A cytological map of the short arm of rye chromosome 1R constructed with 1R dissection stocks of common wheat and PCR-based markers.

The short arm of rye chromosome 1R (1RS) is introduced into many common wheat cultivars because of its agronomic importance. The gametocidal system has been used to produce dissection lines carrying segments of rye chromosome 1R. We focused on establishing more dissection lines for 1RS and on obtaining PCR-based markers specific to 1RS. We established 66 1RS dissection lines carrying 1RS segments of chromosome 1R derived from a common wheat cultivar 'Burgas 2' and obtained 27 markers. We conducted a PCR analysis using the dissection lines and markers, and divided 1RS into 17 regions separated by the breakpoints. Comparison of the 'Burgas 2' 1RS map with another map of 1RS derived from 'Imperial' rye implied a restructuring between the 2 1RS chromosomes.

The gametocidal chromosome as a tool for chromosome manipulation in wheat.

Many alien chromosomes have been introduced into common wheat (the genus Triticum) from related wild species (the genus Aegilops). Some alien chromosomes have unique genes that secure their existence in the host by causing chromosome breakage in the gametes lacking them. Such chromosomes or genes, called gametocidal (Gc) chromosomes or Gc genes, are derived from different genomes (C, S, S(l) and M(g)) and belong to three different homoeologous groups 2, 3 and 4. The Gc genes of the C and M(g) genomes induce mild, or semi-lethal, chromosome mutations in euploid and alien addition lines of common wheat. Thus, induced chromosomal rearrangements have been identified and established in wheat stocks carrying deletions of wheat and alien (rye and barley) chromosomes or wheat-alien translocations. The gametocidal chromosomes isolated in wheat to date are reviewed here, focusing on their feature as a tool for chromosome manipulation.

Stable barley chromosomes without centromeric repeats.

The satellite sequences (AGGGAG)(n) and Ty3/gypsy-like retrotransposons are known to localize at the barley centromeres. Using a gametocidal system, which induces chromosomal mutations in barley chromosomes added to common wheat, we obtained an isochromosome for the short arm of barley chromosome 7H (7HS) that lacked the barley-specific satellite sequence (AGGGAG)(n). Two telocentric derivatives of the isochromosome arose in the progeny: 7HS* with and 7HS** without the pericentromeric C-band. FISH analysis demonstrated that both telosomes lacked not only the barley-specific centromeric (AGGGAG)(n) repeats and retroelements but also any of the known wheat centromeric tandem repeats, including the 192-bp, 250-bp, and TaiI sequences. Although they lacked these centromeric repeats, 7HS* and 7HS** both showed normal mitotic and meiotic transmission. Translocation of barley centromeric repeats to a wheat chromosome 4A did not generate a dicentric chromosome. Indirect immunostaining revealed that all tested centromere-specific proteins (rice CENH3, maize CENP-C, and putative barley homologues of the yeast kinetochore proteins CBF5 and SKP1) and histone H3 phosphorylated at serines 10 and 28 localized at the centromeric region of 7HS*. We conclude that the barley centromeric repeats are neither sufficient nor obligatory to assemble kinetochores, and we discuss the possible formation of a novel centromere in a barley chromosome.

Deletion mapping of homoeologous group 6-specific wheat expressed sequence tags.

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1-6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.

Characterization of genetic variation in and phylogenetic relationships among diploid Aegilops species by AFLP: incongruity of chloroplast and nuclear data.

Intra- and inter-specific genetic variation was investigated in seven diploid Aegilops species using the amplified fragment length polymorphism (AFLP) technique. Of the seven species, the cross-pollinating Aegilops speltoides and Aegilops mutica showed high levels of intraspecific variation whereas the remaining five self-pollinating species showed low levels. Aegilops bicornis, Aegilops searsii and Ae. speltoides formed one cluster in the dendrograms, while Aegilops caudata and Aegilops umbellulata formed another. Relationships among the species inferred were more consistent with the relationships inferred from studies of chromosome pairing in interspecific hybrids, and previous molecular phylogenetic reconstructions based on nuclear DNA, than they were with those based on molecular plasmon analysis, suggesting that the nuclear genome has evolved differently from the cytoplasmic genome in the genus Aegilops.

Transfer of rye chromosome segments to wheat by a gametocidal system.

