ISSN 10227954, Russian Journal of Genetics, 2015, Vol. 51, No. 9, pp. 863–870. © Pleiades Publishing, Inc., 2015. Original Russian Text © E.S. Hajiyev, Z.I. Akparov, R.T. Aliyev, S.V. Saidova, V.I. Izzatullayeva, S.M. Babayeva, M.A. Abbasov, 2015, published in Genetika, 2015, Vol. 51, No. 9, pp. 1009–1017.
Genetic Polymorphism of Durum Wheat (Triticum durum Desf.) Accessions of Azerbaijan E. S. Hajiyev, Z. I. Akparov, R. T. Aliyev, S. V. Saidova, V. I. Izzatullayeva, S. M. Babayeva, and M. A. Abbasov Genetic Resources Institute of Azerbaijan National Academy of Sciences, Baku AZ 1106, Azerbaijan email: [email protected]
Received November 7, 2014
Abstract—The genetic diversity of 110 durum wheat genotypes of Azerbaijan was evaluated by ISSR markers. A total of 107 fragments were determined, ranging from 9 to 18 per locus. ISSR primers have revealed a high level of polymorphism (average 82%) among different durum wheat varieties and botanical varieties. The ISSR markers used in the study were quite informative and made it possible to distinguish all durum wheat accessions from each other. Cluster analysis based on ISSR data classified the accessions into 11 major groups. No linkage was observed between the collection site and genetic structure of the samples. On the other hand, a few accessions were detected as unique genotypes and tended to form separate clusters. The estimated gene diversity value was high, both within the whole collection and within the different groups of botanical varieties. DOI: 10.1134/S1022795415090045
INTRODUCTION Crop cultivation, among which wheat plays the key role, is a highpriority agricultural goal in Azerbaijan. The unique diversity of soils and climatic conditions in this region favored the development of rich plant cover and allowed the inclusion of Azerbaijan on the list of most likely centers of wheat origination . Tradi tional wheat selection has been carried out in Azer baijan since ancient times. Breeds such as Sari bugda, Gara bugda, Ag bugda, and Girmizi bugda have been cultivated for ages. The collection of wheat species, subspecies, and populations that are widespread in Azerbaijan has been successfully performed. The National GenBank presently contains more than 2000 wheat specimens [2, 3]. It is exceptionally interesting to study the DNA polymorphism of durum wheat cul tivated in different regions of Azerbaijan because of the diversity of crop cultures bred in this region. Durum wheat (Triticum durum Desf.) is character ized by a high level of polymorphism. By the diversity and number of ecological types and breeds, it is second only to soft wheat . New breeds containing over whelming genetic diversity that accumulated via thou sands years of selection have been developed. We need some knowledge about its geographical distribution for rational preservation and effective use of this genetic diversity. First, we have to pay special attention to the preservation of unique genotypes. Thus, we need to know in which regions unique species of durum wheat, which stand out against the global geno fond, may be found. Genetically, durum wheat still remains a relatively poorly studied species, because
most genetic studies of crops deal with soft wheat. It is only recently that some studies have reported on the genetic marking of durum wheat breeds carried out on the basis of PCR analysis, as well as their origination and biodiversity [5, 6]. The use of genetic markers may help solve many problems of applied selection in Azerbaijan. It is com mon knowledge that durum wheat selection is extremely laborious, because realization of the genetic potential of a plant very much depends on the condi tions of its cultivation . The use of the most recent achievements of modern genetics, particularly meth ods of genetic marking, will provide more effective selection in order to develop new breeds and improve the properties of the old ones. Moreover, it may help solve a series of fundamental problems, such as the investigation of the genetic diversity of modern breeds, the identification and mapping of agriculturally valu able traits and identification of their carriers as the ini tial material for hybridization, simplification of the finding and removal of duplicates in germoplasm banks, the passport issue, and the classification of plant breeds and genetic monitoring in the selection and genetics of agricultural plants. Molecular genetics methods, which are based on an analysis of DNA polymorphisms (RAPD, AFLP, ISSR, SSR, SNP, etc.) and make it possible to obtain individual characteristics (DNAprofile) of an indi vidual genotype, is considered to be the most promis ing approach to the analysis of genetic diversity [8– 10]. We conducted ISSR analysis (Inter Simple Sequence Repeat) in order to study the interbreed
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polymorphism and reveal the genetic similarities of different breeds of wheat. ISSR analysis is presently widely used to reveal intraspecies polymorphisms, pri marily in closely related genotypes of cultivated plants [11, 12]. ISSR markers are highly polymorphous and may be used in studies of genetic diversity and phylog eny and the molecular mapping of crop plants . ISSR primers may be represented by di, tri, tetra, or pentanucleotide microsatellite motives providing a variety of amplification products [14, 15]. Our goal was to assess the genetic diversity of 110 durum wheat genotypes from Azerbaijan by molecular analysis, to estimate the geographical distribution of genotypes, and to analyze their genetic differentiation. We anticipate that the revealing of genetic similarities and differences between genotypes will allow us to make a more successful choice of pairs for crossbreed ing, which will eventually result in an even faster devel opment of breeds and hybrids with a complex of valu able traits.
