Genetic diversity of Tunisian male date palm (Phoenix dactylifera L.) genotypes using morphological descriptors and molecular markers

Scientia Horticulturae 253 (2019) 24–34

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Genetic diversity of Tunisian male date palm (Phoenix dactylifera L.) genotypes using morphological descriptors and molecular markers

T

Nabila El Kadria, , Mehdi Ben Mimouna, José Ignacio Hormazab ⁎

a

Université de Carthage, Institut National Agronomique de Tunisie, Laboratoire Green-Team LR17AGR01, 43 Avenue Charles Nicolle 1082, Tunis, Tunisia Subtropical Fruit Crops Department, Instituto de Hortofruticultura Subtropical y Mediterránea ‘la Mayora’, (IHSM la Mayora -CSIC-UMA), 29750, Algarrobo-Costa, Málaga, Spain

b

ARTICLE INFO

ABSTRACT

Keywords: Date palm Pollinizer Diversity Tunisia SSR

Twenty-eight morphological characters (consisting of 15 quantitative and 13 qualitative traits) and eight SSR loci were used to study morphological and genetic diversity of 24 male date palm genotypes from Southern Tunisia. The results revealed that fourteen of the fifteen quantitative characters studied showed a high discriminating power to distinguish male date palm trees. UPGMA clustering based on morphological data classified the samples studied in five clusters. The eight SSR loci used showed polymorphism resulting in a total of 42 alleles in the samples analyzed. The number of alleles per locus varied from 3 (mPdCIR35 and mPdCIR63) to 7 (mPdCIR 25 and mPdCIR 32) with a mean of 5.4 alleles per locus. The average of observed heterozygosity (Ho), ranged between 0.13 (mPdCIR78) and 0.79 (mPdCIR25), with a mean of 0.54. The average of expected heterozygosity (He) ranged between 0.32 (mPdCIR78) and 0.74 (mPdCIR57 and mPdCIR63) with a mean of 0.61. Fis values were negative for all the markers excepted for mPdCIR015, mPdCIR035 and mPdCIR078. Fst values varied between 0.04 (mPdCIR015 and mPdCIR063) to 0.29 (mPdCIR063). The dendrogram constructed based on the data from the eight SSR primers revealed 3 clusters. No correlation (r= -0,106, p = 0.075) was obtained between distances based on quantitative morphological traits and genetic distances using the Mantel test. Results indicate a high degree of genetic diversity among date palm pollinizers in southern Tunisia, but the accessions do not group according to geographical origin due probably to the exchange of plant material and seeds among different regions of the country.

1. Introduction Date palm (Phoenix dactylifera L., 2n = 36), is a dioecious, perennial, evergreen plant belonging to the Coryphoideae subfamily in the Arecaceae, and it is considered one of the most widespread perennial crop in arid regions (Chao and Krueger, 2007) and one of the most culturally and economically important crops of the Middle East and North Africa (Hazzouri et al., 2015). In Tunisia, the date palm crop covers an area of 42,000 ha producing annually 241.000 metric tons of dates (GIFruits, personal communication). It contributes significantly to the development of the desert areas in southern Tunisia and has an important social impact, providing a livelihood for nearly 50,000 oasis dwellers, and is one of the main pillars of the regional economy. Actually, Tunisia is the world's leading export value of date crops and the world- leading producer of “Deglet Nour” variety (GIFruits, personal communication). Since the date palm is a dioecious species, pollination is the most

⁎

important stage for producing good quantity and quality of dates. The influence of pollen on improving fruit quality through metaxenia has been proven since 1928 by Nixon (Nixon, 1928). In southern Tunisia, farmers have, commonly, selected and maintained their own pollinizer plants in their farms. Pollinizers are mainly selected according to their overlap in flowering season with female cultivars. Pollination of female plants is currently carried out mixing pollen of the few male plants present in the farm and, usually, farmers also bring pollen from others areas in southern Tunisia to hand-pollinate female trees when there is a lack of local pollen or a poor synchronization in flowering time between male and female cultivars. Genetic diversity of female plants in Tunisia is highly endangered due to the predominance of the female cultivar Deglet Nour, which constitutes most of the commercial date production in the country (Hamza et al., 2012). As a consequence, some works have been performed to improve knowledge about the diversity of Tunisian female date palms (Hamza et al., 2011; Zehdi et al., 2004; Metoui et al., 2017). However, a very limited number of studies have

Corresponding author. E-mail addresses: [email protected] (N. El Kadri), [email protected] (M.B. Mimoun), [email protected] (J.I. Hormaza).

https://doi.org/10.1016/j.scienta.2019.04.026 Received 3 January 2019; Received in revised form 21 March 2019; Accepted 11 April 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

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been carried out on male plants used as pollinizers which are generally propagated by seeds (Racchi et al., 2014). The characterization of date palms in order to evaluate genetic diversity can be based on morphology (leave, spine and fruit characters) and molecular tools (Hammadi et al., 2009; Zehdi et al., 2004). Several studies report the importance of morphological traits in identifying Tunisian date palm cultivars (Hammadi et al., 2009; Salah and Hellali, 2006). However, morphological studies of date palm are always difficult to undertake because they require a large set of phenotypic data (Hammadi et al., 2009), which are, sometimes, variable due to environmental influences (Sedra et al., 1993, 1996). Although morphological characters can still be very useful and good phenotyping is ultimately needed to link molecular markers with traits of interest (Wünsch and Hormaza, 2002), the use of molecular tools can provide more information on the genetic diversity of date plants several of which can be hardly differentiated on morphological basis (Faqir et al., 2017). Thus, in the last decades, important advances in the approaches used to study nucleic acids have taken place resulting in the development of different types of genetic markers (Larranaga and Hormaza, 2015). SSRs (Simple Sequence Repeats) or microsatellites are one of the most used molecular markers for diversity studies in plants. Derived from small DNA sequences tandemly repeated they represent a suitable tool for genotyping because of their particular features such as their codominant nature and their typically high levels of allelic diversity at different loci (Kalia et al., 2011). SSRs have been used to perform genetic diversity analyses, sex determination and gene characterization in date palm (Salomon-Torres et al., 2017) in different countries such as Tunisia (Hamza et al., 2011; Metoui et al., 2017; Zehdi-Azouzi et al., 2015; Zehdi et al., 2004, 2012), Morocco (Bodian et al., 2012), Iraq (Khierallah et al., 2011) and Libya (Racchi et al., 2014). The main objective of this work is to characterize 24 Tunisian male date palm genotypes from different localities of southern Tunisia through morphological and molecular parameters in order to optimize the preservation of the endangered genetic resources of this species and to exploit it to improve the production of this crop.

