RESEARCH ARTICLE

Genetic diversity assessment of Bulgarian durum wheat(Triticum durum Desf.) landraces and modern cultivarsusing microsatellite markers

Ganka Ganeva Æ Victor Korzun Æ Svetlana Landjeva ÆZaprjanka Popova Æ Nikolai K. Christov

Received: 19 July 2008 / Accepted: 30 July 2009 / Published online: 30 August 2009

� Springer Science+Business Media B.V. 2009

Abstract The genetic diversity in a Triticum durum

Desf. collection, consisting of 102 Bulgarian land-

races, nine Bulgarian and 25 introduced cultivars was

studied using 14 highly polymorphic microsatellite

markers. A total of 100 alleles were identified, with an

average of 7.14 alleles per marker. The gene diversity

values (He) of the markers for the total samples ranged

from 0.23 (WMS357 and WMS631) to 0.77 (WMS46),

with an average of 0.52. Within the landraces that were

collected from 18 sites in Southern Bulgaria showed 2–

11 alleles per locus with an average of 6.07. The

microsatellite analysis suggests that the genetic diver-

sity among landraces is lower compared to the

diversity levels for durum wheat in countries close to

the main centers of wheat domestication. Breeding

activities have caused significant reduction of the

allelic polymorphism, elimination of rare alleles, and

increase in the number of common alleles and the

frequency of dominating alleles.

Keywords Genetic diversity � Landrace �Microsatellites � Triticum durum

Introduction

Triticum durum Desf. is the second widespread

Triticum species constituting 10–11% of the world

wheat crop and accounting for about 8% of the total

wheat production. The countries in the areas where

the initial wheat domestication and cultivation took

place (The Mediterranean and Southern Europe, The

Balkans, North Africa, and South-Western Asia) are

still among the leaders of durum wheat production

(Ivanov 1927; Bozzini 1988; Srivastava et al. 1988;

Wang et al. 2007). The durum wheat growing area is

restricted because of the insufficient cold resistance

and spring growth habits of most of the traditional

and modern cultivars (Zhukovsky 1964). One of the

most important sources of genetic material for

improving plant adaptability and grain quality are

the local populations, a product of the natural

selection but also a result of the thorough domesti-

cation and artificial selection carried out by genera-

tions of farmers throughout the years.

In Bulgaria, the durum wheat has been known since

ancient times (Ivanov 1927). Seeds of durum wheat

were present in archaeo-botanical materials dated to

G. Ganeva � S. Landjeva

Institute of Genetics ‘‘Acad. Doncho Kostoff’’, Bulgarian

Academy of Sciences, Bulgaria Tzarigradsko shosse,

13 km, Sofia 1113, Bulgaria

V. Korzun

KWS LOCHOW GMBH, Grimsehl str. 31,

37574 Einbeck, Germany

Z. Popova

Institute for Plant Genetic Resources ‘‘K.Malkov’’,

Sadovo 4122, Bulgaria

N. K. Christov (&)

AgroBioInstitute, 8 Dragan Tsankov Blvd., Sofia 1164,

Bulgaria

e-mail: nikolai_christov@abi.bg

123

Genet Resour Crop Evol (2010) 57:273–285

DOI 10.1007/s10722-009-9468-5

the Early Bronze Age (Popova and Bozilova 1998).

The early studies in Bulgaria already showed that T.

durum consisted of a large number of botanical

varieties (Malkov 1906; Ivanov 1927). Some of the

original populations, used before the introduction of

the modern cultivars have been preserved at the

Institute of Plant Genetic Resources, Sadovo (Popova

2005). A database for reliable differentiation and agro-

biological characterization of those samples is needed

in order to guarantee their further preservation and

utilization in future genetic and breeding programmes.

The analysis of the genetic diversity of these local

and modern accessions is enabled by the development

and successful application of a large number of

molecular markers. Simple sequence repeats (SSRs)

markers (Tautz et al. 1986), or microsatellites, have

shown higher levels of polymorphism compared to

some other marker systems (storage proteins, isoen-

zymes, and RFLPs). In barley and wheat, microsatellite

markers have been used for construction of linkage

maps (Tautz and Renz 1984; Wang et al. 1994; Roder

et al. 1995, 1998; Korzun et al. 1998, 1999), analysis of

genetic diversity and identification of indigenous

landraces and modern cultivars (Donini et al. 2000;

Khanjari et al. 2007; Roder et al. 2002; Huang et al.

2002; Todorovska et al. 2004; Todorovska et al. 2005;

Landjeva et al. 2006; Wang et al. 2007). Numerous

studies have shown that the selective pressure of the

climate changes, the human activities, aiming at

domestication, and the intensive breeding have fixed

specific phenotypic and genotypic diversity amongst

the local wheats. (Donini et al. 2000; Ben Amer et al.

2001; Landjeva et al. 2006; Figliuolo et al. 2007).

The goal of the present study was to evaluate the

microsatellite-based gene diversity among the Bul-

garian landraces, Balgarian and introduced cultivars

as well as to compare genetic diversity within and

between eco-geographic regions.

