A. Fominaya

Identification of C-banded chromosomes in meiosis and the analysis of nucleolar activity in Avena byzantina C. Koch cv ‘Kanota’

SummaryThe Giemsa C-banding technique was used to identify individual meiotic and somatic chromosomes in 21 monosomic lines of Avena byzantina C. Koch cv ‘Kanota’ (genome designation AACCDD). The hexaploid complement is composed of three sets of seven chromosome pairs. The heterochromatin in the putative diploid progenitors is located at the telomeres (genome A), at the centromeric and interstitial regions (genome C), or more evenly spread throughout the set (genome D). Comparisons based on C-banding between A. byzantina and its diploid progenitor species allowed us to allocate individual chromosomes into specific genomes. The C-banding technique may be useful for interspecific chromosome pairing analyses. Nucleolar activity and competition were studied using a silver-staining procedure. Only three chromosome pairs showed nucleolar organizer regions, thus indicating that nucleolar competition occurs naturally in hexaploid oats.

Organization of repeated sequences in species of the genus Avena

SummaryFour repetitive sequences from Avena murphyi have been isolated and their genome organization studied in different species of the genus Avena. A tandem sequence array was found for the Avena species that contain the C genome. Three other dispersed sequences present in the A and C genomes were arranged in a genomespecific manner. The fact that no major differences in the hybridization patterns were found between species with the same basic genome is consistent with the current taxonomy of Avena species.

Discrimination of the closely related A and B genomes in AABB tetraploid species of Avena

Abstract  Fluorescent in situ (FISH) and Southern hybridization procedures were used to investigate the chromosomal distribution and genomic organization of the satellite DNA sequence As120a (specific to the A-genome chromosomes of hexaploid oats) in two tetraploid species, Avena barbata and Avena vaviloviana. These species have AB genomes. In situ hybridization of pAs120a to tetraploid oat species revealed elements of this repeated family to be distributed over both arms of 14 of the 28 chromosomes of these species. Genomes A and B were subsequently distinguished, indicating an allopolyploid origin for A. barbata. This was confirmed by assigning the satellited chromosomes to individual genomes, using the satellite itself and two ribosomal probes in simultaneous and sequential in situ hybridization analyses. Differences between A. barbata and A. vaviloviana genomes were also revealed by both FISH and Southern techniques using pAs120a probes. Whereas two B-genome chromosome pairs were found to be involved in intergenomic translocations in A. vaviloviana, FISH detected no intergenomic rearrangements in A. barbata. When using pAs120a as a probe, Southern hybridization also revealed differences in the hybridization patterns of the two genomes. A 1300-bp EcoRV fragment was present in A. barbata but absent in A. vaviloviana. This fragment was also detected in Southern analyses of A-genome diploid and hexaploid oat species.

Identification of C-genome chromosomes involved in intergenomic translocations in Avena sativa L., using cloned repetitive DNA sequences

Abstract Four anonymous non-coding sequences were isolated from an Avena strigosa (A genome) genomic library and subsequently characterized. These sequences, designated As14, As121, As93 and As111, were 639, 730, 668, and 619 bp long respectively, and showed different patterns of distribution in diploid and polyploid Avena species. Southern hybridization showed that sequences with homology to sequences As14 and As121 were dispersed throughout the genome of diploid (A genome), tetraploid (AC genomes) and hexaploid (ACD genomes) Avena species but were absent in the C-genome diploid species. In contrast, sequences homologous to sequences As93 and As111 were found in diploid (A and C genomes), tetraploid (AC genomes) and hexaploid (ACD genomes) species. The chromosomal locations of the 4 sequences in hexaploid oat species were determined by fluorescent in situ hybridization and found to be distributed over the length of the 28 chromosomes (except in the telomeric regions) of the A and D genomes. Furthermore, 2 C-genome chromosome pairs with the As14 sequence, and 4 with As121, were discovered to beinvolved in intergenomic translocations. These chromosomes were identified as 1C, 2C, 4C and 16C by combining the As14 or As121 sequences with two ribosomal sequences and a C-genome-specific sequence as probes in fluorescence in situ hybridization. These sequences offer new tools for analyzing possible intergenomic translocations in other hexaploid oat species.

A new chromosome nomenclature system for oat (Avena sativa L. and A. byzantina C. Koch) based on FISH analysis of monosomic lines