2.3. Molecular analysis Young leaves were collected and immediately conserved in silica gel for DNA extraction. Genomic DNA was extracted from dried young leaves using the DNeasy Plant Mini Kit (Qiagen SA, Courtaboeuf, France) according to the manufacturer’s protocol. After purification, DNA quantity and quality were checked using a Nanodrop ND-1000 UV–vis spectrophotometer, diluted to 10 ng/ μl with modified TE buffer (1 M Tris−HCl pH 8.0; 0.5 M EDTA) and used for PCR amplification. Then, DNA was diluted to 25 ng/l which was the working concentration. A set of 8 SSR loci (mPdCIR010, mPdCIR 015, mPdCIR 025, mPdCIR 032, mPdCIR 035, mPdCIR 057, mPdCIR 063, mPdCIR 078) was tested in this study. These loci were selected based on their polymorphic information content among SSR loci previously developed in date palm (Billotte et al., 2004). The selected loci were used in 15 μl reactions containing 20 ng of genomic DNA and 1 unit of BioTaq™ DNA polymerase (Bioline, London, UK), 16 mM (NH4)2SO4, 67 mM Tris−HCl pH 8.8, 0.01% Tween® 20, 3 mM MgCl2, 0.1 mM of each dNTP, 0.3 μM of each primer. Amplification was performed in a thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following temperature profile: an initial step at 94 °C for 1 min, 35 cycles at 94 °C for 30 s, followed by 30 s at primer specific annealing temperature and at 72 °C for 1 min, and a final extension step at 72 °C for 5 min. The amplified fragments were separated by capillary electrophoresis in an automatic fragment analyzer CEQ™ 8000 genetic Analysis System (Beckman Coulter, Fullerton, CA, USA) after labeling forward primers with a D2, D3 or D4 fluorescent WellRED dye (Sigma-Aldrich, St. Louis, MO, USA) on the 5′-end. Samples were denaturalized at 90 °C for 120 s, injected at 2.0 kV for 30 s, and separated at 6.0 kV for 35 min. Each PCR reaction and capillary electrophoresis was repeated at least twice to ensure the reproducibility of the results. 2.4. Statistical analysis All morphological parameters were analysed as a completely randomized design with pollinizers as treatments. The analysis of variance (ANOVA) was conducted using IBM SPSS statistics (Statistical Package for the Social Sciences) software (Version 20), IBM, USA, to test the significance of variation between cultivars for each character. When overall cultivar effects were significant, as indicated by F-tests, differences between individual cultivars were determined using Duncan’s multiple range test (Elshibli and Korpelainen, 2009). Mean values of quantitative parameters were subjected to principal component analysis (PCA) in order to identify the parameters that contributed significantly to the variability among pollinizers. Analysis of variance applying descriptive statistics such as correlation coefficient for quantitative traits was calculated. Clustering of genotypes into similar groups was carried out using Ward’s hierarchical method based on Squared Euclidean distances. In order to compare the genetic diversity among genotypes, we estimated a set of genetic diversity parameters: number of alleles per locus (A), allelic frequencies, observed heterozygosity (Ho), expected heterozygosity (He). Wright’s fixation index (Fis, Fst) (Wright, 1965), total diversity (Ht), gene diversity within groups (Hs), the genetic differentiation among groups (Gst) and Polymorphic Information Content (PIC). The computations were performed using the R software version 3.4.2. Polymorphic Information Content (PIC) values were calculated using the CERVUS 3.07 software (Kalinowski et al., 2007; Slate et al., 2000). Genetic relationships among the accessions were studied using UPGMA cluster analysis of the similarity matrix obtained from the proportion of shared amplification fragments (Nei and Li, 1979), and Principal Component Analysis (PCA) using the program NTSYS 2.11 (Exeter Software, Stauket, NY). We also assessed clustering based on the Bayesian approach implemented in STRUCTURE version 2.3.4 (Pritchard et al., 2000) with an initial burn-in period of 50,000

2. Materials and methods 2.1. Plant material The study was conducted on 24 Tunisian date palm pollinizers from 8 different geographical regions and different types of oases in southern Tunisia (Fig. 1). Three pollinizers were chosen from each location and geographical site information for each sample was determined by GPS using a Garmin instrument (Garmin USA) (Table 1). Some climatic parameters such as the average annual temperatures Tmean (°C), the average maximum temperatures Tmax (°C), the average minimum temperatures Tmin (°C) and the average annual total precipitation (mm) for the collection sites for ten years (2007–2017) are presented in Table 2 (INM, 2017). The climate in all collection localities is characterized by aridity and high temperatures. 2.2. Morphologic characteristics of the palm trees For morphological characterization, 28 parameters were studied from the 24 pollinizers as standard descriptors of the date palm (IPGRI, 2005), consisting of 15 quantitative and 13 qualitative characteristics (Table 3). Three leaves (Elhoumaizi et al., 2002; Simozrag et al., 2016) and three male inflorescences from each genotype were used for the description. Leaves were taken from the middle crown during the pollination season. The quantitative traits were measured in all collected samples of each genotype and the average of each trait was calculated. The qualitative traits were recorded for each sample from each location. 25

