African Journal of Biotechnology Vol. 10(72), pp. 16167-16174, 16 November, 2011     
Available online at http://www.academicjournals.org/AJB 
DOI: 10.5897/AJB11.1749 
ISSN 1684–5315 © 2011 Academic Journals  
 
 
 
Full Length Research Paper 
 
Genetic diversity and relationship analysis among 
accessions of Aegilops ssp. in Turkey using amplified 
fragment length polymorphism (AFLP) markers 
 
Ilhan Kaya1,2*, Asude Çallak Kirişözü2, Figen Yildirim Ersoy3,2, Şahin Dere4,2 and Mahinur S. 
Akkaya2 
 
1
Department of Agriculture, Plant Protection, Van Yuzuncuyil University, Van, TR-65080, Turkey. 
2
Department of Chemistry, Biochemistry and Biotechnology Programs, Middle East Technical University,  
Ankara, TR-06531, Turkey. 
3
Department of Biology, Uludag University, Gorukle, Bursa, TR-16059. Turkey. 
4
Department of Field Crops, Agricultural Faculty, Ordu University, Ordu, TR-52200, Turkey. 
 
Accepted 7 September, 2011 
 
Amplified fragment length polymorphism (AFLP) DNA markers were used to assess the genetic 
diversity and relationships between 55 accessions of genus Aegilops, including the species Aegilops 
triuncialis L. (UUCC), Aegilops geniculata Roth (MMUU), Aegilops cylindrica Host (CCDD) and Aegilops 
umbellulata Zhuk (UU). The samples were collected from Aegean region and East Anatolia, Turkey. 16 
AFLP selective primer combinations generated a total of 3200 polymorphic amplification products. 50 
Aegilops accessions were analyzed using the data analysis software, unweighted pair-group method 
arithmetic average (UPGMA) method and numerical taxonomy and multivariate analysis system 
(NTSYSpc-2.02k). The similarity index coefficients were calculated according to simple matching 
coefficient. Using 16 AFLP primer combinations, species from Aegean region and east Anatolia were 
clustered as four major groups. Aegilops species having U genome clustered together and A. cylindrica 
host was out grouped. 
 
Keywords: Aegilops cylindrica, Aegilops triuncialis, Aegilops umbellulata, Aegilops geniculata, amplified 
fragment length polymorphism (AFLP), Li-COR, genetic relationship, unweighted pair-group method arithmetic 
average (UPGMA), principle coordinate analysis.   
 
 
INTRODUCTION 
 
The wild species of Triticeae family, especially the genus (Damania, 1993). Aegilops spreads mainly in central Asia 
Aegilops L. are valuable sources of genetic variation for and Mediterranean region (van Slageren, 1994). Turkey 
wheat improvement since they possess the genetic is the center of diversity for this genus and it is rich in wild 
background of all the cultivated wheat having still un- populations of tetraploid species: Aegilops triuncialis L. 
identified important characters such as resistance to (UUCC), Aegilops geniculata Roth (MMUU), Aegilops 
different biotic and abiotic stresses (Rekika et al., 1998; cylindrica Host (CCDD) and diploid species: Aegilops 
Zaharieva et al., 2004). The genus Aegilops L. has been umbellulata Zhuk. (UU). A. cylindrica and A. triuncialis 
the most intensively studied group of grasses, especially are widely distributed in Turkey adding up to 15 species 
since it is closely related to the cultivated wheat (van of Aegilops (Davis, 1985). Ecogeographic studies are 
Slageren, 1994). Aegilops ssp. is thought to be a genetic necessary as they determine the genetic relationship and 
reserve   for   the   improvement   of   the  wheat  cultivars  also guide conservation programs for the target plant 
 species (Anikster and Noy-Meir, 1991). DNA-based 
 molecular markers are particularly useful both for 
 quantifying genetic diversity within plant species and for 
*Corresponding author. E-mail: ilhank@yyu.edu.tr. Tel: +90 533 identifying and characterizing closely related genotypes 
323 5449. Fax: +90 432 225 1104. (Jasieniuk and  Maxwell,  2001).  Molecular  markers  can  
16168        Afr. J. Biotechnol.  
  
