rapd markers in diversity detection and variety identification of south tunisian barley

Acta Bot. Gallica, 157 (1), 13-23, 2010.

Morphological and molecular variability of some south Tunisian bar-ley accessions

by Ferdaous Guasmi, Nidhal Marzougui, Narjés Sarray, Walid Elfaleh and Ali Ferchichi

Institut des régions arides, 4119 Médenine, Tunisia; [email protected]

Abstract.- A total of 20 accessions of barley (Hordeum vulgare L.) from aridregions of Tunisia were collected and evaluated ex situ in experimental fields(Medenine, South Tunisia). Morphological traits and molecular marker (RandomAmplified polymorphic DNA) were applied to evaluate genetic diversity of theseaccessions. The study showed the existence of a highly significant phenotypicalvariability. Some parameters (production of straw and grain yield) appeared asgood criteria to differentiate particular accessions. Concerning molecular varia-bility, 49 RAPD bands were used to calculate Jaccard’s distance coefficient forcluster analysis using an arithmetic averaging algorithm (UPGMA). The geneticdistance of the 20 studied accessions ranged between 0.25-0.68. The analysisof the hierarchical tree shows the presence of four ramifications thus translatinggreat genetic diversity.

Key words : biodiversity - molecular markers - barley - morphological traits.

Résumé.- Un total de 20 accessions d'orge (Hordeum vulgare L.) collectéesdans des régions arides de la Tunisie ont été évaluées sur la base de traits mor-phologiques et de marqueurs moléculaires (RAPD). L'étude a montré l'existen-ce d'une variabilité phénotypique fortement significative. Certains paramètres(production de paille et de grains) paraissent être de bons critères pour diffé-rencier des accessions particulières. Concernant la variabilité moléculaire, 49bandes RAPD ont été détectées, le regroupement des cultivars grâce à lamoyenne arithmétique (UPGMA) a révélé une hétérogénéité remarquable. Ladistance génétique entre les 20 accessions varie de 0.25 à 0.68. L'analyse del'arbre hiérarchique montre la présence de quatre ramifications traduisant unegrande diversité génétique.

Mots clés : biodiversité - marqueurs moléculaires - orge - traits morpholo-giques.

received October 20, 2008, accepted December 30, 2008

157 (1), 13-23.qxp:modèle SBF.qxp 18/01/10 13:42 Page13

I. INTRODUCTION

Variety identification and genetic relationship estimations have always been importanttasks for breeders. Various methods have been elaborated for this purpose. Pedigree ana-lysis is the most widely used method for estimating the degree of similarity between varie-ties or populations, but the necessary information on ancestry is not always accurate oravailable. The application of these traits is hindered by their limited number and by themodifying effect of environmental factors in some cases.

The spread of DNA markers has allowed the genome to be analysed directly, thus eli-minating errors caused by environmental factors (Liu et al., 1996). Using these markersthe genome can be characterized with great accuracy (Castagna et al., 1997; Mori et al.,1995). The first DNA markers used for genomic analysis were restriction fragment lengthpolymorphisms (rflp), which were used in many species (maize, wheat, durum wheat, bar-ley, rice, etc.) to determine relationships between varieties (Linc et al., 1996; Casas et al.,1998; Bohn et al., 1999; Szucs et al., 2000). However, the complexity and costs of thismethod restricted its use (Manifesto et al., 2001). The introduction of PCR-based markers,including random amplified polymorphisms (rapd), sequence-tagged sites (sts), microsa-tellite markers (SSR) and amplified fragment length polymorphisms (aflp), have facilita-ted the rapid analysis of the genome (Liu et al., 1996).

