Bulbosum Hybrids Research Articles

Interspecific Hybrids of Hordeum marinum ssp. marinum × H. bulbosum Are Mitotically Stable and Reveal No Gross Alterations in Chromatin Properties

Interspecific hybrids between the related wild barley species Hordeum marinum and H. bulbosum were generated and tested regarding their chromosomal stability and chromatin properties. Unlike in H. vulgare × H. bulbosum hybrid embryos, there was no effect of temperature on uniparental chromosome elimination or retention during hybrid seed development and 7 chromosomes from each parent were detected according to genomic in situ hybridization analyses. The centromere-specific histone H3 gene (CENH3) of both parental genomes is active in hybrid plants. Indirect immunostaining of flow-sorted 2C nuclei indicated that no major reorganization of histone H3 methylation (K4, K9 and K27), H3K9 acetylation and histone H2A ubiquitination marks or location of active RNA polymerase II sites occurred after interspecific hybridization.

Elimination of chromosomes in Hordeum vulgare × H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare × H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.

Identification of all chromosome arms and their involvement in meiotic homoeologous associations at metaphase I in 2Hordeum vulgareL. ×Hordeum bulbosumL. hybrids

We have identified all Hordeum bulbosum chromosomes in 2 diploid Hordeum vulgare x Hordeum bulbosum hybrids using suitable probes and fluorescence in situ hybridization. Using the parental idiograms allowed us to carry out a full analysis of chromosome associations among all chromosome arms in the hybrids. Association frequencies were generally lower for the short arms than for the long arms. There were also significant differences among the chromosome arms in association frequencies, partly correlated with the absolute length of the chromosome arm, as well as with the frequency of recombinant lines, which were recovered from partially fertile interspecific hybrids. The H. bulbosum idiogram will be useful for further chromosome association studies and will enable the identification of H. bulbosum chromosomes involved in chromosome addition or substitution lines.

Reduced chromosome association between the short arms of 5H homologues in Hordeum vulgare L. at metaphase I

Rod or ring bivalents at metaphase I (MI) reflect whether crossing-over has occurred on one or both pairs of chromosome arms, respectively, during meiotic prophase (pachytene). In cultivated barley (Hordeum vulgare) the highest frequencies of rod bivalents were found for satellite chromosomes (either 5H or 6H). To distinguish between chromosome 5H and 6H associations, a recombinant line derived from an H. vulgare × H. bulbosum hybrid with a hemizygous introgression from H. bulbosum on the short arm of chromosome 6H was probed with 45S rDNA and pSc119.2 using fluorescent in situ hybridization and compared with non-recombinant H. vulgare. The frequencies of rod bivalents, caused by failure of the short arms of 5H chromosomes (5HS) to associate during meiosis in the recombinant line, were similar to the frequencies of rod bivalents involving the satellite chromosomes in H. vulgare. Hence, rod bivalent formation in H. vulgare satellite chromosomes is probably based on reduced association of 5HS, corresponding to a low frequency of genetic recombination. 5HS is, therefore, more likely to show linkage disequilibrium than the other chromosome arms.

The Chromosome Stability of Hordeum Vulgare L.-H. Bulbosum L. Chromosome Substitution Plants Grown at two Temperatures

The perennial wild species Hordeum bulbosumis closely related to cultivated barley (H. vulgare) and possesses useful traits such as disease resistance. Introgression of desirable genes into barley has recently been achieved by backcrossing fertile triploid hybrids between H. vulgare (2x) and H. bulbosum (4x) to barley. Single and double monosomic chromosome substitution plants were also developed from similar crosses, but consistent chromosome pairing and stability has been difficult to achieve. Since temperature influences chromosome behaviour in H. vulgare×H. bulbosum hybrids, the effect of two temperatures (15 and 21°C) on dividing somatic ovary wall cells and pollen mother cells was investigated. At 21°C, most of the plants contained higher proportions of cells with less than the full chromosome complement, indicating elimination of the H. bulbosum monosomes. However, at 21°C, homoelogous chromosome pairing tended to be greater than at 15T. To promote high chromosome pairing and stability, optimum temperatures should be further determined to enable genetic recombination to take place.

Variable patterns of chromosome synapsis at pachytene in Hordeum vulgare x H. bulbosum hybrids and their parents.

