Gamete formation via meiotic nuclear restitution generates fertile amphiploid F1 (oat×maize) plants.

Hybrid (oat×maize) zygotes developed into euhaploid plants with complete oat chromosome complements without maize chromosomes and into aneuhaploid plants with complete oat chromosome complements and different numbers of retained individual maize chromosomes. The elimination of maize chromosomes in the hybrid embryo is caused by uniparental genome loss during early steps of embryogenesis. Some of these haploid plants set seed in up to 50% of their self-pollinated spikelets. The high fertility was found to be mainly caused by formation of numerically unreduced female and male gametes (nunreduced=3x+0…3=21…24 chromosomes). Gamete formation involves meiotic nuclear restitution. The restitution process is caused by an alternative type of meiosis. It follows the model of levigatum-type semi-heterotypic divisions, but with a formation of the nuclear membrane at the transition from telophase I to interkinesis, which resembles the model of pygaera-type pseudo-homotypic divisions. We propose the name haploid meiotic restitution for this particular process combination. We discuss the use and implications of the specific process of gamete formation in F1 (oat×maize) plants.

Identification of a maize neocentromere in an oat-maize addition line.

We report a neocentromere event on maize chromosome 3 that occurred due to chromosome breakage. The neocentromere lies on a fragment of the short arm that lacks the primary centromere DNA elements, CentC and CRM. It is transmitted in the genomic background of oat via a new centromere (and kinetochore), as shown by immunolocalization of the oat CENH3 protein. Despite normal transmission of the maize fragment in most progeny, neocentromeres appear to vary in size within the same tissue, as shown by fluorescent measurements. A secondary truncation in one line lowered mitotic transmission to 3% and precipitously reduced the size of the chromosome. The results support the view that neocentromere formation is generally associated with major genomic disturbances such as wide species crosses or deletion of an existing centromere. The data further suggest that new centromeres may undergo a period of instability that is corrected over a period of several generations.

Novel inter-series hybrids in Solanum, section Petota.

Sexual hybrids between distantly related Solanum species can undergo endosperm failure, a post-zygotic barrier in inter-species hybridizations. This barrier is explained by the endosperm balance number (EBN) hypothesis, which states that parents must have corresponding EBNs for viable seed formation. Tests for inter-crossability were made involving the Mexican species Solanum pinnatisectum Dunal. (series Pinnatisecta, ApiApi, 1EBN), autotetraploids of this species, Solanum verrucosum Schlechtd. (series Tuberosa, AA, 2EBN), haploids (2x, 2EBN) of the South American S. tuberosum L. (series Tuberosa, A1A1A2A2, 4EBN), and F2 haploid-species hybrids with South American species (AA, 2EBN) S. berthaultii Hawkes, S. sparsipilum (Bitter.) Juz. and Bukasov and S. chacoense Bitter. The development of hybrid endosperms was investigated for these combinations by confocal microscopy with regard to cell-division timing and tissue collapse. Novel sexual diploid (AApi) and triploid (AApiApi) inter-series hybrids were generated from S. verrucosum x S. pinnatisectum crosses by using post-pollination applications of auxin. F1 embryos were rescued in vitro. The hybrid status of recovered plants was verified by microsatellite marker analysis, and the ploidy was determined by chromosome counting. The application of phytohormones in inter-ploidy S. pinnatisectum x S. tuberosum crosses, however, did not delay endosperm collapse, and embryos were not formed. Other diploid, 1EBN species tested in remote hybridizations with Group Tuberosum were S. cardiophyllum Lindl., S. trifidum Correll, and S. tarnii Hawkes and Hjert., series Pinnatisecta, and S. bulbocastanum Dunal., series Bulbocastana. Based on the analysis of post-zygotic reproductive barriers among isolated species of section Petota, we propose strategies to overcome such incompatibilities.

Gametocidal factor-induced structural rearrangements in rye chromosomes added to common wheat.