Materials and methods

Plant material

In total 136 Triticum durum Desf. accessions, repre-

senting a part of the national durum wheat collection and

maintained at the Institute for Plant Genetic Resources

‘‘K. Malkov’’, Sadovo, were evaluated. The set con-

sisted of 102 landraces, grown in Bulgaria before the

introduction of modern cultivars and collected from 18

locations in a region spanning between 41�5 N to

42�29 N and 25 E to 26�50 E and 0 to 326 m above sea-

level (Table 1; Fig. 1), nine Bulgarian and 26 intro-

duced cultivars (Table 2). One accession of each

T. dicoccon and T. timopheevii were added only in

clustering and ordination analyses. For the statistical

analysis, the accessions, that were classified as

Bulgarian landraces, were divided into five spatial

groups according to the place of their sampling.

DNA isolation and microsatellite analysis

For each accession, 4–6 seedlings were pooled and

DNA was isolated by a mini-prep method adapted

from Rogowsky et al. (1991). A set of 14 highly

polymorphic wheat microsatellites (Roder et al.

1998) covering 11 chromosomes were used

(Table 3). Microsatellite designation, composition,

primer sequences and chromosome location of the

amplified loci were reported by Roder et al. (1998).

In general, a PCR protocol according to Roder

et al. (1998) with 35 instead of 45 cycles of 1 min

94�C denaturation, 1 min 50�C (or 55 or 60�C

according to primer) annealing and 1 min of 72�C

extension followed by a final extension step of 5 min

72�C was used.

Data analysis

The allele size at each microsatellite locus was scored

by using Genotyper� software (Applied Biosystem).

The gene diversity, also referred as expected hetero-

zigosity (He), was calculated using the formula:

Di = 1-P

Pij2, where Pij2 is the frequency of the

jth allele for ith locus summed across all alleles in the

locus, as implemented in the Power Marker v3.25

program (Liu and Muse 2005).

For cluster and ordination analyses, the microsatel-

lite markers were scored in binary (presence/absence)

format. The binary data were used to compute pair wise

similarity coefficient (Dice 1945). Dice similarity

matrix was used to perform cluster analysis with the

help of the Unweighted Pair Group Method with

Arithmetic Average (UPGMA) algorithm from

NTSYS (Numerical Taxonomy and Multivariate

Analysis System), ver. 2.29 for PC (Rohlf 2005).

Principal coordinate analysis (PCoA) was conducted

on the same matrix of Dice similarity coeeficients

using the modules DCENTER and EIGEN of the

274 Genet Resour Crop Evol (2010) 57:273–285

123

NTSYS-PC ver. 2.29 and the 3D plot was done using

the MOD3D module of the program.

Analysis of the molecular variance (AMOVA;

Excoffier et al. 1992) was performed to test the

significance of the partitioning of genetic variance

between and within the geographic groups. For this

purpose the populations were divided into four

regions according to their geographic origin with

landraces included into region 1 (Table 2, Fig. 2).

AMOVA was carried out using the software GenAl-

Ex V6 (Peakall and Smouse 2006).

To determine the effects of the local agro-ecological

conditions and the modern breeding, averaged data for

the following groups was calculated: I) the whole

Table 1 List of T. durum Desf. accession, collected from different districts of Bulgaria and used in SSR analyses

Districts Code Germplasm group and

number of accessions

Collection number

1. Group-Yambol

Yambol Y18 21 58E 125/4; 58E 125/5; 58E 125/7; 58E 125/11; 85E 125/13; 85E

125/14; 85E 125/17; 85E 125/19; 85E 125/22; 85E 125/24;

85E 125/25; 85E 126/5; 85E 126/6; 85E 126/10; 85E 128/2;

85E 128/29; 85E 128/30; 85E 128/30; 85E 128/30; 85E 129/9;

85E 129/9;

2. Group-Elhovo

Malomirovo MAL9 6 57E105/3; 57E105/9; 57E106/10; 57E108/30; 57E108/31;

57E108/40

Stefan karadjovo STK14 3 57E109/8; 57E109/13; 57E109/33;

Kamen Brjag KBr7 4 57E 110/5; 57E 110/7; 57E 110/8; 57E 110/15

Boljarovo BOL1 1 57E 111/18

Zlatinitsa ZL5 2 57E 112/4; 57E112/20

Bojanovo BOJ2 5 57E 113/4; 57E 113/7; 57E 113/12; 57E 113/13; 57E 113/13

Karavelovo Kar8 3 57E 114/17; 57E 114/19; 57E 114/23

Total 24

3. Group-Topolovgrad

Topolovgrad TO17 4 57E 103/1; 57E 91/10; 57E 91/18; 57E 91/20;

OrlovDol OrD11 10 57E 92/1; 7E 92/2;57E 92/4; 57E 92/5;57E 92/9; 57E 92/18; 57E

92/25; 57E 93/1; 57E 94/7; 57E 94/13;

