity index was observed for both vesicle thickness and thickness of segment walls (H’ = 0.98). Intermediate values were observed for both fruit axis (H’= 0.49) and pulp firmness (H’ = 0.43). However, fruit shape (H’ = 0.24), shape of fruit apex (H’ = 0.24), and vesicle length (H’ = 0.33) presented the lowest values of diversity index. Current findings will be useful to conserve threatened genotypes ex situ and on farm and also will guide strategic conservation on Citrus genetic resources for future breeding programs.

Keywords

INTRODUCTION

Citrus (Rutaceae family) is one of the most important and ancient crop species domesticated by humans (Krueger & Navarro 2007). Citrus taxonomy and phylogeny are very complicated, controversial, and confusing mainly due to sexual compatibility between Citrus and related genera, the high frequency of bud mutations, the long history of cultivation, and wide dispersion (Nicolosi et al. 2000). The taxonomy of the genus Citrus is controversial as two systems of classification were suggested: Swingle and Reece (1967) distinguished 156 species, whereas Tanaka (1977) only 16 species. It is believed that some Citrus types, including citrons, sour oranges, and lemons, were spread slowly from 500 to 1300 AD through wide areas, including Europe, by successive waves of invaders and travelers of Muslim armies, Arab traders, Crusaders, and others moving along trade routes from other populations to Europe (Moore 2001). Lemon, lime, sour orange, sweet orange, grapefruit, and other edible fruits are apomictically perpetuated biotypes with probable hybrid origin (Kumar et al. 2010).

Lime is a traditional crop in South Asia and the Middle East and comprises a varied group of types of sour and sweet cultivars, different from one to another with distinct fruit characteristics (Nicolosi et al. 2000). Limes hybridize well with other Citrus species. Hybrids could be between lime and lemon or lime and kumquat (Scora 1975), or a tri-hybrid species of citron, pummelo, and Microcitrus (Barrett & Rhodes 1976). In Tunisia, Citrus is one of the most economically important crops. The weather and soil conditions in Tunisia, particularly in the Cap Bon region, are suitable for Citrus production. Currently in Tunisia Citrus plantations extend more than 22,000 ha, and fruit production in the last 5 years increased to 323,000 tones (DGPA 2016). Although limes are classified as a major fruit crop (Mabberley 2008), it is sporadically cultivated in Tunisia. Price of fruit is very high compared with those of sweet orange. Lime is facing different increasing constraints, such as water availability and quality, weather conditions, expansion of diseases, necessity to change the old farming techniques, and urbanization. All these restrictions pose a threat to genetic resources of lime genetic resources in Tunisia. Citrus germplasm is very diverse with many autochthonous cultivars, and it is imperative to implement a strategy for the conservation of genetic resources. For the first time in Tunisia, collecting missions were realized in order to identify and characterize limes’ accessions. In this study, pomological traits were evaluated to determine the genetic diversity of lime. Data collected allowed the establishment of passport data. These findings will enhance both ex situ and on farm genetic conservation program of Citrus germplasm.

MATERIALS AND METHODS
Plant material

Accessions were collected in 2013–2014 and re-collected in 2014–2015 (Table 1) throughout the Cap Bon Nord region east of Tunisia, where citrus cultivation is most widespread. Acquisitions were carried out among a wide range of stakeholders and with the presence of local governmental agencies both in farms and ex situ collections. During the collecting missions, we visited old orchards where limes were cultivated for many decades. Farmers and technical staffs from regional authorities confirmed the names of genotypes. Identification of species was performed with the help of Blondel (1978) classification.

List of species, genotypes studied, abbreviations, acquisition date and accession numbers

SpeciesAbbreviationsDate of first acquisitionAccession ID
Citrus limettioidesLimePal 123/01/2014NGBTUN 757 ARB
LimePal 210/02/2014NGBTUN 992 ARB
Citrus latifoliaLimeBirs04/02/2014NGBTUN 782 ARB
Citrus limettaLime110/02/2014NGBTUN 1032 ARB
Lime223/01/2014NGBTUN 756 ARB
Lime323/01/2014NGBTUN 788 ARB
Lime423/01/2014NGBTUN 793 ARB
Lime523/01/2014NGBTUN 794 ARB
Lime623/01/2014NGBTUN 795 ARB
Lime723/01/2014NGBTUN 797 ARB
Lime804/02/2014NGBTUN 798 ARB
Lime915/01/2014NGBTUN 800 ARB
Lime1015/01/2014NGBTUN 994 ARB
Lime1115/01/2014NGBTUN 823 ARB
Lime1228/04/2014NGBTUN 1033 ARB
Lime1330/01/2014NGBTUN 803 ARB
Lime1430/01/2014NGBTUN 808 ARB
Lime1530/01/2014NGBTUN 812 ARB
Lime1610/02/2014NGBTUN 815 ARB
Lime1710/02/2014NGBTUN 824 ARB
Lime1810/02/2014NGBTUN 759 ARB
Lime1930/01/2014NGBTUN 758 ARB
Lime2030/03/2014NGBTUN 993 ARB
Four-cell analysis (FCA)

The FCA was used to classify the three species under consideration based on the size of the cultivation area and on the number of households, as described by Sthapit et al. (2012).

Pomological characterization

The fully ripe fruits were taken from the four directions of the tree and from the interior and exterior layers of the canopy at the rate of 30 fruits per tree. These fruits were divided into 3 batches of 10 fruits to analyze the quantitative traits (Table 2) and the juice parameters (Table 3). For pomological characterization, analysis has been performed separately for each growing season. Sixteen quantitative traits, including seven parameters dealing with juice description, were measured (Table 4) and correlations among those traits were calculated (Table 5). Nineteen qualitative characters (Table 6) were chosen based on Citrus descriptors (IPGRI 1999). Fresh juice was obtained using a citrus press (Santos Classic N°11, Lyon, France). Subsequently, the juice was filtered through a 1-mm mesh sieve; weighed and volume was measured in a burette. Density was estimated in a sample of 100 ml of juice. Total soluble solids (TSS) content was determined by direct readings on a hand-held refractometer (Toledo, 30 PX) calibrated before use with distilled water. The pH was measured using a pH meter (Toledo, S22) previously calibrated. The titratable acidity (TA) of the juice was evaluated by the determination of citric acid by titration with a NaOH solution (0.1 N). The determination of vitamin C was carried out by titration with iodine solution. Data were obtained in triplicate.

