Molecular and Phenotypic Characterization of Advanced Backcross Lines Derived from Interspecific Hybridization of Durum Wheat

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1 Biotechnology & Biotechnological Equipment ISSN: (Print) (Online) Journal homepage: Molecular and Phenotypic Characterization of Advanced Backcross Lines Derived from Interspecific Hybridization of Durum Wheat Elena Todorovska, Boryana Hadjiivanova, Violeta Bozhanova, Dechko Dechev, Yordan Muhovski, Ivelin Panchev, Nabil Abu-Mhadi, Valentina Peycheva & Albena Ivanova To cite this article: Elena Todorovska, Boryana Hadjiivanova, Violeta Bozhanova, Dechko Dechev, Yordan Muhovski, Ivelin Panchev, Nabil Abu-Mhadi, Valentina Peycheva & Albena Ivanova (2013) Molecular and Phenotypic Characterization of Advanced Backcross Lines Derived from Interspecific Hybridization of Durum Wheat, Biotechnology & Biotechnological Equipment, 27:3, , DOI: /BBEQ To link to this article: Taylor and Francis Group, LLC View supplementary material Published online: 16 Apr Submit your article to this journal Article views: 334 Full Terms & Conditions of access and use can be found at

2 Article A&EB AGRICULTURE AND ENVIRONMENTAL BIOTECHNOLOGY MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ADVANCED BACKCROSS LINES DERIVED FROM INTERSPECIFIC HYBRIDIZATION OF DURUM WHEAT Elena Todorovska 1, Boryana Hadjiivanova 2, Violeta Bozhanova 2, Dechko Dechev 2, Yordan Muhovski 3, Ivelin Panchev 4, Nabil Abu-Mhadi 1, Valentina Peycheva 1, Albena Ivanova 1 1 AgroBioInstitute, Sofia, Bulgaria 2 Field Crops Institute, Chirpan, Bulgaria 3 Walloon Agricultural Research Center (CRA-W), Gembloux, Belgium 4 Sofia University, Faculty of Biology, Sofia, Bulgaria Correspondence to: Elena Todorovska e.g.todorovska@gmail.com ABSTRACT Development, production, identification and evaluation of novel crop plant phenotypes is a fundamental issue addressed to food security, environmental change and bioenergy at a global scale. One of the major approaches used is wide hybridization and introgression breeding. In this study molecular markers and phenotype evaluation were used to characterize eight backcross lines obtained from interspecific hybridization of six Bulgarian durum wheat cultivars (Triticum durum Desf.) (2n = AABB) with eight alien species of the family Gramineae, using a backcross strategy. Thirty-three SSRs mapped on the A, B and D genomes and chromosomes of common wheat and three ISSRs were used to assign the alien chromosomes introgressed in the durum wheat chromosomes. The SSR markers showed introgression of several segments of T. carthlicum, T. dicoccoides, T. dicoccum, T. polonicum, T. macha and T. spelta donor genomes, with the highest number of introgressions from T. polonicum, T. dicoccum, and T. macha. This study reflects easy introgression of the genomes of T. dicoccum, T. polonicum and T. macha in cv. Progress and a full compatibility between their genomes. Only one introgression was found from Ae. tauschii in cv. Victoria with the employed set of SSR markers, while such ones from T. timopheevi were not detected. The ISSR markers (GA) 9 C and (AC) 8 G showed introgression of T. polonicum, T. macha and T. timopheevi chromosome fragments into the genomes of cvs. Progress and Vazhod, respectively. The phenotypic evaluation of the backcross lines and their durum wheat parents was conducted in under field conditions, for several important agronomic and grain quality traits. The studied lines were found to differ statistically from the recurrent parents for some traits either in a positive direction, or in a negative one. Higher grain protein and gluten content was identified in all backcross lines in comparison with the recurrent durum wheat cultivars. Backcross lines BL 3 from the cross with T. dicoccum, BL 4 from the cross with T. polonicum, and BL 7 from the cross with T. macha, and cv. Progress as a recurrent parent possess the best combination of agronomic traits related to spike productivity and grain quality. Biotechnol. & Biotechnol. Eq. 2013, 27(3), Keywords: interspecific hybridization, alien introgression, durum wheat, molecular markers (SSR, ISSR), phenotype evaluation Introduction Common and durum wheat are important cereal crops used for human consumption worldwide. By 2020, the world demand for wheat will be 40 % greater than in the present days. In response to this challenge, as highlighted by Monneveux et al. (58), breeders must enhance the yield and simultaneously reduce the impact of agriculture on the environment. Durum wheat represents about 8 % of the total wheat production but 80 % grows predominantly in Mediterranean climates as well as on the Balkan Peninsula. In these regions the yield is considerably limited due to drought, together with heat, salinity, pests, and diseases. Special efforts have to be made to 3760 increase the tolerance/resistance to biotic and abiotic stresses, and the yield potential in adapted genotypes in which very little genetic variation exist for these traits (58). The low level of genetic variability restricts the further improvement of both productivity and quality of the cultural plants, as well as their resistance to biotic and abiotic stress. Like all other agricultural crops, the present-day cultivated wheat varieties are characterized with similar genetic composition and are mainly obtained by interspecific hybridization and fixation of relatively few preferred genes and their allele variations (89). A more efficient use of biodiversity of wild grasses and domesticated species of the tribe Triticeae in breeding programs is a key for enhancement of genetic variability. Many wild grasses of the tribe Triticeae possess desirable traits such as drought and cold tolerance, resistance to insects and fungal diseases, quality and quantity of grain storage protein as well as nutritive value that have been and are being incorporated