Oreshnik OR12 1 57E /96/7

General Toshevo GT3 7 57E 99/25; 57E 99/26; 57E 99/27; 57E 99/30; 57E 100/5; 57E

101/28; 57E 101/30

Total 22

4. Group-Purvomai

Debur-Purvomai D4 3 58E 120/7; 58E 120/16; 58E 120/26

Tatarevo T16 1 58E 121/4

P. Eftimovo PEft13 2 58E 123/10; 58E 123/14

Izbegli IZ6 4 58E 124/2; 58E 124/3; 58E 124/4; 58E 124/5

Total 10

5. Group-Svilengrad

Sladun SL15 15 57E 73/5; 57E 73/10; 57E 73/17; 57E 74/6; 57E 74/7; 57E 74/11;

57E 74/13; 57E 74/15; 57E 74/25; 57E 74/26; 57E 74/31; 57E

75/4; 57E 75/12; 57E 75/15; 57E 75/24

Momkovo MO10 10 57E 84/16; 57E 84/17; 57E 84/23; 57E 84/27; 57E 84/31; 57E

85/2; 57E 87/5; 57E 87/11; 57E 87/21; 57E 88/4

Total 25

Total for all groups 102

Genet Resour Crop Evol (2010) 57:273–285 275

123

population of Bulgarian landraces; II) accessions from

geographically close locations, the samples being

divided into five groups according to their origin:

(1)—Yambol; (2)—Elhovo; (3)—Topolovgrad; (4)—

Purvomai and Asenovgrad; (5)—Svilengrad

(Table 1); III) modern Bulgarian cultivars; IV) intro-

duced cultivars (Table 2).

Results

Microsatellite polymorphism

By using a set of 14 highly polymorphic microsatellite

markers, a total of 100 alleles with an average of 6.67

per locus and 7.14 per marker were detected in the

Fig. 1 Geographic map showing the collection sites of Bulgarian landraces. The map was produced using Google Earth v.

5.0.11733.9347 with the server kh.google.com

Table 2 List of the used in this study Bulgarian and introduced cultivars of T. durum Desf. and their grouping according to

geographical location of the country where they are created

Cultivars name Country Region

Name and abbreviation Number in AMOVA

Beloslava, Yvor, Impuls, Progress, Zagorka, Lozen 6,

Saturn 1, Wuzhod, Sredets

Bulgaria Southeast Europe (SE_EU) 1

Mauragani iraklion, Moundros-2 Greece Southeast Europe (SE_EU) 1

Miraglia, Ellar, Italliano Rijo, Senatore Cappelli, HJS, Italy Southwest and Western Europe SW_EU 2

Agathe, ARJ76-142 France Southwest and Western Europe SW_EU 2

G8972 AE3-G, G8972 AE3-NG, G8972 AG2-G,

G8972 AG2-NG, G8973 AQ1-G, G8973 AQ1-NG Canada North America (N_America) 3

Langdon, Rugby, Edmore, Lloyd, USA North America (N_America) 3

Kuudusu 1149 Turkey West Asia (W_Asia) 4

Jordan col.86 Jordan West Asia (W_Asia) 4

Korifla Syria West Asia (W_Asia) 4

MJN.NJ2707 China West Asia (W_Asia) 4

Drujba, Hordeiphorme 189 Russia Russia 5

276 Genet Resour Crop Evol (2010) 57:273–285

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studied set of 136 T. durum accessions including both

landraces and advanced improved cultivars (Table 3).

The number of alleles per marker found in the B

genome (7.22) was larger than that in A genome (5.83).

Among the employed set of SSR markers the most

polymorphic was WMS577 (7B) with 18 alleles and

the least polymorphic was WMS165 (4A) with only 2

alleles (Table 3). The highest number of rare alleles

was found in the loci WMS577 (6) and WMS0046 (5),

while markers WMS357, MW1B002 and WMS18 did

not identify any rare alleles. Lack of amplified products

(null alleles) occurred in the WMS155, WMS46 and

WMS577 loci. The total gene diversity (He) calculated

for all markers and accessions was 0.52 on average

(Table 3). Markers WMS357 and WMS631 showed

the lowest He values (0.23 and 0.24, resp.), while

WMS46 and WMS577 were the most informative

(0.77 and 0.73, respectively).

Genetic variations within and among

the agro-ecogeographic regions

To analyse the genetic diversity within the landraces

they were divided into five groups according to their

Table 3 Description of microsatellite markers employed including the number of alleles, rare alleles (with a frequency of\2%), size

range of alleles and gene diversity in all accessions including 102 Bulgarian landrace and 34 Bulgarian and introduced cultivars

Marker Chromosome Total no. of alleles

(rare alleles)

Size range of alleles

(bp)

Gene diversity

(He)