multilocus intermicrosatellite analysis. Electrophore sis of PCR products was carried out in 2% agarose gel supplemented with ethidium bromide. Visualization of the gels was performed in UV light with a geldocu menting system (BioRad, United States). Statistical Analysis Analysis of the amplified fragments was performed with the computer program PAST . To reveal the level of marker polymorphisms between the studied genotypes, the data were represented as a matrix of binary trait conditions in which the “presence” and “absence” of the PCR fragments of amplicons of sim ilar sizes were expressed as 1 and 0 respectively. The number and portion of polymorphous loci, as well as the genetic diversity index, were estimated as indicators of genetic diversity. The genetic diversity coefficient was calculated as described in : n
MATERIALS AND METHODS Plant Material The objects of molecular genetic study were 100 durum wheat specimens obtained from the GeneBank of the Genetic Resources Institute of Azerbaijan National Academy of Sciences (Baku, Azerbaijan) and 10 local breeds (Sherg, Terter, Vugar, Ag bugda, Bereketli 95, Mirbeshir 50, Garagilchig 2, Mugan, Shiraslan, Shirvan) obtained from different regions of Azerbaijan. The largest numbers of specimens were collected for the following species: var. apulicum (12), var. leucomelan (15), var. hordeiforme (19), and 27 gen otypes of var. leucurum (Table 1). Molecular Analysis The DNA was isolated in accordance with the CTAB (cetyltrimethylammonium bromide) protocol provided by Doyle et Doyle  with some modifica tions. The concentration and purity of the obtained DNA was estimated with NanoDrop 2000 (Thermo, United States). DNA amplification was performed in 20 µL of reaction mixture, which contained 2 µL of 10× PCR buffer, 2 µL dNTP mixture (5 mM), 1.5 µL MgCl2 (50 mM), 2 µL of each primer (15 pmole/µL), 0.1 µL Taqpolymerase (1 U/µL), and 2 µL of isolated DNA (50 ng/µL). Optimization was carried out, and the following amplification conditions were chosen: preliminary denaturation at 94°C for 5 min and 35 PCR cycles (denaturation at 94°С for 1 min, primer annealing for 45 s, the temperature of primer annealing depended on the type of the primer used, elongation at 72°C for 5 min, final elongation cycle at 72°C for 10 min). Amplification was performed in a T100 programmed amplifier (Applied Biosystems, United States). Eight polymorphous ISSR primers consisting of 15–18 nucleotides were tested for the
∑p , 2 i
where H is the genetic diversity index and pi is the allelic frequency. Diagrams were composed and the genetic similar ity between the samples was assessed by the Jaccard similarity coefficient. Clusterization was carried out with the PAST program. RESULTS AND DISCUSSION The analysis of polymorphisms of 110 genotypes of durum wheat was performed on 8 of the 20 primers tested, which provided stable amplification and most repeatable results. The main zone of fragment distri bution was located in the diapason of 100–2000 bp. The amount of amplification products sufficient for further analysis of the genetic diversity of durum wheat genotypes was obtained. In total, 107 PCR fragments were identified. The biggest number of DNA amplifi cation products was obtained in the reactions with primers UBC841 and UB857C (17 and 18 respec tively) (Table 2). In ISSR analysis, one primer on aver age initiated the synthesis of 13.3 fragments. Gelelec trophoresis of the PCRprofiles obtained with ISSR primers showed that some genotypes carried specific fragments of different length. Primer UBC841 synthe sized a 110 bp. fragment for the breed Bereketli and var. leucomelan (6145) only. Amplification with the primer UBC112 resulted in the occurrence of a 700bp. ISSR fragment in genotypes var. hordeiforme and Mirbeshir 50. PCR with the primer UBC873 led to the formation of 980bp. amplification fragment in the var. leucomelan (6145) genotype only. Therefore, the genotypes mentioned were identified as unique, and the primers UBC112, UBC841, and UBC873 are considered to be optimal for their identification.