Barley (Hordeum vulgare L.) is one of the most important crop species in the world. Itis a diploid (2n = 2x = 14), largely self- fertilizing species with a large genome (Bennett &Smith, 1976). It‘s a one of the oldest cultivated crops. More than 136 × 106 tons of grainsis produced annually in the world (Gu et al., 2001).The most important use of barley throu-ghout the world is as malt for manufacturing beverages or malt enriched food products. Itis also used as fodder crop for domestic animals, poultry and human food in the form ofpreached grain. Besides these, it is used as industrial purposes, such as medicine andmanufacturing baby food.

In arid regions of Tunisia, barley is cultivated on about 150 000 hectares. During cen-turies, early domestication and local knowledge have generated diverse local barley usedmainly for feed and lowly for food (Vavilov, 1951).

Our main objectives were to detect the genetic diversity of some South Tunisian barleyraces on the basis of morphological traits and RAPD analysis. These methods have beenused by some authors (Sabrina et al., 2007; Pakniyat et al., 1997; Zhang et al., 1993;Saghai-Maroof et al., 1984) and have demonstrated the level of polymorphism within andbetween a number of populations, the distribution of diversity and its relationships withecogeographical factors.

II. MATERIALS AND METHODES

A. Plant material

Twenty barley accessions collected from arid regions of Tunisia were used in this study(Fig. 1). The accessions names and country of origin are listed in Table I.

B. Morphological analysis

This study was carried out in experiment field at Médenine (South Tunisia). This areais characterized by arid conditions. Basic informations (seasonal precipitation, temperatu-re, soil and water) are presented in Table II.

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Seed were sowed at January and the measurement wascarried during March to June and has concerned thevegetative and reproductive stage. During experiment,the irrigation was conduced once every ten days. Thequantity of water of irrigation was the equivalent of 20mm per irrigation.

For measurements at the vegetative stage, the para-meters used were the number, length and width of leaves The reproductive stage (100 days after sowing) is cha-racterized by the number of tillers and speaks, the num-ber, length (cm) and width of leaves (cm; 3 leaves aremeasured/plant), the height of the stem (cm), the leafarea (cm²) and finally the production of straw (g) (TableIII). For each accession 60 plants were randomly selec-ted and measured.

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Fig. 1.- Collectingsites of barleyaccessions.

Fig. 1.- Sites de col-lecte des acces-sions d’orge.

Table I.- Barley accessions stu-died with their origin.

Tableau I.- Accessions d’orgeétudiées avec leur origine.

Designation Origin

1 El morra Tataouine

2 Tataouine ejdida Tataouine

3 Oued el khil 2 Ben Keddache

4 Gasbett gomri Gomrassen

5 Lamaat Tataouine

6 El ferch1 Tataouine

7 Kasar ouled boubaker Tataouine

8 Swittir Medenine

9 Bir ezwai 3 Medenine

10 Bir 30 Tataouine

11 Dkilet toujene Gabes

12 Belkhir Gafsa

13 Gomrassen 2 Tataouine

14 Manzel mgor 3 Ben Keddache

15 El ferch 2 Tataouine

16 Ben gzayel Medenine

17 Mazreet ben slama Gabes

18 Swittir 1 Medenine

19 Pakestany Pakesten

20 Rihane Tunis

Table II.- Trial site characteristics in IRA (ElFjé, Médenine).

Tableau II.- Caractéristiques du site d'es-sai (EL Fjé, Médenine). Mean temp. Mean temp. Soil Water

max °C (winter) max °C (summer) quality

10 30 Sandy Tap water


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C. Genetic diversity analyses

Total DNA was isolated from fresh leaves asdescribed by Doyle & Doyle (1990) with somemodification. DNA concentration was determinedby both spectrophotometry at 260 nm and 2% aga-rose gel electrophoresis.

RAPD reactions were performed with the follo-wing 10-base primers (Operon Technologies Inc.,Almeda, USA): OPA-04 (5’- AATCGGGCTG -3’), OPA-18 (5’- AGGTGACCGT-3’), UBC-490(5’-AGTCGACCTT-3’), UBC- 534 (5’-CACCCCCTGC -3’). More information of the pri-mers is subscribed in Table IV.