MW1A001 1A 7(2) 134–151 0.69 ± 0.14

WMS0357 1AL 5(0) 117–123 0.23 ± 0.28

MW1B002 1B 5(0) 214–247 0.59 ± 0.12

WMS0018 1BS 3(0) 179–181 0.41 ± 0.09

WMS0095 2AS 7(2) 105–124 0.58 ± 0.07

WMS0155 3AL 10(4) Null, 125–147 0.58 ± 0.13

WMS0389 3B 9(4) 116–133 0.64 ± 0.04

WMS0165 4A 2(1) 186–191 0.26 ± 0.23

WMS0165 4B 5(2) 252–264 0.44 ± 0.12

WMS0513 4BL 4(1) 139–144 0.51 ± 0.10

WMS0408 5BL 6(2) 144–180 0.62 ± 0.07

WMS0680 6B 5(2) 119–132 0.57 ± 0.04

WMS0631 7A 4(1) 190–211 0.24 ± 0.08

WMS0046 7BS 10(5) Null, 159–177 0.77 ± 0.07

WMS0577 7BL 18(6) Null, 128–213 0.73 ± 0.06

Total 100(32)

Average per markers Total 7.14 0.52 ± 0.01

A Genome 5.83

B Genome 7.22

Table 4 Total number of alleles, number of alleles per marker, PIC and heterozygosity calculated for 15 microsatellite loci for the

Bulgarian durum wheat landrace collection, originated from five different regions (groups)

Item Region (Group)

Yambol (1) Elhovo (2) Topolovgrad (3) Purvomai (4) Svilengrad (5) Total

No. of accession 21 24 22 10 25 102

Total no. of alleles 56 67 60 45 51 85

No. alleles per marker 4.0 4.8 4.3 4.5 3.6 6.1

Gene diversity (He) 0.58 ± 0.23 0.59 ± 0.22 0.59 ± 0.23 0.45 ± 0.19 0.50 ± 0.19 0.53 ± 0.19

Obs. heterozygosity (Ho) 1.3 1.4 1.2 1.2 1.1 1.24

Genet Resour Crop Evol (2010) 57:273–285 277

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collection sites. The groups of accessions collected

from Yambol (1), Elhovo (2) and Topolovgrad (3)

showed similar average number of alleles per locus

(4–4.8) and He values (0.58–0.59; Table 4). Lower

average allele number per locus (3.6) was found in

the group of accessions from Svilengrad. The lowest

genetic diversity (He = 0.44) was detected in group

4 consisting of samples from Purvomai and Asenov-

grad, followed by the group 5 with mean He value of

0.50. The lower mean He values in the group 4

resulted from the reduced genetic diversity of all loci,

particularly evident in WMS577, WMS18, WMS631

and WMS357 (Table 7). Analysis of molecular

variance (AMOVA) of the microsatellite data showed

significantly higher values of the molecular variance

within the region (76%) as compared to that between

regions (5%) and population/region (19%; Fig. 2).

Amplification of more than one product occurring in

most of the Bulgarian landrace accessions indicated

their high heterogeneity. In total 32 rare alleles with

frequency less than 0.05 were identified in 11 out of

the 14 studied loci.

The data presented in Tables 5, and 6 clearly

demonstrate that the total number of alleles, the

average number of alleles per locus, the number of

rare and null alleles, and the mean heterozygosity are

considerably higher within landraces compared to the

commercial Bulgarian and introduced cultivars. The

highest total number of alleles was detected in

WMS577 locus in both landraces (11) and the

introduced cultivars (8), while in the group of modern

Bulgarian cultivars WMS389 locus showed the

highest number of alleles. The comparison of

Bulgarian landraces and modern cultivars showed

that breeding activities caused a decrease of the

number of private alleles and an increase of the

common alleles (Table 6). The percentage of poly-

morphic loci in modern cultivars was almost twofold

lower in comparison with the landraces. The

decreased number of alleles per locus results from

the small variation in all loci (Table 5). Only 3 loci

(WMS155, WMS46 and WMS577) showed consid-

erably reduced allele number.

The loci MW1B002, WMS408 and WMS680

showed similar pattern of allele size variation in

both Bulgarian and introduced varieties (Table 5).

Among the Bulgarian landraces the alleles 181 bp of

WMS18, 191 and 256 bp of WMS165 and 190 bp of

WMS631 were the most frequently observed (fre-

quency [ 0.80). The loci WM1B002, WMS389,

WMS155, WMS408, WMS46 and WMS577 showed

no clear dominating allele within the group of

landraces, suggesting that no selective pressure had

been exerted in the vicinity of these markers. On the

contrary, in the group of commercial cultivars one or

two alleles were prevailing in majority of loci.

The three groups of accessions showed similar gene

diversity (He = 0.51–0.53, Table 7). In the group of

landraces highest genetic diversity was observed in the

loci MW1A001 (He = 0.75), MW1B002 (He = 0.71)

and WMS155 (0.70), while in the group of modern

Bulgarian cultivars highest diversity was calculated for

the loci WMS46 (He = 0.81), MW1A001 (He =

0.79), WMS577 (He = 0.75). The loci WMS46

(He = 0.80), WMS577 (He = 0.77) were the most

diverse in the group of introduced cultivars. The lowest

He values were estimated for the locus WMS165 (0.11

and 0.15 in landraces and Bulgarian cultivars.