Mean values and significance degree of differences between lime genotypes for fruit characteristics

GenotypesWeight (g)Diameter (mm)Length (mm)Width of skin (mm)Width of epicarp at equatorial plane (mm)Mesocarp thickness (mm)Number of segmentNumber of seedsDiameter of axis (mm)
LimePal 169.0±30e50±0.6d50±0.8cd3.16±0.5bc1.46±0.3d1.81±0.5ab16.46±4a1.93±0.5de6±1de
LimePal 2157.9±34a65±1.3a66.9±0.7a4±1a2.75±0.6a1.33±0.5bc10.26±4b3.8±0.7c13.4±3a
Limebirs87.1±34d60±1a50±0.6cd3.09±0.5c1.8±0.5c1.14±0.3cd8.6±3cd2.26±0.6d8.92±3b
Lime174.3±23ef50±0.4d50±0.7cd3.1±0.4c1.85±0.4c1.38±0.3bc8.46±3cd2.46±0.6d8±3bc
Lime277±17e50±0.4d50±0.9cd3±0.6c2.1±0.7ab1±0.2d9±3c4.2±0.8ab9.4±3b
Lime385.1±29de55±0.6bc60±0.2b4±1a2.6±0.7a1.38±0.4bc8.13±2d1.2±0.2e8.9±3bc
Lime487.3±39d55±0.5bc50±1cd3±0.4c1.75±0.6cd1.4±0.3b8.8±3cd3.2±0.7cd8.25±3bc
Lime573.8±15ef50±0.5d56.8±0.5c3.77±0.7ab2.3±0.7ab1.42±0.4b9.2±3c1.93±0.5e6.71±1d
Lime675.6±39e55±0.7bc55±0.4c3.33±0.6b1.81±0.4c1.5±0.5b9.26±3c3.86±0.7c7.41±2c
Lime764.2±3250±0.6d50±0.5cd3±0.5c1.71±0.6cd1.25±0.1c8.93±3cd5.4±1a9±3b
Lime8106.1±21bc59±0.8ab60±0.6ab3.72±0.7ab1.7±0.5cd2±0.6a8±2d3.73±0.7c9.81±3b
Lime980.3±23de55±0.8bc52.5±1c3.22±0.6bc2±0.4b1.4±0.3b9.33±3c5.06±0.9b8.41±3bc
Lime10111±24b60±0.9a60±0.3b3.38±0.5b1.88±0.4c1.5±0.5b8.6±3cd4.86±1b7.66±2c
Lime1184.6±26de55±0.4bc36.3±0.2f3.63±0.6ab2±0.5b1.14±0.4cd8.13±2d4.78±0.8ab9.58±3b
Lime1256.0±13f50±0.5d50±0.6cd3±0.6c1.57±0.5d1±0.2d10±4b1.86±0.4e8.25±3bc
Lime1347.4±12f45±0.4e50±0.2d3.2±0.6c1.71±0.56cd1.33±0.3bc8.66±3cd3.73±0.7c6.12±1de
Lime1456.6±10f50±0.3d32.6±0.1f3.2±0.4c1.66±0.3d2±0.5a8.93±3cd2.4±0.6d7.88±2c
Lime1582.4±30de50±0.6d55±0.4c3.42±0.5b2±0.4b1±0.2d8.66±3cd2.6±0.6d6.61±1d
Lime1641.6±12h55±0.4bc50±0.4d3.16±0.4bc2±0.5b1±0.1d8.88±3cd3.11±0.7cd7.75±3c
Lime1741.2±16h50±0.5d46.6±0.3e2.57±0.3cd1.37±0.2e1.4±0.3bc8.8±3cd3.93±0.7c5.83±1e
Lime1853.8±28fg46.6±0.2ef50±0.3d3.66±0.8ab2±0.4b1.57±0.4b8±2d3.26±0.7cd7±2c
Lime1947±14g42.5±0.8e50±0.2d2.75±0.1cd1.2±0.1f1±0.1d8.8±3cd3±0.7cd7.5±2c
Lime2090.6±21de56.9±0.5b60±1ab3.6±0.6ab1.83±0.4c1.25±0.3c9.2±3c2.2±0.5de9.66±3b

Note: Data are averaged ±SD; values in each column followed by the same letters are not significantly different according to Duncan's multiple-range test (p < 0.05).

Mean values and significance degree of differences between lime genotypes for juice parameters

GenotypesWeight (g)Volume (ml)Density (g·ml−1)TA (%)TSS (°Brix)pHVit. C (mg·100 mg−1)
LimePal1128.6±40h75±8d101.6bc0.09c11±2a5.7±0.1b48.8±4a
LimePal2349.3±90a150±12a101.3c0.07de9.4±2b5.7±0.1b37.7±3d
LimeBirs184.3±80d117.5±10b101bc0.09c8.3±.5cd5.5±0.1b35.7±3d
Lime1139.3±50g90±9c102ab0.1bc7.5±1d5.1±.01d30.9±2de
Lime2156.3±60f100±9bc100.6cd0.08cd8.5±1bc5.9±0.2b28.8±2ef
Lime3159.6±60f75±12cd100.6cd0.08cd7.8±1d5.8±0.1ab27.9±2ef
Lime4186±85d70±10d100.6cd0.1bc7.4±1d5.5±0.1b26.2±1f
Lime5140.6±50g60±7de102.3ab0.09c8.5±1bc5.6±0.1b31.5±2d
Lime6119.3±50h100±10bc101.3bc0.08cd8.8±1.5bc5.6±0.1b32.7±2d
Lime7112.3±50h65±5de102.6ab0.09c8.7±1bc5.6±0.1b33.3±3d
Lime8205±85b152±10a101.3bc0.08cd7.3±0.5de5.3±0.1bc48.9±4a
Lime9163±6ef75±9d101bc0.06e7.4±0.5de5.8±0.1b30.6±2de
Lime10230.6±80b90±10c101.3bc30.09c8.2±1cd5.6±0.1b32.7±2d
Lime11185±80d85±9c102ab0.11ab9.1±2b5.84±0.1b30.4±2de
Lime12119.6±30i70±9d100.33bc0.08cd8.3±1cd5.9±0.2b45.7±3b
Lime1394±40k55±3f101.3bc0.12a9.4±2b6.05±0.2b28.8±2ef
Lime14105.3±4i67±dde101bc0.09c8.6±1bc5.77±0.1b34.3±3d
Lime15173±7e100±10bc101.3bc0.1bc8.2±1cd5.7±0.1b32.4±2d
Lime1697.6±3k100±12bc102.6ab0.07de7±0.5de5.9±0.1ab29.3±1ef
Lime17102.6±4i67±5de103.6a0.1bc9.5±2b6.3±0.1a40±3c
Lime1889.6±3l50±5e102ab0.1bc8.5±1.5cd5.6±0.1b31.1±2d
Lime1977.6±1m67.5±6de102ab0.11ab8.6±1.5bc5.3±0.1bc27.3±2ef
Lime20184.6±8d95±12bc101bc0.1bc7.45.7±0.1b26.8±1f

Note: See Table 2

Descriptive statistics of quantitative traits studied for Tunisian lime genotypes