The reaction mixture (20 µL) contain 75 ng ofgenomic DNA, 2 μL 10 X Taq DNA polymerasebuffer, 500 nM of a single primers, 100 µM of eachdNTP and 1 U of Taq DNA polymerase.

Amplifications were carried out using aCleaver Scientific thermocycler. The PCR pro-gramme included an initial denaturation step at94 °C for 2 min, followed by 40 cycles of1 min at 94 °C, 37 °C for 2 min, 72 °C for1 min and final extension at 72 °C for 5 min.

After amplification, the DNA fragmentswere separated by electrophoresis for about 2 hunder constant voltage (60 V) in 3% agarosegel submersed in 1x TBE buffer. The gels werestained with ethidium bromide solution andobserved under ultraviolet light. Each gel was photodocumented using the image capturingsystem bio print.

D. Data analysis

The data for different growth parameters were analysed statistically using analysis ofvariance technique (ANOVA). Accessions classification was done with principal compo-nents analysis (PCA) using Stat Box version 6.4 (Grimmersoft, Paris, France) in order toexplore the overall variability at vegetative and reproductive stage.

For molecular analysis, amplified fragments were classified as present (representedwith 1) or absent (0) (Jia et al., 2000; Du et al., 2001). The values of genetic similaritywere obtained with the software RAPDistance Jaccard’s coefficient. The Phylip programwas used for phenogram generation.

III. RESULTS

A. Morphological results

Results from the analysis of variance (ANOVA) prove that for all morphological cha-racters, the F statistics are significant and considerably higher than the F critical (Tables Vand VI).

Table III.- Morphological charactersretained for the study of diversity inbarley.

Tableau III.- Caractères morpholo-giques retenus pour l'étude de ladiversité chez l'orge.

Stage Characters

Number of leaves

Vegetative stage Length of leaves (cm)

Width of leaves (cm)

Number of leaves

Length of leaves (cm)

Width of leaves (cm)

Reproductive stage Number of tillers (cm)

Number of speaks

Height of the stem (cm)

Production of straw (g)

Leaf area (cm²)

Table IV.- Sequence name, repeat motifand annealing temperature for theRAPD primers used.

Tableau IV.- Noms, séquences des motifset températures recommandées pourles amorces de RAPD utilisées.

Primers Sequence name Sequence 5’to 3’ Tm (°C)

1 UBC-490 AGTCGACCTT 35.4

2 UBC-534 CACCCCCTGC 47.7

3 OPA-18 AGGTGACCGT 50

4 OPA-04 AATCGGGCTG 39.5


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At vegetative stage (TableVII), the width of leavesvaries between 0.45 cm and0.68 cm (with an average of0.55 cm). The length variesbetween 12.83 cm and 7.59cm (with an average of 10.41cm). The two characters don’tshow a great variability bet-ween the accessions.However the number ofleaves per plant is the mor-phological descriptor whichshows greatest variabilitywithin the accessions, it variesbetween 5.83 and 14.67 (withan average of 8.37).

The structuring of the 20barley accessions by PCAanalysis according to vegeta-tive characters shows that thetwo axes absorb 94.04% ofthe total variance (TableVIII).The first principal component(F1) is associated with length(0.9) and number of leaves(0.93). F2 accounts for widthof leaves (0.53). Accessionsare classified into four groupsby the first and second vector(Fig. 2). The first groupincludes seven accessions:Gasbett gomri, Lamaat,Rihane, Swittir, ManzelMgor, Dkilet Toujene and ElFerch 2, characterized bynegative values on the axis 1,showing a low performance atvegetative stage. The secondgroup includes eight acces-sions regrouped in the centre,characterized by a mediumperformance at vegetativestage. The third group, cha-racterized by a positive valueson the axis 1 and axis 2,includes Belkir, Pakistany and Mazreet Ben Slama, characterized by a high performanceat vegetative stage. Finally accessions El Ferch1 and Oueld el khil2 represent the fourth

Table V.- Results of one way ANOVA analysis at vegetativestage. F critical = 1.66 at 0.05 and 2.03 at 0.0).