Cluster and ordination analysis

The employed 14 microsatellite markers successfully

distinguished 116 out of the 139 analyzed durum wheat

accessions (Fig. 3). The dendrogram consists of two

clusters and cluster I includes all accessions with the

exception of T. timopheevii which clusters as outgroup

into a second cluster. The cluster I is further divided

into 2 subclusters Ia and Ib. The subcluster Ib consists

of only Bulgarian landraces while Ia includes both

landraces and improved cultivars. Two additional sub

clusters (Ia1 and Ia2) are formed inside subcluster Ia.

The subcluster Ia1 includes the remaining landraces

together with all Bulgarian cultivars and few intro-

duced ones including Canadian cultivars G8972 AE3-

G, G8972 AE3-NG and G8972 AG2-NG and the

Percentages of Molecular Variance

AmongRegions

5%

Among

Pops/Regions

19%

Within Pops

76%

Fig. 2 Analysis of molecular variance among 137 samples

grouped by population and region

278 Genet Resour Crop Evol (2010) 57:273–285

123

French cultivar Agathe while subcluster Ia2 includes

all remaining introduced cultivars. The similarity

between subcluster Ia and Ib was 0.328 and between

subclusters Ia1 and Ia2 0.376. Although the landraces

separated into 2 distinct subclusters Ia and Ib, no

specific clustering according to collection site was

observed even when the SSR data of only landraces

was reanalyzed and a separate dendrogram was

Table 5 Total number of alleles, number of rare alleles, dominating alleles (bp) and their frequencies among the different groups of

T. durum Desf. Bulgarian landraces, Bulgarian cultivars and introduced cultivars

SSR Markers Chromosome Number of alleles

(rare alleles)

Size range of alleles (bp)/dominating

allele (bp) and its frequency

Bulgarian

landraces

Bulgarian

cultivars

Introduced

cultivars

Bulgarian

landraces

Bulgarian

cultivars

Introduced

cultivars

MW1A001 1A 7(2) 4(0) 4(0) 134–151

148(0.62)

142–148

151(0.78)

142–151

148(0.90)

WMS0357 1AL 4(0) 2(0) 2(0) 117–123

119(0.64)

117–119

119(1.00)

117–119

119(0.90)

MW1B002 1B 4(0) 3(0) 3(0) 214–247

247(0.36)

214–247

220(0.89)

214–247

247(0.67)

WMS0018 1BS 2(0) 2(0) 3(0) 179–181

181(0.85)

179–191

179(0.44)

179–181

179(0.67)

WMS0095 2AS 6(2) 4(0) 3(1) 114–124

116(0.60)

118–122

122(0.89)

105–122

118(0.77)

WMS0155 3AL 10(4) 3(0) 4(0) Null, 125–147

128(0.45)

125–141

128(0.78)

125–129

129(0.74)

WMS0389 3B 8(4) 6(0) 3(0) 116–133

127(0.46)

114–129

114;120(0.56)

114–131

127(0.53)

WMS0165 4A 2(1) 2(0) 2(0) 186–191

191(1.00)

186–191

191(0.89)

186–191

191(0.41)

WMS0165 4B 5(2) 2(0) 4(0) 252–264

256(0.85)

248–260

260(1.00)

256–264

260(0.56)

WMS0513 4BL 3(1) 2(0) 4(2) 140–144

142(0.78)

139–144

142(0.67)

142–144

142(0.50)

WMS0408 5BL 6(2) 3(0) 3(0) 144–180

145(0.45)

144–180

148(0.44)

146–180

145;148(0.29)

WMS0680 6B 5(2) 4(0) 3(0) 119–132

122(0.52)

120–132

122(0.78)

120–132

120(0.44)

WMS0631 7A 3(1) 2(0) 2(0) 190–200

190(0.88)

190–211

190(1.00)

190–211

190(0.79)

WMS0046 7BS 9(5) 4(0) 8(0) Null, 159–175

165(0.48)

Null, 163–177

171(0.67)

163–173

173(0.73)

WMS0577 7BL 11(6) 4(0) 8(0) Null, 128–213

141(0.43)

Null, 128–186

140(0.44)

130–211

152(0.35)

Total 85(32) 47(0) 56(5)

Average Total 6.07 3.13 4.00

A genome 4.17 2.83 2.83

B genome 5.89 3.33 4.33

Genet Resour Crop Evol (2010) 57:273–285 279

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produced (data not shown). Principal coordinate anal-

ysis (PCoA) confirmed the results obtained by the

cluster analysis. The landraces are clearly separated in

a distinct group and the modern cultivars also tend to

form specific groups according to their geographic

location (Fig. 4a). The 3D plot of PCoA on Figure 4b

shows no clear separation of the landraces collected

from different sites and confirmed the results obtained

in the cluster analysis.

Discussion

The microsatellite analysis of the durum wheat

collection, comprising Bulgarian landraces and

modern cultivars along with representatives from Italy,

France, Greece, Turkey, Russia, Jordan, USA, Canada,

Algeria and China showed that the average number of

alleles per locus and the average He values were

comparable with the data reported for durum wheat

accessions from various geographic regions (Messele

2001; Alamerew et al. 2004; Maccaferri et al. 2003; Li

et al. 2006; Khanjari et al. 2007; Wang et al. 2007). The

higher polymorphism of the B-genome markers in the

durum wheat from Libya (Ben Amer et al. 2001), Italy

and the Mediterranean (Maccaferri et al. 2003), Oman

(Khanjari et al. 2007) and Ethiopia (Huang et al. 2002;

Alamerew et al. 2004; Yifru et al. 2006b) was also

confirmed in the present study.

