Optimization of chromosome preparation and chromosome number and ploidy in Hemiboea

Chromosome number and genome ploidy are crucial basic information for systematic evolutionary biology and genetics. Hemiboea, a genus of Gesneriaceae, with about 44 species, is mainly distributed in southern China, which have important medicinal and ornamental values. It is difficult to obtain suitable root tips for the chromosome preparation because of their small twining roots and small chromosomes. So far, only the chromosome numbers from three species of Hemiboea have been reported, while the chromosome numbers and ploidy of most species are unknown. The relationship between the pattern of chromosome number evolution and species evolution is also unclear. In this study, in order to explore the optimal conditions for chromosome preparation and the relationship between chromosome numbers and species evolution of Hemiboea, firstly, the root tips of six species (including H. subcapitata, H. longgangensis, H. longzhouensis, H. subacaulis var. jiangxiensis, H. follicularis and H. yongfuensis), which were generated by the method of hydroponic rooting for cutting leaves based on the characteristics of leaf cuttage propagation of plants in Hemboea, were used for the chromosome preparation experiments. Then, the effects of different experimental conditions on chromosome preparation were assessed. Next, the conditions were optimized and the chromosome counting was performed. Finally, the evolutionary history of chromosome numbers in Hemiboea and the related genera including Anna and Loxostigma were traced based on the molecular phylogenetic relationships, and the evolutionary pattern of chromosome numbers and relationships with species evolution were studied. The results were as follows: (1) The optimal conditions for karyotype preparation of the plants in Hemiboea were sampling during 9:30-10:00, dissociating for 10 min, and dyeing for 15 min. (2) All the six species of Hemiboea were diploid and with 32 chromosome numbers (2n=2x=32). (3) Except for a few species in the genus, the chromosome numbers of most species might be 2n=2x=32, and the variation of chromosome numbers might be caused by aneuploidy, which had no obvious relationship with the species evolution. This study sheds light on chromosome preparation of Hemiboea and other groups with similar leaf regeneration characteristics, and provides implications for the classification and phylogeny of this group.