Abstract: Germplasm characterization is essential and molecular markers provide valuable information for breeding programs. Sequence-Related Amplified Polymorphism (SRAP) and phenotypic markers were studied to determine diversity and relationships among 40 subconvares of Triticum durum landraces from the region of North West of Iran and Azerbaijan. The 12 combinations of forward and reverse SRAP primers were used to evaluate the 38 landraces and two cultivars and produced 65 scorable markers, of which 56.730% was polymorphic for all 40 genotypes. As to phenotypic markers, 27 quantitative traits were evaluated in field with 4 replications, 22 of them (81%) were found to be polymorphic. The UPGMA (UN weighted-Pair Group Method Arithmetic average) dendrogram based on the 27 phenotypic markers distinguished all genotypes, but failed to detect any geographic association in durum landraces. The UPGMA dendrogram based on the 12 combinations of SRAP markers distinguished landraces, it was concurs with subconvars grouping characteristic (traits of major gene). SRAP markers are useful for studying diversity and relationships among and have potential in marker-aided selection, linkage mapping and evolutionary studies.

INTRODUCTION

Landraces or traditional cultivars are locally adapted diverse populations, which are the result of natural selection and farmers' cultivation methods. They have contributed genetic material to many breeding programs and constitute important plant genetic resources. Landraces have attracted the scientific interest because of the presence of genetic variability in well-adapted backgrounds. Considerable concerns have emerged, however, because of the quick rate at which landraces disappear due to their replacement by commercial cultivars (Esquinas-Alcazar, 1993). The estimation of genetic diversity of landraces is necessary because it determines significance of the gene pool and the manner in which it can be utilized. Measures of genetic diversity based on the molecular markers can be used on the predicted progeny performance in various crops (Autrique et al., 1996) and the molecular methods can be employed to better characterize genetic reassures. The genetic variability in agronomic traits and morphological characteristics of various durum wheat landraces have been studied (Jaradat, 1991; Autrique et al., 1996; Pecetti et al., 2001). Biochemical markers such as isozymes and seed storage proteins Payne (1987), DNA-based markers, such as Restriction Fragment Length Polymorphisms (RFLPs), Amplified Fragment Length Polymorphism (AFLPs) and Random Amplified Polymorphic DNA (RAPD) have been used to evaluate the genetic diversity among durum wheat landraces (Mantzavinou et al., 2005; Pujar et al., 1999; Pecetti et al., 2001; Autrique et al., 1996). Other molecular markers, such as microsatellites or Simple Sequence Repeats (SSRs) and AFLPs have also been used to study genetic relationships among durum wheat cultivars (Dograr et al., 2000; Soleimani et al., 2002). Among all these molecular markers, RAPDs and SRAPs, which are simple, user friendly, cost-and time-effective (Williams et al., 1990; Gulsen et al., 2007; Fufa et al., 2005), have been used successfully for the evaluation of plant genetic resources in wheat genotypes plant materials (Mukhtar et al., 2002; Mantazavinou et al., 2005). Sequence-Related Amplified Polymorphism (SRAP) markers were recognized as a new and useful molecular marker system for mapping and gene tagging in Brassica (Li and Quiros, 2001). SRAP markers are PCR_based markers that amplify open reading frames and produce a number of co-dominant markers per amplification. SRAPs use forward and reverse primers, 17 or 18 nucleotides long and primers consist of a core sequence of 13 or 14 bases, at the 5’, CCGG in the forward primer and AATT in the reverse primer, targeting open reading frames in genomic sequences. This core sequence is followed by three selective nucleotides at the 3’ end of each primer. SRAP markers are more consistent and repeatable than RAPDs and are less labor-intensive and time-consuming to produce than amplified-fragment length polymorphism techniques (Welsh and McClelland, 1990; Li and Quiros, 2001; Ferriol et al., 2003; Budak et al., 2004a-c; Gulsen et al., 2005, 2007). The SRAP marker technique that uses facile sequencing of selected bands is a simple and reliable method. Further, it targets coding sequences in the genome and results in a moderate number of co-dominant markers (Li and Quiros, 2001). SRAP markers possess multiloci and multi-allelic features, which make them potentially more efficient for genetic diversity analysis, gene mapping and fingerprinting genotypes. However, SRAP markers may not be randomly distributed across the genome (Li and Quiros, 2001). Limited information is available on the chromosomal locations of SRAP markers (Fufa et al., 2005), their linkage with plant traits and the potential of SRAP markers for genetic diversity studies in wheat. Therefore, SRAP markers were employed to examine their potential for genetic diversity analysis in durum wheat.

The objectives of this study, were to evaluate SRAP and phenotypic markers to determine diversities and relationships among subconvars of Durum Wheat landraces in North West of Iran and the Republic of Azerbaijan.