VariableMinimumMaximumMeanStandard deviationCV (%)
Weight (g)41.26157.977.325.132
Fruit diameter (mm)42.56552.8529
Fruit length (mm)32.666.951.77414
Width of fruit skin (mm)2.54.03.30.311
Width of epicarp at equatorial plane (mm)1.22.71.80.3418
Fruit rind (mesocarp) thickness (mm)1.02.01.30.321
Number of segments816.49.11.618
Number of seeds1.25.43.21.135
Diameter of fruit axis (mm)583134081716420
Weight of juice (g)6.569.829.912.7142
Volume of juice (ml)50152802632
Density of juice (g·ml−1)100.3103.6101.50.80.7
TSS (°Brix)7.00118.40.910
pH5.16.25.70.24
TA (%)0.060.10.090.0115
Vitamin C (mg·100 mg−1)26.248.933.56.5719

Correlation matrix based on Person index for the quantitative parameters studied

Fruit weightFruit diameterFruit lengthWidth of fruit skinWidth of epicarp at equatorial areaFruit rind (mesocarp) thicknessNumber of segmentNumber of seedsDiameter of fruit axisWeight of juiceVolumeDensityTATSSpHVit. C
Fruit weight (g)1.0000
Fruit diameter (mm)0.8183**1.0000
Fruit length (mm)0.6129*0.47691.0000
Width of fruit skin (mm)0.6149*0.47550.49081.0000
Width of epicarp at equatorial area (mm)0.6025*0.50520.47360.77561.0000
Fruit rind (mesocarp) thickness (mm)0.14390.1300−.05650.2476−.11511.0000
Number of segment0.0187−0.0460.0322−.1099−.20070.23651.0000
Number of seeds0.14370.1465−.0711−.1708−.0551−.0379−.24091.0000
Diameter of fruit axis (mm)0.7554**0.6867**0.34840.45920.5531−.0981−.16710.18891.0000
Weight of juice (g)0.9735**0.8405**0.55930.54590.59530.04460.00890.15820.7593**1.0000
Volume (ml)0.6208**0.55720.46090.42070.21590.3570−.06350.13010.59070.59511.0000
Density (g · ml−1)−.3851−.2664−.1799−.2582−.2307−.0187−.03110.2690−.3394−.3357−.1086
TA (%)−.3412−.5322−.3556−.2083−.4321−.0898−.1527−.0749−.4175−.3276−.33940.23341.0000
TSS (°Brix)−.0684−.2438−.2031−.1186−.19540.17680.6380**0.0464−.2266−.0631−.13520.23600.27491.0000
pH−.2784−.0593−.1700−.09790.577−.16690.12090.1338−.1677−.1419−.25720.1517−.10320.31291.0000
Vit. C (mg per 100 mg)0.12110.16350.0633−.0395−.28310.43720.5253−.11200.000010.12410.50190.0362−.26190.40610.05551.0000

Correlations are significant at P ≤ 0.05 according to Person correlation

Correlations are significant at P ≤ 0.01 according to Person correlation

Qualitative descriptors used for estimating pomological trait diversity in lime genotypes, their number of classes, proportion (%) of occurrence of each class, and estimated phenotypic diversity index (H’) for each trait

Pomological traitObserved phenotypic classClass*Proportion (%)Shannon–Wiener index (H’)
Fruit shape21 spheroid960.24
2 ellipsoid4
Shape of fruit apex21 mammiform960.24
3 rounded4
Shape of fruit base22 convex650.93
3 truncate35
Fruit skin (epicarp) color32 green-yellow350.96
4 yellow22
5 dark yellow43
Fruit surface texture31 smooth300.8
2 rough9
4 pitted61
Adherence of albedo (mesocarp) to pulp (endocarp)33 weak40.75
5 medium44
7 strong52
Adherence of segment walls to each other33 weak40.72
5 medium35
7 strong61
Thickness of segment walls33 thin350.98
5 medium39
7 thick26
Fruit axis31 solid40.49
2 semi-hollow83
3 hollow13
Cross-section shape of axis11 round1000
Pulp (flesh) color22 green260.82
3 yellow74
Pulp color uniformity11 uniform1000
Pulp firmness25 intermediate90.43
7 firm91
Vesicle length33 short50.33
5 medium4
7 long91
Vesicle thickness33 thin350.98
5 medium26
7 thick39
Seed shape22 clavate740.82
4 ovoid26
Seed surface21 smooth780.76
2 wrinkled22
Seed color42 cream130.84
3 yellowish26
4 green52
5 green (medium)9
Cotyledon color41 white130.65
2 light yellow–cream4
3 light green13
5 green (medium)70

observed class defined based on IPGRI Manual (1999)

Data analysis

For quantitative traits, all analyses were performed using SAS software (version 6.07, 1990). Descriptive statistics were performed and presented as minimum, maximum, mean standard deviation, and coefficient of variation (CV). One-way analysis of variance (ANOVA) was used, and data are presented as mean ± standard deviation (SD). Pearson index was calculated for quantitative traits. Principal component analysis (PCA) was carried out to examine the distribution of genotypes in the first plan of PCA for quantitative parameters. For qualitative data, frequency distributions were computed.

The numbers of phenotypic classes for qualitative parameters that differed for each trait were used to estimate the Shannon–Wiener diversity index (H’). It was used to characterize the phenotypic frequencies of the traits and was defined as H=i=1npilnpiH = \sum\nolimits_{i = 1}^n {pi\,\ln \,pi} where n is the number of phenotypic classes for a character and pi is the proportion of the total number of entries in the ith class. Each value of H was standardized by conversion to a relative phenotypic diversity index (H’) by division by Hmax = ln (n) in order to express the values of H’(H/Hmax) in the range of 0–1 (indicating the absence of diversity and maximum of diversity, respectively). The diversity index was classified as high (H’ ≥ 0.6), intermediate (0.40 ≤ H’ ≤ 0.60), or low (0.1 ≤ H’ ≤ 0.40), as described by Eticha et al. (2005) and Mengistu et al. (2015).

RESULTS AND DISCUSSION

On the basis of pomological traits, we have described the characteristics and the variability of each genotype originating from different orchards. Measurements for both fruit and juice traits are presented in Tables 2 and 3, respectively. LimePal2 represented the highest value of fruit weight, diameter, length, width of skin, and width of epicarp at equatorial area. Lime10 also exhibited high caliber of fruit. The smallest fruit attributes were those of Lime17. LimePal1 had by far the most important number of segments. All genotypes held seeds varying in number from 1.2 to 5.4. The diameter of axis varied widely among the different genotypes (Table 2). Concerning juice attributes, LimPal2 and Lime8 were the juiciest. The sweetest juice was that of LimePal1, LimePal2, and Lime17. Values of pH were the highest for Lime13, Lime16, Lime17, and Lime3. All genotypes exhibited high content of vitamin C. Concentrations varied from more than 48 mg·100 mg−1 (Table 3) for genotypes LimePal1 and Lime8 to about 26 mg·100 mg−1 for Lime20 and Lime4. The recorded data were subjected to statistical analyses as described in Material and Methods that showed the utility of both quantitative and qualitative phenotypic characterizations for the identification of genetic resources of limes.

