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The genomic constitution of Aegilops cylindrica Host (2n = 4x = 28, DcDcCcCc) was analyzed by C-banding, genomic in situ hybridization (GISH), and fluorescence in situ hybridization (FISH) using the DNA clones pSc119, pAs1, pTa71, and pTA794. The C-banding patterns of the Dc- and Cc-genome chromosomes of Ae. cylindrica are similar to those of D-and C-genome chromosomes of the diploid progenitor species Ae. tauschii Coss. and Ae. caudata L., respectively. These similarities permitted the genome allocation and identification of the homoeologous relationships of the Ae. cylindrica chromosomes. FISH analysis detected one major 18S-5.8S-25S rDNA locus in the short arm of chromosome 1Cc. Minor 18S-5.8S-25S rDNA loci were mapped in the short arms of 5Dc and 5Cc. 5S rDNA loci were identified in the short arm of chromosomes 1Cc, 5Dc, 5Cc, and 1Dc. GISH analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The breakpoints in all translocations were non-centromeric with similar-sized segment exchanges.

The distribution of the 5S and 18S-5.8S-26S (18S-26S) ribosomal RNA (rRNA) gene families on chromosomes of all diploid Aegilops species was studied by in situ hybridization with pTa71 (18S-26S rDNA) and pTa794 (5S rDNA) DNA clones. One major 18S-26S rDNA locus was found in the nucleolus organizer region (NOR) of each of the species Aegilops tauschii and Aegilops uniaristata and two loci were detected in the remaining species. In addition to major NORs, from one to nine minor loci were observed; their numbers and chromosomal locations were species-specific. Some minor loci were polymorphic, whereas others were conserved. One or two 5S rDNA loci were observed in the short arms of the chromosomes of groups 1 and 5 of all diploid Aegilops species except Ae. uniaristata, where one 5S rDNA site was located in the distal part of the long arm of chromosome 1N. The 5S rDNA loci were not associated with NORs; however, the relative positions of two ribosomal RNA gene families were diagnostic for chromosomes of homoeologous groups 1, 5, and 6. Implications of these results for establishing phylogenetic relationships of diploid Aegilops species and mechanisms of genome differentiation are discussed. Key words : wheat, Triticum, Aegilops, 5S rRNA, 18S-26S rRNA, in situ hybridization, evolution.

Genome differentiation in 12 diploid Aegilops species was analyzed using in situ hybridization with the highly repetitive DNA sequences pSc119 and pAs1 and C-banding. Chromosomes of all these diploid Aegilops species hybridized with the pSc119 probe; however, the level of hybridization and labeling patterns differed among genomes. Only four species (Ae. squarrosa, Ae. comosa, Ae. heldreichii, and Ae. uniaristata) showed distinct hybridization with pAs1. The labeling patterns were species-specific and chromosome-specific. Differences in in situ hybridization (ISH) patterns, also observed by C-banding, exist between the karyotypes of Ae. comosa and Ae. heldreichii, suggesting that they are separate, although closely related, subspecies. The S genome of Ae. spelioides was most similar to the B and G genomes of polyploid wheats on the basis of both C-banding and ISH patterns, but was different from other species of section Sitopsis. These species had different C-banding patterns but they were similar to each other and to Ae. mutica in the distribution of pSc119 hybridization sites. Two types of labeling were detected in Ae. squarrosa with the pAs1 probe. The first resembled that of the D-genome chromosomes of bread wheat, Triticum aestivum L. em. Thell., while the second was similar to the D genome of some of the polyploid Aegilops species. Relationships among diploid Aegilops species and the possible mechanisms of genome differentiation are discussed. Key words : wheat, Triticum, Aegilops, in situ hybridization, C-banding, evolution.