  
  
provide information needed to select genetically diverse diagnostics GmbH) in a final volume of 25 µl. Preamplification was 
parents for developing breeding and mapping popula- performed in a PTC-100 MJ Research Inc. thermocycler as in the 
following steps: 2 min 95°C, 20 cycles of 30 s at 94°C, 1 min at 
tions, among which the AFLP markers have been 
56°C, 1 min at 72°C and 4 min at 72°C. The preamplification mix 
successfully used to determine genetic diversity in many was diluted to 1/40 and 2 µl was added to the selective 
plant species (Sharma et al., 1996; Pillay and Myers, amplification reaction, containing 1 µM IRDye 700 labeled EcoRI 
1999).  primer A, 1 µM IRDye 800 labeled EcoRI primer B and 1 µM MseI + 
AFLP markers are generated by selective amplification 3 (Table 2), reaction buffer lX, 0.25 mM each dNTPs and 1 unit of 
of a subset of restriction fragments from total genomic Taq DNA polymerase in a final volume of 20 µl. Selective 
amplification was performed on a Stratagene Mx3005P Real Time 
DNA (Vos et al., 1995; Mueller and Wolfenbarger 1999). 
Thermal Cycler as follows: 13 touchdown cycles (- 0.7°C per cycle) 
The reproducibility, heritability, effectiveness and relia- of 30 s at 94°C, 30 s at 65°C, l min at 72°C; 23 cycles of 30 s at 
bility of these amplified DNA products have substantial 94°C, 30 s at 56°C, 1 min at 72°C and 10 min at 72°C. A total of 16 
advantages when compared with other marker systems selective primer combinations were used. 
(Russell et al., 1997). The PCR-based AFLP markers are The PCR products were separated by electrophoresis in a 6.5% 
amenable to automation for high-throughput genotyping polyacrylamide gel using the Li-COR 4300 DNA Analyzer and 
analyzed using the Saga Generation Software. Genetic similarity 
and, being anonymous, do not require any sequence and diversity analysis among 55 Aegilops varieties were performed 
information (Rouf Mian et al., 2002). AFLP fingerprinting using the data analysis software, UPGMA method and NTSYSpc-
is considerably informative, allowing the survey of 2.02k (Rholf, 1997). The similarity index coefficients were 
variation in more than 50 co-amplified restriction frag- calculated according to simple matching (SM) coefficient (Rholf, 
ments in each AFLP reaction (Incirli and Akkaya, 2001; 1997).  
Sudupak et al., 2004; Yildirim and Akkaya, 2006). Li-COR  
2
IR  automated DNA sequencers and associated software  
have been demonstrated to efficiently generate and RESULTS  
analyze complex AFLP patterns of various genomes (Qui  
et al., 1999; Remington et al., 1999). We applied AFLP Turkey is rich in tetraploid species: A. triuncialis L. 
markers to characterize the genetic diversity and (UUCC), A. geniculata Roth (MMUU), A. cylindrica Host 
relationships among different populations of Aegilops in (CCDD) and diploid species: A. umbellulata Zhuk. (UU). 
Turkey using Li-COR instrument.  Thus, it is important to find the genetic diversity of these 
 species in Turkey. 16 selective primer combinations 
 resulted in 3200 polymorphic bands to measure the 
MATERIALS AND METHODS genetic diversity within 55 accessions of Aegilops genus. 
 A dendrogram was generated from the data using the 
The materials of the study consist of A. cylindrica, A. geniculata, UPGMA and the program NTSYSpc 2.02k (Figure 1). 
A.truncialis and A. umbellulata gathered from the Aegean and the Principle coordinate analysis of the data was also 
Eastern Anatolia Regions in 2005. 50 individuals from a total of 11 
determined (Figures 2 and 3).  
populations belonging to these plants were collected (Table 1).  
The genetic diversity within the 55 accessions of A. 
 
triuncialis L. (UUCC), A. geniculata Roth (MMUU), A. 
 