Species classification and estimation of genetic resources status

The participatory FCA was used, while regional agricultural authorities and the farmers were interviewed. It allowed categorizing C. limetta as a threatened species. Although many householders cultivated this species, it was propagated in small area. C. latifolia and C. limettioides were classified as rare species because they were cultivated in small area and by few householders (Fig. 1). Thus, special attention must be paid to these species in order to conserve them and encourage their dissemination. The most cultivated species was C. limetta in contrast to C. latifolia, which is the least cultivated (Table 1). Accessions from different species showed a wide range of variability for all the pomological traits studied. According to a recent research, Curk et al. (2016) have elucidated the origins of limes and lemons based on cytoplasmic and nuclear markers. The survey highlighted that all limes and lemons descend from Citrus medica as the direct male parent in combination with Citrus aurantium for C. limetta and a hybrid (Citrus maxima × Citrus reticulata) for C. limettioides. Among triploid limes, C. latifolia accessions Persian lime types result from the fertilization of a haploid ovule of Citrus limon by a diploid gamete of Citrus aurantifolia. As limes and lemons were vegetatively propagated by apomixes and horticultural practices, the intra-subgroup phenotypic diversity results from asexual variations (Curk et al. 2016). Two classifications of limes have been reported: lime of Pearse known as C. aurantifolia hybrid by Swingle and Reece (1967) and C. latifolia Tan. by Tanaka (1977). Instead, sweet lime of Tunisia is classified as C. limon (L.) Burm. by Swingle and Reece (1967) and C. limetta Risso by Tanaka (1977). The results can be useful for both selection of cultivars and breeding programs aiming the improvement of fruit quality. Snoussi et al. (2012) revealed that both sexual and asexual reproductions of limes cultivated in Tunisia contributed to their genetic diversity.

Fig. 1

Classification of limes species in Tunisia following FCA

Variation among lime species for studied quantitative traits

Table 4 describes the minimum, maximum, mean, standard deviation, and CV for each variable studied. The weight of fruit varied from 41.3 g (Lime16) to 157.9 g (LimePal 2) per fruit with a mean of 77.3 g per fruit. High variability between cultivars was observed for fruit weight, which is confirmed by the relatively high CV (32%). The number of seeds per fruit varied from 1.2 (Lime3) to 5.4 (Lime8) with a mean of 3.3 and a CV of 35%. The weight of juice was highly variable (CV = 42%), ranging from 6.5 (Lime19) to 69.9 (Limpal2) with a mean of 29.9. The volume of juice varied from 50 ml (Lime18) to 152 ml (Lime8), with a mean of 80 ml (CV = 32%). These four parameters were the most discriminant between cultivars based on CVs. The fruit rind thickness (CV = 21%) range from 1 mm (Lime6 and Lime14) to 2 mm (LimPal 2 and Lime3). Diameter of fruit axis (CV = 19%) range from 26.19 (Lime4) to 48.9 mg·dm−3 (LimPal2 and Lime8).

Correlations among studied traits

In order to estimate correlation between quantitative parameters based on the data measured on Tunisian limes, Pearson's correlation coefficients were estimated (Table 5). A significant correlation among several quantitative parameters was observed. Weight of fruit was significantly and positively correlated with fruit diameter (r = 0.81; p ≤ 0.01), diameter of fruit axis (r = 0.75; p ≤ 0.05), weight and volume of juice (r = 0.97; p ≤ 0.01; r = 0.62; p ≤ 0.01, respectively), fruit length (r = 0.61; p ≤ 0.05), width of fruit skin (r = 0.61; p ≤ 0.05), and width of epicarp at equatorial plane (r = 0.60; p ≤ 0.05). Fruit diameter was significantly and positively correlated with the diameter of fruit axis (r = 0.68; p ≤ 0.01) and weight of juice (r = 0.84; p ≤ 0.01). Highly significant and positively correlations were observed between fruit skin width and width of pericarp at equatorial plane (r = 0.77; p ≤ 0.01). Number of segment was significantly and positively correlated with TSS (r = 0.63; p ≤ 0.01); diameter of fruit axis was significantly and positively correlated with weight of juice (r = 0.75; p ≤ 0.01).

Estimation of variation among qualitative traits using the Shannon–Wiener diversity index

Seed color was the most discriminant trait with four different phenotypes (Table 6). Similarly, fruit skin color, fruit surface texture, adherence of albedo to pulp, adherence of segment walls, vesicle length and thickness, fruit axis, and cotyledon color were also more discriminant compared with all the other parameters. Uniformity of pulp color and cross-section shape of axis were monomorphic for all the accessions studied (Table 6). H’ ranged from 0 for both cross-section shape and pulp color uniformity to 0.98 for both thickness of segment walls and vesicle thickness (Table 6) with a mean value of 0.61. Moreover, other parameters showed high values of H’: fruit skin (epicarp) color (H’ = 0.96), shape of fruit base (H’ = 0.93), seed color (H’ = 0.84), pulp (flesh) color (H’ = 0.82), and fruit surface texture (H’ = 0.8). According to the Shannon diversity index, we assume that shape of fruit apex, fruit skin epicarp color, fruit surface texture, thickness of segment walls, pulp (flesh color), vesicle thickness, seed shape, and seed color were the most discriminant qualitative parameters. Pulp (flesh color), fruit skin epicarp color, and fruit surface texture are definitely used as selection criteria throughout the supply and consumption chain. It is well known that genetics, environment, and cultural practices interact to define the eventual main fruit traits (weight, diameter, length, and width of skin).

Principal component analysis

PCA was performed based on fruit and juice quantitative parameters. The results showed that 60.3% of the total variability is accounted for the first three principal components (PCs). The first two PCs account 35.5% and 15.5% of the total variability (Fig. 2 and Table 7). The PC1 positively correlated with weight and diameter of fruit, diameter of axis, and weight of juice. The PC2 positively correlated with fruit rind (mesocarp) thickness, number of segments, and vitamin C content. The projection of lime cultivars in the plan 1–2 of the PCA allows the discrimination of the species C. limettioides and Lime8 from the other genotype (Fig. 2). Regarding C. latifolia and C. limetta, we did not observe any significant discrimination. Accessions from these cultivars were grouped together. Regarding TSS and content of vitamin C, both LimPal1 and Lim-Pal2 exhibited the highest values. Moreover, PCA has also distinguished C. limettioides (LimPal 1 and LimPal 2) and Lime 8 from all the other cultivars. Lime8 genotype, which belongs to C. limetta species, is characterized by large fruit. For this reason, it has been clustered with genotypes LimPal1 and LimPal2, which belongs to C. limettioides species. This species can be selected for breeders in order to improve fruit size (weight and diameter of fruit) and yield of juice. On the basis of the same descriptors of Citrus (IPGRI 1999), similar findings were recorded, referring to Saddoud Debbabi et al. (2013) for the main parameters correlated with the first axis of PCA (weight, diameter, and length of fruit). Phenotypic characterization have shown their efficiency for many crops, such as carrot (Mezghani et al. 2014) and wheat (Mengistu et al. 2015), and for many fruit trees such as fig (Saddoud et al. 2008; Gaaliche et al. 2012), olive (Hannachi et al. 2008), apricot (Ruiz & Egea 2008), apple (Mratinić & Fotirić Akšić 2011), and cornelian cherry (Moradi et al. 2019). This evaluation is necessary to achieve the developmental program and genetic improvement of the lime species. The outcomes of this study will be very useful for Tunisian Gene Bank and for a good identification and documentation of Citrus genetic resources. Although pomological characterization is low cost method and has many advantages, it remains limited in the number of characters and is limited in use. The characterization could be improved through the involvement of molecular markers, for example Simple Sequence Repeat (SSR) that allows the study of molecular diversity and the establishment of fingerprints of the cultivars studied.