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Table 1. List of samples used in the study and their geographical distribution GeneBank number 6152 6101 6304 6109 6123 6086 6157 6136 6148 6087 6133 6154 6158 6089 6231 6114 6134 6150 6129 6141 6108 6088 6308 6118 6142 6153 6131 6093 6116 6110 6103 6091 6127 6100 6105 6315 6166 6312 6305 6161 6126 6111 6311 6140 6106 6162 6313 6139 6125 6165
Sample name var. apulicum var. leucurum var. leucurum var. hordeiforme var. melanopus var. leucurum var. apulicum var. leucomelan var. apulicum var. leucurum var. alborovinciale var. apulicum var. obscurum var. leucurum var. leucurum var. hordeiforme var. alborovinciale var. apulicum var. melanopus var. leucomelan var. hordeiforme var. leucurum var. leucomelan var. hordeiforme var. leucomelan var. apulicum var. murciense var. leucurum var. hordeiforme var. hordeiforme var. hordeiforme var. leucurum var. melanopus var. leucurum var. hordeiforme var. leucurum var. niloticum var. leucurum var. leucurum var. erythromelan var. melanopus var. hordeiforme var. leucomelan var. leucomelan var. hordeiforme var. erythromelan var. murciense var. leucomelan var. melanopus var. niloticum
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Geographical origination Nakhichevan Tovuz Absheron Agdam Tovuz Kazakh Nakhichevan Devichi Ismailli Agdara Dzhalilabad Nakhichevan Zakatala Evlakh Dzhalilabad Evlakh Masalli Mingichavur Nakhichevan Nakhichevan Barda Evlakh Absheron Nakhichevan Masalli Nakhichevan Shamakhi Geokchay Nakhichevan Mingichavur Sheki Khachmaz Barda Barda Terter Absheron Dzhalilabad Absheron Absheron Shamakhi Terter Akstafa Absheron Kazakh Terter Shamakhi Absheron Bilesuvar Mingichavur Agsu Vol. 51
GeneBank number 6144 6138 6130 6309 6135 6121 6160 6310 6156 6314 6119 6128 6124 6098 6318 6099 6147 6232 6155 6159 6094 6117 6115 6112 6096 6145 6095 6146 6163 6316 6122 6120 6143 6104 6307 6102 6090 6113 6164 6137 6317 6306 6092 6097 6319 6132 6107 6085 6151 6149 2015
Sample name var. leucomelan var. leucomelan var. murciense var. leucomelan var. leucomelan var. boeufii var. reichenbachii var. leucomelan var. apulicum var. leucurum var. hordeiforme var. melanopus var. melanopus var. leucurum var. leucurum var. leucurum var. apulicum var. erythromelan var. apulicum var. reichenbachii var. leucurum var. hordeiforme var. hordeiforme var. hordeiforme var. leucurum var. leucomelan var. leucurum var. apulicum var. erythromelan var. caerulescens var. boeufii var. hordeiforme var. leucomelan var. hordeiforme var. hordeiforme var. leucurum var. leucurum var. hordeiforme var. affine var. leucomelan var. eucurum var. leucurum var. leucurum var. leucurum var. alborovinciae var. murciense var. hordeiforme var. leucurum var. apulicum var. apulicum
Geographical origination Lerik Evlakh Saatli Absheron Agdzhabadi Shamakhi Zakatala Absheron Nakhichevan Absheron Nakhichevan Kazakh Geranboy Agdash Absheron Khanlar Agsu Dzhalilabad Nakhichevan Zakatala Agsu Nakhichevan Nakhichevan Shemkir Agsu Nakhichevan Agsu Barda Shamakhi Absheron Masalli Nakhichevan Nakhichevan Terter Absheron Shamakhi Terter Gakh Shamakhi Saatli Absheron Absheron Agdam Agsu Absheron Ismailli Shamakhi Akstafa Nakhichevan Khanlar
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Table 2. ISSR primers and their statistical parameters Primers UBC112 UBC808 UBC811 UBC827 UBC841 UBC857 UBC864 UBC873 Total estimate Average estimate
Sequence (5'–3') (GACA)4 (AG)8C (GA)8C (AC)7G G(ACA)4 (GC)3C (AC)8TT TAG(GT)6GAA (GACA)4
Number of fragments
Percentage of polymorphism
Genetic diversity index
9 13 12 16 17 18 11 11
8 11 10 12 14 16 7 9
88.9 84.6 83.3 75.0 82.4 88.9 63.6 81.8
0.96 0.82 0.97 0.96 0.96 0.98 0.88 0.97