Synaptonemal complexes (SC) have been analysed in barley (Hordeum vulgare), H. bulbosum and two F, hybrids between them. These hybrids show different recombination frequencies and at pachytene show significant differences in the total length of SC formed and in the extent of synapsis. Higher recombination frequency in the hybrids was correlated with a greater total SC length. Differences in SC length were also observed between the parental species with H. bulbosum having a greater SC length than H. vulgare. However, species and hybrid can have similar SC lengths but clearly different recombination frequencies and, therefore, the relationship between SC length and recombination is not clear-cut.

Hordeum bulbosum‐a new source of disease resistance‐transfer of resistance to leaf rust and mosaic viruses from H. bulbosum into winter barley

AbstractAn outline is given of results for the transfer of new resistances against leaf rust and barley mosaic viruses from Hordeum bulbosum into winter barley. Progenies from backcrosses of barley cultivars with H. bulbosum hybrids were tested both in conventional breeding trials and in additional tests under controlled conditions. Resistance to both pathogens proved to be stable and of good heritability, with differences occurring which depended on the combinations used. Lines with resistance to all leaf rust and mosaic viruses tested, or to either one, were selected. Both resistances segregated independently.

Molecular changes at Rrn loci in barley (Hordeum vulgare L.) hybrids with H. bulbosum (L.)

Southern blots of restriction fragments of genomic DNAs from Hordeum vulgare (L.), H. bulbosum (L.), and interspecific hybrids and their derivatives were hybridized with rDNA probe to identify locus-specific modifications at Rrn loci. H. bulbosum rDNA revealed a single EcoRV site per repeat compared with two sites in H. vulgare rDNA repeats. H. bulbosum accessions possessed at least two rDNA repeat lengths, indicating heterozygosity at the Rrn locus. Hybrids possessed both H. vulgare and H. bulbosum rDNA repeats. Two of the hybrid derivatives possessed bulbosum-specific Sau3AI and HaeIII rDNA fragments, while amphiploid and doubled haploid derivatives lacked H. bulbosum rDNA repeat units and (or) fragments. Two hybrid derivatives, one amphiploid and a doubled haploid derived from the same parental combination, lacked the vulgare Rrn2-specific 9.0-kb rDNA repeat. This is the first conclusive evidence for the elimination of vulgare genetic material in vulgare-bulbosum hybrids. The ratios of 9.0- to 9.9-kb vulgare repeats and H. vulgare to H. bulbosum rDNA repeats indicate partial loss of the vulgare-specific 9.0-kb rDNA repeat among the hybrids. Differences in MboI and Sau3AI fragments and the ratios of 9.0 to 9.9 kb vulgare rDNA repeats revealed differential methylation at Rrn1 and Rrn2 loci. Hybrids and derivatives showed differential distribution of methylation of EcoRI, BglII, and SacI sites at the Rrn1 locus. Two of the hybrid derivatives exhibited extensive CpG-biased methylation. Data presented here are indicative of the differences in the onset of events triggered by the interaction of the component genomes and enabled detection of differential methylation among Rrn loci, loss of H. vulgare genetic material, and development of doubled haploids with the Rrn1 locus.

Direct measurement of recombination frequency in interspecific hybrids between Hordeum vulgare and H. bulbosum using genomic in situ hybridization.

Two different genotypes of diploid Hordeum vulgare x H. bulbosum hybrids, which differ in their pattern of meiotic metaphase pairing behaviour, were investigated at MI and AI by genomic in situ hybridization (GISH). One hybrid, 102C2, showed a high frequency of bivalents at metaphase I whereas the other, 103K5, showed a high frequency of univalents. The GISH analysis of both hybrids established that pairing occurred only between chromosomes of different parental genomes and revealed that pairing frequency greatly exceeded recombination. Hybrid 102C2 had a significantly higher recombination frequency than 103K5, but in both hybrids recombination involved only distal chromosome regions. However, an interesting finding is that the ratio of recombination to pairing frequency in 103K5 (1:8.9) is twice as high compared with 102C2 (1:17). The hybrids also differed in chromosome stability; little chromosome elimination occurred in 102C2 but 103K5 showed extensive chromosome loss. It appears that the high frequency of bound arms at MI favours retention of H. bulbosum chromosomes and maintains stability of chromosome numbers in PMCs. Various ideas are put forward to explain the discrepancy between meiotic pairing frequency and recombination in these hybrids.