Fig. 2

PCA biplot of lime cultivars based on quantitative traits

Eigenvectors, the main eigenvalues, and variation in percentage of the two first principal components of PCA

Principal componentsPC1PC2
Cumulative (%)35.5251.06
Proportion (%)35.5215.54
EigenvaluesWeight of fruit (g) (+0.39)Fruit rind (mesocarp) thickness (mm) (+0.37)
Diameter of fruit (mm) (+0.36)Number of segments (+0.49)
Diameter of axis (mm) (+0.34)Vitamin C mg·100 g−1 (+0.55)
Weight of juice (g) (+0.38)
CONCLUSION

The accessions Lime8, LimePal2, and Lime10 had good genetic potential for the important characters: weight and diameter of fruit, diameter of axis, and weight of juice. The collected data point to the need to protect endangered lime species and may help further efforts to portray the diversity of this species in Tunisia.

Fig. 1

Classification of limes species in Tunisia following FCA
Classification of limes species in Tunisia following FCA

Fig. 2

PCA biplot of lime cultivars based on quantitative traits
PCA biplot of lime cultivars based on quantitative traits

Descriptive statistics of quantitative traits studied for Tunisian lime genotypes

VariableMinimumMaximumMeanStandard deviationCV (%)
Weight (g)41.26157.977.325.132
Fruit diameter (mm)42.56552.8529
Fruit length (mm)32.666.951.77414
Width of fruit skin (mm)2.54.03.30.311
Width of epicarp at equatorial plane (mm)1.22.71.80.3418
Fruit rind (mesocarp) thickness (mm)1.02.01.30.321
Number of segments816.49.11.618
Number of seeds1.25.43.21.135
Diameter of fruit axis (mm)583134081716420
Weight of juice (g)6.569.829.912.7142
Volume of juice (ml)50152802632
Density of juice (g·ml−1)100.3103.6101.50.80.7
TSS (°Brix)7.00118.40.910
pH5.16.25.70.24
TA (%)0.060.10.090.0115
Vitamin C (mg·100 mg−1)26.248.933.56.5719

Mean values and significance degree of differences between lime genotypes for juice parameters

GenotypesWeight (g)Volume (ml)Density (g·ml−1)TA (%)TSS (°Brix)pHVit. C (mg·100 mg−1)
LimePal1128.6±40h75±8d101.6bc0.09c11±2a5.7±0.1b48.8±4a
LimePal2349.3±90a150±12a101.3c0.07de9.4±2b5.7±0.1b37.7±3d
LimeBirs184.3±80d117.5±10b101bc0.09c8.3±.5cd5.5±0.1b35.7±3d
Lime1139.3±50g90±9c102ab0.1bc7.5±1d5.1±.01d30.9±2de
Lime2156.3±60f100±9bc100.6cd0.08cd8.5±1bc5.9±0.2b28.8±2ef
Lime3159.6±60f75±12cd100.6cd0.08cd7.8±1d5.8±0.1ab27.9±2ef
Lime4186±85d70±10d100.6cd0.1bc7.4±1d5.5±0.1b26.2±1f
Lime5140.6±50g60±7de102.3ab0.09c8.5±1bc5.6±0.1b31.5±2d
Lime6119.3±50h100±10bc101.3bc0.08cd8.8±1.5bc5.6±0.1b32.7±2d
Lime7112.3±50h65±5de102.6ab0.09c8.7±1bc5.6±0.1b33.3±3d
Lime8205±85b152±10a101.3bc0.08cd7.3±0.5de5.3±0.1bc48.9±4a
Lime9163±6ef75±9d101bc0.06e7.4±0.5de5.8±0.1b30.6±2de
Lime10230.6±80b90±10c101.3bc30.09c8.2±1cd5.6±0.1b32.7±2d
Lime11185±80d85±9c102ab0.11ab9.1±2b5.84±0.1b30.4±2de
Lime12119.6±30i70±9d100.33bc0.08cd8.3±1cd5.9±0.2b45.7±3b
Lime1394±40k55±3f101.3bc0.12a9.4±2b6.05±0.2b28.8±2ef
Lime14105.3±4i67±dde101bc0.09c8.6±1bc5.77±0.1b34.3±3d
Lime15173±7e100±10bc101.3bc0.1bc8.2±1cd5.7±0.1b32.4±2d
Lime1697.6±3k100±12bc102.6ab0.07de7±0.5de5.9±0.1ab29.3±1ef
Lime17102.6±4i67±5de103.6a0.1bc9.5±2b6.3±0.1a40±3c
Lime1889.6±3l50±5e102ab0.1bc8.5±1.5cd5.6±0.1b31.1±2d
Lime1977.6±1m67.5±6de102ab0.11ab8.6±1.5bc5.3±0.1bc27.3±2ef
Lime20184.6±8d95±12bc101bc0.1bc7.45.7±0.1b26.8±1f

List of species, genotypes studied, abbreviations, acquisition date and accession numbers

SpeciesAbbreviationsDate of first acquisitionAccession ID
Citrus limettioidesLimePal 123/01/2014NGBTUN 757 ARB
LimePal 210/02/2014NGBTUN 992 ARB
Citrus latifoliaLimeBirs04/02/2014NGBTUN 782 ARB
Citrus limettaLime110/02/2014NGBTUN 1032 ARB
Lime223/01/2014NGBTUN 756 ARB
Lime323/01/2014NGBTUN 788 ARB
Lime423/01/2014NGBTUN 793 ARB
Lime523/01/2014NGBTUN 794 ARB
Lime623/01/2014NGBTUN 795 ARB
Lime723/01/2014NGBTUN 797 ARB
Lime804/02/2014NGBTUN 798 ARB
Lime915/01/2014NGBTUN 800 ARB
Lime1015/01/2014NGBTUN 994 ARB
Lime1115/01/2014NGBTUN 823 ARB
Lime1228/04/2014NGBTUN 1033 ARB
Lime1330/01/2014NGBTUN 803 ARB
Lime1430/01/2014NGBTUN 808 ARB
Lime1530/01/2014NGBTUN 812 ARB
Lime1610/02/2014NGBTUN 815 ARB
Lime1710/02/2014NGBTUN 824 ARB
Lime1810/02/2014NGBTUN 759 ARB
Lime1930/01/2014NGBTUN 758 ARB
Lime2030/03/2014NGBTUN 993 ARB