Our observations showed that the majority of amplified fragments were characterized by a high level of polymorphism. The frequency of polymorphous fragments varied from 63.6 to 88.9%, and its average value was 82%, which was considered to be rather high. The average number of polymorphous fragments identified with each primer was 10.9. The data are consistent with Sofalian et al. , who studied 39 local wheat breeds in Iran using 15 ISSR markers and revealed a high level of polymorphism (82.2%). It has been suggested that the high level of polymorphism is caused by fast changes in microsatellite sequences due to mistakes that take place during DNA replication and single base modifications . However, a study of 51 breeds of durum wheat belonging to 26 different botanical species in Portugal was carried out by Car valho et al.; the study revealed a low level of polymor phism (42.1%) . Song et al. showed that the polymorphism level increases with an increase in the number of motives with three or four nucleotides contained in the frag ment . Moreover, Nagoaka and Ogihara observed the maximal level of polymorphism with respect to the primers carrying tetranucleotide repeats in hexaploid wheat . In our study, primer UBC811, which contained the (GA)n repeat, revealed a medium polymorphism level (83.3%). The study of soya carried out by Akkaya et al. showed a low polymorphism level with primers con taining the (GA)n repeat . Primers UBC827 and UBC857, which carried the (AC)n repeat, were revealed to have both low and high polymorphism lev els (75 and 88.9% respectively). Nagoaka and Ogihara showed that most polymorphisms may be identified with primers containing the (AC)n repeats. Two prim ers, UBC112 and UBC873, which carried the four nucleotide repeats (GACA)n, revealed high and medium polymorphism levels (88.9 and 81.8% respec tively). Primer UBC864, which carried the (GT)n
repeat, revealed a comparatively low polymorphism level (63.3%). In the present work, we studied the genetic diversity index for each ISSR locus, which varied from 0.82 to 0.98 had an average value of 0.94. The high value of the genetic diversity index may be explained by the involvement of selection material from different geo graphical regions of Azerbaijan. Primers UBC857, UBC811, and UBC873 revealed the highest levels of genetic diversity (Table 2). It is important to mention primer UBC857, which showed an increased poly morphism level (88.9%) along with a high genetic diversity index (0.98). Amplification with this primer also provided the biggest number of PCR loci. In accordance with the above, the prevailing genotypes in the collection were studied: var. leucomelan, var. leucu rum, var. hordeiforme, and var. apulicum. A high genetic diversity index (0.88) was observed in 19 geno types of var. hordeiforme (Fig. 2). var. melanopus dem onstrated quite a high genetic diversity index (0.83), in spite of the small sample of its genotypes. This shows the high genetic diversity of this selection material. The studied samples of var. melanopus were cultivated in certain territories (collection zones) that differed from one another by soil and climatic conditions. This factor apparently facilitated genetic differentiation. Therefore, the primers selected for our study allowed us to identify effectively the genetic diversity of durum wheat genotypes and may be recommended for applied selection. The data are consistent with pre vious ISSR analyses of wheat genotypes. For example, Pasqualone et al.  analyzed the efficacy of ISSR markers, which were used to reveal differences between 30 breeds of Italian local durum wheat and 22 selectively obtained lines. It was shown that the effi cacy of their primers was so high that two primers were enough to reveal the differences among all of the stud ied specimens . Sofalian et al.  showed that ISSR markers can be successfully used to assess the genetic polymorphism of wheat genotypes. Assess
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6166 Terter 6132 Mugan 6305 6161 6126 6111 6311 6140 6106 6162 6313 6139 6125 6165 6144 6138 Mirbeshir 50 6130 6309 6135 6121 6160 6310 6156 6314 6119 6128 6124 6098 6318 6099 6147 6232 6155 6159 6094 6117 6115
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1000 900 800 700 600 500 400 300 200 100
Fig. 1. PCR profiles of durum wheat genotypes obtained with the primer UBC112. The arrow indicates the presence of the frag ment specific for genotypes var. hordeiforme (6305) and Mirbeshir 50.
va r. m ela n
va r. leu cu ru m
va r. ho rd eif or m e va r. leu co m ela n
va r. ap ul ic
Fig. 2. Genetic diversity of durum wheat genotypes revealed by ISSR analysis.