Tableau V.- Résultats d'analyse ANOVA au stade végétatif.

Vegetative stage Sum of df Mean F Sig.

squares square

between groups 1,208 19 0,064 6,235 > 0,000

width of leaves within groups 3,467 340 0,010

total 4,675 359

between groups 965,943 19 50,839 12,798 > 0,000

length of leaves within groups 1350,655 340 3,973

total 2316,598 359

between groups 667,413 19 35,127 3,942 > 0,000

number of leaves within groups 1960,250 220 8,910

total 2627,663 239

Table VI.- Results of one way ANOVA analysis at repro-ductive stage. F critical = 1.66 at 0.05 and 2.03 at 0.0).

Tableau VI.- Résultats d'analyse ANOVA au stade repro-ducteur.

Reproductive stage Sum of df Mean F Sig.

squares square

between groups 2,403 19 0,126 7,919 > 0,000

width of leaves within groups 5,431 340 0,016

total 7,835 359

between goups 726,986 19 38,262 9,945 > 0,000

length of leaves within groups 1308,113 340 3,847

total 2035,100 359

between groups 3863,213 19 203,327 2,293 > 0,002

number of leaves within groups 19512,08 220 88,691

total 23375,29 239

between groups 1459,786 19 76,831 3,096 > 0,000

height of the stem within groups 5459,013 220 24,814

total 6918,799 239

between groups 96,746 19 5,092 2,805 > 0,000

number of tillers within groups 399,417 220 1,816

total 496,163 239

between groups 76,583 19 4,031 2,986 > 0,000

number of speaks within groups 297,000 220 1,350

total 373,583 239

between groups 879,815 19 46,306 5,031 > 0,000

production of straw within groups 2025,092 220 9,205

total 2904,906 239

between groups 1145,764 19 60,303 4,115 > 0,000

leaf area within groups 3224,286 220 14,656

total 4370,050 239


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group characterized by a positive values onthe axis 1 and negative values on axis 2,characterized by an important length andnumber of leaves.

For the reproductive stage (Table IX), themorphological characters which present themost important variability are the number ofleaves (from 6.5 to 31), the production ofstraw (from 0.87 g to 6 g, with an average of3.01 g) and the leaf area (between 4.77 and15.9 cm²). However, the width of the leaves(0.64 cm to 1.00 cm), the length of theleaves (8.16 cm to 13.33 cm) and height ofthe stem (16.97 to 27.5 cm) present a littlevariation. The first two axes explain 49.8%and 16.70% of the total variation, respecti-vely. The first component (Table X) is posi-tively correlated with the number of tillersand speaks length of leaves and the heightof the stem. The second axis is positivelycorrelated with the leaf area and negatively

Table VII.- Average of measurements of themorphological characters during the vege-tative stage.

Tableau VII.- Moyennes des mesures descaractères morphologiques pendant lestade végétatif.

Code accessions width of length of number of

leaves leaves leaves

1 El morra 0,51 9,81 7,67

2 Tataouine ejdida 0,53 9,46 9,33

3 Oued el khil 0,63 12,83 14,67

4 Gasbett gomri 0,49 7,59 6,00

5 Lamaat 0,53 8,86 6,17

6 El ferch 1 0,57 12,77 11,17

7 Ksar ouled boubaker 0,57 10,38 10,00

8 Swittir 0,58 8,12 7,33

9 Bir ezwai 0,52 10,09 6,67

10 Bir 30 0,51 9,82 8,83

11 Dkilet toujene 0,49 8,93 6,50

12 Belkhir 0,61 11,61 10,50

13 Gomrassen 0,56 9,78 7,83

14 Manzel mgor 0,49 8,39 5,67

15 El ferch 2 0,45 8,89 6,50

16 Ben gzayel 0,53 11,51 7,67

17 Mazreet ben slama 0,68 10,57 10,00

18 Switir 1 0,56 10,84 10,33

19 Orge B 0,63 12,10 8,83

20 Orge Rihane 0,55 8,47 5,83

Average 0,55 10,04 8,37

Standard deviation (σ²) 0,056 1,529 2,282

Table VIII.- Dispersion of barley accessionsin planes formed by axes F1 and F2 ofthe PCA at vegetative stage.