Mean values and significance degree of differences between lime genotypes for fruit characteristics

GenotypesWeight (g)Diameter (mm)Length (mm)Width of skin (mm)Width of epicarp at equatorial plane (mm)Mesocarp thickness (mm)Number of segmentNumber of seedsDiameter of axis (mm)
LimePal 169.0±30e50±0.6d50±0.8cd3.16±0.5bc1.46±0.3d1.81±0.5ab16.46±4a1.93±0.5de6±1de
LimePal 2157.9±34a65±1.3a66.9±0.7a4±1a2.75±0.6a1.33±0.5bc10.26±4b3.8±0.7c13.4±3a
Limebirs87.1±34d60±1a50±0.6cd3.09±0.5c1.8±0.5c1.14±0.3cd8.6±3cd2.26±0.6d8.92±3b
Lime174.3±23ef50±0.4d50±0.7cd3.1±0.4c1.85±0.4c1.38±0.3bc8.46±3cd2.46±0.6d8±3bc
Lime277±17e50±0.4d50±0.9cd3±0.6c2.1±0.7ab1±0.2d9±3c4.2±0.8ab9.4±3b
Lime385.1±29de55±0.6bc60±0.2b4±1a2.6±0.7a1.38±0.4bc8.13±2d1.2±0.2e8.9±3bc
Lime487.3±39d55±0.5bc50±1cd3±0.4c1.75±0.6cd1.4±0.3b8.8±3cd3.2±0.7cd8.25±3bc
Lime573.8±15ef50±0.5d56.8±0.5c3.77±0.7ab2.3±0.7ab1.42±0.4b9.2±3c1.93±0.5e6.71±1d
Lime675.6±39e55±0.7bc55±0.4c3.33±0.6b1.81±0.4c1.5±0.5b9.26±3c3.86±0.7c7.41±2c
Lime764.2±3250±0.6d50±0.5cd3±0.5c1.71±0.6cd1.25±0.1c8.93±3cd5.4±1a9±3b
Lime8106.1±21bc59±0.8ab60±0.6ab3.72±0.7ab1.7±0.5cd2±0.6a8±2d3.73±0.7c9.81±3b
Lime980.3±23de55±0.8bc52.5±1c3.22±0.6bc2±0.4b1.4±0.3b9.33±3c5.06±0.9b8.41±3bc
Lime10111±24b60±0.9a60±0.3b3.38±0.5b1.88±0.4c1.5±0.5b8.6±3cd4.86±1b7.66±2c
Lime1184.6±26de55±0.4bc36.3±0.2f3.63±0.6ab2±0.5b1.14±0.4cd8.13±2d4.78±0.8ab9.58±3b
Lime1256.0±13f50±0.5d50±0.6cd3±0.6c1.57±0.5d1±0.2d10±4b1.86±0.4e8.25±3bc
Lime1347.4±12f45±0.4e50±0.2d3.2±0.6c1.71±0.56cd1.33±0.3bc8.66±3cd3.73±0.7c6.12±1de
Lime1456.6±10f50±0.3d32.6±0.1f3.2±0.4c1.66±0.3d2±0.5a8.93±3cd2.4±0.6d7.88±2c
Lime1582.4±30de50±0.6d55±0.4c3.42±0.5b2±0.4b1±0.2d8.66±3cd2.6±0.6d6.61±1d
Lime1641.6±12h55±0.4bc50±0.4d3.16±0.4bc2±0.5b1±0.1d8.88±3cd3.11±0.7cd7.75±3c
Lime1741.2±16h50±0.5d46.6±0.3e2.57±0.3cd1.37±0.2e1.4±0.3bc8.8±3cd3.93±0.7c5.83±1e
Lime1853.8±28fg46.6±0.2ef50±0.3d3.66±0.8ab2±0.4b1.57±0.4b8±2d3.26±0.7cd7±2c
Lime1947±14g42.5±0.8e50±0.2d2.75±0.1cd1.2±0.1f1±0.1d8.8±3cd3±0.7cd7.5±2c
Lime2090.6±21de56.9±0.5b60±1ab3.6±0.6ab1.83±0.4c1.25±0.3c9.2±3c2.2±0.5de9.66±3b

Qualitative descriptors used for estimating pomological trait diversity in lime genotypes, their number of classes, proportion (%) of occurrence of each class, and estimated phenotypic diversity index (H’) for each trait

Pomological traitObserved phenotypic classClass*Proportion (%)Shannon–Wiener index (H’)
Fruit shape21 spheroid960.24
2 ellipsoid4
Shape of fruit apex21 mammiform960.24
3 rounded4
Shape of fruit base22 convex650.93
3 truncate35
Fruit skin (epicarp) color32 green-yellow350.96
4 yellow22
5 dark yellow43
Fruit surface texture31 smooth300.8
2 rough9
4 pitted61
Adherence of albedo (mesocarp) to pulp (endocarp)33 weak40.75
5 medium44
7 strong52
Adherence of segment walls to each other33 weak40.72
5 medium35
7 strong61
Thickness of segment walls33 thin350.98
5 medium39
7 thick26
Fruit axis31 solid40.49
2 semi-hollow83
3 hollow13
Cross-section shape of axis11 round1000
Pulp (flesh) color22 green260.82
3 yellow74
Pulp color uniformity11 uniform1000
Pulp firmness25 intermediate90.43
7 firm91
Vesicle length33 short50.33
5 medium4
7 long91
Vesicle thickness33 thin350.98
5 medium26
7 thick39
Seed shape22 clavate740.82
4 ovoid26
Seed surface21 smooth780.76
2 wrinkled22
Seed color42 cream130.84
3 yellowish26
4 green52
5 green (medium)9
Cotyledon color41 white130.65
2 light yellow–cream4
3 light green13
5 green (medium)70