ment of the genetic differences and similarities of gen otypes may be useful for their differentiation. Najaphy et al. showed that ISSR markers provide sufficient polymorphism for an assessment of the genetic diver sity of wheat genotypes. The observed molecular vari ability, together with agronomic and morphological traits of wheat, may be useful for both traditional and molecular selection programs. It is known that the goals of cluster analysis of wheat are to study genetic relations and identification, to develop databases of agricultural breeds at the molecular genetic level, etc. . We carried out clus ter analysis and reconstructed a tree that demonstrates the genetic relations among the specimens (Fig. 3). Grouping of the durum wheat specimens in the accepted tree allowed us to form 11 basic clusters and revealed the complex character of the distribution of the studied breeds and variations. Each cluster united different variations of genotypes collected in different regions of the country. Cluster 8 was the largest. It RUSSIAN JOURNAL OF GENETICS
united 36.6% of all of the studied specimens. Of 40 genotypes in cluster 8, var. leucurum, var. hordei forme, and var. apulicum were the prevailing. More over, five independent clusters were identified. Geno types var. apulicum from Khanlar region (6149) and Agsu region (6147) formed clusters 1 and 11 respec tively, var. alborovinciale from Absheron region formed cluster 2, var. leucurum from Agsu region was sepa rated into cluster 5, var. murciense from Shamakhy region was located in cluster 9. This demonstrates the uniqueness of these genomes among the genotypes studied. The most remote was var. apulicum from Agsu region (6147). It stands completely apart from all of the other specimens in this cluster. The smallest clus ter was cluster 6, which was represented by two geno types only: var. erythromelan (6161) from Shamakhy region and var. melanopus (6126) from Terter region. It is noteworthy that these territories are characterized by similar soil and climatic conditions. This, in our opinion, may favor similarity of their genetic organi 2015
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Cluster 1 Cluster 2
6149 6319 6156 6119 6128 6124 6138 6155 6159 6232 6099 6166 Bereketli 95 6104 6143 6146 6122 6316 6163 6112 6096 6137 6095 6094 6161 6126 Mirbeshir 50 6144 6309 6139 6313 6165 6314 6130 6305 6098 6318 6121 6160 6135 6310 6125 Мugan 6312 6162 6106 6140 6111 6311 Garagilchig 2 6152 6101 6148 6158 6118 6088 6141 6308 6116 6100 Akrugda 6105 Shirvan 6150 6142 6133 6154 6087 6089 6231 6153 6093 6103 6108 Sherg 6091 6127 6110 6134 6114 6129 6136 6157 6123 6109 6086 6315 Terter 6304 6131 6151 6085 6107 6132 6145 6117 6115 6102 Vugar 6120 6307 Shiraslam 6317 6306 6090 6097 6194 6113 6092 6147
Cluster 4 Cluster 5 Cluster 6
Cluster 9 Cluster 10
Fig. 3. Dendrogram composed on the basis of ISSR analysis and Jaccard similarity coefficient. RUSSIAN JOURNAL OF GENETICS
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zation. We observed some tendency toward an associ ation of variations originating from the same region. For example, variations of var. leucurum (6317 and 6115) originating from Absheron region were included into cluster 10, which is considered to be quite logical. The same situation was observed for the specimens of var. hordeiforme (6117 and 6115), which originated from Nakhichevan region. Apart from the variations, each cluster united different breeds of durum wheat. Cluster 8 grouped 5 of 10 breeds of durum wheat (Garagilchig, Ag bugda, Sherg, Shirvan and Terter). Other breeds were spread throughout different clus ters. This apparently may be explained by special fea tures of the selection process, because ISSR loci were not the object of the selection that we carried out. Therefore, based on the above, the ISSR dendrogram may be characterized as follows. (1) The genotype distribution has a complex character. (2) There is no clear connection between the genetic structure of genotypes and their geographical distribution. In other words, the differentiation of variations and breeds of durum wheat observed in our study did not have a geographical trend. (3) High genetic diversity is observed, both in the whole collection and inside the clusters formed. (4) Durum wheat samples with unique genetic organization were identified. Genetic relations between different genotypes of wheat were observed in many studies. For example, Carvalho et al.  analyzed 48 breeds of soft wheat from the Old Portuguese Collection by 18 ISSR mark ers. It was found that most cultures that belonged to the same botanical species were grouped in one cluster . These results are also consistent with the data of Malik et al., who conducted a cluster analysis of 27 genotypes of soft wheat . It was shown that all gen otypes were united in one group in accordance with their origination. Therefore, we may conclude that ISSR markers may be effectively used to assess the genetic diversity of durum wheat genotypes. Genotypes found by cluster analysis are considered to be the most remote from other specimens. These genotypes may be recom mended for selectionists in order to obtain genetically diverse populations characterized by the maximal spectrum of variation in the hybrid progeny. ACKNOWLEDGMENTS This work was supported by Fund for Scientific Development under the support of the President of the Republic of Azerbaijan, project no. EI·F20122(6) 39/03/3. REFERENCES 1. Vavilov, N.I., Nauchnye osnovy selektsii pshenitsy (Sci entific Bases of Wheat Breeding), Leningrad: Nauka, 1967. RUSSIAN JOURNAL OF GENETICS
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Translated by M. Bibov