Tableau VIII.- Dispersion des accessionsd’orge dans le plan formé par les deuxpremiers axes F1 et F2 d’ACP au stadevégétatif.

Principal component F1 F2

Inertie % 79,99 14,05

Cumulative % 79,99 94,04

Contributions of characters (%) LngF (0,90) LrgF (0,53)

NF (0,93)

Fig. 2.- Plots of 20 barley acces-sions on the first two principalaxes at vegetative stage.

Fig. 2.- Groupement des 20accessions d’orge sur les deuxpremiers axes de l’ACP pour lestade végétatif.


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correlated with width of leaves. The plotobtained according to axes 1-2 (66.5% oftotal inertia) shows two groups (Fig. 3).Thefirst group includes seven accessions: ManzelMgor3, El Ferch2, Bir Ezwai, Gasbett Gomri,Dkilet Toujene, Bir30 and Lamaat, characte-rized by negative values on the axis 1, sho-wing a low performance at reproductivestage. The second group characterized by apositive value on the axis 1 includes BenGzayel, Gomrassen, Oued El khil, Pakistanyand El Ferch1, characterized by a high per-formance for all reproductive characters.

Data projection on plans as defined byinertia axes of PCA from barley accessions do not show significant geographic polymor-phism between accessions (Fig. 2 and 3). Morphological characters display a minimalvariability with regard to the geographical origin. Figures 2 and 3 clearly reflect that acces-sions appear to be individual without any particular geographical structuring.

B. RAPD analysis

DNA from leaves was assayed for RAPD-PCR. After screening, four primers, UBC-490, UBC-534, OPA-018 and OPA-04 (Table XI), give reproducible amplification pat-terns. Of 49 amplified fragments, 42 are polymorphic.

Table IX.- Average of measurements of the morphological characters during the reproducti-ve stage.

Tableau IX.- Moyennes des mesures des caractères morphologiques pendant le stadereproducteur.

Code accessions width of length of number height of number number leaf production