Table 7 The gene diversity (He) among study groups of T.durum Desf. Bulgarian landraces which were collected from

the region of Yambol (group 1); Elhovo (group 2),

Topolovgrad (group 3), Purvomai (group 4), Svilengrad (group

5) and Bulgarian and introduced cultivars

Marker Bulgarian landraces (He) Bulgarian

cultivars (He)

Introduced

cultivars (He)Region (Group) Average

Yambol

(1)

Elhovo

(2)

Topolovgrad

(3)

Purvomai

(4)

Svilengrad

(5)

MW1A001 0.79 0.77 0.83 0.65 0.72 0.75 ± 0.07a 0.79 0.53

WMS0357 0.55 0.65 0.65 0.29 0.57 0.54 ± 0.15b 0.01 0.13

MW1B002 0.86 0.68 0.74 0.63 0.64 0.71 ± 0.09a 0.48 0.58

WMS0018 0.35 0.30 0.35 0.16 0.38 0.31 ± 0.09c 0.43 0.49

WMS0095 0.73 0.56 0.74 0.61 0.60 0.65 ± 0.08a 0.58 0.51

WMS0155 0.72 0.79 0.77 0.61 0.63 0.70 ± 0.08a 0.60 0.44

WMS0389 0.71 0.58 0.77 0.61 0.71 0.67 ± 0.08a 0.66 0.59

WMS0165 0.20 0 0 0.36 0 0.11 ± 0.16d 0.15 0.53

WMS0165 0.09 0.51 0.44 0.20 0.34 0.32 ± 0.17c 0.44 0.55

WMS0513 0.36 0.53 0.47 0.28 0.42 0.41 ± 0.10 0.50 0.61

WMS0408 0.74 0.72 0.65 0.60 0.53 0.65 ± 0.06a 0.67 0.54

WMS0680 0.65 0.66 0.49 0.58 0.51 0.52 ± 0.08 0.58 0.60

WMS0631 0.52 0.48 0.42 0.16 0.29 0.31 ± 0.15 0.16 0.26

WMS0046 0.74 0.83 0.75 0.56 0.55 0.69 ± 0.12 0.81 0.80

WMS0577 0.66 0.80 0.82 0.44 0.58 0.66 ± 0.16 0.75 0.77

Total 0.58 ± 0.23 0.59 ± 0.22 0.59 ± 0.23 0.44 ± 0.19 0.50 ± 0.19 0.53 ± 0.19 0.51 ± 0.24 0.53 ± 0.17

Table 6 Total for Binary Band Pattern by Groups of Bulgar-

ian landraces and Bulgarian and introduced cultivars

Item Bulgarian

landraces

Bulgarian

cultivars

Introduced

cultivars

No. alleles 85 47 56

No. alleles freq. C5% 53 47 55

No. private alleles 31 2 1

No. common alleles

(B25%)

18 13 8

No. common alleles

(B50%)

31 24 18

Mean heterozygosity 0.135 0.117 0.082

SE of mean heterozygosity 0.015 0.016 0.015

Polymorphic loci (%) 76.36 40.00 22.97

280 Genet Resour Crop Evol (2010) 57:273–285

123

In our study, markers WMS577, WMS155 and

WMS46, which have been reported to be highly

polymorphic (Messele 2001; Yifru et al. 2006a, b)

showed highest allele number (WMS577-18 alleles;

WMS155 and WMS46-10 alleles).