A gametocidal chromosome derived from Aegilops triuncialis (3C) induces chromosome mutations in gametes lacking the 3C chromosome in common wheat (Triticum aestivum L.). We combined 3C with chromosome 1R of rye (Secale cereale L.) in a common wheat line to know how efficiently 3C induces transfers of small 1R segments to wheat. In the 811 progeny of this wheat line, we found five wheat chromosomes (2A, 2D, 3D, 5D and 7D) carrying segments of the 1R satellite. Wheat plants carrying these translocations were tested for the presence of a storage protein locus Sec-1 and a cluster of resistance genes for wheat rust diseases, Sr31, Lr26 and Yr9. The 2A and 2D translocations had the Sec-1 and three rust resistance loci. The 3D and 5D translocations had Sr31, Lr26 and Yr9 but not Sec-1. The 7D translocation lacked Sec-1, Lr26 and Yr9, but the presence of Sr31 in this translocation was not determined. This showed that the translocation points fell into three regions of the 1R satellite, namely, proximal to Sec-1, between Sec-1 and the rust resistance loci, and distal to the rust resistance loci. Thus, the 3C gametocidal system was demonstrated to be effective in transferring small rye chromosome segments.

Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat.

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).

Barley chromosome arms longer than half of the spindle axis interfere with nuclear divisions.

We have tested the influence of recombinantly-elongated chromosome arms on nuclear divisions in barley and confirmed a rule according to which half the length of the average spindle axis defines the upper tolerance limit for chromosome arm length. A slightly longer chromosome arm caused incomplete separation of sister chromatids in approximately 70% of mitotic telophase cells and >2.5% of daughter cells showing a micronucleus, due to disruption of non-separated sister chromatids by the newly forming cell wall. In homozygous condition, this elongated chromosome mediated a slower growth and reduced fertility of the carrier plants. Its meiotic transmission was not impaired because of the larger spindle dimensions in meiocytes as compared to those in mitotic cells.

Large-scale selection of lines with deletions in chromosome 1 B in wheat and applications for fine deletion mapping.

Terminal deletions of chromosome 1B in common wheat were selected on a large scale. The gametocidal gene of Aegilops cylindrica was used as the inducer of chromosome breakage. First, genes for endosperm storage proteins located on both arms of chromosome 1B were used as the selection markers. However, it was found that the chromosome breakage occurred during female gametogenesis, causing genotypic inconsistency between the embryo and endosperm. Thus, we isolated plants with terminal deletions in chromosome 1B by C-banding. Of 1327 plants examined, 128 showed aberrations in chromosome 1B: 47 in the short arm, 76 in the long arm, and 5 in both arms. The present deletions tended to have the breakpoint at more proximal regions than those produced previously by T.R. Endo and B.S. Gill. Using 33 deletion lines produced in this study and 34 lines previously produced, we mapped 39 RFLP loci and a nucleolar organizer region (NOR) on a specific region of chromosome 1B. The NOR was found to consist of two subregions with different repetitive units, which were termed NOR-Bld and NOR-Blp. Based on this fine deletion map and genotypic inconsistency between embryo and endosperm, the features of the gametocidal gene are discussed.

Barley chromosome addition lines of wheat for screening of AFLP markers on barley chromosomes.

We conducted AFLP (Amplified Fragment Length Polymorphism) analysis with the six wheat-barley chromosome addition lines of common wheat cultivar Chinese Spring. We analyzed the AFLP fingerprints generated by 36 combinations of selective-amplification primers to find 103 markers specific to the barley chromosomes (2.9 markers per combination on average). The numbers of AFLP markers mapped to the barley chromosomes varied (one to 16) depending of the primer combinations. Each barley chromosome had 10 to 27 AFLP markers (17.2 markers on average). We identified the chromosome arms in which these markers are located using the barley telocentric addition lines (one to 20 markers per chromosome arm). The AFLP markers were not distributed evenly among chromosomes and chromosome arms. We could not determine the chromosome-arm locations for some of the barley-specific markers, either because such markers were found in both the short- and long-arm telocentric lines, or in neither line.

Identification of AFLP markers on the satellite region of chromosome 1BS in wheat.

The satellite region on the short arm of chromosome 1B in wheat (Triticum aestivum L., 2n = 6x = 42) carries many agronomically important genes; i.e., genes conferring fungal disease resistance, seed storage proteins, and fertility restoration. To find molecular markers located on the satellite region, we applied the fluorescent AFLP (amplified fragment length polymorphism) technique to aneuploids and deletion stocks of the cultivar T. aestivum 'Chinese Spring'. Out of 6017 fragments amplified with 80 primer combinations in normal 'Chinese Spring', 24 were assigned to 1BS. Twelve of them clustered within a small region of the satellite known to be rich in RFLP (restriction fragment length polymorphism) markers. AFLPs in 1BS and in the whole genome were calculated between 'Chinese Spring' and T. spelta var. duhamelianum. The polymorphism rates in the satellite region (58.3%) and in the 1BS arm (45.8%) were much higher than the average rate for the whole genome (10.7%). Seven of the 12 AFLP markers in the satellite region were revealed to be specific to 'Chinese Spring' and could potentially be useful for genetic mapping in a segregation population of 'Chinese Spring' x T. spelta.