leaves leaves of leaves the stem of tillers of speaks area of straw

1 El morra 0,71 8,90 13,17 23,25 2,00 1,50 6 9,93

2 Tataouine ejdida 0,82 9,41 20,17 18,08 3,17 1,67 4,33 5,42

3 Oued el khil 0,93 11,05 27,50 23,50 4,00 2,33 4,33 6,70

4 Gasbett gomri 0,71 8,36 14,50 18,55 2,83 1,17 2,27 8,03

5 Lamaat 0,77 9,81 15,17 20,57 2,00 1,33 1,60 4,77

6 El ferch 1 0,83 10,30 19,83 26,07 3,67 3,17 1,93 9,42

7 Ksar ouled boubaker 0,78 9,41 16,83 21,33 3,00 2,50 3,67 12,28

8 Swittir 0,95 11,77 15,67 17,33 1,83 1,17 2,33 15,9

9 Bir ezwai 0,73 8,31 13,67 18,62 1,83 1,33 1,93 7,47

10 Bir 30 0,64 9,04 19,67 17,37 3,33 1,67 4,00 6,98

11 Dkilet toujene 0,78 9,12 11,83 21,58 2,33 1,67 1,43 7,25

12 Belkhir 0,80 11,74 31 27,5 4,5 3 0,87 12,20

13 Ghomrassen 0,76 11,27 24,17 22,50 3,83 2,33 2,67 7,12

14 Manzel mgor 0,69 8,16 12,00 16,97 2,17 1,17 1,27 6,41

15 El ferch 2 0,70 8,56 12,17 17,57 1,83 1,33 2,67 9,98

16 Ben gzayel 0,77 11,66 22,17 22,75 3,17 2,00 3,67 6,12

17 Mazreet ben slama 1 11,05 16,50 19,38 2,83 1,33 4,00 13,25

18 Switir 1 0,71 10,46 20,83 20,92 3,50 1,50 4,00 9,13

19 Orge B 0,87 13,33 26,67 24,42 4,00 2,00 3,00 9,95

20 Orge Rihane 0,76 10,23 6,5 24,95 1,33 1,17 4,33 8,15

Average 0,77 9,89 18 21,16 2,85 1,76 3,01 8,47

Standard deviation (σ²) 0,092 1,425 6,1576 3,157 0,902 0,612 1,330 2,852

Table X.- Dispersion of barley accessions inplanes formed by axes F1 and F2 of thePCA at reproductive stage.

Tableau X.- Dispersion des accessionsd’orge dans le plan formé par les deuxpremiers axes F1 et F2 d’ACP au stadereproducteur.

Principal component F1 F2

Inertia % 49,80 16,70

Cumulative % 49,80 66,50

Contributions of characters (%) LngF (0,87) LrgF (-0,46)

NF (0,88) SF (0,74)

Ht (0,75)

NT (0,89)

NE (0,84)


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C. Cluster analysis

The genetic distance bet-ween studied accessionsranges from 0.25 to 0.68.39.5% of the accessions have awide genetic distance (morethan 0.6) and 60.5% are cha-racterized by a distance rangedbetween 0.3-0.6. AccessionRihane and El Ferch2, origina-ting respectively from Tunisand Tataouine, are the mostgenetically distant from the others, they are characterized by their lower performance forall morphological characters. The genetic relations between cultivars are represented by a

Fig. 3.- Plots of 20 barley acces-sions on the first two principalaxes at reproductive stage.

Fig. 3.- Groupement des 20accessions d’orge sur les deuxpremiers axes de l’ACP pour lestade reproducteur.

Table XI.- Characteristics of RAPD banding profiles pro-duced in Tunisian barley.

Tableau XI.- Caractéristiques des profils RAPD produitschez les accessions d'orge.

Primers Number of Number of % of Wight

amplified bands polymorphic bands polymorphic min - max (pb)

0PA -04 9 9 100 431-1083

0PA-18 13 11 84,61 162-1000

UBC-490 14 10 71,42 103-975

UBC-534 13 12 92,3 160-1150

Total 49 42 85.71 103-1150

Mean 12.25 10.5 87.08

Fig. 4.- Phylogenetictree of 20 barleyaccessions using anarithmetic averaging(UPGMA) algorithm(the length of thebranches corres-ponds on a relativegenetic distance; thenumbers correspondto the cultivars) and agenetic distance withan ancestor.

Fig. 4.- Arbre phylogé-nétique de 20 culti-vars d’orge établiselon la méthodeUPGMA.


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phenogram based on the Jaccard’s coefficient (Fig. 4). The analysis of this tree shows thepresence of five ramifications thus translating great genetic diversity. The first noted « a »is represented by the two accessions El Ferch2 and Rihane which are considered most dis-tant (distance equal to 0.50 and 0.49 respectively). The second noted « b » includes adichotomic ramification formed by two cultivars, El Morra and Tataouine Ejdida, fromwhich are detached cultivars Ksar Ouled Boubakar and Wad el Khil2. This branch is consi-dered the nearest (distance not excess 0.28). The third branch noted « c » presents tworamifications: one dichotomic contains two cultivars, Gasbett Gomri and Lamaat; acces-sions El Ferch1 and Bir30 are detached from the last ramification formed of the cultivarsBir Ezwai, Swittir and Dkilet toujen. The branch « d » includes also two dichotomic rami-fications, each one contains two cultivars, respectively Manzel Mgor3 and Mazreet BenSlama for the first, Swittir1 and Pakistan for the second, from which are detached theaccession Ben Gzayel. The latest branch « E » includes Belkir and Gomrassen.