3 into polyploid wheats: durum, or macaroni, wheat (Triticum turgidum L. subsp. durum [Desf.]) (2n = 4x = 28, AABB) and bread wheat (Triticum aestivum L.) (2n = 6x = 42, AABBDD). Genes of resistance to various pests and diseases are present in durum wheat related species. Twelve of the 40 known genes for leaf rust resistance and 20 of the 41 known genes for stem rust resistance originated in Triticum species other than the cultivated ones (55, 56, 58). Some related species, such as Ae. tauschii, Ae. umbellulata, Ae. speltoides, T. turgidum and T. dicoccum have been found to be good sources for improving abiotic stress tolerance (drought, cold, salinity) (58); Ae. tauschii for increasing biomass and yield potential and Ae. longissima, Ae. kotschyi, Ae. peregrina, Ae. cylindrica, Ae. ventricosa, Ae. geniculata for micronutrient biofortification (79). The genetic variation from alien species can be exploited both directly, i.e. target genes can be transferred into the adapted durum wheat leading to the development of superior varieties, and indirectly, i.e. genes from alien species can be used to determine the genetic control of target traits and the pathways involved in their expression (1, 2, 3, 70). Among the methods used, the backcross strategy allows introgression of desirable characters from one species into another by conducting negative phenotypic and/or genotypic selection against unfavorable genes originating from the donor parent. This approach allows the frequency of the donor-parent genome in each of the advanced backcross lines to be reduced and the masking effect of deleterious wild recessive alleles for characters such as sterility, seed shattering, undesirable growth habit to be avoided (78). Current investigations of the efficiency of the single backcross breeding strategy through computer simulation found that this strategy allows more than 60 % of the favorable genes from wild donor plants to be transferred and, at the same time, the adaptation of the recurrent cultivars to be improved (85). Despite of the strategy used for introgression of useful genes from wild relatives or unadapted germplasm (BILs, F2, F3, BC1, etc.), one of the major problems is the selection of recombinant plants derived from the crosses between adapted durum wheat and its wild relatives (38). The approach to identify introgression could be based on cytological, morphological or molecular comparisons (4). Chromosome banding and in situ hybridization are commonly used to screen lines containing alien chromatin. These techniques, however, are labour intensive, highly technical and require advanced cytogenetic skills, and are not adequate in breeding programs that require rapid screening of a large number of genotypes. Morphological screening is also commonly used by breeders for detection of introgression but is also labour intensive and requires year-to-year repetition for selection of appropriate genotypes. Recent advances in molecular biology, principally the development of PCR-based DNA marker systems, have provided powerful tools for characterizing and evaluating the genetic diversity of cereal crops, evolution, and alien germplasm introgression. In durum wheat, DNA markers have been used for studying genetic relationships between species, populations and cultivars. RAPDs (88), SSRs (69), ISSRs (99), AFLPs (84) and recently developed SNPs are the most widely used markers to identify genetic relationships among durum wheat accessions, estimate genetic diversity, genome mapping and marker-assisted selection for agronomically important traits (17, 48, 49, 50, 51). Among the markers used, microsatellites, also referred to as sequence tagged microsatellite sites (STMSs) or simple sequence repeats (SSRs), are an effective tool for the investigation of cereal genomes and specifically in evolution studies, genotype assignment, marker assisted breeding, genetic mapping, and diversity assessment because of the high level of polymorphism they reveal (23, 69). SSRs can be used in wide hybridization of wheat to monitor and map desirable alien genes in segregating populations as they are locus-specific and are inherited in a codominant manner (73). A large number of the primer sequences of wheat SSRs are publicly available. However, the de novo development of new SSR markers for wild relatives of wheat is time-consuming and costly. The flanking regions of microsatellites show sufficient homology between closely related species, allowing the primers of wheat SSR markers to be successfully used in studies of other Poaceae species. This transferability has been successfully employed for cultivated (barley, oats, rye) (92) and wild (Elymus, Aegilops) species (52, 93). ISSRs (Inter Simple Sequence Repeats) have been also proved to be useful in cultivar identification, genetic diversity and alien gene introgression studies (9, 46, 62, 98). ISSRs detect polymorphisms in inter-microsatellite loci, using a single primer composed of an SSR sequence anchored at the 3 or 5 end by 1 4 arbitrary nucleotides. ISSR markers are superior to SSRs and AFLPs in that they are low-cost, highly polymorphic and well reproducibile. It is also not necessary to know the DNA sequence for ISSR primer design. In Bulgaria, the breeding of durum wheat has a long tradition. A large number of T. durum botanical varieties have been identified among the local landraces (31, 53). Some of the original local populations have been preserved at the Institute of Plant Genetic Resources (IPGR, Sadovo) (65). However, these populations are not homogenous and are mostly a mixture of landraces. Artificial selection aimed at development of homogeneous and highly productive cultivars suitable for modern agricultural technologies, was initiated 90 years ago in the Field Crops Institute, FCI formerly Institute of Cotton and Durum Wheat, Chirpan. The first durum wheat cultivars were obtained by individual selection from the collected materials from local populations. In the subsequent breeding stages interspecific, intercultivar hybridization as well as experimental mutagenesis were successfully applied to develop short-stem and lodging resistant cultivars with higher productivity and grain quality (5, 10, 11, 18, 81, 90). The number of released Bulgarian durum wheat cultivars, however, is lower compared to that of the bread wheat. This explains the relatively lower diversity in the present-day T. durum gene pool. Several microsatellite-based studies have 3761

4 shown moderate to low level of genetic diversity in cultivated durum wheat (22, 80), highlighting the necessity to improve the present genetic pool by means of wide hybridization. Phenotypic screening of a large collection of wild and related species of the genus Triticum and Aegilops from the gene bank of IPGR, Sadovo, showed the presence of useful variation for grain quality parameters as well as resistance to biotic and abiotic stress factors (7, 82). This variation was recently exploited for development of new breeding lines with improved characteristics by application of wide hybridization (intergeneric and interspecific) in a durum wheat breeding program (8, 28). The aim of the study was to identify lines with improved characteristics appropriate for future durum wheat breeding programs through molecular and phenotypic characterization of backcross lines derived from interspecific hybridization of durum wheat. Materials and Methods Plant material Eight lines obtained through interspecific hybridization of six durum wheat genotypes (Triticum durum Desf.) (2n = AABB) with eight alien species of the family Gramineae by a backcross strategy were used in this study. The Bulgarian durum wheat cultivars Gergana, Vazhod, Progress, Beloslava, Victoria and one breeding line developed