Fluorescent in situ hybridization (FISH) with multiple probes was used to analyze mitotic and meiotic chromosome spreads of Avena sativa cv ‘Sun II’ monosomic lines, and of A. byzantina cv ‘Kanota’ monosomic lines from spontaneous haploids. The probes used were A. strigosa pAs120a (a repetitive sequence abundant in A-genome chromatin), A. murphyi pAm1 (a repetitive sequence abundant in C-genome chromatin), A. strigosa pITS (internal transcribed spacer of rDNA) and the wheat rDNA probes pTa71 (nucleolus organizer region or NOR) and pTa794 (5S). Simultaneous and sequential FISH employing pairs of these probes allowed the identification and genome assignation of all chromosomes. FISH mapping using mitotic and meiotic metaphases facilitated the genomic and chromosomal identification of the monosome in each line. Of the 17 ‘Sun II’ lines analyzed, 13 distinct monosomic lines were found, corresponding to four monosomes of the A-genome, five of the C-genome and four of the D-genome. In addition, 12 distinct monosomic lines were detected among the 20 ‘Kanota’ lines examined, corresponding to six monosomes of the A-genome, three of the C-genome and three of the D-genome. The results show that 19 chromosomes out of 21 of the complement are represented by monosomes between the two genetic backgrounds. The identity of the remaining chromosomes can be deduced either from one intergenomic translocation detected on both ‘Sun II’ and ‘Kanota’ lines, or from the single reciprocal, intergenomic translocation detected among the ‘Sun II’ lines. These results permit a new system to be proposed for numbering the 21 chromosome pairs of the hexaploid oat complement. Accordingly, the A-genome contains chromosomes 8A, 11A, 13A, 15A, 16A, 17A and 19A; the C-genome contains chromosomes 1C, 2C, 3C, 4C, 5C, 6C and 7C; and the D-genome consists of chromosomes 9D, 10D, 12D, 14D, 18D, 20D and 21D. Moreover, the FISH patterns of 16 chromosomes in ‘Sun II’ and 15 in ‘Kanota’ suggest that these chromosomes could be involved in intergenomic translocations. By comparing the identities of individually translocated chromosomes in the two hexaploid species with those of other hexaploids, we detected different types of intergenomic translocations.

Studies of isozymes in oat species

SummaryStarch and polyacrylamide gel electrophoresis of seven isozyme systems was investigated as a means of identifying wild and cultivated species of Avena with different ploidy levels. By examining the characteristic isoenzymatic patterns, it was shown that there was considerable variability within the different species. However, it was nevertheless possible to unequivocally identify the species of wild oats and to distinguish between the different species belonging to the same genomic set, thus providing a definitive reference technique for the identification of Avena species in seed-testing laboratories. The relationships between Avena species were inferred from the electrophoresis data. The divergence of the A. maroccana — A. murphyi complex and its contribution to the AACC genomes are emphasized.

Microdissection and microcloning of plant chromosomes

Cytogenetic and molecular tools play an increasingly important role in plant genome research. A number of interesting applications that involve chromosome painting, the relationship between specific chromosomes and specific linkage groups, the relationships between physical and genetic distances on linkage maps, and the isolation of genes of interest, have been the subjects of recently published research. The aim of this paper is to review the different techniques available for chromosome microdissection and microcloning, and their use for the study of plant genomes. The quality of chromosomal DNA obtained is considered, and some recent results from our laboratory are presented.

Use of Tyramide-Fluorescence in situ Hybridization and Chromosome Microdissection for Ascertaining Homology Relationships and Chromosome Linkage Group Associations in Oats

The physical mapping of single locus sequences by tyramide-fluorescence in situ hybridization (Tyr-FISH) and the analysis of sequences obtained from microdissected chromosomes were assayed as potential tools for (1) determining homology and homoeology among chromosome regions of Avena species, and (2) establishing associations between linkage groups and specific chromosomes. Low copy number probes, derived from resistance gene analogues (RGAs) and 2.8–4.5 kb long, successfully produced hybridization signals on specific chromosomes. Four sets of homoeologous chromosome regions were identified in the hexaploids using 3 probes that produced 4 single locus markers in A. strigosa and 2 in A. eriantha. Laser capture microdissection of metaphase I cells of A. sativa monosomic lines allowed the isolation of critical univalents. Sequences derived from 2 RGAs were successfully amplified in DNA extracted from univalents. In one instance, it was possible to map a nucleotide polymorphism specific for 1 chromosome. An association was established between this chromosome and its linkage groups in 2 hexaploid genetic maps. The results indicate that Tyr-FISH is useful in the characterization of homoeologous chromosome segments in hexaploids, whereas chromosome microdissection, as employed in this work, needs to be improved before it can routinely be used with meiotic chromosomes.

Cytological and molecular characterization of a chromosome interchange and addition lines in Cadet involving chromosome 5B of wheat and 6Ag of Lophopyrum ponticum

Efforts to transfer wheat curl mite (Eriophyes tulipae Keifer) resistance from Lophopyrum ponticum 10X (Podb.) Love to bread wheat (Triticum aestivum L.) have resulted in the production of a number of cytogenetic stocks, including an addition line of 6Ag, a “ditelo” addition line, and a wheat-Lophopyrum translocation line. Characterization of these lines with C-banding, in situ hybridization with a Lophopyrum species-specific repetitive DNA probe (pLeUCD2), and Southern blotting with pLeUCD2 and a 5S ribosomal DNA probe (pScT7) confirmed that the distal portion of the short arm of 6Ag was translocated onto the distal portion of 5BS (5BL. 5BS-6AgS). It was also determined that the “ditelo” addition was an acrocentric chromosome of 6AgS.

A possible effect of B-chromosomes on metaphase I homologous chromosome association in rye

The metaphase I association of rye chromosomes was examined both in normal plants (2n = 14) and in plants with rye B chromosomes (2n = 14 + Bs) using the C-banding technique. It was found that B chromosomes (Bs) affect homologous chromosome association. B isochromosomes (iso-Bs) were present in several plants; their presence was deduced, in addition to the morphology of chromosomes at mitosis, from the meiotic association between the two arms of the B and it was confirmed by its C-banding pattern. The strongest increase in rye metaphase association (measured as bound arms) was produced by the presence of the iso-B short (iso-BS) chromosome. The comparison of chromosome association between plants with and without Bs seems to point to the existence of a promoting effect on meiotic chromosome association by Bs, and in particular by the short arm.