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We have constructed a cytogenetically based physical map of chromosome 1B in common wheat by utilizing a total of 18 homozygous deletion stocks. It was possible to divide chromosome 1B into 17 subregions. Nineteen genetic markers are physically mapped to nine subregions of chromosome 1B. Comparison of the cytological map of chromosome 1B with an RFLP-based genetic linkage map of Triticum tauschii revealed that the linear order of the genetic markers was maintained between chromosome 1B of hexaploid wheat and 1D of T. tauschii. Striking differences were observed between the physical and genetic maps in relation to the relative distances between the genetic markers. The genetic markers clustered in the middle of the genetic map were physically located in the distal regions of both arms of chromosome 1B. It is unclear whether the increased recombination in the distal regions of chromosome 1B is due to specific regions of increased recombination or a more broadly distributed increase in recombination in the distal regions of Triticeae chromosomes.Key words: common wheat, chromosome 1B, homozygous deletion lines, physical map, RFLP markers.

A strategy is described for rapid chromosome region-specific mapping in hexaploid wheat (Triticum aestivum L. em. Thell., 2n=6x=42, AABBDD). The method involves allocation of markers to specific chromosome regions by deletion mapping and ordering of probes by high resolution genetic mapping in Triticum tauschii, the D-genome progenitor species. The strategy is demonstrated using 26 chromosome deletion lines for wheat homoeologous group-6. Twenty-five DNA probes from the T. tauschii genetic linkage map and six wheat homoeologous group-6 specific probes were mapped on the deletion lines. Twenty-four of the 25 probes from 6D of T. tauschii also mapped on wheat homoeologous group-6 chromosomes, and their linear order in wheat is the same as in T. tauschii. A consensus physical map of wheat group-6 was constructed because the linear order and the relative position of the probe loci was the same among the three group-6 chromosomes. Comparison of the consensus physical map with the genetic map demonstrated that most of the recombination occurs in the distal ends of the wheat chromosomes. Most of the loci mapped in the distal regions of the chromosomes. The probes were mostly either PstI genomic clones or cDNA clones indicating that the undermethylated single-copy sequences are concentrated in the distal ends of the wheat chromosomes. Fifteen loci are uniformly distributed in the distal 11% of the group-6 chromosomes. Physically, the region spans only 0.58 μm, which in wheat translates to about 40 Mb of DNA. The average distance between the markers is, therefore, less than 2.7 Mb and is in the range of PFGE (pulsed-field gel electrophoresis) resolution. Any gene present in the region can be genetically ordered with respect to the markers since the average recombination frequency in the region is very high (>90 cM genetic distance).

Restriction fragment length polymorphism (RFLP) markers linked to genes controlling Hessian fly resistance from Triticum tauschii (Coss.) Schmal. were identified for two wheat (Triticum aestivum L.) germ plasm lines KS89WGRC3 (C3) and KS89WGRC6 (C6). Forty-six clones with loci on chromosomes of homoeologous group 3 and 28 clones on those of group 6 were surveyed for polymorphisms. Eleven and 12 clones detected T. tauschii loci in the two lines, respectively. Analysis of F2 progenies indicated that the Hessian fly resistance gene H23 identified in C3 is linked to XksuH4 (6.9 cM) and XksuG48 (A) (15.6 cM), located on 6D. The resistance gene H24 in C6 is linked to XcnlBCD451 (5.9 cM), XcnlCD0482 (5.9 cM) and XksuG48 (B) (12.9 cM), located on 3DL.

The recent advances in alien gene transfer from distantly-related species into wheat are reviewed in the present paper. The main achievements during the last ten years include the great expansion of the range of wide hybridization and development of new techniques for production and characterization of wheat-alien chromosome translocations. Updated results of wide hybridization since 1983 and comprehensive characterization of wheat-alien translocation lines in our laboratory are compiled. The future outlook for alien gene transfer in wheat is also discussed.

Radiation-induced wheat-rye chromosome translocation lines resistant to Hessian fly, Mayetiola destructor (say), were analyzed by in situ hybridization using total genomic and highly repetitive rye DNA probes pSc119 and pSc74. In situ hybridization analysis revealed the exact locations of the translocation breakpoints and allowed the estimation of the sizes of the transferred rye segments. T6BS·6BL-6RL and T4BS· 4BL-6RL are terminal translocations with either most of the complete long arm of rye chromosome 6R or only the distal 57% of the 6RL arm attached to the long arms of wheat chromosomes 6B and 4B, respectively. The breakpoint in T6BS·6BL-6RL is located at a fraction length (FL) of 0.11 in the long arm of T6BS 6BL-6RL and at FL 0.46 in the long arm of T4BS·4BL-6RL. Ti4AS·4AL-6RL-4AL is an intercalary translocation with the breakpoint located at FL 0.06 in the long arm of wheat chromosome 4A. The inserted 6RL segment, with the Hessian fly resistance gene, has a size of 0.7 μm, and is the smallest and, so far, the first radiation-induced intercalary translocation identified in wheat.