Association of leaf rust and powdery mildew resistance in a recombinant derived from a Hordeum vulgare × Hordeum bulbosum hybrid

AbstractWhile studying powdery mildew resistance in a recombinant line (code 81882) derived from a Hordeum vulgare (cv. ‘Vada’) ×Hordeum bulbosum hybrid, a low infection type of resistance to leaf rust was observed. To determine the mode of inheritance of the leaf rust resistance and whether there was linkage between the two resistances, F2 and F3 progenies from crosses between 81882 and ‘Vada’ were inoculated with the leaf rust and powdery mildew pathogens. Southern blots were prepared using restricted DNA extracted from leaves of 82 F2 plants and four chromosome 2HS sequences were hybridized with the blots to define the length of the introgression. The leaf rust resistance appears to be inherited as a single dominant gene on chromosome 2HS, which co‐segregates with the powdery mildew resistance. There was an almost complete association between the resistances and the respective molecular markers, but it is likely that the strong linkage results from the frequent inheritance of the introgressed H. bulbosum DNA as an intact segment of chromatin with only low levels of recombination within the segment.

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.

Anther Culture‐Derived Regenerants from Hordeum vulgare × H. bulbosum Crosses

Abstract Hordeum bulbosum has several desirable attributes, including disease resistance, which would be worthwhile transferring to H. vulgare. Despite homoeologous chromosome pairing in the interspecific hybrids, there have been few reports of successful gene introgression between the two species. A possible explanation for this is that recombinant male gametes are at a competitive disadvantage with normal balanced gametes during post‐pollination events. To circumvent this problem, the possibility of obtaining plants directly from immature pollen grains was investigated. Anthers from diploid, triploid and tetraploid H. vulgare × H. bulbosum hybrids were cultured on defined media. Only hybrids with dehiscent anthers in vivo responded in culture, and after transfer of calli and embryoids to regeneration medium, 36 albino and 12 green plants were obtained. Seven of the green regenerants survived, one of which contained 15 H. vulgare chromosomes (including one acrocentric chromosome) and one H. bulbosum chromosome. Another regenerant (Ac166) resembled a diploid H. vulgare × H. bulbosum hybrid but had partial anther dehiscence and a slightly modified chromosome constitution. Mostly normal H. vulgare progeny were obtained from crosses between H. vulgare cv.‘Emir’ and Ac166, but three plants involved chromosome additions and substitutions.

Chromosome elimination at mitosis and protein synthesis ability in interspecific hybrids between barley and Hordeum bulbosum L.

Elimination of chromosomes, which is detected by the presence of laggards at telophase, occurs frequently in the spike primordia, but not in the root meristems of the hybrids between H. vulgare cv. Amsel and H. bulbosum L. Cordycepin (10-5M), cycloheximide (10-5M) and colchicine (0.1%) were applied for 30 min to the root meristems of H, vulgare L. cv. Amsel and H. bulbosum L. (4x), and their triploid hybrid E159. Colchicine did not induce laggards at telophase in Amsel, H. bulbosum and hybrid E159, although C-metaphases were observed. Spindle disruption may not be involved in the chromosorne elimination of the hybrids. Cordycepin and cycloheximide induced frequently laggards at telophase in the hybrids at 60 min after the end of the treatments, while no laggards were observed in Amsel and H. bulbosum. Hybrid E159 appears to have the partial suppression of RNA or protein syntheses related to the centromere function, and is sensitive to cordycepin and cycloheximide. Cordycepin and cycloheximide may enhance the suppression of the centromere activity and results in the laggards at telophase. RNA and protein synthesis during G2 or prophase is involved in the chromosome elimlnation in H. vulgre-H. bulbosum hybrids. Cd-staining method, which was reported to distinguish active centromeres from inactive ones of human chromosomes, were modified for chromosomes of Hordeum species. The modified Cd-staining revealed clear centromere dots only in the centromeres. However, both normally separating chromosomes and laggards showed centromere dots. The modified Cd-staining may not stain the centromere structures related to the centromere-microtubule interaction.

