CYTOLOGICAL VARIATION OF (AAG)7 REPEAT ON LETTUCE CHROMOSOMES BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Cytological studies using fluorescence in situ hybridization (FISH) technique provides phylogenetical information in closely related taxa and have been widely applied for karyotyping and studying chromosomal organization and evolution in plant species. In the present study, FISH using a microsatellite sequence of (AAG)7 as the probe was performed in order to discriminate the chromosomes in four Lactuca species, i.e., L. sativa, L. serriola, L. saligna, and L. virosa. The experiment was carried out in April to September 2018 at Laboratory of Genetic and Plant Breeding of Breeding of Graduate School of Horticulture, Chiba University, Japan. Different distribution patterns of (AAG)n signals were shown on the chromosomes in the four Lactuca species studied, In L. sativa and L. serriola, FISH with (AAG)7 sequences revealed dispersed distribution patterns with one pair of bright signals, respectively. While in L. saligna and L. virosa, distinct signals with different intensities were observed in two pairs of chromosomes of L. saligna and five pairs of chromosomes of L. virosa. In conclusion, the AAG repeat signals could be used as cytogenetic landmarks for chromosome identification in Lactuca species.

A universal karyotypic system for hexaploid and diploid Avena species brings oat cytogenetics into the genomics era

Background The identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species. Results Tandem repetitive sequences were predicted and physically located on chromosomal regions of the released Avena sativa OT3098 genome assembly v1. Eight new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of A. brevis, A. nuda, A. wiestii, A. ventricosa, A. fatua, and A. sativa species. We established a high-resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations in A. sativa. Conclusions This new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

Distribution of FISH oligo-5S rDNA and oligo-(AGGGTTT)3 in Hibiscus mutabilis L.

Hibiscus exhibits high variation in chromosome number both within and among species. The Hibiscus mutabilis L. karyotype was analyzed in detail using fluorescence in situ hybridization (FISH) with oligonucleotide probes for (AG3T3)3 and 5S rDNA, which were tested here for the first time. In total, 90 chromosomes were counted in prometaphase and metaphase, and all exhibited similarly intense (AG3T3)3 signals at both ends. (AG3T3)3 showed little variation and thus did not allow discrimination among H. mutabilis chromosomes, but its location at both ends confirmed the integrity of each chromosome, thus contributing to accurate counting of the numerous, small chromosomes. Oligo-5S rDNA marked the proximal/distal regions of six chromosomes: weak signals on chromosomes 7 and 8, slightly stronger signals on chromosomes 15 and 16, and very strong signals on chromosomes 17 and 18. Therefore, 5S rDNA could assist in chromosome identification in H. mutabilis. Metaphase chromosome lengths ranged from 3.00 to 1.18 μm, indicating small chromosomes. The ratios of longest to shortest chromosome length in prometaphase and metaphase were 2.58 and 2.54, respectively, indicating karyotype asymmetry in H. mutabilis. These results provide an exact chromosome number and a physical map, which will be useful for genome assembly and contribute to molecular cytogenetics in the genus Hibiscus.

A universal karyotypic system for hexaploid and diploid Avena species brings oat cytogenetics into the genomics era

Background The identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species. Results Tandem repetitive sequences were predicted and physically located on chromosomal regions of the Avena sativa genomes. Thirteen new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of eleven hexaploid and diploid Avena accessions. We established a high resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations. Conclusion This new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

Chromosome inheritance and meiotic stability in allopolyploidBrassica napus

Homoeologous recombination, aneuploidy, and other genetic changes are common in resynthesized allopolyploid Brassica napus. In contrast, the chromosomes of cultivars have long been considered to be meiotically stable. To gain a better understanding of the underlying mechanisms leading to stabilization in the allopolyploid, the behavior of chromosomes during meiosis can be compared by unambiguous chromosome identification between resynthesized and natural B. napus. Compared with natural B. napus, resynthesized lines show high rates of nonhomologous centromere association, homoeologous recombination leading to translocation, homoeologous chromosome replacement, and association and breakage of 45S rDNA loci. In both natural and resynthesized B. napus, we observed low rates of univalents, A–C bivalents, and early sister chromatid separations. Reciprocal homoeologous chromosome exchanges and double reductions were photographed for the first time in meiotic telophase I. Meiotic errors were non-uniformly distributed across the genome in resynthesized B. napus, and in particular homoeologs sharing synteny along their entire length exhibited multivalents at diakinesis and polysomic inheritance at telophase I. Natural B. napus appeared to resolve meiotic errors mainly by suppressing homoeologous pairing, resolving nonhomologous centromere associations and 45S rDNA associations before diakinesis, and reducing homoeologous cross-overs.

Generation of microdissected DNA probes from metaphase chromosomes when chromosome identification by routine staining is impossible

Application of microdissected DNA libraries and DNA probes in numerous and various modern molecular cytogenetic studies showed them as an efficient and reliable tool in the analysis of chromosome reorganization during karyotypic evolution and in the diagnosis of human chromosome pathology. An important advantage of DNA probe generation by metaphase chromosome microdissection followed by sequence-independent polymerase chain reaction in comparison with the method of DNA probe generation using chromosome sorting is the possibility of DNA probe preparation from chromosomes of an individual sample without cell line establishment for the production of a large number of metaphase chromosomes. One of the main requirements for successful application of this technique is a possibility for identification of the chromosome of interest during its dissection and collection of its material from metaphase plates spread on the coverslip. In the present study, we developed and applied a technique for generation of microdissected DNA probes in the case when chromosome identification during microdissection appeared to be impossible. The technique was used for generation of two sets of Whole Chromosome Paints (WCPs) from all chromosomes of two species of free-living flatworms in the genus Macrostomum, M. mirumnovem and M. cliftonensis. The single-copy chromosome technique including separate collection of all chromosomes from one metaphase plate allowed us to generate WCPs that painted specifically the original chromosome by Chromosome In Situ Suppression Hybridization (CISS-Hybridization). CISS-Hybridization allowed identifying the original chromosome(s) used for DNA probe generation. Pooled WCPs derived from homologous chromosomes increased the intensity and specificity of chromosome painting provided by CISS-Hybridization. In the result, the obtained DNA probes appeared to be good enough for application in our studies devoted to analysis of karyotypic evolution in the genus Macrostomum and for analysis of chromosome rearrangements among the worms of laboratory cultures of M. mirumnovem.