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Fig. 1. Geographic situation of the 24 date palm pollinizers analysed in this work. Studied regions are marked with a red spot. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

replicates and 50,000 Markov Chain Monte Carlo (MCMC) iterations in order to assign date palm genotypes to different groups. Ten independent simulations were run for each K value (Chaluvadi et al., 2014). To identify the number of populations that best reflect the

structure of our samples, the average K value was calculated from the ten runs and Delta K was calculated by a web-based program, Structure Harvester (Earl and Vonholdt, 2012), as in Evanno et al. (2005). Among all 10 independent runs, the one with the highest Ln Pr (X|K) value (log 26

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Table 1 Male date palm genotypes analyzed in this study. Locations, pollinizer names, abbreviations (ABVR), origin, geographical characteristics and type of oases of date palm pollinizers used in the manuscript are shown. location

No

Pollinizer name

ABRV

Latitude N

Longitude E

Altitude (m)

Collection site

Governorate

Type of oasis

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Jerba1 Jerba2 Jerba3 Gataya1 Gataya2 Gataya3 Hazoua1 Hazoua2 Hazoua3 Jemna1 Jemna2 Jemna3 Gtar1 Gtar2 Gtar3 Zarat1 Zarat2 Zarat3 Midés1 Midés2 Midés3 BenGhilouf1 BenGhilouf2 BenGhilouf3

jerba1 jerba2 jerba3 GTY1 GTY2 GTY3 HZ1 HZ2 HZ3 jemna1 jemna2 jemna3 gtar1 gtar2 gtar3 zarat1 zarat2 zarat3 mides1 mides2 midés3 BG1 BG2 BG3

33°43.88' 33°41.326' 33°41.243' 33°40.902 33°10.903' 33°40.906' 33°43.847' 33°43.845' 33°43.838' 33°36.014' 33°35.960' 33°35.937' 34°19.607' 34°19.592' 34°19.594' 33° 30.244' 33°40.238' 33° 40.260' 34°24.446' 34°24.422' 34°24.864' 33°53.284' 33°53.286' 33°53. 690'

10°50.338' 10°53.609' 10°53.843' 8°52.605' 8°52.601' 8°52.595' 7°35.407 7°35.417' 7°35.413' 9°.239' 9°.241' 9°262' 8°57.642' 8°57.648' 8°57.521' 10° 21.242' 10°21.250' 10°21.168' 7°55.043' 7°55.187' 7°55.081' 9°37.206' 9°37.203' 9 36.668'

0 4 4 13 9 0 25 31 25 29 39 39 225 219 227 25 21 26 348 360 361 30 30 25

Jerba. South- East. Tunisia

Medenine

Island oasis

Gataya. South- West. Tunisia

Kebili

Continental oasis

Hazoua. south -West. Tunisia

Tozeur

Continental oasis

Jemna. south -West.Tunisia

kebili

Continental oasis

Gtar. South- West. Tunisia

Gafsa

Mountainous oasis

Zarat. south –East. Tunisia

Gabes

Coastal oasis

Mides. South- West. Tunisia

Tozeur

Mountainous oasis

Benghilouf. South –East. Tunisia

Gabes

Continental oasis

2 3 4 5 6 7 8

Table 2 Climatic parameters in the collection sites from 2007 to 2017: Tmean (°C): average annual temperature; Tmax (°C): average maximum temperatures; Tmin (°C): average minimum temperatures; PP(mm): average annual total precipitation. (INM, 2017).

Kébili Tozeur Gafsa Gabes Jerba

Tmin

Tmax

Tmean

PP (mm)

16,86 15,20 13,78 14,87 17,24

28,62 28,52 27,03 26,27 25,82

22,68 22,57 20,48 20,49 21,42

97,12 96,87 148,94 105,31 240,12

Table 3 Qualitative traits measured in 24 male date palm genotypes collected from Southern Tunisia, according to the IPGRI descriptors (2005). Palm part

Qualitative characters

Abbreviation

Pattern

Code

Tree

Trunk shape

FS

Presence of wadding mane

CB

Cylindric Conical Spheric No

1 2 3 1

Rotating of the palm

RP

Color of petiole

CP

Rigidity

RE

Number of spines per type of grouping

NG

Yes Yes No Yellowish Brown Blackened Flexible Medium Rigid In 1

2 1 2 1 2 3 3 5 7 1

Color

PE

Disposition of the pinneaes

DP

In 2 In 3 Yellowish Green Olive green Bleu green Interne

2 3 1 2 3 1

Shape of spathe

SS

Color of floral stem

CS

Intermediate Externe Lonceolate Fusiform Inflated Green Yellow Yellowish Orange Orange Dark orange Light Medium High Light Strong White Yellow

2 3 1 2 3 1 2 3

Leaf

probability or log likelihood) was chosen and represented as bar plots. The Mantel test (Mantel, 1967) using XLSTAT (version 2018) was performed to infer a possible correlation between matrices of the Euclidean distance matrix based on morphological variables and Nei and Li distance matrices obtained with SSR markers.

Spines

3. Results

Pinneaes

3.1. Morphological analyses Results of the morphological analyses, among the studied male date palm genotypes, collected from different localities with different climatic characteristics, showed significant differences in all studied characteristics of leaves and male inflorescences. The pollinizer BG2 showed the lowest inflorescence length (40,00 cm) and width (10,00 cm), the lowest number of spikelets per inflorescence (96), and the lowest length of the apical pinnae (0,83 cm) compared with all other samples. Gataya 3 produced the largest inflorescences (23,70 cm) with the highest average number of spikelets per inflorescence (413,33) between the studied genotypes. Gatr 2 showed the longest inflorescence (124,00cm). On the other hand, Gataya 1 presented the longest leaves (426,66 cm), while Jemna 2 showed the shortest ones (204,33 cm). Midés 1 and BG3 showed significatively highest rachis thickness (5,33 and 7,16 cm respectively) compared with all other samles. Hezoua 1 was the pollinizer with the longest spines (43,66 cm). This diversity shown in Table 4 was strongly supported by the