in the Field Crops Institute (FCI, Chirpan) were used as maternal parents in the hybridization. Accessions of the following alien species: Aegilops tauschii Coss. (2n = DD), Triticum carthlicum Nevski (2n = AABB), Triticum dicoccoides Korn (2n = AABB), Triticum timopheevii Zhuk. (2n = AAGG), Triticum macha Dek. et Men. (2n = AABBDD) and Triticum spelta L. (2n = AABBDD) were used as male parents, while Triticum dicoccum Schrank (2n = AABB) and Triticum polonicum L. (2n = AABB) as Backcross lines obtained through interspecific hybridization and a backcrossing strategy maternal ones. The accessions were kindly provided from the Gene Bank collection of the Institute of Plant Genetic Resources (IPGR, Sadovo, Bulgaria). The cross combinations and breeding phase during the analysis of interspecific hybrids are presented in Table 1. Interspecific crosses Durum wheat genotypes were grown in the experimental field of FCI, while alien species, earlier generations of hybrid plants and the backcross progenies, in the greenhouse. The F 1 generations of interspecific hybrids were produced by the methods of emasculation and pollination followed by an embryo rescue technique (Table 1). Three days prior to anthesis, durum wheat spikes were emasculated and bagged to avoid pollination with other wheat plants. Crosses were carried out with fresh pollen from the wild relatives. The hybrid seeds were removed from the spikelets 16 days to 20 days after pollination and embryo rescue methods were applied according to the protocols described by Hadzhiivanova et al. (28). The in vitro regenerated F 1 plants were cultivated in greenhouse conditions to maturity. All F 1 hybrids were single backcrossed to recurrent durum wheat cultivars: Progress, Vazhod or Victoria, used as male parents. Lines BL 4 and BL 6 were obtained through two rounds of backcrossing (Table 1). Each backcross was followed by one or two generations of selfing. Progenies from each of the selfed generations, along with durum wheat parents, were screened for several traits at the field of FCI, Chirpan, during several growing seasons ( ). Strict and repeated selection of plants with durum wheat phenotype was performed in the segregation populations. The selection was carried out using the bulk-population method. Phenotypic evaluation Phenotypic evaluation of the advanced-generation lines originating from interspecific hybridization and the TABLE 1 Cross combinations Year of F 1 release Breeding phase during investigation Molecular Morphological analysis evaluation BL 1 (7383 Triticum carthlicum) F 2 Victoria 2007 F 1 F 3 BL 2 (Vazhod х Triticum dicoccoides) F 1 Vazhod 2007 F 1 F 3 BL 3 (Triticum dicoccum Triticum durum) F 1 Progress 2006 F 2 F 4 BL 4 (Triticum polonicum Triticum durum) F 2 Triticum durum) Progress 2005 BC 2 F 1 BC 2 F 3 BL 5 (Vazhod Victoria) F 1 Tr. timopheevii) F 1 Vazhod 2006 F 3 F 5 BL 6 (Gergana Vazhod) F 1 Aegilops tauschii) F 1 Tr. durum) Victoria) 2006 BC 2 F 2 BC 2 F 4 BL 7 (Triticum durum Triticum macha) F 1 Progress) F F 4 F 6 BL 8 (Progress х Beloslava) F 1 Triticum spelta) F 1 Vazhod) F F 4 F

5 corresponding durum wheat parents was performed during Plants were grown in experimental plots at a 5 cm plant-to-plant distance and 20 cm distance between rows. Phenotypic observations were carried out on 20 plants of each cross combination and durum wheat parents. Several important agronomic traits were recorded: plant height, heading date (days to heading from 1 st May), flag leaf parameters, spike length, number of spikelets per spike, grain number per spike, grain weight per spike, 1000 grain weight, grain protein and gluten content. Grain protein content (N 5.7 % dm basis) was determined by the Kjeldahl method. Gluten content was measured according to the Bulgarian State Standard (BDS and BDS ). Statistical analysis All phenotypic data (morphological, agronomical and grain quality traits) were processed by one-way ANOVA. Duncan s test was used for comparing the means at the detected significant differences (P < 0.05). The software STATISTICA 7 (76) was used. Microsatellite marker assay DNA was extracted from leaves of bulked samples (5 plants per sample) from the parents involved in interspecific hybridization and each of the selected backcross lines grown in a greenhouse, using a modified CTAB method according to Murray and Tompson (61). The microsatellite markers Xgwm (69) and Xwmc (24, 67) were used for characterization of both parental and backcross lines, as well as the additional genotypes included in the interspesific crosses (Table 1). For analysis, at least one marker per chromosome was chosen. The following markers were used: Xgwm 136-1S, Xwmc 24-1L, Xgwm 99-1L, Xgwm 95-2S, Xgwm 312-2L, Xgwm 5-3L, Xgwm 165-4S, Xwmc 327-5L, Xgwm 639-5L, Xgwm 233-7S, Xwmc 83-7S, Xwmc 9-7L and Xgwm 282-7L mapped on the A genome; Xgwm 268-1L, Xgwm 285-3S, Xgwm 165-4L, Xgwm 234-5S, Xgwm 639-5L, Xgwm 644-6S, Xgwm 400-7S, Xgwm 46-7S, Xgwm 644-7L and Xgwm 302-7L on the B genome; Xgwm 642-1L, Xgwm 484-2S, Xgwm 261-2S, Xgwm 456-3L, Xgwm 165-4L, Xgwm 190-5S, Xgwm 639-5L, Xgwm 469-6S, Xgwm 325-6S and Xgwm 437-7L on the D genome. PCR reactions were performed in a final volume of 10 μl in a Gene Amp 2900 thermocycler. The reaction mixture contained 250 nmol/l of each primer, 0.2 mmol/l of each deoxynucleotide, 1.5 mmol/l of MgCl 2, and 0.8 U Taq polymerase. The development of the microsatellite markers, primer sequences, chromosome location and the annealing temperature are presented by Röder et al. (69) for Xgwm and by Prasad et al. (67) for Xwmc. Fragment sizes were calculated with Fragment Manager (Pharmacia) software by comparing with internal size standards added to each lane in the loading buffer. The repeat number of alleles was calculated according to the fragment sizes and number of repeat units at the corresponding locus in cv. Opata or Chinese spring. In cases where no amplification product was observed, PCR was repeated with newly isolated DNA. For ISSR analysis three ISSR primers (GA) 9 C, (CT) 9 G and (AC) 8 G were used. ISSR amplification was conducted in a 20 μl volume containing 75 ng of genomic DNA, 1.25 U Taq DNA polymerase, 1.5 mmol/l MgCl 2, 0.2 mmol/l dntps, and 0.1 μmol/l primer. The PCR protocol consisted of initial denaturation at 94 ºC for 4 min, followed by 40 cycles of 94 ºC for 45 s, annealing at 54 ºC ((GA) 9 C) or 58 ºC ((CT) 9 G; (AC) 8 G) for 45 s, 72 ºC for 1 min, and a final extension step of 72 ºC for 10 min. All PCR reactions were carried out in a Thermal Cycler QB-96 (LKB). PCR products were separated in 1.2 % agarose gels, stained with GelRedTM (Biotium, USA) and photographed under UV light with EC3 Imaging System (Ultra-Violet Products Ltd, Cambridge, UK). SSR and ISSR data analysis Peaks of SSR markers and bands of ISSR profiles were scored as 1 (present) and 