Genetic induction of chromosomal rearrangements in barley chromosome 7H added to common wheat.

Chromosome 2C of Aegilops cylindrica induces chromosomal rearrangements in alien chromosome addition lines, as well as in euploid lines, of common wheat. To induce chromosomal rearrangements in barley chromosome 7H, reciprocal crosses were made between a mutation-inducing common wheat line that carries a pair of 7H chromosomes and one 2C chromosome and a 7H disomic addition line of common wheat. Many shrivelled seeds were included in the progeny, which was an indication of the occurrence of chromosome mutations. The chromosomal constitution of the viable progeny was examined by FISH (fluorescence in situ hybridization) using the barley subterminal repeat HvT01 as a probe. Structural changes of chromosome 7H were found in about 15% of the progeny of the reciprocal crosses. The aberrant 7H chromosomes were characterized by a combination of N-banding, FISH and genomic in situ hybridization. Mosaicism for aberrant 7H chromosomes was observed in seven plants. In total, 89 aberrant 7H chromosomes were identified in 82 plants, seven of which had double aberrations. More than half of the plants carried a simple deletion: four short-arm telosomes, one long-arm telosome, and 45 terminal deletions (23 in the short arm, 21 in the long arm, and one involving both arms). About 40% of the aberrations represented translocations between 7H and wheat chromosomes. Twenty of the translocations had wheat centromeres, 12 the 7H centromere, with translocation points in the 7HS (five) and in the 7HL (seven), and the remaining four were of Robertsonian type, three involving 7HS and one with 7HL. In addition, one translocation had a barley segment in an intercalary position of a wheat chromosome, and two were dicentric. The breakpoints of these aberrations were distributed along the entire length of chromosome 7H.

Detection of the Sec-1 locus of rye by a PCR-based method.

The structural genes for the omega-secalins of rye (Secale cereale) are located in the Sec-1 locus on the short arm of rye chromosome 1R. We applied PCR (polymerase chain reaction) to detect the Sec-1 locus in a wheat genomic background. A primer set we designed based on a published sequence of a omega-secalin gene amplified not only the omega-secalin sequence, but also a putative omega-gliadin sequence. We determined partial sequences of both PCR-amplified fragments and designed different primers for the specific amplification of the omega-secalin sequence. One of the new primer sets amplified DNA fragments only in rye and wheat lines carrying chromosome 1R or telosome 1RS; no amplification occurred in either euploid wheats or 1RS deletion lines. This PCR-based method would provide efficient screening for the Sec-1 locus in progeny of wheat lines carrying chromosome 1R.

Identification and high-density mapping of gene-rich regions in chromosome group 1 of wheat.

We studied the distribution of genes and recombination in wheat (Triticum aestivum) group 1 chromosomes by comparing high-density physical and genetic maps. Physical maps of chromosomes 1A, 1B, and 1D were generated by mapping 50 DNA markers on 56 single-break deletion lines. A consensus physical map was compared with the 1D genetic map of Triticum tauschii (68 markers) and a Triticeae group 1 consensus map (288 markers) to generate a cytogenetic ladder map (CLM). Most group 1 markers (86%) were present in five clusters that encompassed only 10% of the group 1 chromosome. This distribution may reflect that of genes because more than half of the probes were cDNA clones and 30% were PstI genomic. All 14 agronomically important genes in group 1 chromosomes were present in these clusters. Most recombination occurred in gene-cluster regions. Markers fell at an average distance of 244 kb in these regions. The CLM involving the Triticeae consensus genetic map revealed that the above distribution of genes and recombination is the same in other Triticeae species. Because of a significant number of common markers, our CLM can be used for comparative mapping and to estimate physical distances among markers in many Poaceae species including rice and maize.

Identification and high-density mapping of gene-rich regions in chromosome group 5 of wheat.

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed < 18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.

Variation of starch granule proteins and chromosome mapping of their coding genes in common wheat.