The gametocidal factor on the Aegilops cylindrica chromosome 2Cc was used to induce and analyze the nature of chromosomal rearrangements in rye chromosomes added to wheat. For this purpose we isolated plants disomic for a given rye chromosome and monosomic for 2Cc and analyzed their progenies cytologically. Rearranged rye chromosomes were identified in 7% of the progenies and consisted of rye deficiencies (4.6%), wheat rye dicentric and rye ring chromosomes (1.8%), and terminal translocations (0.6%). The dicentric and ring chromosomes initiated breakage-fusion-bridge cycles (BFB) that ceased within a few weeks after germination as the result of chromosome healing. Of 56 rye deficiencies identified, after backcrossing and selfing, only 33 were recovered in either homozygous or heterozygous condition covering all rye chromosomes except 7R. The low recovery rate is probably caused by the presence of multiple rearrangements induced in the wheat genome that resulted in poor plant vigor and seed set, low transmission, and an underestimation of the frequency of wheat rye dicentric chromosomes. Genomic in-situ hybridization (GISH) analysis of the 33 recovered rye deficiencies revealed that 30 resulted from a single break in one chromosome arm followed by the loss of the segment distal to the breakpoint. Only three had a wheat segment attached distal to the breakpoint. Although some of the Gc-induced rye rearrangements were derived from BFB cycles, all of the recovered rye rearrangements were simple in structure. The healing of the broken chromosome ends was achieved either by the de-novo addition of telomeric repeats leading to deficiencies and telocentric chromosomes or by the fusion with other broken ends in the form of stable monocentric terminal translocation chromosomes.

Fate of multicentric and ring chromosomes induced by a new gametocidal factor located on chromosome 4Mg of Aegilops geniculata.

A new gametocidal (Gc) factor was identified on chromosome 4Mg of Aegilops geniculata Roth. When transferred to Chinese Spring wheat, monosomic and disomic Triticum aestivum-Ae. geniculata chromosome 4Mg addition plants undergo regular first and second meiotic divisions. Male gametogenesis in disomic 4Mg addition plants also is normal. However, chromosome breakage and anaphase bridges were observed at ana/telophase of the first (29%) and second (11%) pollen mitosis in monosomic 4Mg addition plants. Gc-induced multicentric and ring chromosomes can be transmitted to the offspring and initiate breakage fusion bridge (BFB) cycles in dividing root tip meristem cells of the derived sporophytes. The fate of multicentric and ring chromosomes was analyzed in root meristems at different time intervals after seed germination. The majority of the BFB cycles ceased about 32 days after germination. Broken chromosome ends were healed either by the fusion of a centric and an acentric fragment forming terminal translocation chromosomes or as deficiencies or telocentric chromosomes. Lack of cytologically detectable telomeric repeats at the stabilized newly broken termini suggests that chromosome healing by addition of telomeric repeats may be a gradual process.

Characterization by RFLP analysis and genomic in situ hybridization of a recombinant and a monosomic substitution plant derived from Hordeum vulgare L. x Hordeum bulbosum L. crosses.

Interspecific crosses in Hordeum have been made with the aim of transferring desirable traits, such as disease resistance, from a wild species, Hordeum bulbosum, into cultivated barley (Hordeum vulgare). Interspecific recombinants have previously been identified using several methods, but there are limitations with all the techniques. We improved our ability to characterize progeny from H. vulgare x H. bulbosum crosses by using genomic in situ hybridization (GISH). The plant material comprised a recombinant and a monosomic alien substitution plant derived from H. vulgare x H. bulbosum crosses. The recombinant possesses a pubescent leaf sheath conferred by a gene transferred from H. bulbosum into barley cultivar Golden Promise. The use of GISH on a plant homozygous for the pubescence gene confirmed the presence of H. bulbosum DNA located distally on two barley chromosomes and we mapped the introgression to barley chromosome 4HL using RFLP analysis. Furthermore, by means of an allelism test we found that the transferred gene for pubescence is allelic or closely linked to a gene for pubescence (Hs) located on barley chromosome 4HL. The presence of a single H. bulbosum chromosome in the monosomic substitution plant was confirmed by GISH. A distal introgression of H. bulbosum DNA was also observed on one barley chromosome, which was located on chromosome 3HL by RFLP analysis.