0 (absent). The alien genome introgression levels in the progeny from SSR data equaled the number of loci showing alien alleles in the introgression line divided by the total number of polymorphic SSR loci. Similarly, the alien genome introgression levels in the progeny from ISSR data were calculated as follows: the number of alien specific markers present in the backcross individual/total number of the polymorphic markers between both parents. Results and Discussion Alien introgression is considered to be a useful tool for enriching the genetic diversity by introducing new traits and for germplasm development (29, 63). Obtaining ample collections of monosomic substitution and deletion lines of wheat is a key objective for cytogeneticists as well as geneticists. At the same time, wheat breeders have created a number of strategies for improving the wheat germplasm based on alien introgression. For this purpose, a large variety of wild and cultivated wheat relatives, e.g. rye, Triticum spp., Aegilops spp., Thinopyron spp., have been employed in alien introgression programs (14, 19, 25, 30, 33, 37, 59). Effective alien introgression depends on achieving high rates of homoeologous recombination between host and alien chromosomes, or on using large population sizes and many generations, in order to diminish any unwanted linkage drag from the alien species, which may otherwise bring about transfer of genetic load. For example, Jenkins et al. (34) demonstrated that differences in DNA content (total DNA content or repetitive sequences) do not compromise the recombination between homoeologous chromosomes within the plant genomes, also including cereal ones. The backcross strategy has been widely used in the breeding programs aiming at introduction of alien germplasm into cultivated wheat. Some breeding programs have adopted advanced backcross inbred line (BIL) populations as a method for identification and introgression of useful genes from wild relatives or unadapted germplasm with the potential to improve 3763

6 the agronomic performance of elite cultivated lines (20, 77). As Gadaleta et al. (21) point out, the potential benefits of these lines versus more balanced populations (e.g. F2, F3, BC1, etc.) depends on the possibility of reducing the introgressed chromosomal regions and fine mapping loci linked with quantitative traits (QTLs). The backcrossing method has found limited application in the improvement of quantitative traits mainly because of the low heritability of these characters and the difficulty of the simultaneous transfer of a relatively large number of genes. Unlike other common approaches in wheat breeding, the backcross inbred line method (87), produces BILs that can be tested in replicated trials over environments prior to selection. BILs are characterized by the low proportion of the donor parent in each of the population members, which makes them ideal for mapping interspecific variation (15). However, the strategy based on single backcrossing with selected bulk breeding (SBBS s) has advantages in retaining or improving the adaptation of the recurrent parents, and at the same time transferring most of the desired donor genes in a wide range of scenarios and has been successfully used in wheat improvement at CIMMYT breeding strategy (85). The Triticeae tribe is formed from species with three levels of polyploidy (diploids, tetraploids and hexaploids) showing high degree of crossability between each other, which has allowed the natural transferability of genes. This process has been a useful tool in the breeding of cultivated species of this tribe (54). As a result of wide hybridization between Bulgarian durum wheat cultivars and species with a different ploidy level: diploids (Ae. tauschii Coss., 2n = DD), tetraploids (T. carthlicum Nevski, 2n = AABB; T. dicoccoides Korn, 2n = AABB; T. dicoccum Schrank, 2n = AABB; T. polonicum L., 2n = AABB; T. timopheevii Zhuk., 2n = AAGG) and hexaploids (T. macha Dek. et Men., 2n = AABBDD; T. spelta L., 2n = AABBDD), and a backcross strategy, several lines were selected and used in this study. Molecular characterization of introgression lines Better knowledge of genome segment introgression in interspecific hybridization would facilitate breeding programs by the recognition of lines with desirable introgressed traits and the development of new cultivars. The detection of genome introgression in interspecific hybridization can be further assisted by the recent advances in molecular marker based techniques. Among various markers, SSRs have been proved as an efficient tool due to a high level of polymorphism because of variation in the number of tandem repeats within microsatellites at certain loci and chromosome specificity. SSRs are highly sensitive in detecting DNA structural differentiation in plant genomes (86). Sequence-specific SSR primer pairs designed from common wheat (T. aestivum L.) can be used to detect SSRs in related species, basically known as transferability. It is possible since the T. aestivum is an allopolyploid species, which is made up of three diploid genomes: A, B, and D, by polyploidization, after intercrossing between T. turgidum and Ae. tauschii. Microsatellites from common wheat have also been shown to give amplification in many related or alien species (Ae. umbellulata, Ae. longissima, Ae. kotschyi, T. timopheevii, etc.) with genomes different from that of Ae. tauschii and T. turgidum and vice versa (26, 27, 40, 64, 72, 75, 91, 96). In this study microsatellite and ISSR markers were used to follow the transmission of introgressed segments from alien species of the family Gramineae into more adapted and agronomically acceptable Bulgarian durum wheat germplasm. Detection of introgression events by SSR markers In order to cover the wheat genome searching for chromosomal regions introgressed from the wild parents, a total of 29 microsatellite markers amplifying 33 SSR loci were selected on the basis of already published maps (24, 69, 74). Thirteen microsatellite loci mapped on the A genome, 10 on the B genome, and 10 loci on the D genome were analysed to identify polymorphic markers for each of the 14 chromosomes of the A, B, as well as for the additional D and G genomes of both parental lines. The polymorphic markers were then tested in the backcross lines at different stages of the selection process as shown in Table 1. Introgression from tetraploid alien species with AABB and AAGG. Twenty-three SSR markers mapped on all 14 chromosomes of the A and B genomes, excluding 2B and 6A, and 10 SSR markers on the D genome were employed for the genotyping of four alien species with AABB genomes (T. carthlicum, T. dicoccoides, T. dicoccum and T. polonicum) and three durum wheat cultivars (Victoria, Vazhod and Progress) used as recurrent parents in the interspecific crosses. Out