Stem rust (Puccinia graminis f. sp. tritici Eriks.E. Henn.) (the causal agent of wheat stem rust) race Ug99 (also designated TTKSK) and its derivatives have defeated several important stem rust resistance genes widely used in wheat (Triticum aestivum L.) production, rendering much of the worldwide wheat acreage susceptible. In order to identify new resistance sources, a large collection of wheat relatives and genetic stocks maintained at the Wheat Genetic and Genomic Resources Center was screened. The results revealed that most accessions of the diploid relative Dasypyrum villosum (L.) Candargy were highly resistant. The screening of a set of wheat-D. villosum chromosome addition lines revealed that the wheat-D. villosum disomic addition line DA6V#3 was moderately resistant to race Ug99. The objective of the present study was to produce and characterize compensating wheat-D. villosum whole arm Robertsonian translocations (RobTs) involving chromosomes 6D of wheat and 6V#3 of D. villosum through the mechanism of centric breakage-fusion. Seven 6V#3-specific EST-STS markers were developed for screening F(2) progeny derived from plants double-monosomic for chromosomes 6D and 6V#3. Surprisingly, although 6D was the target chromosome, all recovered RobTs involved chromosome 6A implying a novel mechanism for the origin of RobTs. Homozygous translocations (T6AS·6V#3L and T6AL·6V#3S) with good plant vigor and full fertility were selected from F(3) families. A stem rust resistance gene was mapped to the long arm 6V#3L in T6AS·6V#3L and was designated as Sr52. Sr52 is temperature-sensitive and is most effective at 16°C, partially effective at 24°C, and ineffective at 28°C. The T6AS·6V#3L stock is a new source of resistance to Ug99, is cytogenetically stable, and may be useful in wheat improvement.

The emergence of a new highly virulent race of stem rust (Puccinia graminis tritici), Ug99, rapid evolution of new Ug99 derivative races overcoming resistance of widely deployed genes, and spread towards important wheat growing areas now potentially threaten world food security. Exploiting novel genes effective against Ug99 from wild relatives of wheat is one of the most promising strategies for the protection of the wheat crop. A new source of resistance to Ug99 was identified in the short arm of the Aegilops searsii chromosome 3S(s) by screening wheat- Ae. searsii introgression libraries available as individual chromosome and chromosome arm additions to the wheat genome. For transferring this resistance gene into common wheat, we produced three double-monosomic chromosome populations (3A/3S(s), 3B/3S(s) and 3D/3S(s)) and then applied integrated stem rust screening, molecular maker analysis, and cytogenetic analysis to identify resistant wheat-Ae. searsii Robertsonian translocation. Three Robertsonian translocations (T3AL·3S(s)S, T3BL·3S(s)S and T3DL·3S(s)S) and one recombinant (T3DS-3S(s)S·3S(s)L) with stem rust resistance were identified and confirmed to be genetically compensating on the basis of genomic in situ hybridization, analysis of 3A, 3B, 3D and 3S(s)S-specific SSR/STS-PCR markers, and C-banding. In addition, nine SSR/STS-PCR markers of 3S(s)S-specific were developed for marker-assisted selection of the resistant gene. Efforts to reduce potential linkage drag associated with 3S(s)S of Ae. searsii are currently under way.

Background A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat. Results Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed. Conclusions In a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.

Background Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. Results We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. Conclusions The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.

Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries. Conclusions The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.

L'article original est publié par The American Society of Plant Biologists; To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.

The distribution of the telomeric repeats in common wheat and their role in the healing of broken ends of deleted chromosomes was studied. In situ hybridization to mitotic chromosomes was carried out using a synthetic probe that was derived from the sequence of the telomeric repeats of Arabidopsis thaliana. Sites of hybridization were visualized as double dots at both ends of each wheat chromosome. Variation in the strength of the signal that was detected among chromosome arms might be due to the variable number of telomeric repeats of each chromosome end. While signals were absent on normal chromosomes at the pericentric and intercalary regions, hybridization sites were detected at the broken chromosome ends of all deleted chromosomes included in the study. All telocentric chromosomes of multitelocentric lines of 'Chinese Spring' showed a strong signal at the centromeric region. The results suggest that a de novo chromosome healing mechanism exists in wheat involving the addition of the telomeric sequences to the ends of broken chromosome. Further evidence indicated that the healing of broken ends is probably intrinsic to replication during gametogenesis.Key words: in situ hybridization, telomeric sequences, deleted chromosomes, chromosome healing, telosome.

C-banded karyotypes of Agropyron intermedium (2n=6x=42, E1E2X), a partial amphiploid Triticum aestivum -Ag. intermedium (2n=8x=56, TAF46), and six derived chromosome addition lines, were analyzed. In Ag. intermedium, diagnostic C-bands were present on 14 pairs of chromosomes, designated from A to N, while the remaining seven pairs, designated O to U, either lacked, or had only faint, C-bands and were not always identified unambiguously. All seven Ag. intermedium chromosome pairs of the partial amphiploid TAF46, and the added Ag. intermedium chromosomes present in the six derived addition lines, were identified by their characteristic C-banding patterns. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent Ag. intermedium accession, suggesting that these chromosomes were not structurally rearranged. In-situ hybridization, using a 18s.265s rDNA probe, showed that the Ag. intermedium chromosomes 1Ai-1 and 5Ai-l present in the addition lines L3 and L5 were carrying actively transcribed nucleolus organizer regions. The results are discussed with respect to the genomic relationships of these chromosomes.