The genetic diversity among Bulgarian landraces,

cultivated before the introduction of modern culti-

vars, has been influenced by various factors, in

particular, peoples migration, seed trade, and long-

term selection, carried out for centuries by farmers

under the specific environmental conditions,

characteristic for the Balkan Peninsula. During the

second half of the 7th millennium B.C. some tribes

left the Near East and moving across Anatolia had

settled on the Balkan Peninsula. It is well known that

around the beginning of the 6th millennium certain

agricultural practices were widely used in Greece and

by the end of the same millennium cereal stands

occupied the region of Starchevo, a Bulgarian village

near Danube. T. durum seeds were found in archaeo-

botanical samples together with T. monococcum and

T. dicoccon seeds (Popova and Bozilova 1998).

The local populations were described as a mixture of

botanical varieties instead of being homogenous

(Malkoff 1906; Ivanov 1927). In our sample, consist-

ing of 102 preserved ancient landrace accessions,

grown in 18 locations of Southern Bulgaria, we found

85 alleles, their number varying between 2 and 11 with

an average of 6.07 alleles per marker. This number is

lower than the average number of alleles per locus,

reported for durum wheat collections in Ethiopia

Fig. 3 UPGMA cluster analysis of all studied T. durumaccessions

BG_landraceSE_EUN_AmericaSW_EUW_AsiaRussiaT. dicocconT. timopheevii

0.33 0.33 0.08 0.08 PCoord.-2 PCoord.-2

-0.17 -0.17 -0.42 -0.42 -0.67 -0.67 -0.51 -0.51 -0.56 -0.56

-0.28 -0.28

-0.05 -0.05 -3 PCoord.-3

0.18 0.18

0.41 0.41

- -0.27PCoord.-1 0.02

0.32 0.61

Pop. 5Pop. 3Pop.2 Pop. 4Pop. 1

0.45 0.45 0.23 0.23 PCoord.-2 PCoord.-2

0.02 0.02 -0.20 -0.20 -0.45 -0.45 -0.42 -0.42 -0.56

-0.20 -0.20

0.04 0.04 PCoord.-3 PCoord.-3

0.29 0.29

0.53 0.53

-0.270.03PCoord.-1

0.320.61

a

b

Fig. 4 Principal Coordinates Analysis (PCoA) of a all 139

accessions including Bulgarian landraces, Bulgarian and

foreign durum wheat varieties and b only Bulgarian landrace

accessions

Genet Resour Crop Evol (2010) 57:273–285 281

123

(7.9–11.0), Oman (7.1) and Anatolia, Turkey (Dograr

et al. 2000; Messele 2001; Alamerew et al. 2004; Yifru

et al. 2006a; Khanjari et al. 2007). It is, however, higher

than that estimated for durum wheat from Libya

(4.5, Ben Amer et al. 2001) and Italy (4.3, Figliolo

et al. 2007), and similar to that characteristic for the

Mediterranean durum wheat germplasm (Maccaferri

et al. 2003). The gene diversity of the Bulgarian durum

wheat varies from 0.11 to 0.75 per marker (0.53 on

average) and is less than the corresponding values for

the Ethiopian (0.72, Yifru et al. 2006b), Anatolian

(0.76, Dograr et al. 2000), Tunisian (0.68, Medini et al.

2005) and Oman (0.68, Khanjari et al. 2007) durum

wheats, yet it is similar to that of the durum wheat

accessions from the Mediterranean region (0.56,

Maccaferri et al. 2003). All Bulgarian durum wheat

accessions, including both landraces and cultivars,

showed lower total number of alleles and genetic

diversity, especially at loci WMS18, WMS357 and

WMS408, as compared to samples from Ethiopia

(Yifru et al. 2006a, b).

According to Vavilov (1966), there is a global

irregular distribution of the species diversity. The

diversity is less expressed in Central and Northern

Europe. The comparative analysis of T. aestivum

microsatellite diversity in 8 geographical regions

showed greater diversity among the samples from the

Near and Middle East as well as Southern Europe

(Huang et al. 2002). A collection consisting of 349

T. durum accessions from all over the world, with loci

encoding 13 enzymes was assessed by Asins and

Carbonell (1989) and their results corresponded to

what was expected for the primary and secondary

centers of the genetic diversity described by Vavilov

(1951). According to Asins and Carbonell (1989), the

genetic variation among the durum wheats in the

Near East, a region considered to be the center of

primary evolution, proved to be greater than the one

in the secondary centers, namely the Mediterranean

(Egypt, Greece, Tunisia, Spain, France and former

Yugoslavia) and Central Asia. The presented refer-

ences on genetic diversity, together with our data,

obtained through microsatellite analysis, confirmed

the trend of polymorphism decrease in the durum

wheat germplasm when moving north-westwards

from the original center of domestication. Despite

this trend of decreasing the diversity being away from

the centre of wheat domestication, the old Bulgarian

durum wheat landraces were described as highly

heterogeneous populations (Ivanov 1927). This sug-

gestion was recently supported by the data on gliadin

polymorphism (Melnikova et al. 2007) and diversity

of spike morphology (Ganeva et al. 2005). The results

of the present study also indicate higher molecular

variance (76%) in the estimated regions than among

them (5%) or population /region (19%). However, a

trend for regional differentiation of the populations is

evident. The accessions from the regions of Purvomai

and Asenovgrad (group 4) had reduced genetic

diversity (He = 0.44). In the case of the samples

from Svilengrad (group 5) the microsatellite analysis

revealed lower polymorphism and an average number

of alleles for locus similar to that characteristic for

the Italian ancient durum wheat accessions (Figliuolo

et al. 2007). Asins and Carbonell (1989) reported a

surprisingly low diversity in durum wheat from

Israel, a region situated close to the original area of

domestication, as well as in durum wheat from Italy

and Cyprus—the main regions of cultivation. The

domestication is a fast process, which is a part of the

human historical development and is strongly deter-

mined by the selective pressure of the environment

and the farming techniques. Higher levels of genetic

diversity in a particular region are probably associ-

ated with a relatively stronger presence of rare alleles

resulting from restricted artificial selection applied in

those areas (Roussel et al. 2004). It can be assumed

that the reduced genetic diversity in countries with

traditional durum wheat agriculture is due to both

agro-ecological factors and more efficient and con-

sistent selection, performed by the farmers through-

out the years, including their efforts to improve

uniformity and productivity of local populations. It is

highly probable that the lower genetic diversity and

the increased allele frequency at some of the micro-

satellite markers reflect the selective impact of the

agro-ecological conditions and the directed selection.