Correlation matrix based on Person index for the quantitative parameters studied

Fruit weightFruit diameterFruit lengthWidth of fruit skinWidth of epicarp at equatorial areaFruit rind (mesocarp) thicknessNumber of segmentNumber of seedsDiameter of fruit axisWeight of juiceVolumeDensityTATSSpHVit. C
Fruit weight (g)1.0000
Fruit diameter (mm)0.8183**1.0000
Fruit length (mm)0.6129*0.47691.0000
Width of fruit skin (mm)0.6149*0.47550.49081.0000
Width of epicarp at equatorial area (mm)0.6025*0.50520.47360.77561.0000
Fruit rind (mesocarp) thickness (mm)0.14390.1300−.05650.2476−.11511.0000
Number of segment0.0187−0.0460.0322−.1099−.20070.23651.0000
Number of seeds0.14370.1465−.0711−.1708−.0551−.0379−.24091.0000
Diameter of fruit axis (mm)0.7554**0.6867**0.34840.45920.5531−.0981−.16710.18891.0000
Weight of juice (g)0.9735**0.8405**0.55930.54590.59530.04460.00890.15820.7593**1.0000
Volume (ml)0.6208**0.55720.46090.42070.21590.3570−.06350.13010.59070.59511.0000
Density (g · ml−1)−.3851−.2664−.1799−.2582−.2307−.0187−.03110.2690−.3394−.3357−.1086
TA (%)−.3412−.5322−.3556−.2083−.4321−.0898−.1527−.0749−.4175−.3276−.33940.23341.0000
TSS (°Brix)−.0684−.2438−.2031−.1186−.19540.17680.6380**0.0464−.2266−.0631−.13520.23600.27491.0000
pH−.2784−.0593−.1700−.09790.577−.16690.12090.1338−.1677−.1419−.25720.1517−.10320.31291.0000
Vit. C (mg per 100 mg)0.12110.16350.0633−.0395−.28310.43720.5253−.11200.000010.12410.50190.0362−.26190.40610.05551.0000

Eigenvectors, the main eigenvalues, and variation in percentage of the two first principal components of PCA

Principal componentsPC1PC2
Cumulative (%)35.5251.06
Proportion (%)35.5215.54
EigenvaluesWeight of fruit (g) (+0.39)Fruit rind (mesocarp) thickness (mm) (+0.37)
Diameter of fruit (mm) (+0.36)Number of segments (+0.49)
Diameter of axis (mm) (+0.34)Vitamin C mg·100 g−1 (+0.55)
Weight of juice (g) (+0.38)

Barrett H.C., Rhodes A.M. 1976. A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives. Systematic Botany 1: 105–136. DOI: 10.2307/2418763.BarrettH.C.RhodesA.M.1976A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relativesSystematic Botany110513610.2307/2418763Open DOISearch in Google Scholar

Blondel L. 1978. Classification botanique des espèces du genre Citrus. Fruits 33(11): 695–720. [in French]BlondelL.1978Classification botanique des espèces du genre CitrusFruits3311695720[in French]Search in Google Scholar

Curk F., Ollitrault F., Garcia-Lor A., Luro A., Navarro L., Ollitrault P. 2016. Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Annals of Botany 117(4): 565–583. DOI: 10.1093/aob/mcw005.CurkF.OllitraultF.Garcia-LorA.LuroA.NavarroL.OllitraultP.2016Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markersAnnals of Botany117456558310.1093/aob/mcw005Open DOISearch in Google Scholar

DGPA 2016. Direction Générale de la Production Agricole. Tunisian Ministry of Agriculture. www.agriculture.tn (accessed June 2016)DGPA2016Direction Générale de la Production AgricoleTunisian Ministry of Agriculturewww.agriculture.tn (accessed June 2016)Search in Google Scholar

Eticha F., Bekele E., Belay G., Börner B. 2005. Phenotypic diversity in durum wheat collected from Bale and Wello regions of Ethiopia. Plant Genetic Resources 3(1): 35–43. DOI: 10.1079/pgr200457.EtichaF.BekeleE.BelayG.BörnerB.2005Phenotypic diversity in durum wheat collected from Bale and Wello regions of EthiopiaPlant Genetic Resources31354310.1079/pgr200457Open DOISearch in Google Scholar

Gaaliche B, Saddoud O., Mars M. 2012. Morphological and pomological diversity of fig (Ficus carica L.) cultivars in Northwest of Tunisia. ISRN Agronomy, Article ID 326461, 9 p. DOI: 10.5402/2012/326461.GaalicheBSaddoudO.MarsM.2012Morphological and pomological diversity of fig (Ficus carica L.) cultivars in Northwest of TunisiaISRN Agronomy, Article ID 326461,910.5402/2012/326461Open DOISearch in Google Scholar

Hannachi H., Breton C., Msallem M., Ben El Hadj S., El Gazzah M., Bervillé A. 2008. Differences between native and introduced olive cultivars as revealed by morphology of drupes, oil composition and SSR polymorphisms: A case study in Tunisia. Scientia Horticulturae 116: 280–290. DOI: 10.1016/j.scienta.2008.01.004.HannachiH.BretonC.MsallemM.Ben El HadjS.El GazzahM.BervilléA.2008Differences between native and introduced olive cultivars as revealed by morphology of drupes, oil composition and SSR polymorphisms: A case study in TunisiaScientia Horticulturae11628029010.1016/j.scienta.2008.01.004Open DOISearch in Google Scholar

IPGRI 1999. Descriptors for citrus. International Plant Genetic Resources Institute, Italy, 66 p.IPGRI1999Descriptors for citrusInternational Plant Genetic Resources InstituteItaly66Search in Google Scholar

Krueger R.R., Navarro L. 2007. Citrus germplasm resources. In: Khan I.A. (Ed.), Citrus genetics, breeding and biotechnology. CAB International, pp. 45–140. DOI: 10.1079/9780851990194.0045.KruegerR.R.NavarroL.2007Citrus germplasm resourcesIn:KhanI.A.(Ed.),Citrus genetics, breeding and biotechnologyCAB International4514010.1079/9780851990194.0045Open DOISearch in Google Scholar

Kumar S., Jena S.N., Nair N.K. 2010. ISSR polymorphism in Indian wild orange (Citrus indica Tanaka, Rutaceae) and related wild species in North-east India. Scientia Horticulturae 123: 350–359. DOI: 10.1016/j.scienta.2009.10.008.KumarS.JenaS.N.NairN.K.2010ISSR polymorphism in Indian wild orange (Citrus indica Tanaka, Rutaceae) and related wild species in North-east IndiaScientia Horticulturae12335035910.1016/j.scienta.2009.10.008Open DOISearch in Google Scholar

Mabberley D.J. 2008. Mabberley's plant-book: A portable dictionary of plants, their classifications, and uses, 3rd ed. Cambridge University Press, UK, 1021 p.MabberleyD.J.2008Mabberley's plant-book: A portable dictionary of plants, their classifications, and uses3rd ed.Cambridge University PressUK1021Search in Google Scholar

Mengistu D.K., Kiros A.Y., Pè M.E. 2015. Phenotypic diversity in Ethiopian durum wheat (Triticum turgidum var. durum) landraces. Crop Journal 3: 190–199. DOI: 10.1016/j.cj.2015.04.003.MengistuD.K.KirosA.Y.M.E.2015Phenotypic diversity in Ethiopian durum wheat (Triticum turgidum var. durum) landracesCrop Journal319019910.1016/j.cj.2015.04.003Open DOISearch in Google Scholar