Alloplasmic euploid wheat lines with the cytoplasm of Elymus trachycaulus (2n = 4x = 28, StStHtHt) and Elymus ciliaris (2n = 4x = 28, ScScYcYc) are male sterile and have reduced vigor. Fertile alloplasmic wheat plants are also recovered, but they always contain complete or partial chromosome additions of 1Ht or 1St (in E. trachycaulus derived lines) or 1Yc (in E. ciliaris derived lines) with specific Elymus gliadin genes (Gli-Ht1, Gli-St1, Gli-Yc1) and fertility restoration (Rf) genes. The Rf genes on 1Ht 1St and 1Yc were named as Rf-Ht1, Rf-St1, and Rf-Yc1, respectively. In this study, we crossed different disomic addition lines with one another to produce double monosomic additions either in E. trachycaulus or E. ciliaris cytoplasm. The chromosome constitution, transmission, and fertility of the selfed and backcrossed progenies of three double monosomic additions with 21″ + 3BS∙1YcS′ + 1HtS∙1HtS′ (cytoplasm from E. trachycaulus), 21″ + 3BS∙1YcS′ + 1HtS∙1HtS′ (cytoplasm from E. ciliaris), and 21″ + 3BS∙1YcS′ + 7AL∙S-1StS′ (cytoplasm from E. ciliaris) were analyzed. The results indicated that (i) Rf-Ht1 and Rf-St1 on 1Ht and 1St restored fertility to wheat with E. ciliaris cytoplasm; (ii) Rf-Yc1 on 1Yc restored fertility to wheat with E. trachycaulus cytoplasm; (iii) cytoplasms of E. ciliaris and E. trachycaulus are closely related; and (iv) certain E. trachycaulus and E. ciliaris chromosomes show preferential transmission.Key words: Elymus, Rf genes, wheat–Elymus hybrids, cytoplasmic genetics, genome evolution.

Although the genetic linkage map of Triticum tauschil, the D-genome progenitor of wheat its available, its use for linkage analysis of hexaploid wheat chromosome regions is hampered by the lack of polymorphism in wheat. Here we describe a strategy to identity probes that detect a high degree of polymorphism in wheat. The strategy involves the use of DNA probes that detect null alleles. About 16% of the Pstl genomic clones from Triticum tauschil detect null alleles in the species. The probes that detect null alleles reveal high degree of polymorphism among hexaploid wheat cultivars. The probes selected following this strategy are expected to detect null alleles throughout the tribe Triticeae, therefore, reveal high degree of polymorphism.

Homogeneity between the chromosome lines of an aneuploid set is necessary to avoid supplemental effects of the genetic background. The objective of this study was to determine the homogeneity of the Wihita' (WI) winter wheat (Triticum aestivum L.) monosomic set for grain quality and agronomic traits. Disomics were produced from 20 chromosome lines of the WI monosomic set and 2B disomics from Cheyenne' monosomic 2B (...)

A disomic F 2 substitution study was conducted to elucidate the chromosomal location of genes influencing the high grain-protein concentration (GPC) in Plainsman V' (PV) wheat (Triticum aestivum L.) and to give preliminary information on genes influencing its dough mixogram peak time and tolerance. Plainsman V has been used extensively in the Kansas State University wheat breeding program and is included in the pedigree of many lines because of its high GPC and strong gluten (...)

A generalized C-banded karyotype of Triticum tauschii (Aegilops squarrosa) was established based on chromosome analysis of 15 accessions of diverse origin, including the two varieties eusquarrosa (with the former varieties typica, anathera, and meyeri) and strangulata. The C-banding pattern of T. tauschii chromosomes was similar to the D-genome chromosomes of cultivated bread wheat, T. aestivum, thus permitting their unequivocal identification and homoeologous chromosome designations. Whereas only minor variation in C-banding pattern was observed within the accessions, a large amount of polymorphic variation was found among the different accessions. However, this polymorphic variation did not prevent chromosome identification in these lines. One accession (TA 2462) was found to be homozygous for a reciprocal translocation involving the complete arms of chromosomes 1D and 7D. In situ hybridization using the D-genome specific probe pAS1 confirmed the presence of T1DS-7DL and T7DS.1DL translocation in the accession TA 2462.Key words: Triticum tauschii, C-banding, in situ hybridization.

C-banding patterns of the seven `Imperial' rye chromosomes and 11 derived rye telocentrics added to `Chinese Spring' were analyzed and compared with in situ hybridization (ISH) patterns using biotin labeled highly repetitive rye DNA sequences pSc119, pSc74, and total rye genomic DNA as probes. C-banding and ISH analyses allow the identification of all individual rye chromosomes and most chromosome arms with these probes. A C-banded karyotype of the added rye chromosomes was constructed and data on chromosome lengths, arm ratios, and fraction lengths of diagnostic C-bands are given following the nomenclature system recently recommended for wheat. Similar telomeric and interstitial localized ISH patterns were observed with pSc119, pSc74, and total rye genomic DNA probes. In addition, pSc119 and total rye genomic DNA labels rye chromatin over their entire lengths, also permitting the detection of unbanded euchromatic rye chromatin transfers in wheat. All localized ISH sites correspond to dark C-banded regions; however, not all C-bands show up as hybridization sites.

The cryptic wheat–alien translocation T5DL·5DS-5MgS(0.95), with leaf rust and stripe rust resistance genes Lr57 and Yr40 transferred from Aegilops geniculata (UgMg) into common wheat, was further analyzed. Molecular genetic analysis using physically mapped ESTs showed that the alien segment in T5DL·5DS-5MgS(0.95) represented only a fraction of the wheat deletion bin 5DS2-0.78-1.00 and was less than 3.3 cM in length in the diploid wheat genetic map. Comparative genomic analysis indicated a high level of colinearity between the distal region of the long arm of chromosome 12 of rice and the genomic region spanning the Lr57 and Yr40 genes in wheat. The alien segment with genes Lr57 and Yr40 corresponds to fewer than four overlapping BAC or PAC clones of the syntenic rice chromosome arm 12L. The wheat–alien translocation breakpoint in T5DL·5DS-5MgS(0.95) was further localized to a single BAC clone of the syntenic rice genomic sequence. The small size of the terminal wheat–alien translocation, as established precisely with respect to Chinese Spring deletion bins and the syntenic rice genomic sequence, further confirmed the escaping nature of cryptic wheat–alien translocations in introgressive breeding. The molecular genetic resources and information developed in the present study will facilitate further fine-scale physical mapping and map-based cloning of the Lr57 and Yr40 genes.