of 33 SSR markers, 4 failed to produce any amplification product in T. carthlicum, 10 in T. dicoccoides, 9 in T. dicoccum, and 9 in T. polonicum. Among the markers amplifying alleles in alien species with AABB genomes, several ones were found which had been preliminarily mapped on the D genome of common wheat: Xgwm 642-1D, Xgwm 261-2D, Xgwm 639-5D, Xgwm 190-5D, Xgwm 325-6D, Xgwm 437-7D. The remaining markers could fully discriminate both alien and cultivated durum wheat genotypes by amplifying completely different sizes of SSR products or give products of similar size in parents involved in interspecific crosses. SSR markers amplifying similarly sized products in both parents were excluded from further experiments. The observed transferability of SSRs raises the question of how SSR markers in common wheat could be referred to the specific chromosome/s and genomes in alien species. In our study primer pairs specific for the B and D genome SSR loci amplified in T. timopheevii, the tetraploid donor of the AG genome (7/33 = 21.2 %). Similarly, primer pairs for some SSR loci located on the D genome of common wheat amplified tetraploid species with AABB genomes: T. cartlicum (6/33 = 18.2 %), T. dicoccoides (2/33 = 6.1 %), T. dicoccum (2/33 = 6.1 %) and T. polonicum (1/33 = 3 %) as has been shown for T. urartu (donor of the wheat A genome) or Aegilops speltoides or its related species (the probable donor of the wheat B genome) (94). Among the SSR markers mapped on the D genome, 3764

7 only Xgwm 642-1D showed amplification in the genome of cultivated wheat cvs. Victoria, Vazhod and Progress. Similarly, the studies on SSR loci of newly synthesized hexaploid wheat and its donor species (a tetraploid wheat and the diploid Ae. tauschii) with 21 D-genome specific primers and a primer set of 66 SSRs specific to the A/B genomes of common wheat showed that some primer pairs specific to the D genome of wheat could amplify SSR products in tetraploid wheat with AABB genomes and over 70 % of A/B genome specific SSR markers could amplify SSR products in Ae. tauschii with the D genome (94, 95). These reports drew attention to the fact that some amplification products in tetraploid wheat disappeared in the synthesized hexaploid wheat background and emphasized on the difficulty of determining the origin (from the D genome or from A/B genomes) of some amplified SSR fragments in synthetic hexaploid wheat. Studies on the evolution of common wheat (16, 41, 45, 47) have shown that some of the SSR loci found in a particular genome of a diploid species had been either lost, or mutated in the primer binding site, which led to the failure of SSR amplification in common wheat. Other studies involving artificially synthesized allopolyploids of Triticeae revealed allopolyploidization-induced, rapid DNA sequence changes (including also DNA elimination) in new synthetic allopolyploids. However, several reports (26, 27, 72) suggest that the locus specificity of SSRs in common wheat is most probably due to mutations in primer binding sites, rather than to loss of SSRs themselves in related genomes during polyploidization events (94, 95). The above-mentioned observations suggested that caution should be taken when using SSR markers to genotype new synthetic hexaploid wheat or other interspecific wheat hybrids (95). Among the parents involved in the interspecific crosses in our study, the highest level of polymorphism (78.8 %) was observed between Victoria and T. carthlicum and between Progress and T. dicoccum (69.7 %) a value much higher than that found among the cultivated wheat implicated in the crosses (25 % to 35 %) because of the genetic distance between the two parental lines. In comparison, the percentage of the polymorphism between Vazhod and T. dicoccoides and between cv. Progress and T. polonicum was slightly lower: 60.6 % and 57.6 %, respectively (Table 1S and Table 2S in the Online Supplementary Appendix, com/bbeq). The polymorphic markers between alien species and cultivated durum wheat genotype were further used to detect alien genome introgression in the backcross lines. The analysis of durum wheat T. dicoccum and durum wheat T. polonicum backcross lines, using the selected polymorphic Xgwm and Xwmc SSR markers, led to the identification of introgressed segments of dicoccum and polonicum chromatin in the A and B genomes of the backcross lines BL 3 and BL 4 (Table 2, and Fig. 1S C, D in the Online Supplementary Appendix, Introgressed chromatin segments of T. dicoccum were found with markers Xgwm 136 (12 cm) and Xwmc 24 (48 cm) mapped on 1AS according to the published genetic map of Somers et al. (74), while introgressed chromatin segments of T. polonicum were identified with markers on 5B (Xgwm 234-5BS [38 cm] and Xgwm 408-5BL [117 cm]) as well as with markers Xgwm 46 (56 cm) and Xgwm 302 (86 cm) on 7BS and 7BL chromosomes (Table 2, and Fig. 1S D in the Online Supplementary Appendix, Additional introgressions of T. dicoccum chromatin were observed with markers located on different chromosomes, e.g. with Xgwm 5-3AS, Xgwm 165-4AS, Xgwm 234-5BS, Xwgm 644-6BL, and Xwmc 83-7AS, while such introgressions from T. polonicum were found with Xgwm 165-4AS, Xwmc 327-5AL, and Xwmc 9-7AL. This suggested that the mechanism of alien segment transmission is mostly based on genetic recombination through crossover but substitutions of a whole or large segments of chromatin from both alien species in the cultivated durum wheat is also possible. Introgressions of T. carthlicum and T. dicoccoides chromatin segments in the backcross lines BL 1 and BL 2, were found only with markers mapped on the A and D genomes, but not on the B genome. Introgressed genome segments from T. carthlicum were observed on 5A, 5D and 7A, while these from T. dicoccoides were found on the chromosomes 2A, 4A and 5D (Table 2). Gadaleta et al. (21), using backcross inbred lines (BILs) and a set of 138 polymorphic SSR markers, have identified introgression of several segments of Triticum turgidum var. dicoccoides donor genome in cv. Latino. The authors estimated an average percentage of the introgressed dicoccoides genome of 6.3 %, which is an indication for easy introgression of the genome of dicoccoides and a full compatibility between the two parental genomes. In our study, the average percentage of introgressions of the T. dicoccoides donor genome was 15 % and they were detected only with markers mapped on the A and D genome (Xgwm 312-2AL, Xgwm 165-4AS, and Xgwm 639-5DL), as mentioned above. Interestingly, among the markers mapped on the D genome of common wheat, the marker Xgwm 639-5D was the only one that showed introgressions of genome segments from the tetraploid alien species T. carthlicum, T. diccocoides and T. dicoccum in the genome of cultivated wheat cvs. Victoria, Vazhod and Progress (Fig. 1S B in the Online Supplementary Appendix, Introgression of A or G chromosome segments of T. timopheevii (AAGG) in the genome of cv. Vazhod (backcross line BL 5 ) was not detected with the employed set of the 33 SSR markers mapped on the A/B and D genomes of common wheat, despite of the high level of polymorphism (66.7 %) between the donor genome T. timopheevii and the recipient cv. Vazhod (Table 2S in the Online Supplementary Appendix, This is in accordance with earlier observations (71) which suggest a limited efficiency of wheat SSR markers for characterization of G, U, and M genomes (57). 3765