Inflorescence (spathe)

Pollen

27

Pollen productivity

PP

Pollen odour

OP

Color of pollen

PR

4 5 3 4 7 3 7 1 2

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Table 4 Mean values of quantitative morphological traits of 24 male date palm genotypes collected from Southern Tunisia of 24 male date palm genotypes collected from Southern Tunisia. LT: Leaf length (cm); LR: Maximum width (cm); ER: Rachis thickness (cm); NE: Spine number; EE: Maximum thickness of Medium spine (cm); LE: Maximum length of medium spine (cm); AP: Maximum width of pinnae in the middle of the palm; LP: Maximum length of pinnae in the middle of the palm; LA: Length of the apical pinnae; GA: Maximum width of the apical pinnae; LS: Total spathe length; GS: Maximum width of spathe; NP: Number of spikelets per inflorescence; LL: Length of the longest spikelet; LC: Length of shortest spikelet. poll

GS

LS

NP

LL

LC

LT

LR

ER

NE

EE

LE

AP

LP

LA

GA

Jerba1 Jerba2 Jerba3 GTY 1 GTY 2 GTY 3 HZ 1 HZ 2 HZ 3 Jemna1 Jemna 2 Jemna 3 Gtar1 Gtar2 Gtar3 Zarat1 Zarat 2 Zarat3 Mides1 Mides2 Midés 3 BG1 BG2 BG3 ANOVA P

20.48e-g 10.80ab 17.88b-g 20.16d-g 15.40a-f 23.70g 13.80a-c 17.00a-g 14.30a-e 11.33ab 16.66a-g 12.00ab 19.16c-g 19.66c-g 13.00a-c 20.00d-g 11.30ab 15.23a-f 19.33c-g 21.66f-g 17.66b-g 14.76a-f 10.00a 12.73a-c 0.000

80.16b 65.66ab 73.50ab 82.00bc 83.33bc 75.66b 66.33ab 71.33ab 72.66ab 68.66ab 53.00ab 74.00ab 113.33 cd 124.00d 65.00ab 83.33bc 56.66ab 82.00bc 86.00bc 73.00ab 79.00b 58.33ab 40.00a 65.33ab 0.001

366.00jk 243.33e-h 190.33b-f 197.00b-f 347.00i-k 413.33k 147.66a-d 170.33a-e 140.66a-c 158.33a-e 290.00h-j 158.00a-e 200.00b-g 154.66a-e 216.00c-h 237.00d-h 162.66a-e 286.66 g-j 221.33c-h 270.33f-i 266.33f-i 131.00a-c 96.00a 117.33ab 0.000

27.33d-f 16.66ab 25.33c-d 28.06d-f 32.16f 27.83d-f 27.06d-f 27.00d-f 27.33d-f 28.73d-f 33.00f 28.46d-f 31.73ef 28.46d-f 23.50b-d 46.86g 15.41a 30.66d-f 32.00f 27.66d-f 30.73d-f 18.73a-c 12.50a 23.93c-e 0.000

5.73a-c 5.80a-c 8.41a-c 7.00a-c 6.00a-c 5.80a-c 5.46a-c 7.00a-c 10.06b-c 8.60b-c 26.00d 12.06c 9.46b-c 7.13a-c 1.50a 9.60b-c 3.13ab 3.66ab 6.85a-c 5.16a-c 4.00ab 5.13a-c 6.50a-c 7.66a-c 0.000

326.33e-g 245.00ab 275.66b-d 426.66j 356.00f-h 335.66e-g 355.66f-h 411.00ij 395.00h-j 392.50h-j 204.33a 293.33c-e 353.33f-h 326.66e-g 315.00d-f 325.00e-g 240.00ab 334.00e-g 338.33e-g 372.00 g-i 331.00e-g 316.66d-f 260.00bc 359.66f-h 0.000

63.33e-i 26.00a 31.66ab 67.33h-j 66.66 g-j 51.00c-e 71.00ij 61.33 d-i 62.00 e-i 58.00d-i 57.50d-i 50.66c-e 62.33e-i 64.66f-i 66.00 g-i 78.66j 38.66bc 54.00d-h 60.00d-i 53.33d-g 51.33d-f 48.33 cd 61.66d-i 64.66f-i 0.000

3.40b-f 2.66ab 2.60ab 4.00e-g 4.10f-g 3.33b-f 3.43bf 3.66c-g 2.66ab 2.05a 1.93a 3.00b-d 3.70c-g 3.96e-g 3.93e-g 3.30b-f 3.16b-e 3.58c-g 5.33h 3.43b-f 3.86d-g 3.30b-f 4.33 g 7.16i 0.000

11.66a 27.66c-e 50.00 g 31.66de 13.66a 13.66a 24.66 cd 26.00 cd 32.00de 48.33 g 48.66 g 40.66f 12.33a 11.33a 22.00bc 15.66ab 27.66c-e 17.33ab 12.66a 25.33 cd 15.33ab 34.33e 31.66de 32.00de 0.000

0.50ab 0.50ab 0.80b-d 1.30gh 1.00d-g 0.60a-c 0.73a-d 1.23f-h 1.40 h 0.70a-d 0.86c-e 1.30gh 1.30gh 1.00d-g 0.96d-f 0.90c-e 0.43a 1.16e-h 0.80b-d 0.86c-e 0.83 cd 0.60a-c 0.90c-e 0.76b-d 0.000

17.50a-e 13.33a-c 18.00b-e 25.50e-i 16.66a-d 27.33 g-i 43.66j 24.83d-i 28.66hi 17.00a-d 10.60ab 18.66c-e 25.50e-i 27.00f-i 31.66hi 24.00d-i 13.66a-c 23.33d-h 19.00c-f 21.00c-h 19.33c-g 25.33e-i 18.33b-e 9.70a 0.000