Maize individualized chromosome and derived radiation hybrid lines and their use in functional genomics.

The duplicated and rearranged nature of plant genomes frequently complicates identification, chromosomal assignment and eventual manipulation of DNA segments. Separating an individual chromosome from its native complement by adding it to an alien genetic background together with the generation of radiation hybrids from such an addition line can enable or simplify structural and functional analyses of complex duplicated genomes. We have established fertile disomic addition lines for each of the individual maize chromosomes, except chromosome 10, with oat as the host species; DNA is available for chromosome 10 in a haploid oat background. We report on instability and transmission in disomic additions of maize chromosomes 1, 5, and 8; the chromosome 2, 3, 4, 6, 7, and 9 additions appear stable. The photoperiodic response of the two recovered maize chromosome 1 addition lines contrasts to the long-day flowering response of the oat parents and the other addition lines. Only when grown under short days did maize chromosome 1 addition lines set seed, and only one line transmitted the maize chromosome 1 to offspring. Low resolution radiation hybrid maps are presented for maize chromosomes 2 and 9 to illustrate the use of radiation hybrids for rapid physical mapping of large numbers of DNA sequences, such as ESTs. The potential of addition and radiation hybrid lines for mapping duplicated sequences or gene families to chromosome segments is presented and also the use of the lines to test interactions between genes located on different maize chromosomes as observed for ectopic expression of cell fate alterations.

Chromosome healing by addition of telomeric repeats in wheat occurs during the first mitotic divisions of the sporophyte and is a gradual process.

Alien gametocidal chromosomes cause extensive chromosome breakage prior to S-phase in the first mitotic division of gametophytes lacking the alien chromosome. The broken chromosomes may be healed either by addition of telomeric repeats in the gametophyte or undergo fusions to form dicentric or translocation chromosomes. We show that dicentric chromosomes undergo breakage fusion-bridge (BFB) cycles in the first few mitotic divisions of the sporophyte, are partially healed before the germ line differentiation regimen, and are healed completely in the ensuing gametophytic stage. The gametocidal factor on chromosome 4Mg of Aegilops geniculata was used to induce dicentrics involving the satellite chromosomes1B and 6B of wheat, Triticum aestivum. The dicentrics 1BS x 1BL-2AL x 2AS and 6BS x 6BL-4BL x 4BS initiated BFB cycles that ceased 2 to 4 weeks after seed germination. At the end of the BFB cycles, we observed deficient 1B and 6B chromosomes with breakpoints in proximal regions of the 1BL and 6BL arms. The process of chromosome healing was analyzed in root tip meristems, at meiotic metaphase I, and in the derived progenies by fluorescence in-situ hybridization analysis using a telomeric probe pAtT4. The results show that chromosome healing in wheat occurs during very early mitotic divisions in the sporophyte by de-novo addition of telomeric repeats and is a gradual process. Broken chromosome ends have to pass through several cell divisions in the sporophyte to acquire the full telomeric repeat length.

A maize chromosome 3 addition line of oat exhibits expression of the maize homeobox gene liguleless3 and alteration of cell fates.