8 TABLE 2 Introgressions of chromosome segments from tetraploid (AABB), diploid (DD) and hexaploid (AABBDD) alien species into the genome of cultivated durum wheat (cvs. Victoria, Vazhod, Progress) and newly appearing alleles in their backcross lines Cross combinations Introgressions New alleles Cross combinations Introgressions New alleles T. carthlicum (AABB) BL 1 Victoria Xwmc 327-5AL Xgwm 639-5DL Xwmc 9-7AL Xgwm 268-1BL Xgwm 285-3BS Xgwm 165-4BL Xgwm 234-5BS Xgwm 282-7AL T. timopheevii (AAGG) NONE BL 5 Vazhod Xgwm 136-1АS Xgwm 285-3BS Xgwm 165-4BL Xgwm 644-6BL Xgwm 233-7AS Xwmc 9-7AL Xgwm 302-7BL Total 3 5 Total 0 7 % % T. dicoccoides (AABB) BL 2 Vazhod Xgwm 312-2AL Xgwm 165-4AS Xgwm 639-5DL Xwmc 24-1AS Xgwm 285-3BS Xwmc 327-5AL Xgwm 639-5BL Xgwm 644-6BL Xgwm 46-7BS Xgwm 233-7AS Ae. tauschii (DD) BL 6 Victoria Xgwm 639-5DL Xgwm 268-1BL Xgwm 644-6BL Xwmc 83-7AS Total 3 7 Total 1 3 % % T. diccocum (AABB) BL 3 Progress Xgwm 136-1АS Xwmc 24-1AS Xgwm 5-3AS Xgwm 165-4AS Xgwm 234-5BS Xgwm 639-5DL Xgwm 644-6BL Xwmc 83-7AS Xgwm 136-1АS Xgwm 99-1AL Xgwm 268-1BL Xgwm 312-2AL Xgwm 285-3BS Xgwm 639-5BL Xgwm 233-7AS Xwmc 9-7AL Xgwm 46-7BS Xgwm 302-7BL T. macha (AABBDD) BL 7 Progress Xgwm 136-1АS Xgwm 99-1AL Xgwm 285-3BS Xgwm 165-4AS Xgwm 233-7AS Xwmc 83-7AS Xwmc 9-7AL Xgwm 282-7AL Xwmc 24-1AS Xgwm 268-1BL Xgwm 234-5BS Xgwm 233-7AS Xgwm 46-7BS Xgwm 302-7BL Total 8 10 Total 8 6 % % T. polonicum (AABB) BL 4 Progress Xgwm 165-4AS Xwmc 327-5AL Xgwm 234-5BS Xgwm 408-5BL Xwmc 9-7AL Xgwm 46-7BS Xgwm 302-7BL Xgwm 136-1АS Xgwm 268-1BL Xgwm 285-3BS Xgwm 234-5BS Xgwm 408-5BL Xgwm 644-6BL T. spelta (AABBDD) BL 8 Vazhod Xwmc 24-1AS Xgwm 408-5BL Xgwm 469-6DS Xgwm 302-7BL Xgwm 136-1AS Total 7 6 Total 4 1 % % Intogression from hexaploid alien species with AABBDD genomes. Of a total of 33 SSR markers mapped on the A, B and D genomes of wheat, 30 SSRs revealed polymorphism between T. macha (AABBDD) and cv. Progress (90.9 %) and between T. spelta (AABBDD) and cv. Vazhod (90.1 %) (Table 3S in the Online Supplementary Appendix, www. diagnosisnet.com/bbeq). Genotyping of the backcross line BL 7 of the interspecific cross T. macha Progress did not reveal introgressed segments from the D genomes of alien species into the cultivated durum wheat. Most of the introgressions from the T. macha donor genome were on the chromosomes 7A and 1A (Table 2, and Fig. 2S A in the Online Supplementary Appendix, According to the published map of Somers et al. (74), the largest introgressed chromatin of T. macha was found on chromosome 7A of BL 7 with the markers Xgwm 233 (4 cm), Xwmc 83 (55 cm), Xwmc 9 (72 cm), and Xgwm 282 (100 cm). This is a clear evidence for introgression/substitution of a whole or almost a whole 3766

9 7A chromosome in the recipient genotype Progress with the homeoelogous chromosome of T. macha. Introgression events were also observed on chromosome 1A with the markers Xgwm 136 (12 cm) and Xgwm 99 (126 cm) but the large distance between the two markers is an indication of single introgression events rather than of introgression of one large segment. In comparison to the interspecific cross T. macha Progress, T. spelta (AABBDD) introgressions, albeit smaller in number, were found in loci mapped on the A, B and D genomes of the backcross line BL 8 (Xwmc 24-1AS, Xgwm 408-5BL, Xgwm 469-6DS, Xgwm 302-7BL) (Fig. 2S B in the Online Supplementary Appendix, com/bbeq). Only one marker with location on the D genome showed an introgressed DNA fragment from T. spelta. Introgression from diplod alien species with DD genomes. Introgression of D genome segments from the donor parent Ae. tauschii (DD) was found in BL 6 only with the SSR marker Xgwm 639-5DL, despite of the high level of polymorphism (78.8 %) between the alien species and cv. Victoria. This is basically due to the fact that some of the primer pairs for specific loci on the chromosomes of A/B genomes of common wheat amplified loci in Ae. tauschii (14/33 = 42.4 %), the diploid progenitor of the wheat D genome (Table 2, and Table 2S, Fig. 2S C in the Online Supplementary Appendix, However, the set of polymorphic SSR markers employed here was not sufficient to detect more introgressions from the donor genome of Ae. tauschii. Therefore, more SSR markers would be needed for a more comprehensive genome analysis of parental lines involved in the interspecific crosses, and hence, for a more accurate estimation of genome introgressions of the progenies. Comparison of the compatibility between the genomes of cultivated durum wheat genotypes and alien species. Despite of the low number of SSR markers used, we were able to find several segment introgressions from alien species. The experiments clearly showed high average percentage of introgressions in cv. Progress from the donor genomes of T. polonicum (AABB), T. dicoccum (AABB), and T. macha (AABBDD) (36.8 %, 34.8 %, and 26.7 %, respectively) (Table 2). This suggested a high compatibility between the genomes of parental lines and, respectively, high level of chromosome exchanges. The level of introgressions in cv. Vazhod from T. diccocoides and T. spelta was 15 % and 13.3 %, respectively; and those in cv. Victoria from the donor genomes of T. carthlicum and Ae. tauschii, 11.5 % and 3.8 %. Newly appearing alleles in the backcross lines. In addition to introgressions, new alleles, which did not correspond correctly to the alleles of both parents, were detected in the backcross lines with the selected set of polymorphic markers in each of the crosses screened here. Table 2 shows the highest level of new alleles in the introgression lines BL 3 (43.5 %), BL 2 (35 %), BL 5 (31.8 %) and BL 4 (31.6 %) of the crosses T. diccocum Progress, T. diccocoides Vazhod, T. timopheevi Vazhod and T. polonicum Progress, respectively, even though no introgressed SSR alleles were detected in the cross T. timopheevii Vazhod. As seen from Table 2, most of the new alleles in the backcross lines discussed here were found in loci Xgwm 268-1B and Xgwm 285-3B with 5 alleles, as well as in Xgwm 136-1A, Xgwm 644-6B and Xgwm 233-7A with 4 alleles. The lowest number of new alleles was observed in the backcross lines BL 6 (Ae. tauschii Victoria) and BL 8 (T. spelta Vazhod): three and one, respectively. The presence of new alleles is