Artificial selection aiming at developing homoge-

neous, highly productive and suitable for modern

agricultural technologies cultivars caused variation in

the degree and nature of the genetic diversity in the

34 modern Bulgarian and introduced cultivars

included in the present study. Reliable information

about the genetic erosion is afforded by the genetic

diversity index, combined with the characterization

of the allele polymorphism, specific and rare alleles

(Figliuolo et al. 2007). According to Maccaferri et al.

(2003), the large number of rare alleles implies

282 Genet Resour Crop Evol (2010) 57:273–285

123

genetic introgression, which in turn leads to an

increase in the germplasm diversity. Our study also

showed relatively large number (32 %) of rare alleles

amongst the samples of the Bulgarian durum wheat

landraces. Large number of rare alleles with fre-

quency below 0.05 was also reported by Maccaferri

et al. (2003) for durum wheat from Italy and the

Mediterranean (43%), by Wang et al. (2007) for

wheat samples from seven different countries

(36.78%) and by Yifru et al. (2006a, b), Roder

et al. (2002), Huang et al. (2002), and Roussel et al.

(2004) for samples from Ethiopia. Large number of

rare alleles was reported also for some Bulgarian

bread wheat cultivars (Landjeva et al. 2006; Tod-

orovska et al. 2005). The modern breeding has

eliminated those alleles in the newer cultivars thus

decreasing the level of polymorphism of the micro-

satellite markers among the commercial durum

cultivars. According to Frankel et al. (1995) the loss

of specific alleles is a direct indicator for genetic

erosion, indicating at the same time indirectly the

formation of unique gene combinations, related to the

process of adaptation.

Artificial breeding has not significantly altered the

genetic diversity (He = 0.51) as compared with the

landrace populations (He = 0.53; Table 6). How-

ever, in some microsatellite loci the effects of

selection pressure were evident. For example, the

He value at the locus WMS357 is considerably lower

in the modern Bulgarian (0.01) and introduced (0.13)

cultivars in comparison with the landraces (0.54). On

the contrary, the He values at loci WMS46 and

WMS577 are higher in the group of cultivars.

Artificial breeding was initiated in Bulgaria 80 years

ago with the first durum wheat cultivars being selected

from a local population. Later interspecific and inter-

cultivar hybridization as well as experimental mutagen-

esis was successfully applied (Filev 1990; Tsvetkov

1992; Yanev 2006; Bozhanova and Dechev 2002;

Bozhanova et al. 2004; Dechev 1996). The new cultivars

possess short stem and are resistant to lodging. The

number of durum wheat cultivars, released in Bulgaria is

much lower compared to the bread wheat, this explain-

ing the lower degree of diversity in T. durum. The

reported number of alleles per marker in some com-

mercial Bulgarian bread wheat cultivars (Todorovska

et al. 2005; Landjeva et al. 2006) is higher compared to

our results on durum wheat. An important difference

between the bread and the durum wheats is given by the

fact that the bread wheat contains a large number of rare

alleles. It is apparent that the newly created hard wheat

cultivars suffer from erosion in the genetic diversity and

the old forms are the important source for improving it.

This is particularly true for the grain quality and the

ecological adaptability.

The cluster and ordination analyses allowed to

separate the Bulgarian and introduced cultivars

according to agro-ecological conditions of their

region of origin. Although the genetic similarity

between Bulgarian cultivars and landraces was higher

compared to the introduced ones, the PCoA success-

fully separated the landraces into a distinct group,

suggesting introgression of introduced materials

during the breeding process. However, despite the

separation of the Bulgarian landraces into 2 subclus-

ters, nether cluster nor PCoA analyses revealed any

eco-geographic grouping within landraces. The latest

could be explained by the relatively small geographic

distances between the collection sites with similar

climatic condition and/or the possibility of trade and

exchange of seeds between the farmers. Huang et al.

(2002) and Khanjari et al. (2007) reported that not all

accessions from the same geographic region cluster in

the same group even for the regions larger than

described here. On the other hand Ben Amer et al.

(2001) showed that clustering of Libian genotypes

can be strongly related to the geographic region.

Taken together the above data one can speculate that

clustering according to geographic region could be

related not only to the eco-geografic conditions but

also to ability of people to freely migrate and trade

between the regions.

The results presented in this study confirm the

applicability of the microsatellite analysis for evalu-

ating the genetic diversity in wheat. This information

might contribute to the better preservation of the local

Bulgarian accessions and their further utilization in

the genetic and breeding programmes for improve-

ment of durum wheat. The study might be also useful

as a basis for further investigations on the origin,

domestication, and distribution pathway of T. durum

and its variability under certain ecological conditions.

Acknowledgments The investigation was supported by the

Bulgarian NCSR, contract CC-1415/04. The technical

assistance of Mrs K. Prokopova is greatly acknowledged.

Genet Resour Crop Evol (2010) 57:273–285 283

123

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