Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora

Abstract

ABSTRACT. 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 F 1 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 2149©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 9 (4): 2148-2155 (2010) Cytological preparations for FISH in Passiflora 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.

Origin of the cultivated passion fruit Passiflora edulis f. flavicarpa and genomic relationships among species of the subgenera Decaloba and Passiflora

“…The GISH technique followed the protocol proposed by Schwarzacher & Haslop‐Harrison (2000) and Souza et al . (2010), with modifications by Melo et al . (2015).…”

Karyotypic characterization of melon accessions

“…Slide preparation followed the protocol proposed by Schwarzacher & Heslop-Harrison (2000), modified by Souza et al (2010). Slides containing the cytological preparations were oven-dried at 37 °C for a minimum time of 1 h. After application of 50 μL RNase (1 μg/mL) (Sigma ®) in 2× SSC buffer (0.3 M sodium chloride, Sigma®, 0.03 M sodium citrate, Sigma®), slides were incubated in a humid chamber for 1 h at 37 °C.…”

“…Cytogenetic information has allowed comparison between taxa and the identification of inter-and intraspecific chromosomal variations (Souza et al, 2010;Melo et al, 2011;Guerra, 2013;Melo et al, 2014;Melo et al, 2015;Coelho et al, 2016;Melo et al, 2017). More detailed information on chromosome structure has been developed in recent studies demonstrating the potential of cytogenetic analysis between taxa for comparisons of genetic and cytogenetic maps among different species and genera (Snowdon et al, 1997).…”

Establishment of the genomic in situ hybridization (GISH) technique for analysis in interspecific hybrids of Passiflora

“…GISH application followed the protocols for in situ hybridization that were proposed by Schwarzacher and Haslop-Harrison (2000) and Souza et al (2010) with adaptations. Slides containing the cytological preparations were oven-dried at 37°C for at least 1 h and were then subjected to treatment with 100 μg/mL RNase (Sigma, United States, R5125) in 2X SSC buffer [0.3 M sodium chloride (Sigma, United States, S3014) and 0.03 M sodium citrate (Sigma, United States, S5941)]; the slides were then incubated in a humid chamber for 1 h at 37°C.…”