probably an indication of active recombination between the genomes of alien species and durum wheat cultivars, rather than a residual heterosigosity in parental genotypes. Such results have been also described in the work of Wang et al. (86), who studied the genome introgression of Festuca mairei into Lolium perenne by SSR and RAPD markers. The authors reported that out of 13 polymorphic primers between both parents, two did not amplify the expected fragments in the F 1 and backcrossed progeny. The possible explanations of these results are: 1) heterozygosity in the parents, rather than recent mutations which have led to varied alleles; 2) variation resulting from the colchicine treatment for chromosome doubling; or 3) recombination events that could potentially change the SSR length by unequal crossing-over or by gene conversion (32, 68). The possible influence of replication slippage during recombination-dependent DNA repair has also been suggested as one of the reasons for variance in the number of alleles and repeat size at SSR loci (42, 43, 44). Genome introgression detected by ISSR markers Three ISSR markers (GA) 9 C, (AC) 8 G and (CT) 9 G were selected on the basis of their wide application in studies on cultivar discrimination in wheat and its close relatives (62, 98). ISSR primer (GA) 9 C generated clear amplification profiles in all the 24 genotypes included in our study, comprising 230 reproducible bands with an average of 9.98 bands per genotype. The size of the amplified fragments ranged from 440 bp to 2800 bp. ISSR primer (AC) 8 G amplified a total of 220 bands in all 24 genotypes with an average of 9.16 bands per genotype. The size of the PCR fragments varied between 175 bp and 3000 bp. The third primer, (CT) 9 G, generated a less complex profile in the studied genotypes. Of the three ISSR primers, two (GA) 9 C and (AC) 8 G, respectively, amplified polymorphic bands in 3 out of a total of 8 alien species, not found in the cultivated durum wheat. ISSR primer (GA) 9 C generated a polymorphic band of 700 bp in T. polonicum and 680 bp in T. macha. Such a band was not found in cv. Progress, which was used as a parent in the interspesific crosses with both alien species. The polymorphic fragment was also amplified in the backcross lines BL 4 (T. polonicum Progress) and BL 7 (T. macha Progress), which is evidence for stable introgression of the alien chromatin into the genome of cultivated durum wheat. ISSR primer (AC) 8 G produced 4 polymorphic bands with a length of 300 bp, 3767

10 600 bp, 750 bp and 1000 bp in T. timopheevii as compared to cv. Vazhod. The polymorphic fragments were successfully transmitted in the backcross line BL 5 (Fig. 3S in the Online Supplementary Appendix, In addition to SSR markers, the application of ISSR markers allowed the detection of introgressed fragments from several alien species. Such introgressed fragments were found in the genome of cv. Progress from T. polonicum and T. macha, thus confirming the high compatibility between each of the two parental genomes. The several introgressed genome segments of T. timopheevii (AAGG) in BL 5 observed with the ISSR primer (AC) 8 G is an evidence for the usefulness of a large set of distinct markers for complete genome analysis of parental lines involved in the interspecific hybridization and their further use in molecular characterization of backcross progenies. Phenotypic evaluation of advanced generation of backcross lines The lines with proven genome segment introgressions from alien parents were phenotypically evaluated and chosen for further selection in late generations. The results of the phenotypic evaluation of eight backcross lines with proven genome introgressions from wild durum wheat relatives and their recurrent durum wheat parents are presented in Table 3. The field experiment in the year 2011 showed obvious variation for the studied agronomic and grain quality traits. The backcross lines differ statistically from the corresponding recurrent parents for some traits either in a positive direction, or in a negative one. In comparison to recurrent parents, the studied lines were late flowering (days to heading from 1 st May) with the exception of two lines: BL 2 from the cross combination with T. dicoccoides, and BL 5 from the cross combination with T. timopheevii, which flowered earlier than cultivar Vazhod. All lines were shorter (75.6 cm to 98.6 cm) than the recurrent cultivars (88.2 cm to cm), with the exception of line BL 2 from the hybrid combination with T. dicoccoides, which was taller than cultivar Vazhod. The flag leaf plays an important role during grain filling. Among the morphological characters, obvious variation in the flag leaf parameters of the backcross lines was observed. The flag leaf length ranged from 20.7 cm to 27.4 cm in the backcross lines, while the variation in this parameter was lower in the recurrent durum wheat parents (from 19.6 cm to 23.8 cm). Four of the lines were with a longer flag leaf, while two were with a shorter one, in contrast to recurrent cultivars. Five lines were found to have a wider flag leaf. Four lines were with a larger flag leaf area. Spike-related traits are very important for grain yield formation. The studied lines were with a shorter spike length than the corresponding recurrent parent, with the exception of line BL 6 from the cross combination with Ae. tauschii. The number of spikelets per spike showed similar values in both recurrent cultivars (from 20.9 to 21.1) and introgression lines (from 18.8 to 22). Two lines, BL 4 from the cross combination with T. polonicum, and BL 7 from the cross combination with T. macha, possess a higher number of spikelets per spike in comparison to the recurrent cultivar Progress. The number of grains per spike varied to a greater extent among the introgression lines and ranged from 27 to 51 as compared to in the recurrent cultivars. The number of grains was higher in lines BL 3 (T. dicoccum), BL 4 (T. polonicum) and BL 7 (T. macha) as compared to the recurrent cultivar Progress, which is characterized with the highest mean for this trait among the recurrent durum wheat cultivars. The grain weight TABLE 3 Agronomical and grain quality trait characteristics of the backcross lines obtained through wide hybridization and their recurrent durum wheat parents (years ) Backcross lines Heading date (days since May 1) Plant height (cm) Flag leaf parameters (cm) Length Width Leaf area Length (cm) Spike parameters Number of spikelets Number of grains Grain weight (g) 1000 grain weight 3768 Protein (%) Gluten % BL 1 20 c* 98.6 de 22.7 bc 1.78 b bcd 19 a 27 a 1.1 a 40.7 a d BL 2 20 c 91 cde 23.2 bcd 2.11 d bcd 21.2 ab 43.6 bc 1.7 ab 39 a g BL 3 23 e 75.6 a 21.3 ab 1.98 cd d 20.8 ab 51.4 c 2.2 cd 42.8 ab f BL 4 20 c 94.4 cde 20.7 a 1.94 cd ab 22 bc 50.2 bc 2.0 bcd 40 a e BL 5 19 b 80.2 ab 22.7 ab 1.92 cd bcd 19.2 a 46.8 bc 1.96 bcd 42.7 ab e BL 6 21 d 79.2 ab 22.7 ab 2.1 d bcd 20.8 ab 43.6 bc 1.98 bcd 45.9 abc d BL 7 26 g 86.8 abc 27.4 f 1.98 cd ab 22 bc 51 c 2.5 d cd f BL 8 24 e 80.6 ab 25.9 ef 1.63 a d 18.8 a 37.6 b 1.7 ab 45.3 abc g Progress 18 a 97.4 de 23.8 cd 1.84 bc a 20.9 ab 46.4 bc 2.34 cd 51.9 cd 15.9 b Vazhod 21 d 88.2 bcd 23.3 bcd 1.95 cd abc 21.1 ab 42 bc 1.98 bcd 45.5 abc c Victoria 19 b e 19.6 a 1.92 cd bcd 21 ab 40 bc 1.78 bc 47.3 bcd a * Means with the same letter are not significantly different (Duncun s multiple range test). wet dry