The wheat leaf-rust resistance gene Lr21 was first identified in an Iranian accession of goatgrass, Aegilops tauschii Coss., the D-genome donor of hexaploid bread wheat, and was introgressed into modern wheat cultivars by breeding. To elucidate the origin of the gene, we analyzed sequences of Lr21 and lr21 alleles from 24 wheat cultivars and 25 accessions of Ae. tauschii collected along the Caspian Sea in Iran and Azerbaijan. Three basic nonfunctional lr21 haplotypes, H1, H2, and H3, were identified. Lr21 was found to be a chimera of H1 and H2, which were found only in wheat. We attempted to reconstitute a functional Lr21 allele by crossing the cultivars Fielder (H1) and Wichita (H2). Rust inoculation of 5876 F2 progeny revealed a single resistant plant that proved to carry the H1H2 haplotype, a result attributed to intragenic recombination. These findings reflect how plants balance the penalty and the necessity of a resistance gene and suggest that plants can reuse “dead” alleles to generate new disease-resistance specificity, leading to a “death–recycle” model of plant-resistance gene evolution at simple loci. We suggest that selection pressure in crop–weed complexes contributes to this process.

Cytogenetics is the correlated study of genetics and cytology. In cereals, five phases of cytogenetic research can be recognized: (i) meiotic pairing analysis of F1 hybrids; (ii) aneuploidy. (iii) molecular cytogenetics (C-banding and in situ hybridization); (iv) deletion bin mapping; and (v) flow cytogenetics. We review here the first four phases of cytogenetic research with special reference to chromosome analysis of wheat and rye. Meiotic pairing analysis revealed genomic relationships among diploid and polyploid species. Aneuploidy opened possibilities of chromosome/arm and comparative mapping. C-banding and in situ hybridization allowed rapid identification and analysis of heterochromatic and euchromatic components of wheat and rye chromosomes. The isolation of deletion stocks and their use to study the structure and function of the expressed portion of the wheat genome further revealed structural and functional differentiation of wheat chromosomes into proximal gene-poor/low recombination and distal gene-rich/high recombination compartments. The abovementioned structural and functional differentiation may have been driven by chromosome behavior at meiosis. As DNA sequence information becomes available and with the application of techniques such as Fiber FISH and others that close the gap between DNA level and chromosome level observations, we can truly begin to understand the biological meaning of the superimposed structural, functional, and behavioral differentiation and organization of cereal chromosomes.

A set of four repetitive DNA clones, pEt1, pEt2, pCb1, and pCb3, were isolated from SH-genome polyploid Elymus trachycaulus and H-genome diploid Critesion bogdanii. The clone Et1 represents a tandemly arranged telomeric sequence. Et2 represents tandem repeats interspersed along the entire length of individual chromosomes. The Cb1 sequence was more evenly dispersed. The Et1 clone shared homology with a 350 base pair family of rye sequences. The Cb3 sequence was evenly distributed in S- and H-genome species. All the repetitive DNA sequences were excellent markers for the specific detection and genomic affinity of Elymus chromatin added to wheat. All clones showed intragenomic variation in copy number and chromosomal location. Based on the analysis of this variation, we conclude that E. trachycaulus most probably originated from putative diploid H- and S-genome species resembling Critesion californicum and Pseudoroegneria spicata, respectively.Key words: wheatgrass, wheat–Elymus hybrid, addition lines, polyploidy, restriction fragment length polymorphism.

The bottleneck restricting introgression of useful genes directly from diploid into hexaploid wheats is the low number of BC1F1 seeds obtained. In crosses between hexaploid wheat (Triticum aestivum L.; AABBDD) and Aegilops squarrosa L. (DD) or T. urartu Thum. (AA), this bottleneck may be overcome simply by pollinating a sufficient number of F1 spikes. However, hybrids between hexaploid wheat cultivars (T. aestivum) and T. monococcum L. (AA) generally are highly female-sterile, often having no pistils. One T. monococcum accession, PI 355520, when crossed with T. aestivum, produced hybrids with female fertility in the same range as that of T. aestivum/A. squarrosa or T. aestivum/T. urartu hybrids, ca. 0.5 to 1.0 backcross seed per spike. We found that female fertility was controlled by two duplicate genes in PI 355520, and that this accession can be used as a bridging parent to introgress genes from other T. monococcum accessions into hexaploid wheat. Pairing of homologous chromosomes was less frequent and weaker in such crosses than in T. aestivum/A. squarrosa crosses, but homoeologous bivalents occurred at a rate of almost 0.5 II per cell. Restitution division was detected in crosses involving all three diploid species and was confirmed cytologically in crosses with PI 355520. Chromosome numbers of BC1F1 plants ranged from 35 to 67; plants with 49 or more chromosomes occurred at frequencies of 0.09 to 0.21 among progeny of A. squarrosa and T. urartu and 0.29 in progeny of T. aestivum/T. monococcum crosses involving PI 355520. These results are consistent with those of previous studies, demonstrating the potential of direct Hexaploid/diploid crosses for rapidly introgressing useful genes into Hexaploid wheat with minimum disturbance of the background genotype.

