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

<br>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 (<i>Prospero</i>, Hyacinthaceae) and dicots (<i>Melampodium</i>, 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 <i>Prospero autumnale</i> 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 <i>Melampodium nayaritense</i> (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.