Nine families of bread wheat (TC5, TC6, TC7, TC8, TC9, TC10, TC14, 5395-(243AA), and 5395) with resistance to barley yellow dwarf virus and containing putative translocations between wheat and a group 7 chromosome of Agropyron intermedium (L1 disomic addition line, 7Ai#1 chromosome) induced by homoeologous pairing or tissue culture were analyzed. C-banding, genomic in situ hybridization (GISH), and restriction fragment length polymorphism (RFLP) in combination with repetitive Agropyron-specific sequences and deletion mapping in wheat were used to determine the relative locations of the translocation breakpoints and the size of the transferred alien chromatin segments in hexaploid wheat-Agropyron translocation lines. All homoeologous compensating lines had complete 7Ai#1 or translocated 7Ai#1-7D chromosomes that substitute for chromosome 7D. Two complete 7Ai#1 (7D) substitution lines (5395-(243AA) and 5395), one T1BS-7Ai#1S∙7Ai#1L addition line (TC7), and two different translocation types, T7DS-7Ai#1S∙7Ai#1L (TC5, TC6, TC8, TC9, and TC10) and T7DS∙7DL-7Ai#1L (TC14), substituting for chromosome 7D were identified. The substitution line 5395-(243AA) had a reciprocal T1BS∙1BL-4BS/T1BL-4BS∙4BL translocation. TC14 has a 6G (6B) substitution. The RFLP data from deletion mapping studies in wheat using 37 group 7 clones provided 10 molecular tagged chromosome regions for homoeologous and syntenic group 7 wheat or Agropyron chromosomes. Together with GISH we identified three different sizes of the transferred Agropyron chromosome segments with approximate breakpoints at fraction length (FL) 0.33 in the short arm of chromosome T7DS-7Ai#1S∙7Ai#1L (TC5, TC6, TC8, TC9, and TC10) and another at FL 0.37 of the nonhomoeologous translocated chromosome T1BS-7Ai#1S∙7Ai#1L (TC7). One breakpoint was identified in the long arm of chromosome T7DS∙7DL-7Ai#1L (TC14) at FL 0.56. We detected some nonreciprocal translocations for the most proximal region of the chromosome arm of 7DL, which resulted in small duplications. Key words : C-banding, genomic in situ hybridization (GISH), physical mapping, translocation mapping, RFLP analysis.

Genomic in situ hybridization to somatic metaphase chromosomes of hexaploid wheat cv. Chinese Spring using biotinylated maize genomic DNA as a probe revealed the existence of amplified maize DNA sequences in five pairs of chromosomes. The in situ hybridization sites were located on chromosomes 1A, 7A, 2B, 3B, and 7B. One pair of in situ hybridization sites was also observed in hexaploid oat. The locations and sizes of in situ hybridization sites varied among progenitor species.

Triticum araraticum Jakubz. (2n = 4x = 28, AtAtGG), a wild progenitor of the polyploid cultivated wheat T. timopheevii, shows extensive chromosome translocation polymorphism in natural populations from the Middle East and Transcaucasia. From an extensive survey, eight intergenomic translocation types were observed and their breakpoints analyzed by genomic in situ hybridization. The previously reported species-specific 6At-1G-4G cyclic translocation was found in all accessions studied. In four translocation types, the breakpoints were in interstitial regions of chromosomes and the other four arose via centric-breakage-fusion. A model is presented on the mechanism of origin and the adaptive significance of translocations with centromeric and noncentromeric breakpoints.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.

A targeted mapping strategy using representational difference analysis (RDA) was employed to isolate new restriction fragment length polymorphism probes for the long arm of chromosome 6 in rye (6RL), which carries a gene for resistance to Hessian fly larvae. Fragments from the 6RL arm were specifically isolated using a 'Chinese Spring' (CS) wheat - rye ditelosomic addition line (CSDT6RL) as tester, and CS and (or) CS4R as the driver for the genomic subtraction. Three RDA experiments were performed using BamHI amplicons, two of which were successful in producing low-copy clones. All low-copy clones were confirmed to have originated from 6RL, indicating substantial enrichment for target sequences. Two mapping populations, both of which are derived from a cross between two similar wheat-rye translocation lines, were used to map five RDA probes as well as five wheat probes. One of the populations was prescreened for recombinants by C-banding analysis. Fifteen loci, including seven new RDA markers, were placed on a map of the distal half of 6RL. The Hessian fly resistance gene was localized by mapping and C-banding analysis to approximately the terminal 1% of the arm. The utility of RDA as a method of targeted mapping in cereals and prospects for map-based cloning of the resistance gene are discussed.