MATERIALS AND METHODS

Plant materials: Subconvars of durum wheat were defined and identified by Dorofeev et al. (1979), which was based on spike characteristics. In this study, two cultivars and 38 subconvars of durum wheat landraces from North West of Iran and Republic of Azerbaijan were evaluated (Table 1). For SRAP marker study seeds of one Spike were germinated and its leaf was used, while seeds from each genotype were used for phenotypic marker study.

Phenotypic traits: The seeds were planted on the 22 of October, 2005 in the Agricultural research station of AZAD University in Ardabil city, located at 38°5' North latitude, 48°17' East longitude was 20x5 cm. Experimental design was (RCBD) with 4 replications. Fourty durum wheat genotypes were evaluated for 27 morphological traits including phonological (days to tiller, stems elongation, heading, pollination and physiological maturating) morphological (plant height, length of spike, seed density in spike and total number of tillers), yield and yield components (1000 kernels weight, number of seeds in spike, number of fertile tiller and harvest index) and flour quality (Hectoliter Weight, Suni Bug Damaged Kernals, Yellow Berries, Protein percent, Sedimentation Test, Hardness Index, Wet Gluten, Gluten Elasticity, Gluten Index, Dry Gluten, Semolina Percent, Disc Pressure Test and Quality Marker) characteristics.

DNA extraction and SRAP amplification conditions: Total genomic DNA was isolated from young frozen leaf tissue of individual genotypes using the method of Dellaporta et al. (1983). Twelve different combinations were employed using four forward and four reverse primers (Table 2). Each 25 μL reaction mixture was consisted of 0.4 μM of each primer, 200 μM of each dNTPs, 2.5 μL 10x PCR Buffer, 1.5 mM MgCl₂ as a final concentration and 1, unit of Taq polymerase (Qiagen), 25 ng DNA templates.

Table 1:	The genetic material (Durum wheat landraces) used

Table 2:	Primer sequences used for SRAP analysis

Eppendorf Mstercycler Gradiant was used and cycling parameters included: one cycle of 5 min at 94°C, 5 cycles of three steps: 1 min of denaturation at 94°C, 1 min at 35°C and 2 min elongation at 72°C in the following 30 cycles the annealing temperature was increased to 50°C, with a final elongation step of 5 min at 72°C. PCR products were separated on 12% polyacrylamide gels at 400 V for 11 h and gels were dried overnight.

Data analysis: Presence or absence of each SRAP was coded as 1 and 0, respectively. Where, 1 indicated the presence of specific allele and 0 indicated, its absence. The distance matrix and dendrograms were constructed using the numerical taxonomy multivariate analysis system (NTSYS-pc ver. 2.1) software package. Genetic polymorphism, a similarity matrix and Nei's gene diversity index were used to compute Neil's standard genetic distance coefficients (Nei and Li, 1979) and to construct an Unweighted Pair-Group Method with Arithmetic Averages (UPGMA) dendrogram (Sneath and Sokal, 1973) using SPSS (ver. 10.0) software package.

In order to see how well a cluster analysis represents the distance matrix, COPH module was used to transform the tree matrix to a matrix of ultrametric distances (a matrix of distances implied by the cluster analysis). Finally, MXCOMP module was used to compare these ultra-metric distances and distance matrix produced for UPGMA analysis.

RESULTS AND DISCUSSION

Analysis of the two cultivars and 38 subconvars of durum wheat landrace from Iran and the republic of Azerbaijan with 12 SRAP primer combinations identified a total of 65 reproducible fragments (Table 3).

Among them 56.37% were polymorphic, ranging in size from 150-653 bp. The number of fragments detected by an individual primer combination ranged from 1-12, with an average of 8.

Based on the percentage of polymorphic fragments, different levels of polymorphism ranged from 25% (ME1/EM2) to 100% (ME7/EM5).

Table 3:	Primer (forward and reveres) combinations used for SRAP analysis

Table 4:	Number of total bands and number of polimorphysim band on the subconvars of T. durum

This finding concurs with the work done by Ferriol et al. (2003) in the Cucurbita pepo collection Gene diversity ranged from 0.1 (ME1/EM2) to 0.3 (ME7/EM5), with an average of 0.23 per primer combination (Table 3). Ferriol et al. (2003) also reported that ME1/EM2 and ME8/EM3 combined primer had the lowest genetic diversity in C. orifera sp. The highest average number of polymorphic bands was found among different subconvars in hordeiforme and lowest number of polymorphic bands in Boeuffi and Albiproviancale subconvars (Table 4). Fufa et al. (2005) in a study of genetic diversity in 30 Red winter cultivars by SRAP DNA marker reported that 23 SRAP markers produced 468 amplified fragments with an average genetic diversity of 0.418, which ranged from 0.1-0.9. They also, showed the diversity estimates for SRAP marker from 0.11-0.677 with average value of 0.357. As such, the SRAP markers provided more conservative estimates of genetic diversity than SSR markers. Also, SRAP Markers were used for the evaluation of genetic diversity in Buffalograss (Budak et al., 2004a-c), Okra Germplasm (Gulsen et al., 2007), Collection germplasm of Cucurbit pepo (Ferriol et al., 2003) and Brasica (Li and Quires, 2001).