One hundred and seventy-eight loci have been mapped in Triticum tauschii (Coss.) Schmal. (2n = 14, DD) and Triticum aestivum L. em. Thell. (2n = 42, AABBDD). Thirty-five loci were mapped by aneuploid analysis in T. aestivum. One hundred and fifty-two loci, including 143 restriction fragment length polymorphisms (RFLPs), 8 proteins, and 1 leaf rust resistance gene, were mapped in an F2 population (60 plants) of T. tauschii. One hundred and twenty-seven loci were placed in linkage groups belonging to seven D-genome chromosomes of T. tauschii. The source of the probes was a PstI genomic library of T. tauschii, which gave 13% single-low copy clones. Four restriction endonucleases (DraI, EcoRI, EcoRV, HindIII) gave 75% polymorphism between the two parents. Nineteen clones detected multiloci ranging from two to nine in number. Deletions–insertions and point mutations were equally important for generating RFLPs. A hypervariable sequence was identified, which may have potential use in varietal fingerprinting. One marker was found to be linked to a rust-resistance gene. The map will be useful for determining genetic relationships in the Triticeae and for tagging genes of economic importance.Key words: restriction fragment length polymorphism, Triticum aestivum, leaf rust, isozymes, Aegilops squarrosa.

The chromosome constitutions of eight wheat streak mosaic virus (WSMV)-resistant lines, three of which are also greenbug resistant, derived from wheat/ Agropyron intermedium/Aegilops speltoides crosses were analyzed by C-banding and in situ hybridization. All lines could be traced back to CI15092 in which chromosome 4A is substituted for by an Ag. intermedium chromosome designated 4Ai-2, and the derived lines carry either 4Ai-2 or a part of it. Two (CI17881, CI17886) were 4Ai-2 addition lines. CI17882 and CI17885 were 4Ai-2-(4D) substitution lines. CI17883 was a translocation substitution line with a pair of 6AL.4Ai-2S and a pair of 6AS.4Ai-2L chromosomes substituting for chromosome pairs 4D and 6A of wheat. CI17884 carried a 4DL.4Ai-2S translocation which substituted for chromosome 4D. CI17766 carried a 4AL.4Ai-2S translocation substituting for chromosome 4A. The results show that the 4Ai-2 chromosome is related to homoeologous group 4 and that the resistance gene(s) against WSMV is located on the short arm of 4Ai-2. In addition, CI17882, CI17884, and CI17885 contained Ae. speltoides chromosome 7S substituting for chromosome 7A of wheat. The greenbug resistance gene Gb5 was located on chromosome 7S.

In situ hybridization in conjunction with deletion mapping was used to map physically the 18S.26S multigene rRNA family in Triticum aestivum L. cv. Chinese Spring. Using in situ hybridization, we report a new locus in the 7DL arm of Chinese Spring and Aegilops squarrosa, and also confirm the nucleolus organizing region (Nor) locus in the short arm of chromosome 1A at the telomeric end in Chinese Spring. Based on in situ hybridization labeling patterns, we show that rDNA exists as condensed rDNA (heterochromatic) at each end and diffused rDNA within the secondary constriction region of the Nor-B1 (1B), Nor-B2 (6B) and Nor-D3 (5D) loci. In Nor-B1, 80% of the condensed rDNA domain lies in the proximal end and 20% in the distal end joined by diffuse rDNA threads. In Nor-B2, condensed rDNA is distributed evenly at each end joined by diffuse rDNA in the middle. In Nor-D3, the base of the satellite contains a greater concentration of condensed rDNA than the tip of the short arm. On the basis of these observations, we support the model that the usual state of rDNA is inactive (facultatively heterochromatic; Hilliker and Appels 1989). A small fraction of rDNA at a specific location (usually in the middle in wheat) exists as a diffuse region (active) in condensed chromosomes.

Chromosome pairing and chiasma frequency were studied in bread wheat euhaploids (2n = 3x = 21; ABD genomes) with and without the major pairing regulatorPh1. This constitutes the first report of chromosome pairing relationships among the A, B, and D genomes of wheat without the influence of an alien genome. AllPh1 euhaploids had very little pairing, with 0.62-1.05 rod bivalents per cell; ring bivalents were virtually absent and mean arm-binding frequency (c) values ranged from 0.050 to 0.086. In contrast, theph1b euhaploids had extensive homoeologous pairing, with chiasma frequency 7.5-11.6 times higher than that in thePh1 euhaploids. They had 0.53-1.16 trivalents, 1.53-1.74 ring bivalents, and 2.90-3.57 rod bivalents, withc from 0.580 to 0.629. N-banding of meiotic chromosomes showed strongly preferential pairing between chromosomes of the A and D genomes; 80% of the pairing was between these genomes, especially in the presence of theph1b allele. The application of mathematical models to unmarked chromosomes also supported a 2∶1 genomic structure of theph1b euhaploids. Numerical modeling suggested that about 80% of the metaphase I association was between the two most related genomes in the presence ofph1b, but that pairing under Ph1 was considerably more random. The data demonstrate that the A and D genomes are much more closely related to each other than either is to B. These results may have phylogenetic significance and hence breeding implications.

A terminal deletion in the short arm of chromosome 4B was obtained in the progeny of an alien monosomic addition line of common wheat (Triticum aestivum (L.) emend. Thell) with a chromosome from Aegilops cylindrica Host. Common wheat plants homozygous for the deletion were completely male sterile. The breakpoint was at about 84% of the length of the short arm from the centromere. Therefore, the male-fertility gene was located in the distal 16% region of the chromosome 4B short arm. This terminal deletion practically suppressed meiotic pairing between the short arms of the normal 4B and the deletion 4B chromosomes.