Maize chromosome addition lines of oat offer the opportunity to study maize gene expression in oat and the resulting phenotypes. Morphological examination of a maize chromosome 3 addition line of oat showed that this line exhibited several morphological abnormalities including a blade-to-sheath transformation at the midrib region of the leaf, a hook-shaped panicle, and abnormal outgrowth of aerial axillary buds. Dominant mutations in the maize liguleless3 (lg3) homeobox gene result in a blade (distal)-to-sheath (proximal) transformation at the midrib region of the leaf. Ectopic expression of the dominant mutant Lg3 allele is believed to cause the phenotype. Therefore, we suspected that the maize lg3 gene, which is located on maize chromosome 3, was involved in the phenotypes observed in the maize chromosome 3 addition line of oat. Genetic analyses of an oat BC1F2 family segregating for maize chromosome 3 showed that the presence of a stable maize chromosome 3 was required for the expression of these cell fate abnormalities. RNA expression analysis of leaf sheath tissue from oat plants carrying maize chromosome 3 demonstrated that maize LG3 transcripts accumulated in oat, indicating that this expression is associated with the blade-to-sheath transformation, hook-shaped panicle and outgrowth of aerial axillary bud phenotypes. Our results demonstrate that the maize chromosome addition lines of oat are useful genetic stocks to study expression of maize genes in oat.

Molecular cytogenetic analysis of Aegilops cylindrica host.

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

Molecular cytogenetic characterisation of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale).

The terminal heterochromatic segments of the long arms of 20 rye B-chromosomes were isolated by means of laser microdissection technology. Also the remaining portions of the long arms, along with the short arms of the same chromosomes were isolated. Each sample was used for degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplification reactions. The resulting products were used as probes for chromosome in situ hybridisation experiments, and in Southern hybridisation to digests of 0B and +B DNA. Competition hybridisation of these probes with 0B DNA allowed the detection of B-specific sequences. The terminal heterochromatin of the rye B-chromosome contains both B-specific sequences and sequences also present on the A-chromosomes of rye. The B-specific D1100 family is the major repeat species located in the terminal heterochromatin. Primers designed to the cloned sequence (E1100) were used to search for related low copy sequences in 0B DNA. The sequences of the PCR products revealed no similarities to that of the clone E1100 except for the primer sequences. The possible origin of this sequence is discussed in the context of models for the evolution of the rye B-chromosome.

Mapping maize sequences to chromosomes using oat-maize chromosome addition materials.

Oat- (Avena sativa) maize (Zea mays) chromosome additions are produced by crossing maize and oat. During early embryo development maize chromosomes are preferentially eliminated, and oat plants are often recovered that retain a single maize chromosome. Each of the 10 maize chromosomes recently has been isolated as a separate oat-maize addition. We describe here the mapping of 400 maize sequences to chromosomes using polymerase chain reaction and DNA from the oat-maize addition material. Fifty of the sequences were from cloned markers that had been previously mapped by linkage analysis, and our results were consistent with those obtained using Southern-blot analysis. Previously unmapped expressed sequence tags and sequence tagged sites (350) were mapped to chromosomes. Maize gene sequences and expression data are rapidly being accumulated. Coupling this information with positional information from high throughput mapping programs provides plant biologists powerful tools for identifying candidate genes of interest.

A complete set of maize individual chromosome additions to the oat genome.

All 10 chromosomes of maize (Zea mays, 2n = 2x = 20) were recovered as single additions to the haploid complement of oat (Avena sativa, 2n = 6x = 42) among F(1) plants generated from crosses involving three different lines of maize to eight different lines of oat. In vitro rescue culture of more than 4,300 immature F(1) embryos resulted in a germination frequency of 11% with recovery of 379 F(1) plantlets (8.7%) of moderately vigorous growth. Some F(1) plants were sectored with distinct chromosome constitutions among tillers of the same plant and also between root and shoot cells. Meiotic restitution facilitated development of un-reduced gametes in the F(1). Self-pollination of these partially fertile F(1) plants resulted in disomic additions (2n = 6x + 2 = 44) for maize chromosomes 1, 2, 3, 4, 6, 7, and 9. Maize chromosome 8 was recovered as a monosomic addition (2n = 6x + 1 = 43). Monosomic additions for maize chromosomes 5 and 10 to a haploid complement of oat (n = 3x + 1 = 22) were recovered several times among the F(1) plants. Although partially fertile, these chromosome 5 and 10 addition plants have not yet transmitted the added maize chromosome to F(2) offspring. We discuss the development and general utility of this set of oat-maize addition lines as a novel tool for maize genomics and genetics.