The analysis of genetic similarity coefficients (Nei and Li, 1979) for pairs of genotypes (subconvars) showed (no reported) that the mean value of genetic similarity was 0.73 with a range from 0.01-0.97. Subconvars of leucumelam landraces from Naxcivan and Lerik (Azerbaijan) and Langan3 (Iran) regions were the least related to any other subconvars used in this study. The genetic similarity coefficients among those that subconvars and other genotypes ranged from 0.01-0.34, with the average of 0.302. The subconvars of albiprovienciale (Quba) from Azerbaijan and Africanum (Tabriz 2) from Iran were the highest related to other genotypes, which ranged from 0.380-0.889 with the average of 0.77.

Cluster analysis based on SRAPs markers: A cluster analysis was performed using the Nei and Li (1979) genetic diversity and UPGMA method. The dendrogram grouped the different accessions in two major clusters (Fig. 1). The cluster 1 included the subconvars of

Leucumelan, Boeuffi, Apulicum and Mursiens, while cluster 2 included other subconvars of T. durum landraces such as Hordeiforme, Leucurum, Niloticum, Africanum, Erythromelan and Albiprovincial. In the within-group on the cluster 1 there is high similarity among the subconvars of Landrace from different regions. This grouping concurs with subconvars grouping of landrace durum wheat by Dorofeev et al. (1979) methods. This might be due to the fact that the grouping done by Dorofeev et al. (1979) was based on the major gene effects.

In this study, there was no correlation between genetic diversity and geographical distribution.

Cluster analysis based on phenotypic markers: Twenty five phenotypic markers were scored for the 40 genotype of T. durum landrace and 81.0% of them were found to be polymorphic. The ratio of polymorphic phenotypic marker (81.0%) is higher than that of polymorphic molecular markers (56.37%) in the same landrace.

Fig. 1:	An Unweighted Pair-Group Method with Arithmetic Average (UPGMA) Dendrogram of genetic relationships among 40 subconvars of durum wheat calculated on the basis analysis by means of 12 SRAP primer combinations

Fig. 2:	An Unweighted Pair-Group Method with Arithmetic Average (UPGMA) Dendrogram based on dissimilarity matrix costracted from the 27 phenotypic markers scored for 40 subconvars of durum wheat from Iran and Azerbaijan

Molecular and phenotypic marker-based analysis produced two different clustering patterns in this study. This may be caused by quantitative control of phenotypic traits studied and/or fluctuations in environmental conditions, having potential effect on phenotypic performances (Gulsen et al., 2007). This is due to the fact that the estimated distance based on phonotypical traits and SRAP markes had a low non significant correlation (r = 0.12).

Fufa et al. (2005) also, reported non significant correlation coefficients among distance estimates of morphological and SRAP markers. Because they beloved the SRAP markers tended to have low correlation with the other genetic diversity estimates, they may provide different and unique insights into genetic diversity.

Few studies of SRAP markers have been reported in wheat. SRAP markers may be used for improving cultivars, understanding relationships, establishing germplasm collections and integrating markers into genetic linkage maps. Therefore, the use of these markers is commended for the investigation of genetic diversity of durum wheat landraces because SRAP markers were replicable and they correlated with major gene traits, for example with the grouping indicators of Dorofeev et al. (1979).

CONCLUSION

The 12 combinations of forward and reverse SRAP primers were used to evaluate the 38 landraces and two cultivars and produced 65 scorable markers, of which 56.730% was polymorphic for all 40 genotypes. As to phenotypic markers, 27 quantitative traits were evaluated in field with 4 replications, 22 of them (81%) were found to be polymorphic. The UPGMA (UN weighted-pair group method arithmetic average) dendrogram based on the 27 phenotypic markers distinguished all genotypes, but failed to detect any geographic association in durum landraces. The UPGMA dendrogram based on the 12 combinations of SRAP markers distinguished landraces, it was concurs with subconvars grouping characteristic (traits of major gene). SRAP markers are useful for studying diversity and relationships among and have potential in marker-aided selection, linkage mapping and evolutionary studies.

ACKNOWLEDGMENTS

This research was supported in part by I.A. University Ardabil branch. The researchers thank Prof. M. A. Saghai-maroof. Dr. Sh. Behtari, Dr. M. Abassov and Sh. Jamaati-e-Somarin for their critical review and suggestions to improve this manuscript.