DNA isolation  cylindrica Host (CCDD) and A. umbellulata Zhuk were 
 calculated using 16 selective primer combinations which 
The seeds of Aegilops were germinated and DNA was isolated from resulted in 3200 polymorphic bands (UU). In the tree, the 
the seedlings of two weeks old leaves starting with 200 mg young species sharing the U genome formed a main cluster and 
leaf tissue using a minor modified cetyl trimethyl ammonium A. cylindrica which is intensely associated with A. 
bromide (CTAB) method.  
squarrosa (DD), was clearly different from the other 
 
species, due to the influence of the presence of distant D 
 
AFLP analysis genome. This genome has undergone less divergence 
 than other diploid genomes during evolution and 
AFLP analysis was carried out according to Vos et al. (1995) using therefore appears to be less modified and is well 
fluorescently labeled primers and bands were detected using a Li- separated within the Triticeae (Damania, 1993; Badaeva 
COR automated sequencer (model 4300). All the chemicals and et al., 1996). 
enzymes apart from 10X reaction buffer and Taq DNA polymerase AFLP based UPGMA dendrogram of Aegilops acces-
were present in the kit provided by Li-COR (IRDye Fluorescent 
AFLP Kit for Large Plant Genome Analysis). Genomic DNA (200 sions is presented in Figure 1, in which there are four 
ng) was double digested with 1.5 units each of EcoRI and MseI main clusters: A. cylindrica (Ac), A. triuncialis (At), A. 
(MBI Fermentas) in a final volume of 20 µl and incubated at 37°C umbellulata (Au) and A. geniculata (Ag). The closest 
for 2 h. 7.5 and 75 pmols of adaptors EcoRI and MseI, respectively, genetic similarity between genotypes (0.980 simple 
were ligated to the resulting fragments (20 µl of the digestion mix) matching coefficient) was determined between Ag9 and 
using 1 unit of T4 DNA ligase (Roche diagnostics GmbH) in a final 
Ag10 genotypes. Other genetic similarity results are res-
volume of 25 µl buffer ligase 1X and incubated for 2 h at 25°C. The 
ligation mix was diluted 1/10 and 2.5 µl were added to the pectively as follows: the similarity between Au5 and Au6 
preamplification reaction containing AFLP Pre-amp primer mix, 1X (0.958 simple matching coefficient), the similarity between 
PCR    reaction   buffer,   2.5  U   Taq    DNA   polymerase    (Roche  At9   and  At10  (0.948  simple  matching  coefficient), the  
Kaya et al.         16169 
 
 
 
Table 1. The species of Aegilops L. collected from Turkey: sample numbers, locations, species and genomes. 
 
