Cultivated sweetpotato (Ipomoea batatas (L.) Lam.) from the family Convolvulaceae is a hexaploid species with 2n = 6x = 90, and has been controversial regarding its nature as an autopolyploid arising within a species or allopolyploid forming between species. Here, we developed oligonucleotide-based painting probes for two chromosomes of I.nil, a model diploid Ipomoea species. Using these probes, we revealed pairing behavior of homoeologous chromosomes in I. batatas and its two possible polyploid ancestral species, tetraploid I. tabascana (2n = 4x = 60) and hexaploid I. trifida (2n = 6x = 90). Chromosome painting analysis revealed a high percentage of quadrivalent formation in zygotene-pachytene cells of I. tabascana, which supported that I. tabascana was an autotetraploid likely derived by doubling of structurally similar and homologous genomes rather than a hybrid between I. batatas and I. trifida (2x). The high frequency of hexavalent/bivalent and tetravalent pairing was observed in I. trifida (6x) and I. batatas. However, the percentage of hexavalent pairing in I. trifida (6x) was far higher than that in I. batatas. Thus, present results tended to support that I. trifida (6x) was an autohexaploid, while I. batatas was more likely to be a segmental allohexaploid.

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