Bread wheat (*Triticum aestivum*), a staple crop, suffers from low genetic diversity due to its recent origin from a limited number of hybridization events and subsequent domestication. This restricts its adaptability to changing environmental conditions and pathogen evolution. To overcome this, wheat breeding relies on intra- and interspecific hybridization to introduce novel variation. Emmer wheat (*T. turgidum* subsp. *dicoccum* and *dicoccoides*), a progenitor of bread wheat, possesses various beneficial traits, including disease resistance, drought tolerance, and improved end-use quality. Similarly, *Aegilops* species, particularly those containing the D genome (*Ae. tauschii*), offer valuable sources of genetic diversity, having been utilized for decades in wheat improvement due to the ease of introgression of D genome segments. However, other D genome-containing *Aegilops* species, such as *Ae. crassa*, *Ae. cylindrica*, and *Ae. ventricosa*, have remained largely unexploited because of crossing barriers, potential deleterious genetic drag, and limitations in molecular techniques for differentiating between the exotic and bread wheat D-genome chromosomal segments. Advances in whole genome sequencing now offer opportunities to efficiently introgres desirable traits from these underutilized resources. This study aimed to generate novel genetic resources by incorporating the genetic diversity of these less-exploited *Aegilops* species and diverse *T. turgidum* and *T. timopheevii* genotypes into bread wheat through hybridization.

The A and B genomes of emmer wheat are derived from *Triticum urartu* and a *Sitopsis* species, likely *Aegilops speltoides*, respectively, hybridizing around 0.36-0.5 million years ago to form wild emmer wheat (*T. turgidum* subsp. *dicoccoides*). Cultivated emmer wheat was domesticated approximately 10,000 years ago. Natural hybridization between cultivated emmer and *Ae. tauschii* led to the emergence of common wheat. The limited genetic variation in common wheat necessitates continuous development of new varieties to combat environmental stresses and evolving pathogens. Previous studies have highlighted the useful traits present in emmer wheat and *Aegilops* species, such as cold and salt tolerance, resistance to rusts and nematodes, but many allopolyploid *Aegilops* species containing a copy of the D genome remain largely unexploited due to hybridization barriers and genetic drag. Existing literature emphasizes the potential of using *Aegilops* species to enhance the genetic diversity of bread wheat through the production and subsequent use of amphiploids.

This research involved creating 192 different cross combinations between diverse genotypes of emmer wheat, durum wheat, *T. timopheevii*, and D genome-containing *Aegilops* species (*Ae. tauschii*, *Ae. crassa*, *Ae. cylindrica*, and *Ae. ventricosa*) over three consecutive years. Eleven emmer wheat landraces from Kurdistan province, Iran, and 12 emmer wheat genotypes from the Seeds and Plant Improvement Institute of Iran (SPII) were used. Additional genotypes of durum wheat and *T. timopheevii* were obtained from ICARDA and CIMMYT. *Aegilops* genotypes originated from the IPK gene bank (Germany) and the SPII. Hand pollination was performed between May and June, with the two outermost florets of spikelets pollinated. For crosses involving *Ae. tauschii*, in vitro embryo rescue was employed to overcome seed shrivelling. Shriveled F1 seeds were surface sterilized, the embryo extracted, and cultured on ½ MS media. Seedlings were then transferred to soil and subsequently to the field. Hybrids between tetraploid *Aegilops* species and wheat genotypes did not require embryo rescue as their seeds possessed endosperm. Pollen viability was assessed using Alexander's solution. Fluorescence in situ hybridization (FISH) was used to identify chromosomes, using pTa535-1 and (GAA)10 oligonucleotide probes. C-banding was also used for chromosomal analysis. Phenotypic evaluation of the amphiploid lines included measurements of plant height, spike length, awn length, spikelets per spike, nodes number, flag leaf width and length, flowering time, and peduncle length. Iron and zinc content in the grains was determined using atomic absorption spectrophotometry. Statistical analysis was performed using R software. Completely randomized design was used to analyze pollen viability, and Pearson's correlation was used to assess correlations between seed set rate and viable gamete rate. Principal component analysis was conducted for morphological data.

From the 192 cross combinations, 56 distinct synthetic hexaploid and octaploid F2 lines were successfully recovered using in vitro embryo rescue and spontaneous F2 seed production. These lines exhibited diverse genome compositions (AABBDD, AABBDDDD, AAGGDD, D'D'XXAABB, D'D'C'C'AABB, and D'D'N'N'AABB). Analysis of parental lines revealed high morphological variability, particularly for spike morphology, flag leaf width, spikelets per spike, spike length, flowering time, seed iron and zinc content. Crossability between tetraploid wheat and *Ae. tauschii* was moderate, with embryo rescue often needed for successful hybrid production. Crossability was high between tetraploid *Aegilops* species and tetraploid wheat with mostly plump seeds. Pollen viability in the F1 hybrids varied significantly, ranging from 0% to 24.81%. A positive correlation was found between unreduced gamete rate and plump seed set. FISH analysis confirmed the chromosomal constitution of the synthetic lines, revealing some chromosomal rearrangements or translocations induced by polyploidization in some lines. Phenotypic analysis of the amphiploids revealed high variation for morphological traits and some lines exhibited high iron and zinc content. Principal component analysis grouped amphiploids with similar genome compositions together.

The successful generation of diverse synthetic hexaploid and octaploid wheat lines demonstrates the potential of utilizing emmer wheat and underutilized *Aegilops* species to broaden the genetic base of bread wheat. The high variability observed in both the parental lines and the resulting amphiploids highlights the success of incorporating genetic diversity. The use of in vitro embryo rescue proved crucial in overcoming hybridization barriers, particularly in crosses involving *Ae. tauschii*. The observed chromosomal rearrangements suggest potential for generating novel genetic combinations. The high variation in morphological and nutritional traits in the amphiploids opens possibilities for selecting lines with improved agronomic characteristics and enhanced nutritional value. The correlation between unreduced gamete rates and plump seed set provides further insights into the mechanisms of polyploidization in wheat and *Aegilops* hybrids. These findings contribute significantly to wheat improvement efforts by providing new germplasm resources for breeding programs.

This study successfully produced a diverse set of synthetic hexaploid and octaploid wheat lines and amphiploids by crossing tetraploid wheat with D genome-containing *Aegilops* species. The resulting lines exhibited significant variation in genome constitution, morphology, and nutritional content. These findings demonstrate the value of utilizing the genetic diversity of emmer wheat and *Aegilops* species to enhance bread wheat breeding programs. Future research should focus on evaluating the agronomic performance and disease resistance of these lines, and explore the potential for further genetic improvement through backcrossing and marker-assisted selection.

The study was conducted under specific environmental conditions at the University of Kurdistan research farm. The generalizability of the findings might be limited as specific environmental effects or interactions between genotypes and environment could affect the results. The sample size of some cross combinations and the limited number of generations evaluated may affect the conclusions regarding the stability and inheritance of certain traits. The cytogenetic analysis was done for a sample of the materials, hence some other chromosomal arrangements might exist for other lines.