Starch granule proteins (SGPs) of common wheat (Triticum aestivum L.) were analyzed by two electrophoretic techniques: sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2D-PAGE). These analyses identified three kinds of SGPs which were tentatively designated SGP-1, SGP-2 and SGP-3. SDS-PAGE resolved the products of three homoeologous genes for SGP-1 into three protein fractions, SGP-A1, -B1 and -D1. While SDS-PAGE resolved SGP-3 into one fraction, 2D-PAGE separated it into three protein fractions encoded by homoeologous genes Sgp-A3, B3 and -D3. SGP-2 was detected as one protein by SDS-PAGE and was present as one protein on 2D-PAGE. Aneuploid (nullisomic-tetrasomic and ditelosomic) analyses in the cultivar Chinese Spring showed that the genes for two SGPs (SGP-1 and -3) were located on the short arms of group-7 chromosomes. The results obtained from deletion lines for chromosome arms 7AS, 7BS and 7DS suggested that the gene order along the arms is 'centromere-Sgp-1-Sgp-3-Wx'. An electrophoretic survey of wheat germ plasm identified a few cultivars lacking one of the proteins SGP-A1, -B1, -D1, SGP-A3 and -B3. The null alleles Sgp-A1b, Sgp-B1b and Sgp-D1b will be useful for the production of a variant wheat lacking SGP-1.

Cytologically based physical maps of the group-2 chromosomes of wheat.

We have constructed cytologically based physical maps (CBPMs), depicting the chromosomal distribution of RFLP markers, of the group-2 chromosomes of common wheat (Triticum aestivum L. em Thell). Twenty-one homozygous deletion lines for 2A, 2B, and 2D were used to allocate RFLP loci to 19 deletion-interval regions. A consensus CBPM was colinearily aligned with a consensus genetic map of group-2 chromosomes. The comparison revealed greater frequency of recombination in the distal regions. Several molecularly tagged chromosome regions were identified which may be within the resolving power of pulsed-field gel electrophoresis. The CBPMs show that the available probes completely mark the group-2 chromosomes, and landmark loci for sub-arm regions were identified for targeted-mapping.

Characterization of deletions in common wheat induced by an Aegilops cylindrica chromosome: detection of multiple chromosome rearrangements.

An Aegilops cylindrica chromosome induces terminal deletions of chromosomes in wheat as identified by C-banding. We are constructing high-density physical maps of wheat chromosomes and have detected additional chromosome rearrangements. Among 63 lines with chromosomal subarm deletions in group 7 chromosomes, 7 lines (11.1%) were shown to harbor additional chromosome rearrangements. Two other lines were also omitted from the physical mapping because of the nature of the breakpoint calculations. The presence or absence of chromosome-specific restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) markers indicated that additional interstitial deletions are present in 3 lines (4.8%) with deletions in the short chromosome arms and in 4 lines (6.3%) with deletions in the long chromosome arms. We also used chromosome pairing analysis of F1 plants of deletion lines with double ditelosomic lines of 'Chinese Spring' wheat to detect small terminal deletions. The deletion of the most distal 1% of chromosome arm 7AL was associated with a pairing reduction of 60%.

Comparison of wheat physical maps with barley linkage maps for group 7 chromosomes.

Comparative genetic maps among the Triticeae or Gramineae provide the possibility for combining the genetics, mapping information and molecular-marker resources between different species. Dense genetic linkage maps of wheat and barley, which have a common array of molecular markers, along with deletion-based chromosome maps of Triticum aestivum L. will facilitate the construction of an integrated molecular marker-based map for the Triticeae. A set of 21 cDNA and genomic DNA clones, which had previously been used to map barley chromosome 1 (7H), were used to physically map wheat chromosomes 7A, 7B and 7D. A comparative map was constructed to estimate the degree of linkage conservation and synteny of chromosome segments between the group 7 chromosomes of the two species. The results reveal extensive homoeologies between these chromosomes, and the first evidence for an interstitial inversion on the short arm of a barley chromosome compared to the wheat homoeologue has been obtained. In a cytogenetically-based physical map of group 7 chromosomes that contain restriction-fragment-length polymorphic DNA (RFLP) and random amplified polymorphic DNA (RAPD) markers, the marker density in the most distal third of the chromosome arms was two-times higher than in the proximal region. The recombination rate in the distal third of each arm appears to be 8-15 times greater than in the proximal third of each arm where recombination of wheat chromosomes is suppressed.

Cytologically based physical maps of the group 3 chromosomes of wheat.

Cytologically based physical maps for the group 3 chromosomes of wheat were constructed by mapping 25 Triticum aestivum deletion lines with 29 T. tauschii and T. aestivum RFLP probes. The deletion lines divide chromosomes 3A, 3B, and 3D into 31 discrete intervals, of which 18 were tagged by marker loci. The comparison of the consensus physical map with a consensus RFLP linkage map of the group 3 chromosomes of wheat revealed a fairly even distribution of marker loci on the long arm, and higher recombination in the distal region.