2.76a-e 2.23ab 2.13ab 3.40c-f 2.70a-d 2.93b-f 2.96b-f 2.46a-c7 2.93b-f 3.70e-g 3.60d-g 3.60d-g 3.00b-f 3.20c-f 2.93b-f 3.00b-f 2.73a-d 4.30g 3.83fg 2.93b-f 3.00b-f 1.96a 2.23ab 2.60a-c 0.000

48.33e-g 26.66a 32.33ab 59.66h 55.00 gh 44.33d-f 43.66c-f 51.66f-h 33.66ab 49.00e-g 34.00ab 48.66e-g 42.00b-e 40.33b-e 40.00b-e 38.33b-d 37.66b-d 44.33d-f 40.00b-e 37.00b-d 32.33ab 38.00b-d 34.33a-c 46.33d-g 0.000

28.00f-j 18.33bc 15.33b 34.00K-m 30.00h-k 30.33h-k 31.00i-l 25.66d-i 21.33 cd 36.00 lm 18.50bc 36.66 m 24.66d-h 29.33 g-k 33.00i-m 27.33e-j 23.33c-f 22.66c-f 22.00c-e 24.66d-h 22.00c-e 23.66c-g 8.66a 18.33bc 0.000

1.83d-f 1.63c-f 0.96ab 1.66c-f 1.80d-f 2.13e-g 1.43a-e 2.00d-g 1.73c-f 2.13e-g 2.06e-g 2.00d-g 2.03d-g 2.6g 2.16 fg 1.66c-f 1.93d-g 2.16 fg 1.50b-f 1.10a-c 1.76c-f 2.00d-g 0.83a3 1.33a-d 0.000

Means followed by different letter(s) are significantly different at 5% level of probability.

Fig. 2. Principal component analysis projection of 24male date palm genotypes collected from Southern Tunisia generated by 1–2 axis. A significant opposition of Gataya 1, Gtar 3, Hezoua 2 and Hezoua 1 pollinizers to Jemna 2 Jerba 2 and Jerba 3 pollinizers was registered according to the first axis.

results of PCA analysis, which showed that the first three axes accounted for 59.06% of the total variability among pollinizers. The first component (24.7% of the total variability) is mainly and positively

correlated with the following parameters: leaf length (LT), leaf width (LR), spine width (EE), spine length (LE), apical pinnae length (LA) and middle pinnae length (LP). The second axis (19.8% of the total 28

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Table 5 Correlation matrix between the different morphological traits of the 24 male date palm genotypes collected from Southern Tunisia analyzed in this study.

GS LS NP LL LC LT LR ER NE EE LE AP LP LA GA

GS

LS

NP

LL

LC

LT

LR

ER

NE

EE

LE

AP

LP

LA

GA

1 .569** .579** .568** .047 .321 .234 −.469* .030 .089 .206 .158 .173 .184 .101

1 .186 .523** −.093 .380 .260 −.575** .133 .367 .244 .320 .240 .362 .435*

1 .385 .017 −.059 −.041 −.441* −.182 −.218 −.116 .206 .120 .185 .213

1 .343 .318 .538** −.279 −.067 .351 .143 .526** .248 .371 .237

1 −.316 .083 .495* −.336 .215 −.323 .252 −.104 −.136 .082

1 .515* −.240 .280 .461* .437* .181 .628** .475* .069

1 −.358 .352 .406* .418* .298 .502* .354 .121

1 −.388 −.003 −.306 −.094 −.131 −.154 −.246

1 .055 −.079 −.059 .258 −.171 −.252

1 .287 .352 .327 .181 .139

1 .048 .140 .410* .164

1 .307 .417* .414*

1 .662** .264

1 .572**

1

* Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

genotypes, showed yellow pollen color (PP) excepted BG3. All genotypes from cluster 4 were characterized by the presence of a wadding mane (CB). Only Jemna 2 has a number 3 as a number of spine per type of grouping (NG) which is different from all the other pollinizers.

variability) is mainly and positively influenced by the spathe width (GS), the spathe length (LS), spikelet number per inflorescence (NP), length of the longest spikelet (LL) and the maximum width of the apical pinnae (GA) traits. The spine number (NE) is negatively correlated with this principal component. Fig. 2 presents the projection of the studied cultivars on the plot as defined by the two first principal component axes 1–2. The third axis (13.17%) is also positively correlated with the length of the shortest spikelet (LC) and the spine number (NE). This principal component is negatively influenced by rachis width (ER). The correlation matrix (Table 5) of the studied characters showed that inflorescence traits [spathe width (GS), spathe length (LS), number of spikelets per inflorescence (NP) and length of the longest spikelet (LL)] are correlated. The maximum length of pinnae in the middle of the palm (LP) was significantly correlated with the leaf length (LT) and with the length of the apical pinnea (LA). Also the length of the apical pinnea is correlated with the maximum width of the apical pinnae (GA). However, a negative significant correlation was found between rachis thickness (ER) and male spathe length (LS). In the dendrogram based on morphological data using the Ward’s Method (Fig. 3) all pollinizers from the Mides region were in cluster 1 with Zarat 1, Zarat 3 and Gtar 3. Cluster 2 groups two genotypes, Jemna 2 and Jerba 2, and showed the highest SD for the longest spikelet length (11.55), the shortest spikelets length (14.28), leaf width (22.27), spine number (14.85) and the maximum width of pinnae in the middle of the palm (0.97) (Table 6). Cluster 3 groups two pollinizers from the Gataya region (GTY 2 and GTY 3) and Jerba 1 and showed highest SD only for spathe width (4.18). Highest SD values for spikelet number (35.71), spine width (0.33), length of the apical pinnae (10.50) and the maximum width of the apical pinnae (0.59) were registered for cluster 4 which groups Jemna 3, Jerba 3, Zarat 2 and two samples from Ben Ghilouf (BG1 and BG2). The largest cluster was cluster 5 that groups all pollinizers from Hezoua, two from Gtar, Gataya 1, Jemna 1 and BG 3. It shows the highest SD values for spathe length (22.80) leaf length (33.88), rachis thickness (1.51), spine length (9.74), and the maximum length of pinnae in the middle of the palm (7.87). The qualitative characters of all pollinizers were arranged in Table 7 according to the 5 clusters obtained based on quantitative characters. Analysis showed that all pollinizers from cluster 1 and 3 show seasonal flowering periods. Those from cluster 3 show a green spathe color (CS). Cluster 2 contains two pollinizers with different flowering periods. This cluster is characterized by a leaf rotation (RP), cylindrical trunk shape (FS), dark orange color of the spathe and yellow pollen which is different from all other clusters. The petiole color (CP) for all pollinizers is yellow except for the pollinizers from Jemna even when they belong to different clusters (Jemna 2, Jemna 3 and Jemna 1). Pollen odor (OP) is similar in clusters 1 and 4. Cluster 5, with the largest number of

