Euchromatic Supernumerary Chromosomal Segments—Remnants of Ongoing Karyotype Restructuring in the Prospero autumnale Complex?

Supernumerary chromosomal segments (SCSs) represent additional chromosomal material that, unlike B chromosomes, is attached to the standard chromosome complement. The Prospero autumnale complex (Hyacinthaceae) is polymorphic for euchromatic large terminal SCSs located on the short arm of chromosome 1 in diploid cytotypes AA and B7B7, and tetraploid AAB7B7 and B6B6B7B7, in addition to on the short arm of chromosome 4 in polyploid B7B7B7B7 and B7B7B7B7B7B7 cytotypes. The genomic composition and evolutionary relationships among these SCSs have been assessed using fluorescence in situ hybridisation (FISH) with 5S and 35S ribosomal DNAs (rDNAs), satellite DNA PaB6, and a vertebrate-type telomeric repeat TTAGGG. Neither of the rDNA repeats were detected in SCSs, but most contained PaB6 and telomeric repeats, although these never spanned whole SCSs. Genomic in situ hybridisation (GISH) using A, B6, and B7 diploid genomic parental DNAs as probes revealed the consistently higher genomic affinity of SCSs in diploid hybrid B6B7 and allopolyploids AAB7B7 and B6B6B7B7 to genomic DNA of the B7 diploid cytotype. GISH results suggest a possible early origin of SCSs, especially that on chromosome 1, as by-products of the extensive genome restructuring within a putative ancestral P. autumnale B7 genome, predating the complex diversification at the diploid level and perhaps linked to B-chromosome evolution.

Formamide-Free Genomic in situ Hybridization Allows Unambiguous Discrimination of Highly Similar Parental Genomes in Diploid Hybrids and Allopolyploids

Polyploidy and hybridization play an important role in plant diversification and speciation. The application of genomic in situ hybridization (GISH) allows the identification of parental genomes in hybrids, thus elucidating their origins and allowing for analysis of their genomic evolution. The performance of GISH depends on the similarity of the parental genomes and on the age of hybrids. Here, we present the formamide-free GISH (ff-GISH) protocol applied to diploid and polyploid hybrids of monocots (Prospero, Hyacinthaceae) and dicots (Melampodium, Asteraceae) differing in similarity of the parental genomes and in chromosome and genome sizes. The efficiency of the new protocol is compared to the standard GISH protocol. As a result, ff-GISH allowed efficient labeling and discrimination of the parental chromosome sets in diploid and allopolyploid hybrids in Prospero autumnale species complex. In contrast, the standard GISH protocol failed to differentiate the parental genomes due to high levels of similar repetitive DNA. Likewise, an unambiguous identification of parental genomes in allotetraploid Melampodium nayaritense (Asteraceae) was possible after ff-GISH, whereas the standard GISH hybridization performance was suboptimal. The modified method is simple and non-toxic and allows the discrimination of very similar parental genomes in hybrids. This method lends itself to modifications and improvements and can also be used for FISH.