Wheat (Triticum aestivum L.) yield is limited by wheat streak mosaic virus which is vectored by the wheat curl mite (WCM) Aceria tosicheilla (Keifer). Host resistance to WCM has reduced losses. This study was conducted to evaluate the effectiveness of resistance in wheat to WCM collected from various locations in the Great Plains. Collections of WCM from Montana, Nebraska, South Dakota, Texas, Alberta, Canada, and eight locations in Kansas were compared for their ability to survive and reproduce in the greenhouse on seven lines of wheat and wheat relatives previously identified as resistant. The lines and their sources of resistance were: AC PGR16635 (Aegilops tauschii Coss., Cmc1), PI525452 (Thinopyrum ponticum (Podp.) Liu and Wang, Cmc2), KS96WGRC40 (Ae. tauschii and Secale cereale L.), TA920 (Triticum timopheevii (Zhuk.) Zhuk spp. armenidcum), PI 475772 (S. cereale), TAM 107' (S. cereale), PI 222655 (T. aestivum). KS96WGRC40 and TA920 were the only entries that were resistant to all WCM collections. Other sources of resistance were effective against WCMs from some but not all locations. PI 222655 was resistant to WCMs from Nebraska and central Kansas but not to mites from most other locations. WCMs that were virulent to TAM 107 generally were also virulent to PIs 222655 and 475772 but avirulent to Cmc2. The WCMs from western Kansas, where TAM 107 is widely grown, were generally more virulent to that cultivar than WCM from central Kansas where the hectarage of TAM 107 is smaller. WCMs collected at different times or locations may vary in their responses to different sources of resistance; therefore, testing mites for their response to resistance genes advanced in breeding programs may be needed before resistant cultivars are deployed in the field.

Effects of polyploidy in allohexaploid wheat (Triticum aestivum L.) have primarily been ascribed to increases in coding sequence variation and potential to acquire new gene functions through mutation of redundant loci. However, regulatory variation that arises through new promoter and transcription factor combinations or epigenetic events may also contribute to the effects of polyploidization. In this study, gene expression was characterized in a synthetic T. aestivum line and the T. turgidum and Aegilops tauschii parents to establish a timeline for such regulatory changes and estimate the frequency of nonadditive expression of homoeologous transcripts in newly formed T. aestivum. Large-scale analysis of nonadditive gene expression was assayed by microarray expression experiments, where synthetic T. aestivum gene expression was compared to additive model values (mid-parent) calculated from parental T. turgidum and Ae. tauschii expression levels. Approximately 16% of genes were estimated to display nonadditive expression in synthetic T. aestivum. A certain fraction of the genes (2.9%) showed overdominance or underdominance. cDNA–single strand conformation polymorphism analysis was applied to measure expression of homoeologous transcripts and further verify microarray data. The results demonstrate that allopolyploidization, per se, results in rapid initiation of differential expression of homoeologous loci and nonadditive gene expression in T. aestivum.

Threshability is an important crop domestication trait. The wild wheat progenitors have tough glumes enveloping the floret that make spikes difficult to thresh, whereas cultivated wheats have soft glumes and are free-threshing. In hexaploid wheat, the glume tenacity gene Tg along with the major domestication locus Q control threshability. The Q gene was isolated recently and found to be a member of the AP2 class of transcription factors. However, only a few studies have reported on the tough glume trait. Here, we report comparative mapping of the soft glume (sog) gene of diploid Triticum monococcum L. and tenacious glume (Tg) gene of hexaploid T. aestivum L. using chromosome-specific SSR and RFLP markers. The sog gene was flanked by Xgwm71 and Xbcd120 in a 6.8 cM interval on chromosome 2A(m)S of T. monococcum whereas Tg was targeted to a 8.1 cM interval flanked by Xwmc503 and Xfba88 on chromosome 2DS of T. aestivum. Deletion bin mapping of the flanking markers assigned sog close to the centromere on 2AS, whereas Tg was mapped to the most distal region on 2DS. Both 2AS and 2DS maps were colinear ruling out the role of chromosome rearrangements for their non-syntenic positions. Therefore, sog and Tg are not true orthologues suggesting the possibility of a diverse origin.

An alloplasmic wheat line, TA5536, with the “zebra” chromosome z5A was isolated from an Elymus trachycaulus/Triticum aestivum backcross derivative. This chromosome was named “zebra” because of its striped genomic in situ hybridization pattern. Its origin was traced to nonhomologous chromosome 5A of wheat and 1Ht of Elymus; four chromatin segments were derived from chromosome 1Ht and five chromatin segments including the centromere from 5A. In this study, our objective was to determine the mechanism of origin of chromosome z5A, whether by nonhomologous recombination or by multiple translocation events. Different crossing schemes were used to recover recombinants containing various Elymus chromatin segments of the z5A chromosome. In addition, one z5AL telocentric chromosome and three z5AL isochromosomes were recovered. The dissection of the Elymus segments into different stocks allowed us to determine the chromosomal origin of the different chromosome fragments on the basis of the order of the RFLP markers employed and suggested that the zebra chromosome originated from nonhomologous recombination. We present a model of possible mechanism(s) of chromosome evolution and step changes in chromosome number applicable to a wide range of organisms.