11 per spike was observed to vary within a narrow range in both recurrent cultivars ( ) and backcross lines ( ). Line BL 7 from the cross between T. macha and Progress as a recurrent parent is distinguished by the highest value of this trait among the studied genotypes. All studied lines were with lower 1000 grain weight in comparison to the recurrent durum wheat cultivars. The protein and gluten content in grains as well as gluten strength are the basic quality parameters for durum wheat used as raw material for pasta production and, therefore, their improvement is an important factor for durum wheat breeding programs. Higher protein content was identified in the grain of all studied lines in comparison with the recurrent durum wheat cultivars. The protein content varied from 16.9 % in line BL 1 of the cross with T. carthlicum, to 22.2 % in BL 5 from the cross with T. timopheevii, while in durum wheat it was lower: between 14.9 % in cv. Victoria to 16.5 % in cv. Vazhod. In addition, the results showed that the studied lines are characterized with a higher wet and dry gluten content than the recurrent durum wheat cultivars. Lines BL 2 from the cross with T. dicoccoides, BL 7 from the cross with T. macha and BL 8 from the cross with T. spelta were the genotypes with the highest gluten content among the studied ones. The study showed that most of the backcross lines are characterized with improved agronomic characters: shorter stem, longer flag leaves, and larger flag leaf area; and improved spike related traits: higher number of spikelets, higher grain number as well as higher protein and gluten content. Among the backcross lines BL 3 from the cross with T. dicoccum, BL 4 from the cross with T. polonicum, and BL 7 from the cross with T. macha and cv. Progress as a recurrent parent, possess the best combination of agronomic traits related to spike productivity and grain quality. It should be noted that in these lines only the molecular analyses revealed the highest level of introgressions from the donor genomes of the above-mentioned wild species. These lines should be further used in durum wheat breeding programs. The involvement of wide hybridization in practical durum wheat breeding programs still remains limited, regardless of the advancements in the protocols used for hybridization, genetic transfer and molecular identification of introgression (60). The tetraploid species T. dicoccoides and T. dicoccum have been widely used for durum wheat improvement in contrast to other tetraploids: T. polonicum, T. timopheevii, T. carthlicum; hexaploids: T. macha and T. spelta, and diploids: Ae. tauschii (54). These wild and related wheat species have been exploited mainly for transfer of genes responsible for biotic and abiotic stress resistance (54). However, earlier studies reported a loss of bread-making quality in wheat after transfer of these traits. The most frequently mentioned example of the detrimental effects is the use of the 1BL/1RS rye wheat translocation. The presence of this translocation is associated with superior grain yield and poor bread-making qualities, including low sedimentation volume and reduced dough strength (97). There are several reports indicating that the tetraploid species T. dicoccoides, T. dicoccum and T. polonicum are sources of protein with good quality (6, 7, 13, 35). The studies of Joppa et al. (36) and Kovacs et al. (39) also show that durum wheat-dicoccoides 6B substitution lines are a valuable source for improving the protein content in higher yielding cultivars, without negative effect on pasta cooking quality. The most promising traits of the backcross lines evaluated here are the higher protein and gluten content compared to the recurrent cultivars. Line BL 2 from the cross combination with T. dicoccoides is the genotype with the highest gluten content among all studied lines. These results confirmed the abovementioned observations that tetraploid species T. dicoccoides, T. dicoccum, T. polonicum and T. timopheevii could be used as a source of genes for high protein quantity. Since the protein content is not always related to the protein quality, additional investigations on gluten strength and the presence of certain gliadin and glutenin alleles for dough technological properties are in progress. The lines studied here do not significantly differ in a negative direction from the durum wheat cultivars due to the conducted phenotypic selection against unfavorable agronomic traits originating from the alien parents. The results indicate that the backcross strategy used is appropriate for transferring of novel alleles from alien species into durum wheat elite cultivars without a significant reduction in their valuable agronomic qualities. Our investigation supports the computer simulated data of Wang et al. (85) showing that the single backcrossing breeding strategy allows a considerable amount of favorable genes from wild donor plants to be transferred and simultaneously the adaptation of the recurrent cultivars to be improved. Conclusions The wild and cultivated durum wheat relatives are a rich source of genetic variability for virtually all important traits, e.g. yield, resistance to unfavorable climatic conditions, recombination frequency and distribution, etc. This variability could be exploited for improvement of cereal crops through introgression of desirable genetic characters from wild relatives to cultivated varieties, which could be aided by careful monitoring of alien gene introgression at the molecular level (66). The present study demonstrates the efficiency of SSR and ISSR technologies for polymorphism detection and analysis of introgression in durum wheat cultivars with high degree of genetic similarity, thereby offering a wide scope of applications in marker assisted breeding programs of wheat. Our results further showed that the polymorphism identified in the backcross lines was therefore a consequence of introgressive hybridizations involving different alien species and presentday durum wheat cultivars. Introgressed lines with improved single agronomic traits or combinations of several important ones related to spike productivity and grain quality were selected on the basis of phenotypic evaluation which could be 3769