Mezghani N., Zaouali I., Bel Amri W., Rouz S., Simon P.W., Hannachi C. et al. 2014. Fruit morphological descriptors as a tool for discrimination of Daucus L. germplasm. Genetic Resources and Crop Evolution 61: 499–510. DOI: 10.1007/s10722-013-0053-6.MezghaniN.ZaoualiI.Bel AmriW.RouzS.SimonP.W.HannachiC.2014Fruit morphological descriptors as a tool for discrimination of Daucus L. germplasmGenetic Resources and Crop Evolution6149951010.1007/s10722-013-0053-6Open DOISearch in Google Scholar

Moore G.A. 2001. Oranges and lemons: clues to the taxonomy of Citrus from molecular markers. Trends in Genetics 17: 536–540. DOI: 10.1016/s0168-9525(01)02442-8.MooreG.A.2001Oranges and lemons: clues to the taxonomy of Citrus from molecular markersTrends in Genetics1753654010.1016/s0168-9525(01)02442-8Open DOISearch in Google Scholar

Moradi Y., Khadivi A., Salehi-Arjmand H. 2019. Morphological and pomological characterizations of cornelian cherry (Cornus mas L.) to select the superior accessions. Scientia Horticulturae 249: 208–218. DOI: 10.1016/j.scienta.2019.01.039.MoradiY.KhadiviA.Salehi-ArjmandH.2019Morphological and pomological characterizations of cornelian cherry (Cornus mas L.) to select the superior accessionsScientia Horticulturae24920821810.1016/j.scienta.2019.01.039Open DOISearch in Google Scholar

Mratinić E., Fotirić Akšić M. 2011. Evaluation of phenotypic diversity of apple (Malus sp.) germplasm through the principle component analysis. Genetika 43(2): 331–340. DOI: 10.2298/gensr1102331m.MratinićE.Fotirić AkšićM.2011Evaluation of phenotypic diversity of apple (Malus sp.) germplasm through the principle component analysisGenetika43233134010.2298/gensr1102331mOpen DOISearch in Google Scholar

Nicolosi E., Deng Z.N., Gentile A., La Malfa S., Continella G., Tribulato E. 2000. Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theoretical and Applied Genetics 100: 1155–1166. DOI: 10.1007/s001220051419.NicolosiE.DengZ.N.GentileA.La MalfaS.ContinellaG.TribulatoE.2000Citrus phylogeny and genetic origin of important species as investigated by molecular markersTheoretical and Applied Genetics1001155116610.1007/s001220051419Open DOISearch in Google Scholar

Ruiz D., Egea J. 2008. Phenotypic diversity and relationships of fruit quality traits in apricot (Prunus armeniaca L.) germplasm. Euphytica 163(1): 143–158. DOI: 10.1007/s10681-007-9640-y.RuizD.EgeaJ.2008Phenotypic diversity and relationships of fruit quality traits in apricot (Prunus armeniaca L.) germplasmEuphytica163114315810.1007/s10681-007-9640-yOpen DOISearch in Google Scholar

Saddoud O., Baraket G., Chatti K., Trifi M., Marrakchi M., Salhi-Hannachi A., Mars M. 2008. Morphological variability of fig (Ficus carica L.) cultivars. International Journal of Fruit Science 8: 35–51. DOI: 10.1080/15538360802365921.SaddoudO.BaraketG.ChattiK.TrifiM.MarrakchiM.Salhi-HannachiA.MarsM.2008Morphological variability of fig (Ficus carica L.) cultivarsInternational Journal of Fruit Science8355110.1080/15538360802365921Open DOISearch in Google Scholar

Saddoud Debbabi O., Bouhlal R., Abdelaali N., Mnasri S., Mars M. 2013. Pomological study of sweet orange (Citrus sinensis L. Osbeck) cultivars from Tunisia. International Journal of Fruit Science 13: 274–284. DOI: 10.1080/15538362.2012.679186.Saddoud DebbabiO.BouhlalR.AbdelaaliN.MnasriS.MarsM.2013Pomological study of sweet orange (Citrus sinensis L. Osbeck) cultivars from TunisiaInternational Journal of Fruit Science1327428410.1080/15538362.2012.679186Open DOISearch in Google Scholar

Scora R.W. 1975. On the history and origin of citrus. Bulletin of the Torrey Botanical Club 102: 369–375. DOI: 10.2307/2484763.ScoraR.W.1975On the history and origin of citrusBulletin of the Torrey Botanical Club10236937510.2307/2484763Open DOISearch in Google Scholar

Snoussi H., Duval M.F., Garcia-Lor A., Belfalah Z., Froelicher Y., Risterucci A.M. et al. 2012. Assessment of the genetic diversity of the Tunisian citrus root-stock germplasm. BMC Genetics 13; 16, 12 p. DOI: 10.1186/1471-2156-13-16.SnoussiH.DuvalM.F.Garcia-LorA.BelfalahZ.FroelicherY.RisterucciA.M.2012Assessment of the genetic diversity of the Tunisian citrus root-stock germplasmBMC Genetics131612 p.10.1186/1471-2156-13-16332342622429788Open DOISearch in Google Scholar

Sthapit B., Rana R., Subedi A., Gyawali S., Bajracharya J., Chaudhary P. et al. 2012. Participatory four-cell analysis (FCA) for understanding local crop diversity. In: Sthapit B., Shrestha P., Upadhyay M. (Eds.), On-farm management of agricultural biodiversity in Nepal. Good Practices, revised edition, pp. 13–16. NARC/LI-BIRD/Bioversity International, Nepal.SthapitB.RanaR.SubediA.GyawaliS.BajracharyaJ.ChaudharyP.2012Participatory four-cell analysis (FCA) for understanding local crop diversityIn:SthapitB.ShresthaP.UpadhyayM.(Eds.),On-farm management of agricultural biodiversity in NepalGood Practices,revised edition1316NARC/LI-BIRD/Bioversity InternationalNepalSearch in Google Scholar

Swingle W.T., Reece P.C. 1967. The botany of citrus and its wild relatives. In: Reuther W., Batchelor L.D., Webber H.J. (Eds.), The citrus industry, vol. 1. History, world distribution, botany and varieties, revised edition. University of California Press, USA, pp. 190–430.SwingleW.T.ReeceP.C.1967The botany of citrus and its wild relativesIn:ReutherW.BatchelorL.D.WebberH.J.(Eds.),The citrus industry1History, world distribution, botany and varieties,revised editionUniversity of California PressUSA190430Search in Google Scholar

Tanaka T. 1977. Fundamental discussion of citrus classification. Studia Citrologica 14: 1–6. [in Japanese]TanakaT.1977Fundamental discussion of citrus classificationStudia Citrologica1416[in Japanese]Search in Google Scholar

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