The diminutive "midget" chromosome derived from rye (Secale cereale) was analyzed by C-banding and fluorescence in situ hybridization (FISH) using DNA probe pSau3A9 that is located in the centromeres of cereal chromosomes. FISH signals were detected at one end and overlapped one of the two telomeres of the midget, indicating that the midget is a telocentric chromosome. The FISH and C-banding results show that the centromere of the midget chromosome is smaller than those of normal wheat and rye chromosomes. These results indicate that one of the breakpoints occurred in the middle of the centromere of rye chromosome 1R during generation of the midget.Key words: Secale cereale, midget chromosome, centromere, telomere

Fusarium head blight (FHB) resistance was identified in the alien species Leymus racemosus, and wheat-Leymus introgression lines with FHB resistance were reported previously. Detailed molecular cytogenetic analysis of alien introgressions T01, T09, and T14 and the mapping of Fhb3, a new gene for FHB resistance, are reported here. The introgression line T09 had an unknown wheat-Leymus translocation chromosome. A total of 36 RFLP markers selected from the seven homoeologous groups of wheat were used to characterize T09 and determine the homoeologous relationship of the introgressed Leymus chromosome with wheat. Only short arm markers for group 7 detected Leymus-specific fragments in T09, whereas 7AS-specific RFLP fragments were missing. C-banding and genomic in situ hybridization results indicated that T09 has a compensating Robertsonian translocation T7AL·7Lr#1S involving the long arm of wheat chromosome 7A and the short arm of Leymus chromosome 7Lr#1 substituting for chromosome arm 7AS of wheat. Introgression lines T01 (2n = 44) and T14 (2n = 44) each had two pairs of independent translocation chromosomes. T01 had T4BS·4BL-7Lr#1S + T4BL-7Lr#1S·5Lr#1S. T14 had T6BS·6BL-7Lr#1S + T6BL·5Lr#1S. These translocations were recovered in the progeny of the irradiated line Lr#1 (T5Lr#1S·7Lr#1S). The three translocation lines, T01, T09, and T14, and the disomic addition 7Lr#1 were consistently resistant to FHB in greenhouse point-inoculation experiments, whereas the disomic addition 5Lr#1 was susceptible. The data indicated that at least one novel FHB resistance gene from Leymus, designated Fhb3, resides in the distal region of the short arm of chromosome 7Lr#1, because the resistant translocation lines share a common distal segment of 7Lr#1S. Three PCR-based markers, BE586744-STS, BE404728-STS, and BE586111-STS, specific for 7Lr#1S were developed to expedite marker-assisted selection in breeding programs.

Seed color is on important trait affecting flour yield and quality in wheat. Seed color also is either tightly linked to or pleiotropically controls seed dormancy in wheat, because most of the red-seeded wheats are tolerant to preharvest sprouting in comparison to white-seeded wheats. Seed color in hexaploid wheat is controlled by the dominant red seed color genes R-A1, R-B1, and R-D1 located in orthologous positions on chromosome arms 3AL, 3BL, and 3DL, respectively. By using wheat ESTs and synteny with rice, we identified one STS marker and one EST marker flanking R-A1 in a 4.4-cM interval by using an RIL and F 2 populations of Langdon (LDN)/LDN-DIC3A (disomic substitution of T. turgidum subsp. dicoccoides chromosome 3A for 3A of LDN). Physical mapping of the R-A1 gene using tightly linked markers on a set of deletion lines specific to the long arms of group-3 chromosomes indicated that the red seed color genes are located in the distal region (less than 10% of the chromosome arm 3L), which is a high-recombination, generich region in wheat. Comparative genomic analysis indicated that, except for a very minor rearrangement of gene sequences in wheat relative to rice, macrocolinearity is well conserved between the consensus distal deletion bin of wheat 3L-0.80-1.00 and rice chromosome arm 1L. The R-A1 gene of wheat was targeted into a single PAC of rice using colinear flanking markers.

Direct introgression of genes from diploid Aegilops squarrosa L into hexaploid wheat (Tnticum aestivumL.) is an efficient way of developing stable, hexaploid lines with unique, useful genes. 39 such lines derived front direct crosses to determine the effects of Ac. squarrosa germplasm were field-tested on productivity. Whereas some lines were agronomically inferior to the recurrent parent, others periormed at an equall or better level. In addition to the larget traits (Hessian-fly and leaf-rust resistance), other traits, including soilborne-mosaic-virus resistance and protein concentration, were improved.

A set of four normal chromosomes (1D, 2D, 3D, and 6D), and three translocation chromosomes (4DS·5DS, 5DL·7DS, and 7DL·4DL) involving all 14 chromosome arms of the D-genome were obtained as monosomic additions from Aegilops squarrosa (genome D, n=7) in Triticum durum Desf. cv 'PBW114' (genome AB, n=14). The cyclical translocation occurred during the synthesis of the amphiploid probably as a result of misdivision and reunion of the univalents during meiosis of the F1 hybrid T. durum x A. Squarrosa. The amphiploid was backcrossed twice with the durum parent to obtain monosomic addition lines. The monosomic addition chromosomes were identified by C-banding and associated phenotypic traits. All monosomic addition lines were fertile. The development of disomic and ditelosomic addition lines is underway, which will be useful for cytogenetic analysis of individual D-genome chromosomes in the background of T. Durum.