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.

The chromosome 1R of rye, or the midget chromosome, is necessary for plump, viable seed development and fertility restoration in the alloplasmic line with rye cytoplasm and a hexaploid wheat nucleus. The midget chromosome of rye represents 1/15th of the physical length of the chromosome 1R of rye. C-banding analysis indicated that the centromeric and pericentric region (approximately 30% physical length) of the midget chromosome is heterochromatic and the distant 70% physical length is euchromatic. These data suggest that the midget chromosome may represent the pericentric region of the long arm of chromosome 1R. In contrast with earlier reports, our results indicate that an array of rye-specific repeated sequences (both dispersed and tandem) are present on the midget chromosome. Various rye-specific repeated DNA sequences that are present on the midget chromosome will be useful in constructing a long-range map and studying the genomic organization of the midget chromosome. It is unclear if any of these repeated DNA sequences are involved in the origin of the midget chromosome.

C-banding polymorphism was analyzed in 17 accessions of Triticum longissimum from Israel and Jordan, and a generalized idiogram of this species was established. C-banding analysis was further used to identify two sets of disomic T. aestivum - T. longissimum chromosome addition lines and 13 ditelosomic addition lines and one monotelosomic (6S1L) addition line. C-banding was also used to identify T. aestivum - T. longissimum chromosome substitution and translocation lines. Two major nucleolus organizing regions (NORs) on 5S1 and 6S1 and one minor NOR on 1S1 were detected by in situ hybridization using a 18S-26S rDNA probe. Sporophytic and gametophytic compensation tests were used to determine the homoeologous relationships of T. longissimum chromosomes. The T. longissimum chromosomes compensate rather well and fertility was restored even in substitution lines involving wheat chromosomes 2A, 4B, and 6B that contain major fertility genes. Except for the deleterious gametocidal genes, T. longissimum can be considered as a suitable donor of useful genes for wheat improvement.

Different combinations of chromosome N- or C-banding with in situ hybridization (ISH) or genomic in situ hybridization (GISH) were sequentially performed on metaphase chromosomes of wheat. A modified N-banding-ISH/GISH sequential procedure gave best results. Similarly, a modified C-banding - ISH/GISH procedure also gave satisfactory results. The variation of the hot acid treatment in the standard chromosome N- or C-banding procedures was the major factor affecting the resolution of the subsequent ISH and GISH. By the sequential chromosome banding - ISH/GISH analysis, multicopy DNA sequences and the breakpoints of wheat-alien translocations were directly allocated to specific chromosomes of wheat. The sequential chromosome banding- ISH/GISH technique should be widely applicable in genome mapping, especially in cytogenetic and molecular mapping of heterochromatic and euchromatic regions of plant and animal chromosomes.

Alloplasmic euploid wheat with the cytoplasm of Elymus ciliaris (2n = 4x = 28, ScScYcYc) is male sterile and has reduced vigor. However, alloplasmic plants with E. ciliaris chromosomes 1Sc or 1Yc marked by gliadin genes Gli-Sc1 and Gli-Ycl, respectively, are vigorous and fertile. The Rf genes on 1Sc and 1Yc are named Rf-Sc1 and Rf-Yc1. Two chromosome translocations involving 1Yc were isolated. The first involved the short arm of 1Yc translocated to the short arm of wheat chromosome 3B. The second involved the short arm of 1Yc translocated to the short arm of a chromosome, designated L, of E. ciliaris. The second line also has another E. ciliaris chromosome designated A and lacks wheat chromosome 6A. This line is resistant to Puccinia recondita. The relationship between fertility restoration and nucleolar organizing regions is discussed. Key words : Triticum aestivum, Elymus ciliaris, chromosome addition, Rf genes, nucleolar organizing regions.