3.2. Molecular analyses All the 8 microsatellites analyzed showed a good quality of DNA amplification for proper genotyping. A total of 42 alleles were identified with the 8 SSR loci selected for this study. The number of alleles per locus varied from 3 (mPdCIR35 and mPdCIR63) to 7 (mPdCIR025 and mPdCIR032) with a mean of 5.4 alleles per locus (Table 8). The average of observed heterozygosity (Ho), ranged between 0.13 (mPdCIR078) and 0.79 (mPdCIR025), with a mean of 0.54. HO showed high values except for mPdCIR035 and mPdCIR078. The average of expected heterozygosity (He) ranged between 0.32 (mPdCIR078) and 0.74 (mPdCIR057 and mPdCIR063) with a mean of 0.61 indicating the high degree of genetic diversity among date palm pollinizers in southern Tunisia. A heterozygosity excess (Hobs > Hexp) was observed for mPdCIR010, mPdCIR025, mPdCIR057 and mPdCIR063 loci whereas a heterozygosity deficit was observed in the other loci. Fis values were negative for all the loci except for mPdCIR015, mPdCIR035 and mPdCIR078. The Fst value varied between 0.04 (mPdCIR015 and mPdCIR063) and 0.29 (mPdCIR063). The highest PIC value was obtained for mPdCIR032 (0.71) and the lowest for mPdCIR035 (0.29) with an average of 0.56. The average of the total rate of heterozygosity (Ht) by primer for all cultivars was very high (0.62). The dendrogram constructed based on the data from the eight SSRs primers revealed 3 clusters (Fig. 4). Cluster 1 was the largest and includes 17 samples. It groups all samples from Hezoua, Gtar, Jemna and Ben Ghilouf regions, 2 samples from Zarat (Zarat 1 and Zarat 2) and 2 samples from Gataya (GTY1 and GTY2). Jerba 1 belongs also to this cluster, while Jerba 2, Jerba 3 were in cluster 2. Cluster 3 groups all samples from Mides region, 1 sample from Gataya (GTY3) and 1 sample from Zarat (Zarat3). The three clusters detected by the dendrograme based on molecular data may be observed in the projection of the genotypes analyzed by the two first components 1–2 (Fig. 5). The STRUCTURE-derived populations did not show clustering based on the geographical origin of the samples. However, the analysis of population structure led us to keep three sub-populations (Fig. 6) based on Evanno statistics (Fig. 2A). 3.3. Correlation between phenotypic and molecular data According to the Mantel test, in this study, the SSR data were not 29

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Fig. 3. Dendrogram of 24 male date palm genotypes collected from Southern Tunisia genotypes collected from Southern Tunisia based on morphological data using the Ward’s method. The 24 pollinizers were classified into 5 main phenotypically related groups.

correlated with the morphological data (r= -0,106, p = 0.075). In spite of this, comparing molecular and morphological characterization, the results show that all the cultivars from the Hezoua (HZ 1, HZ 2, and HZ 3), and Mides (Mides 1, Mides 2 and Mides 3) regions were in the same clusters in the dendrogram based on morphological and molecular data. Also, Zarat 3 was clustered with samples from Mides region for morphological and molecular data. However Jerba 2 and Jerba 3, very close geographically and genetically, were clustered in two different morphological clusters.

distinguish date palm cultivars including male genotypes (Djerouni et al., 2015; Elsafy et al., 2015; Faqir, 2016; Iqbal et al., 2011; Salem et al., 2008; Soliman and Al-Obeed, 2013). Discrimination between the studied male genotypes could be assigned according to the following characters: leaf length (LL) leaf width (LW), spine width (EE), spine length (LE), apical pinnae length (LA) and middle pinnae length (LP), spathe width (GS), the spathe length (LS), spikelets number per inflorescence (NP), Length of the longest spikelet (LL), the maximum width of the apical pinnae (GA), spine number (NE), the length of the shortest spikelet (LC), the spine number (NE) and rachis width (ER). Significant correlations were shown among some morphological traits as proven by previous studies (Faqir, 2016; Salem et al., 2008). Overall, the analyses of selected morphological characteristics show the utility of morphological markers in studying the genetic diversity of male date palm diversity in southern Tunisia. Some pollinizers were classified according to their geographical location. On the other hand, some genotypes were placed in different clusters while they were

4. Discussion This study shows the use of phenotypic traits and molecular markers to characterize and evaluate the diversity of male date palms from different localities in Southern Tunisia. Morphological traits showed a great variability among the male date palm genotypes studied. In fact, several studies have shown that morphological traits can be used to

Table 6 Mean (M) and standard deviation (SD) of quantitative characters according to clusters in 24 male date palm genotypes collected from Southern Tunisia. cluster number

