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.

Fine physical mapping of Ph1, a chromosome pairing regulator gene in polyploid wheat.

The diploid-like chromosome pairing in polyploid wheat is controlled by the Ph1 (pairing homoeologous) gene that is located on chromosome arm 5BL. By using a combination of cytogenetic and molecular techniques, we report the physical location of the Ph1 gene to a submicroscopic chromosome region (Ph1 gene region) that is flanked by the breakpoints of two deletions (5BL-1 and ph1c) and is marked by a DNA probe (XksuS1). The Ph1 gene region is present distal to the breakpoint of deletion 5BL-1 but proximal to the C-band 5BL2.1. Two other DNA probes (Xpsr128 and Xksu75) flank the region-Xpsr128 being proximal and Xksu75 being distal. The estimated size of the region is less than 3 Mb. The chromosome region around the Ph1 gene is high in recombination as the genetic distance of the region between 5BL-1 breakpoint and C-band 5BL2.1 (not resolved by the microscope) is at least 9.3 cM.

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.

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.

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.

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.

Induction of Chromosomal Structural Changes by a Chromosome of Aegilops cylindrica L. in Common Wheat.

Chromosomal structural changes-deletions and translocations-occurred in almost half the progeny of a monosomic addition line of common wheat, Triticum aestivum (2n = 42, AABBDD), which had a chromosome from Aegilops cylindrica (2n = 28, CCDD). Most of the progeny with chromosomal structural changes lacked the A. cylindrica chromosome. Chromosome breaks were observed in various regions of all the wheat chromosomes and the A. cylindrica chromosome. Chromosome aberrations occurred far less frequently both in the self-progeny of the disomic addition plants and in the F1 monosomic addition plants derived from reciprocal crosses with normal common wheat. These findings suggest that when the A. cylindrica chromosome was in the sporophytes, chromosome breakage was likely to occur in the gametophytes that lacked it but that the gametophytes were still functional, resulting in the production of offspring with chromosomal structural changes.

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.

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.

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.

A cytogenetic ladder-map of the wheat homoeologous group-4 chromosomes.

We report the results of chromosome maps of wheat homoeologous chromosomes 4A, 4B, and 4D using 40 RFLP markers and 39 homozygous deletion lines. Deletion breakpoints divide the chromosomes into 45 subarm intervals with 32 intervals distinguished by molecular markers. The chromosome maps confirm the homoeology of arms 4AS to 4BL and 4DL, and 4AL to 4BS and 4DS. The chromosome map of 4A reveals novel information concerning the 4AL-5AL-7BS cyclical translocation. The presence of homoeologous group-4 long-arm markers, Xksu G10 and Xpsr 1051, intervening between the translocated 5AL and 7BS chromosome segments in 4AL suggests that the translocation events are more complex than was earlier believed. Chromosome maps confirm a pericentric inversion in Chinese Spring chromosome 4B. The consensus chromosome map is compared to the genetic map of wheat to construct a cytogenetic ladder-map (CLM). The CLM reveals an unequal distribution of recombination along the length of the chromosome arms. Recombination is highest in the distal half, and low in the proximal half, of the chromosome arms.

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.

Waxy protein deficiency and chromosomal location of coding genes in common wheat.

Deficiency of the wheat waxy (Wx) proteins (Wx-A1, Wx-B1 and Wx-D1) was studied in 1,960 cultivars derived from several countries. Gel electrophoretic analyses revealed that the null allele for the Wx-A1 protein occurred frequently in Korean, Japanese and Turkish wheats but was relatively rare in cultivars from other countries and regions. About 48% of the wheats deficient for the Wx-B1 protein were from Australia and India. One Chinese cultivar lacked the WxD1 protein. While 9 Japanese cultivars were deficient in both the Wx-A1 and Wx-B1 proteins, no cultivars lacked both the Wx-A1 and Wx-D1 proteins, both the Wx-B1 and Wx-D1 proteins or all three Wx proteins. Two-dimensional gel electrophoresis revealed polymorphisms of the three Wx proteins that varied according to isoelectric points or molecular weight. The Wx-A1 gene coding the Wx-A1 protein and the Wx-B1 gene coding the Wx-B1 protein were localized in the distal regions of chromosome arms 7AS and 4AL, respectively, by deletion mapping using the deletion lines developed in the common wheat cultivar 'Chinese Spring'.

Toward a cytogenetically based physical map of the wheat genome.

Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the genome, buffered by polyploidy, can tolerate structurally and numerically engineered chromosomes for analysis over infinite generations. This feature of polyploidy can be used in developing a high-resolution, cytogenetically based physical map of the wheat genome. We show that numerous deletions, observed in the progeny of a monosomic addition of a chromosome from Triticum cylindricum in wheat, result from single breakpoints and a concomitant loss of distal fragments. Breakages occurred in euchromatic and heterochromatic regions. Forty-one deletions for chromosomes 7A, 7B, and 7D, and a set of genetically mapped DNA probes, were used to construct physical maps. Recombination was low in proximal chromosomal regions and very high toward the distal ends. Deletion mapping was more efficient than genetic mapping in resolving the order of proximal loci. Despite variation in size and arm ratio, relative gene position was largely conserved among chromosomes 7A, 7B, and 7D and a consensus group 7 physical map was constructed. Several molecularly tagged chromosome regions (MTCRs) of approximately one to a few million base pairs were identified that may be resolved by long-range mapping of DNA fragments. Thus, a cytogenetically based physical map may be used to integrate chromosome and DNA-based maps. The MTCRs may simplify strategies for cloning of agronomically useful genes despite the genetic complexity and the large genome size of wheat.

Further discovery of alien cytoplasms inducing haploids and twins in common wheat.

In addition to the already known Aegilops caudata cytoplasm, the cytoplasms of five Aegilops species, all belonging to the section Polyeides, were found to induce haploids (11-56%) and twins (0.5-15%) in a common wheat, Salmon, at high frequencies. The great majority of the twin pairs were of the diplo-haplo type. The origin of both the haploids and twins was ascribed to the induction of parthenogenesis in Salmon by the alien cytoplasm. Pollen parents produced some differences in haploid frequency. The distribution of the parthenogenesis-inducing cytoplasms in the genus Aegilops is discussed in relation to the phylogeny of the donor species.

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.

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.

Comparison of genetic and physical maps of group 7 chromosomes from Triticum aestivum L.

We present a high density physical map of homoeologous group 7 chromosomes from Triticum aestivum L. using a series of 54 deletion lines, 6 random amplified polymorphic DNA (RAPD) markers and 91 cDNA or genomic DNA clones from wheat, barley and oat. So far, 51 chromosome segments have been distinguished by molecular markers, and 54 homoeoloci have been allocated among chromosomes 7A, 7B and 7D. The linear order of molecular markers along the chromosomes is almost identical in the A- B- and D-genome of wheat. In addition, there is colinearity between the physical and genetic maps of chromosomes 7A, 7B and 7D from T. aestivum, indicating gene synteny among the Triticeae. However, comparison of the physical map of chromosome 7D from T. aestivum with the genetic map from Triticum tauschii some markers have been shown to be physically allocated with distortion in more distal chromosome regions. The integration of genetic and physical maps could assist in estimating the frequency and distribution of recombination in defined regions along the chromosome. Physical distance did not correlate with genetic distance. A dense map facilitates the detection of multiple rearrangements. We present the first evidence for an interstitial inversion either on chromosome arm 7AS or 7DS of Chinese Spring. Molecularly tagged chromosome regions (MTCRs) provide landmarks for long-range mapping of DNA fragments.