S/N Location (city) Coordinate Species Genome 
1 Uşak 38° 40.507N, 029° 18.648E, 928 m A. cylindrica CCDD 
2 Uşak 38°40.507N, 029° 18.648E, 928 m A. cylindrica CCDD 
3 Uşak 38°40.507N, 029° 18.648E, 928 m A. cylindrica CCDD 
4 Uşak 38° 40.507N, 029° 18.648E, 928 m A. cylindrica CCDD 
5 Uşak 38° 40.507N, 029° 18.648E, 928 m A. cylindrica CCDD 
6 Van 38° 33.794N, 043° 17.839E, 1672 m A. cylindrica CCDD 
7 Van 38° 33.794N, 043° 17.839E, 1672 m A. cylindrica CCDD 
8 Van 38° 33.794N, 043° 17.839E, 1672 m A. cylindrica CCDD 
9 Van 38° 33.794N, 043° 17.839E, 1672 m A. cylindrica CCDD 
10 Van 38° 33.794N, 043° 17.839E, 1672 m A. cylindrica CCDD 
11 Van 38° 31.868N, 043° 20.808E, 1671 m A. cylindrica CCDD 
12 Van 38° 31.868N, 043° 20.808E, 1671 m A. cylindrica CCDD 
13 Van 38° 31.868N, 043° 20.808E, 1671 m A. cylindrica CCDD 
14 Van 38° 31.868N, 043° 20.808E, 1671 m A. cylindrica CCDD 
15 Van 38° 31.868N, 043° 20.808E, 1671 m A. cylindrica CCDD 
16 Uşak 38° 40.507N, 029° 18.648E, 928 m A. triuncialis UUCC 
17 Uşak 38° 40.507N, 029° 18.648E, 928 m A. triuncialis UUCC 
18 Uşak 38° 40.507N, 029° 18.648E, 928 m A. triuncialis UUCC 
v
19 Uşak 38  40.507N, 029° 18.648E, 928 m A. triuncialis UUCC 
20 Uşak 38° 40.507N, 029° 18.648E, 928 m A. triuncialis UUCC 
v
21 Van 38  25.543N, 043° 15.695E, 1664 m A. triuncialis UUCC 
22 Van 38° 25.543N, 043° 15.695E, 1664 m A. triuncialis UUCC 
23 Van 38° 25.543N, 043° 15.695E, 1664 m A. triuncialis UUCC 
24 Van 38° 25.543N, 043° 15.695E, 1664 m A. triuncialis UUCC 
25 Van 38° 25.543N, 043° 15.695E, 1664 m A. triuncialis UUCC 
26 Van 38° 33.794N, 043° 17.839E, 1672 m A. triuncialis UUCC 
27 Van 38° 33.794N, 043° 17.839E, 1672 m A. triuncialis UUCC 
28 Van 38° 33.794N, 043° 17.839E, 1672 m A. triuncialis UUCC 
29 Van 38° 33.794N, 043° 17.839E, 1672 m A. triuncialis UUCC 
30 Van 38° 33.794N, 043° 17.839E, 1672 m A. triuncialis UUCC 
0
31 Van 38  25.544N, 043° 15.697E, 1664 m A. umbellulata UU 
32 Van 38° 25.544N, 043° 15.697E, 1664m A. umbellulata UU 
33 Van 38° 25.544N, 043° 15.697E, 1664m A. umbellulata UU 
34 Van 38° 25.544N, 043° 15.697E, 1664m A. umbellulata UU 
35 Van 38° 25.544N, 043° 15.697E, 1664m A. umbellulata UU 
v
36 Van 38  31.868N, 043° 20.808E, 1671m A. umbellulata UU 
37 Van 38° 31.868N, 043° 20.808E, 1671m A. umbellulata UU 
v 0
38 Van 38  31.868N, 043  20.808E, 1671m A. umbellulata UU 
39 Van 38° 31.868N, 043° 20.808E, 1671m A. umbellulata UU 
40 Van 38° 31.868N, 043° 20.808E, 1671m A. umbellulata UU 
41 Uşak 38° 40.476N, 029° 16.412E, 902 m A. umbellulata UU 
42 Uşak 38° 40.476N, 029° 16.412E, 902 m A. umbellulata UU 
43 Uşak 38° 40.476N, 029° 16.412E, 902 m A. umbellulata UU 
44 Uşak 38° 40.476N, 029° 16.412E, 902 m A. umbellulata UU 
45 Uşak 38° 40.476N, 029° 16.412E, 902 m A. umbellulata UU 
46 Izmir 38° 31.229N, 026° 37.433E, 16 m A. geniculata MMUU 
47 Izmir 38° 31.229N, 026° 37.433E, 16 m A. geniculata MMUU 
48 Izmir 38° 31.229N, 026° 37.433E, 16 m A. geniculata MMUU 
49 Izmir 38° 31.229N, 026° 37.433E, 16 m A. geniculata MMUU 
50 Izmir 38° 31.229N, 026° 37.433E, 16 m A. geniculata MMUU 
51 Uşak 38° 40.620N, 029° 22.638E, 904 m A. geniculata MMUU 
16170        Afr. J. Biotechnol. 
 
 
 
Table 1. Contd 
 
52 Uşak 38° 40.620N, 029° 22.638E, 904 m A. geniculata MMUU 
53 Uşak 38° 40.620N, 029° 22.638E, 904 m A. geniculata MMUU 
54 Uşak 38° 40.620N, 029° 22.638E, 904 m A. geniculata MMUU 
55 Uşak 38° 40.620N, 029° 22.638E, 904 m A. geniculata MMUU 
 
 
 
Table 2. MseI and IRDye 700 labeled EcoRI primers used in selective 
amplification reaction.  
 
Primer Flourescent label Sequence (5’-3’) 
M-CAA - GATGAGTCCTGAGTAACAA 
M-CAC - GATGAGTCCTGAGTAACAC 
M-CAG - GATGAGTCCTGAGTAACAG 
M-CAT - GATGAGTCCTGAGTAACAT 
M-CTA - GATGAGTCCTGAGTAACTA 
M-CTC - GATGAGTCCTGAGTAACTC 
M-CTG - GATGAGTCCTGAGTAACTG 
M-CTT - GATGAGTCCTGAGTAACTT 
E-AAC IRDye 700 GACTGCGTACCAATTCAAC 
E-AAG IRDye 700 GACTGCGTACCAATTCAAG 
E-ACA IRDye 700 GACTGCGTACCAATTCACA 
E-ACT IRDye 700 GACTGCGTACCAATTCACT 
E-ACC IRDye 800 GACTGCGTACCAATTCACC 
E-ACG IRDye 800 GACTGCGTACCAATTCACG 
E-AGC IRDye 800 GACTGCGTACCAATTCAGC 
E-AGG IRDye 800 GACTGCGTACCAATTCAGG 
 