IV. DISCUSSION

From the studied morphological parameters, production of straw and grain yield (numberof speaks) can be considered as good criteria to differentiate some accessions of barley.Accessions Ksar Ouled Boubaker, Belkir and El Ferch 1 can be selected for good grainyield production (> 2.5 speaks/plant), whereas accessions Switter, Mazreet Ben Slama,Belkir and Ksar Ouled Boubaker present the highest productions of straw (production ofstraw/plant = 12.5 g).

The importance of the character « production of straw » was announced by Khazaal etal. (2004) who studied the importance of straw in diets for ruminants in the Lebanon andthe Middle East. These authors suggest that selection of new cereal varieties should takeinto consideration the nutritive value of the straw as well as the quantities of seed and strawproduced. Others studies have demonstrated the importance of barley straw to suppressalgal blooms in small water bodies (Newman & Barrett, 1993). For grain yield, Shakhatrehet al. (2001) have established a correlation between the drought susceptibility and grainyield to selected barley lines for drought tolerance. However, although the agronomic para-meters are important to study diversity and select genotypes with high potential, they arelimited.

The use of RAPD markers shows that this method is informative to determine the phy-logenetic relationships between accessions. This method is used by many authors in theobjective to identify genetic variability in many barley cultivars (Kochieva et al., 2001;Molnar et al., 2000). Zeng et al. (2002) use a specific RAPD fragment to select barleyvarieties; of the 48 fragments generated by five selected primers, 44 appeared to be poly-morphic (92%). The 23 varieties were divided into two groups for high and low β-glucancontent. In our study, because of many habitats of barley accessions (Medénine, Tataouine,Gabès, Gafsa, Tunis, Pakesten), 39.5% of the accessions have a wide genetic distance(more than 0.6) and 60.5% are characterized by a distance ranged between 0.3-0.6. Thegenetic relations between barley accessions were represented by a phenogram based on theJaccard’s coefficient; the analysis of this tree shows the presence of four ramifications thustranslating great genetic diversity. The accessions clustered independently from their geo-graphic origin: the branches « a » and « d » are considered the most distant (distance >0.40) compared to the branch « b » who is considered the nearest (distance = 0.28).


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V. CONCLUSION

The study of the genetic diversity of south Tunisian barley collection using morphologicalparameters referring to the leaves and seeds made it possible to appreciate genetic varia-bility of intra and inter accessions. Some of these parameters production of straw and grainyield (number of speaks) can be considered as good criteria to differentiate some acces-sions. However, data projection on plans as defined by inertia axes of PCA from barleyaccessions do not show significant geographic polymorphism between accessions.

The aim of the present article was to use RAPDs to examine the genetic diversity inTunisian barley. The ecotypes clustered independently from their geographic origin.Accessions 3, 6, 12, 13, 16 and 19 were significantly better than all other accessions fortotal yield. Accessions 3, 6, and 13 were collected from Tataouine and Ben Keddache,these regions are the nearest area to Medenine (Table II) and it have almost the same eda-phic experimental field, but accessions 12 and 19 were collected respectively from Gafsaand Pakesten with different edaphic and climatic conditions than Medenine; these twogermplasms can be choosing for breeding new barley variety more tolerant and adapted toarid environnement.

This low geographic differentiation can be explained by the anthropogenetic diffusionwith extremely low heterozygosity of genetically homogeneous reproductive material asearly as the first exploration. For this reason, we are considering using other approachessuch as simple DNA sequence repeats (microsatellites) to better characterize genetic diver-sity of tunisian barley accessions.

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rapd markers in diversity detection and variety identification of south tunisian barley

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