GS

LS

NP

LL

LC

LT

LR

ER

NE

EE

LE

AP

LP

LA

GA

1

17.82 3.22 13.73 4.15 19.86 4.18 13.19 3.15 16.02 3.42

78.06 7.79 59.33 8.96 79.72 3.85 60.50 14.06 82.96 22.80

249.61 28.88 266.67 33.00 375.44 34.16 147.60 35.71 160.75 27.90

31.91 7.94 24.83 11.55 29.11 2.66 20.09 6.69 27.79 2.18

5.13 2.81 15.90 14.28 5.84 0.14 7.05 3.40 7.80 1.50

335.89 19.45 224.67 28.76 339.33 15.17 277.13 29.58 377.56 33.88

60.56 10.37 41.75 22.27 60.33 8.25 46.20 11.53 63.92 3.98

3.88 0.77 2.30 0.52 3.58 0.45 3.24 0.66 3.83 1.51

18.06 4.72 38.17 14.85 13.00 1.15 36.87 8.73 27.29 11.91

0.92 0.13 0.68 0.26 0.70 0.26 0.81 0.33 1.05 0.29

23.06 4.68 11.97 1.93 20.50 5.93 18.80 4.18 25.23 9.74

3.33 0.59 2.92 0.97 2.80 0.12 2.53 0.66 3.03 0.40

38.67 3.97 30.33 5.19 49.22 5.39 38.20 6.31 45.79 7.87

25.28 4.30 18.42 0.12 29.44 1.26 21.53 10.50 27.54 6.14

1.72 0.40 1.85 0.30 1.92 0.18 1.54 0.59 1.86 0.41

2 3 4 5

M SD M SD M SD M SD M SD

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Table 7 Variation of qualitative characters within the clusters obtained using the UPGMA method in 24 male date palm genotypes collected from Southern Tunisia. Cluster 1

pollinizers

maturity

FS

CB

RP

CP

NG

RE

PE

DP

SS

CS

PP

OP

RP

1

zarat3 midés3 midés2 Zarat1 mides1 gtar3 jemna 2 jerba2 jerba1 GTY 2 GTY 3 jemna 3 BG1 jerba3 zarat 2 BG2 gtar1 gtar2 GTY 1 HZ 2 jemna1 HZ 3 BG3 HZ 1

seasonal seasonal seasonal seasonal seasonal seasonal early late seasonal seasonal seasonal early seasonal seasonal seasonal seasonal seasonal seasonal seasonal seasonal late late early seasonal

1 1 1 2 1 2 1 1 2 2 2 1 1 2 1 1 2 2 2 1 1 1 1 1

1 1 1 2 2 2 2 1 1 1 2 2 2 2 2 2 2 1 2 2 1 2 2 1

2 2 1 2 1 2 1 1 1 2 1 2 1 1 2 1 1 1 2 1 1 1 1 1

1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1

2 2 2 1 2 2 3 2 1 2 1 2 2 2 2 2 1 2 1 1 2 1 1 2

5 7 7 5 5 5 5 7 3 3 3 7 5 3 5 3 5 7 7 7 5 7 3 7

2 1 2 2 1 2 1 1 2 2 2 1 3 2 2 3 2 2 2 3 1 3 3 3

2 1 3 3 3 1 2 2 3 1 3 2 1 2 3 1 3 3 1 3 3 1 1 1

2 1 3 2 3 1 3 2 1 1 3 1 3 7 2 1 2 2 2 3 1 2 1 1

4 1 4 1 1 4 5 5 1 1 1 5 1 4 1 1 5 1 4 4 5 4 5 4

4 4 4 3 4 4 7 4 4 7 7 7 7 7 7 3 7 4 7 4 7 7 4 4

3 3 3 3 3 3 7 3 7 3 7 3 3 3 3 3 3 3 7 3 3 3 3 3

2 1 1 1 1 1 2 2 2 1 1 2 2 2 1 1 2 2 2 2 2 2 1 2

2 3 4

5

Table 8 Number of alleles per locus (A), allelic frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Wright’s fixation index (Fis and Fst), Polymorphism Information Content (PIC) for 24 male date palm genotypes collected from southern Tunisia. locus

allelic richness

Size (bp)

A

Ho

He

PIC

Ht

Fst

Fis

Hs

Gst

F

mPdCIR010 mPdCIR015 mPdCIR025 mPdCIR032 mPdCIR035 mPdCIR057 mPdCIR063 mPdCIR078 Mean

5.44 4.93 6.86 6.82 2.98 5.67 3.94 4.68

113-161 120-136 200-239 289-302 186-196 250-286 119-151 117-138

6 5 7 7 3 6 3 5

0.58 0.54 0.79 0.75 0.13 0.58 0.75 0.21 0.54

0.67 0.44 0.66 0.64 0.65 0.74 0.74 0.32 0.61

0.59 0.61 0.70 0.71 0.29 0.60 0.60 0.41 0.56

0.66 0.67 0.76 0.76 0.33 0.64 0.69 0.45 0.62

0.16 0.04 0.10 0.05 0.26 0.14 0.04 0.29 0.14

−0.04 0.16 −0.15 −0.03 0.50 −0.04 −0.13 0.38 0.08

0.57 0.63 0.71 0.73 0.23 0.57 0.68 0.31 0.55

0.14 0.07 0.06 0.04 0.31 0.12 0.02 0.31 0.13

0.06 0.11 −0.04 0.01 0.43 0.03 −0.09 0.43

Fig. 4. UPGMA dendrogram of the similarity matrix obtained from the proportion of shared amplification fragments (Nei and Li, 1979) of 24 date palm pollinizers collected from Southern Tunisia based on 8 SSR loci.