Four wheat-rye lines derived from a cross between hexaploid wheat 'ND 7532' and 'Chaupon' rye were homogeneous for resistance to biotype L of the Hessian fly,Mayetiola destructor. Because the wheat parent was susceptible and the rye parent was resistant to larval feeding, resistance was derived from rye. Resistance of 'Chaupon' and the wheat-rye lines was expressed as larval antibiosis. First-instar larvae died after feeding on plants. Chromosomal analyses using C- and N-banding techniques were performed on plants of each line to identify genomes and structural changes of chromosomes. Results showed that two of the resistant lines were chromosome addition lines carrying either the complete rye chromosome,2R, or only the long arm of2R. The other two resistant lines were identified as being2BS/ 2RL wheat-rye translocation lines. It was concluded, therefore, that the long arm of rye chromosome2R carries a gene or gene complex that conditions antibiosis to Hessian fly larvae and, in the2BS/2RL translocation lines, this rye chromatin is cytologically stable and can be used directly in wheat breeding programs.

Resistance in the common wheat cultivar «Seneca» has been reported to be controlled by two independent, partially dominant genes that exhibit duplicate epistasis. Monosomic stocks were crossed to Seneca in an attempt to assign these genes, H7 and H8, to their respective chromosomes

A combination of cytological and biochemical methods were used to isolate and identify six Triticum aestivum 'Chinese Spring' – Elymus trachycaulus (= Agropyron trachycaulum, 2n = 28, genomes StStHtHt) Ht genome disomic and ditelosomic chromosome addition lines. Protein and morphological markers indicated that Elymus chromosomes 1Ht, 1Htp, 5Ht, 6Ht, 7Ht, and 7Htp have been added to the wheat genome. Two alloplasmic addition lines, 1Ht and 1Htp, were determined to have favorable nucleocytoplasmic interactions by the presence of vegetative vigor and fertility restoration. The gene(s) for vigor and fertility restoration were located on the short arm of 1Ht. The Elymus chromosomes of each line were found to affect plant morphology and fertility, with the exception of disomic addition 6Ht, which appeared similar to 'Chinese Spring'. Phenotypic differences between each line may be attributed to the expression of genes from specific Elymus chromosomes, the cumulative dosage of homoeoalleles, or nucleocytoplasmic interactions. These morphological traits, in combination with biochemical markers, provide evidence of the gene synteny relationships between the Elymus and Triticum species. Knowledge of the homoeologous relationships among wheat and Elymus chromosomes may be useful for the eventual transfer of disease-resistance genes from Elymus to wheat.Key words: Elymus, wheat, addition line, polyploidy.

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.

Segregation distortion genes are widespread in plants and animals and function by their effect on competition among gametes for preferential fertilization. In this study, we evaluated the segregation distortion of molecular markers in multiple reciprocal backcross populations derived from unique cytogenetic stocks involving the durum cultivar Langdon (LDN) and wild emmer accessions that allowed us to study the effects of chromosome 5B in isolation. No segregation distortion of female gametes was observed, but three populations developed to analyze segregation of male gametes had genomic regions containing markers with skewed segregation ratios. One region of distortion was due to preferential transmission of LDN alleles over wild emmer alleles through male gametes. Another region required the presence of LDN 5B chromosomes in the female for preferential fertilization by male gametes harboring LDN alleles indicating that the corresponding genes in the female gametes can govern genes affecting segregation distortion of male gametes. A third region of distortion was the result of preferential transmission of wild emmer alleles over LDN alleles through male gametes. These results indicate the existence of different distorter/meiotic drive elements among different genotypes and show that distortion factors along wheat chromosome 5B differ in chromosomal location as well as underlying mechanisms.

Sears (1956) pioneered plant chromosome engineering 50 years ago by directed transfer of a leaf rust resistance gene from an alien chromosome to a wheat chromosome using X-ray irradiation and an elegant cytogenetic scheme. Since then many other protocols have been reported, but the one dealing with induced homoeologous pairing and recombination is the most powerful, and has been extensively used in wheat. Here, we briefly review the current status of homoeologous recombination-based chromosome engineering research in plants with a focus on wheat, and demonstrate that integrated use of cytogenetic stocks and molecular resources can enhance the efficiency and precision of homoeologus-based chromosome engineering. We report the results of an experiment on homoeologous recombination-based transfer of virus resistance from an alien chromosome to a wheat chromosome, its characterization, and the prospects for further engineering by a second round of recombination. A proposal is presented for genome-wide, homoeologous recombination-based engineering for efficient mining of gene pools of wild relatives for crop improvement.

Wheat genes for pathogenesis-related (PR-)proteins, chitinase and beta-1,3-glucanase, under the control of maize ubiquitin promoter-intron were used for transforming the spring wheat 'Bobwhite', using a biolistic approach. Twenty of the 24 primary transgenic lines expressing the PR-protein genes in the T0 generation were silenced in either the T1 or T2 generations. Two apparently genetically identical regenerants arising from a single callus co-bombarded with chitinase and beta-1,3-glucanase transgene combinations, but differing in the expression of the transgenes were selected for further characterization. In one homozygous line, transgene silencing was observed in the T3 plants, while the other line homozygous for the transgene loci stably expressed and inherited the transgenes to at least the T4 generation. Southern blot analyses of genomic DNA from the two lines using the isoschizomeric methylation-sensitive enzymes, MspI and HpaII, revealed a higher degree of methylation of CCGG sequences in the line with the silenced transgene locus. Analysis by reverse transcriptase-polymerase chain reaction, Northern blotting and Western blotting detected stable expression of the transgenes in the line with a lesser extent of methylation, whereas the line with a higher level of CCGG methylation had no transgene expression by the T3 generation. The germination of seeds from the silenced plants in the presence of a cytidine analogue, 5-azacytidine (azaC), did not lead to a reversion of this phenotype.