 
 
similarities between Au3 and Au4, Ag7 and Ag9 (0.946 DISCUSSION 
simple matching coefficient). The least genetic similarity  
between genotypes was found between Au8 and Au9 Periodical genetic diversity assessments of all kinds of 
(0.800 simple matching coefficient). Christiansen et al. wild type plant species are very important for many 
(2002) and Lage et al. (2003) showed similar results and reasons, but it is more crucial for the wild types of wheat 
generally followed the trend that increased geographical since they are genome donors of cultivated wheat. Since 
distance correlates with increased genetic distance. the cultivated wheat has a very narrow genetic diversity, 
In the analysis of the dendrogram, with 2 and 3 for crop improvement, we always need to investigate the 
dimensional scaling; it was detected that the genotypes traits hidden in the wild types or the ancestors of the 
of Ag2, Ag5, Ag6, Au8 and Au9 had a different branching wheat, so that we can maintain the sustainable agri-
pattern from the one expected. Ac3, Ac13, At1, At2 and culture to feed ever increasing human population. Thus, 
At15 had different branching patterns and positions from by continual diversity assessment in nature, we can 
other genotypes. It was found that genotypes with high maintain the most diverse species in gene banks for 
similarity (Ag6, Ag7, Ag9 and Ag10) and genotypes conservation and crop improvement purposes. The 
gathered from similar populations (Au5, Au6 and At9, effects of environmental changes or climate fluctuations 
At10) had the same branching pattern and positions in on the natural diversity can also be traced by continual 
general (Figures 2 and 3). Since the genus Aegilops L. is analyses. This is especially the case for Aegilops species 
a valuable source of genetic variation, the data presented in Turkey since the land is the center of origin for these 
here might have advantage in various studies in future. Aegilops species.  
 
 
Kaya et al.         16171 
  
 
   
Ac1 
Ac2 
Ac3 
Ac4 
Ac5 
Ac6 
Ac7 
Ac8 
Ac9 
Ac10 
Ac11 
Ac12 
Ac13 
Ac14 
Ac15 
At1 
At2 
At3 
At4 
At5 
At6 
At7 
At8 
At9 
At10 
At11 
At14 
At12 
At13 
At15 
Au1 
Au2 
Au3 
Au4 
Au5 
Au6 
Au7 
Au10 
Au9 
Au11 
Au12 
Au13 
Au14 
Au15 
Ag1 
Ag2 
Ag3 
Ag6 
Ag7 
Ag9 
Ag10 
Ag8 
Ag4 
Ag5 
Au8 
 
Coefficient  
  
  
Figure 1. AFLP-based UPGMA dendrogram of Aegilops accessions. 
16172        Afr. J. Biotechnol. 
 
 
 
 
 
Figure 2. 2D plot generated by principle coordinate analysis. 
Kaya et al.         16173 
 
 
 
 
 
Figure 3. 3D plot of the principal coordinate analysis of the AFLP data generated by NTSYS. 
 
 
 
AFLP marker system to test the genetic diversity is one of molecular biology and genomics technologies. Weed Sci. 49: 257-
mostly used marker system since, it is multi-locus, thus 265. 
Lage J, Warburton ML, Crossa J, Skovmand B, Andersen SB (2003). 
highly polymorphic, reproducible and high through-put Assessment of genetic diversity in synthetic hexaploid wheats and 
instrumentation is available, such as Li-Cor. Thus, in this their Triticum dicoccum and Aegilops tauschii parents using AFLPs 
study, we preferred to perform analysis using AFLP and agronomic traits. Euphytica, 134: 305-317.  
highly polymorphic marker system. We believe the data Mueller UG, Wolfenbarger LL (1999). AFLP genotyping and 
fingerprinting. Trends Ecol. Evol. 14: 389-394.  
presented here will be a tool for other wheat researchers.  Pillay M, Myers GO (1999). Genetic diversity in cotton assessed by 
 variation in ribosomal RNA genes and AFLP markers. Crop Sci. 39: 
 1881-1886. 
Qui J, van Santen E, Tuzun S, Campos-Andrada M (1999). Genetic 
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