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mean of 4.14) (Al-Faifi et al., 2016). Similar results (42 alleles) to those obtained in our work were obtained when using 6 SSR markers to study 14 female cultivars in Nigeria but with an average of 7 alleles per locus (Yusuf et al., 2015). Other works have reported a higher number of alleles per locus such as those found by Zehdi et al. (2012, 2004) when studying diversity of 101 and 49 Tunisian date palm accessions respectively, using 14 SSR markers, probably due to the use of a higher number of accessions and microsatellite loci. However, it should be pointed out that the results obtained in this work are difficult to compare with results obtained in other studies since, on the one hand, the number of varieties or loci analyzed and/or the detection method is different and, on the other hand, most of the studies carried out so far in date palm only involve female genotypes. Regarding He, the value found in this work (0.61) is similar to the values reported by other studies for Tunisian date palm cultivars varying from 0.61 (Metoui et al., 2017) to 0.63 (Hamza et al., 2011) and 0.67 for Saudian cultivars (Al-Faifi et al., 2016). On the other hand a higher He (0.731) was found when studying the diversity of 74 female cultivars and 27 male from southern Tunisia using 14 SSR loci (Zehdi et al., 2012). However the mean value of Ho (0.54) found in our study was lower than findings of other studies conducted in Tunisia, 0.67 (Metoui et al., 2017) and 0.70 (Hamza et al., 2011). An excess of heterozygosity manifested by negative Fis values was observed for most loci in this study as shown also by Bodian et al. (2012; 2014) studying date palm accessions from Morocco. On the whole, high values of He and Ho indicate, mainly, that the date palm samples studied in this work have a high genetic diversity. This could be due to several factors such as: seed propagation (Bodian et al., 2012), the use of mixed pollen of various males for pollination, environmental effects and interactions between environmental and genetic factors or the exchange of offshoots, which are a mix of vegetative and seed-propagated materials (Elmeer and Mattat, 2015), among growers. All the Fst values found in this study were positive. The average Fst value was 0.14 which means moderate differentiation of male trees (Wright, 1978) and that only about 14% of the genetic variation was present among populations. Results showed that for all the 8 loci studied the total gene diversity (Ht) and estimates of the mean gene diversity within groups (Hs) are nearly similar which indicates that the maximum of variability is locally maintained, and the low values of the genetic differentiation among groups (Gst) with an average of 0.13 confirm this hypothesis as found by Zehdi et al. (2004). Most loci studied showed PIC values greater than 0.5 and, consequently, those would have a higher power for analyzing the genetic variability of date palm cultivars. The mean PIC value (0.56) recorded in this study is higher than that found by Metoui et al. (2017) (0.33) studying female date palm cultivars in southern Tunisia. However, some comparable PIC values were found when studying 1066 date palms, from 411 cultivars in 12 countries using 255 SSR that were used in recent studies, who recognized PIC values 0.62, 0.63, 0.66, and 0.67 respectively for Qatar, Algeria, Iran and Iraq. However, this same study showed higher PIC values than found by us for Libya Nigeria and Morocco (0.81, 0.83 and 0.93, respectively) (Salomon-Torres et al., 2017). All these high PIC values could be due to the dioecious nature of the date palm (Arabnezhad et al., 2012). Regarding the Bayesian clustering, results of this study did not show clustering based on the geographic origin of the samples. However, the analysis of population structure led us to keep three sub-populations (Fig. 6) based on Evanno statistics (Fig. 2A). Our results are in line with those found by Zehdi-Azouzi et al. (2015) who confirmed that the Tunisian groups showed minimal admixture and were clearly maintained for all K values in their investigation of 295 date palm genotypes diversity from several countries including 38 male and female Tunisian date palm genotypes. Further investigations with a larger number of samples will be needed to analyze population structure of Tunisian date palm genotypes. When correlating morphologic with genetic data, this study

Fig. 5. Representation of date-palm variables of 24 male genotypes collected from Southern Tunisia on the plane 1–2 of principal component analysis based on molecular data using the UPGMA method.

geographically close; one example are the three samples from Jemna which are placed in three different clusters according to morphologic data. The total number of SSR alleles found in this study (42, varying from 3 to 7 with an average of 5.4 alleles per locus) is slightly higher than those found studying the genetic diversity of 12 female cultivars of date palms from southern Tunisia with the same SSR markers (39 alleles, varying from 3 to 5 with an average mean of 4.33 alleles per locus) (Metoui et al., 2017). The number of alleles is also higher than that found when using 22 SSR markers to study genetic relationships among 32 date palms representing common cultivars grown in different geographical regions in Saudi Arabia (2 to 6 alleles per locus with a 32

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Fig. 6. Population structure analysis of 24 male date palm genotypes collected from Southern Tunisia using STRUCTURE 2.3.4. A. Determination of the most appropriate value of K for STRUCTURE analysis calculated by Structure Harvester (Earl and Vonholdt, 2012) as in Evanno et al. (2005). B. STRUCTURE-based neighbor joining tree showing relative distances between inferred clusters. C Populations derived from STRUCTURE analysis are color coded. Each accession is represented by a vertical colored bar. Individuals of the same color belong to the same cluster. Individuals with several different colors show the percentage of the genome that was inherited from each cluster in K = 3 clusters.

confirms that, for the male date quantitative morphological traits distances, a similar observation to cultivars in Tunisia (Hamza et al.,

palms studied, distances based on were not correlated with genetic that reported for female date palms 2011).

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5. Conclusion The present study provides a database of Tunisian male date palm genotypes based on morphological and genetic traits. The set of morphological parameters and SSR markers used shows a great variation among the studied genotypes. This high genetic diversity provides a good potential for further selection of male genotypes to optimize fruit quality when used for pollination. In fact, the association between morphological and molecular analyses can be a useful tool to distinguish good pollinizers and also the trees most able to adapt to climate and environmental changes. However, no clear relationship was found between geographical origin and genetic composition, suggesting an exchange of seeds and offshoots among different regions. Additional studies with male trees from other regions, a larger number of pollinizers and even other countries should be performed in order to have a clear picture of overall male date palms genetic diversity and optimize collaborative pollinizer genetic resource management and conservation in date palm. Acknowledgements This research was supported by Ministerio de Economía y Competitividad – European Regional Development Fund, European Union (AGL2016-77267-R). We thank Yolanda Verdún for help with the molecular analyses. 33

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