Karyotype evolution and phylogenetic analyses in the genus Cardiospermum L. (Paullinieae, Sapindaceae).

Cardiospermum L. belongs to the Paullinieae tribe (Sapindaceae) and comprises 16 species. Of these, 12 species are present in South America and all occur in Brazil. Cardiospermum shows the most variable chromosome number of the tribe. Phylogenetic relationships within the genus Cardiospermum, especially with other species of the tribe, are poorly understood. This research focuses on characterisation of the karyotypic features of Cardiospermum using conventional cytogenetic methods, CMA/DAPI chromosome banding and fluorescence in situ hybridisation (FISH). To elucidate the phylogeny of the genus, the nuclear markers ITS1 and ITS2 were sequenced and analysed using maximum parsimony and Bayesian inference. Cardiospermum shows important diversity in basic numbers, with x = 7, 9, 10, 11 and 12. All species studied have metacentric and submetacentric chromosomes, some species have subtelocentric chromosomes, while telocentric chromosomes are absent. The interphase nuclei differentiate the Cardiospermum species into two groups. The CMA(3) /DAPI chromosome banding revealed the presence of an AT-rich terminal region in C. corindum, C. grandiflorum and C. urvilleoides, whereas GC-rich regions were found in C. grandiflorum, C. halicacabum var. halicacabum, C. halicacabum var. microcarpum, C. heringeri and C. integerrimum. FISH revealed syntenic and non-syntenic distribution of the 18-5.8-26S and 5S rDNA. The syntenic distribution always occurred in the short arms of the same chromosome in all of the species. The phylogenetic relationships reveal, in part, the taxonomic arrangement of the genus Cardiospermum.

Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora.

Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F1 progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.

Karyotypes, heterochromatin, and physical mapping of 18S-26S rDNA in Cactaceae.

Karyotype analyses in members of the four Cactaceae subfamilies were performed. Numbers and karyotype formula obtained were: Pereskioideae = Pereskiaaculeata(2n = 22; 10 m + 1 sm), Maihuenioideae = Maihuenia patagonica (2n = 22, 9 m + 2 sm; 2n = 44, 18 m + 4 sm), Opuntioideae = Cumulopuntia recurvata(2n = 44; 20 m + 2 sm), Cactoideae = Acanthocalycium spiniflorum (2n = 22; 10 m + 1 sm),Echinopsis tubiflora (2n = 22; 10 m + 1 sm), Trichocereus candicans (2n = 22, 22 m). Chromosomes were small, the average chromosome length was 2.3 mum. Diploid species and the tetraploid C. recurvata had one terminal satellite, whereas the remaining tetraploid species showed four satellited chromosomes. Karyotypes were symmetrical. No CMA(-)/DAPI(+) bands were detected, but CMA(+)/DAPI(-) bands associated with NOR were always found. Pericentromeric heterochromatin was found in C. recurvata, A. spiniflorum, and the tetraploid cytotype of M. patagonica. The locations of the 18S-26S rDNA sites in all species coincided with CMA(+)/DAPI(-) bands; the same occurred with the sizes and numbers of signals for each species. This technique was applied for the first time in metaphase chromosomes in cacti. NOR-bearing pair no.1 may be homeologous in all species examined. In Cactaceae, the 18S-26S loci seem to be highly conserved.