Chromosome elimination in Hordeum vulgare x H. bulbosum hybrids

Both diploid and tetraploid hybrids of Hordeum vulgare x H. bulbosum exhibit chromosome instability during plant development and generally lose the H. bulbosum chromosomes by the next generation. However an amphidiploid involving the cultivar 'Vada' was found to retain the bulbosum chromosomes through a sexual cycle. Comparisons were made between this stable amphidiploid and a second, unstable amphidiploid involving the cultivar 'Emir'. At meiosis there was a wide range of chromosome numbers in the 'Emir' amphidiploid but only a narrow range in the 'Vada' amphidiploid. Degraded chromosomes were commonly found in the PMCs of the 'Emir' amphidiploid but none was found in the stable 'Vada' amphidiploid. The two amphidiploids were reciprocally crossed and the chromosome numbers of the progeny checked. The H. bulbosum chromosomes had been retained in many of the progeny and both triploid and tetraploid hybrids were found. It is proposed that a gene(s) exists in 'Vada' capable of preventing the elimination of bulbosum chromosomes from H. vulgare x H. bulbosum hybrids.

Intergeneric hybridization between Agropyron tsukushiense and Hordeum bulbosum (4X).

In order to obtain polyhaploids in genus Agropyron by the bulbosum method, nine species were pollinated by Hordeum bulbosum (4x). Among progenies obtained by embryo culture, six plants did not show elimination of any chromosomes derived from H. bulbosum and were intergeneric hybrids between A. repens or three ecotypes of A. tsukushiense and H. bulbosum. Out of six hybrids obtained, two plants of A. tsukushiense EE-4 or ST-1× H. bulbosum (4x) (2n=34 or 2n=35) were examined morphologically and cytologically. These hybrids were shorter, but had numerous tillers. They were completely sterile. The spike morphology resembled female parent rather than intermediate between parents. Several characteristics of pollen parent were also observed in these hybrids. The average chromosome pairing per cell at MI of PMCS in A. tsukushiense EE-4 and ST-1×H. bulbosum hybrids was 2.8II+28.4I and 4.6II+25.8I, respectively. The loosely associated bivalent formation, a rare occurrence of cells with more than seven bivalents, and absence of multivalent association in these hybrids suggest a lack of segmental homology among chromosome complements of three genomes of A. tsukushiense and two of H. bulbosum.The production of these intergeneric hybrids suggests that genome balance hypothesis proposed in interspecific hybrids of Hordeum can not beapplicable to intergeneric hybrids between Agropyron and H. bulbosum.

Chromosome instability in Hordeum vulgare x H. bulbosum hybrids

Chromosome number in Hordeum vulgare x H. bulbosum hybrids ranged between the haploid and diploid number but with peaks in frequency occurring at the 14 and 7 chromosome level. This was reflected in a gradual change from hybrid morphology to that of haploid H. vulgare. The rate of chromosome elimination differed significantly between hybrids, while within each hybrid, differences in mean chromosome number were recorded between and within individual tillers. An increase in temperature from 25–30° C caused a significant increase in the rate of elimination of H. bulbosum chromosomes.

THE CYTOLOGY OF THREE HYBRIDS INVOLVING HORDEUM JUBATUM L.: THE CHIASMA DISTRIBUTIONS AND THE OCCURRENCE OF PSEUDO RING-BIVALENTS IN GENETICALLY INDUCED ASYNAPSIS

Three hybrids, two of Hordeum jubatum L. × Secale cereale L. var. Prolific, and one of H. jubatum × H. bulbosum L., were examined cytologically. The two genomes of H. jubatum showed autosyndesis in the H. jubatum × S. cereale hybrids. It was concluded, therefore, that H. jubatum has two closely related genomes.One of the H. jubatum × S. cereale hybrids displayed weak asynapsis. The H. jubatum × H. bulbosum hybrid, on the other hand, showed strong asynapsis, although the prophase pairing was almost complete. It was concluded that the asynapsis of this latter hybrid was due to interaction of genes of the H. jubatum and the H. bulbosum genomes.The chiasma distributions of the hybrids were tested for randomness by Gaul's formula. It was found that the chiasma distributions of the H. jubatum × S. cereale hybrids followed a random pattern, whereas in the H. jubatum × H. bulbosum hybrid the distribution was disturbed by a secondary effect. Cytological observations and theoretical considerations led to the conclusion that the occurrence of pseudo ring-bivalents had disturbed the apparent chiasma distribution in the H. jubatum × H. bulbosum hybrid. A theory of the origin of pseudo bivalents and an evaluation of Gaul's formula is presented.