A Comparative Analysis of Multi-Probe Fluorescence <i>In Situ</i> Hybridisation (FISH) Karyotypes in 26 <i>Pinus</i> Species (Pinaceae)

Abstract

Summary Every species has a unique karyotype, but certain genera have common karyptypes among species. The markers (chromosome length and centromere position) used in traditional karyotyping do not distinguish all chromosome pairs in the genus Pinus. However, the application of multi-probe fluorescence in situ hybridisation (FISH) procedures allowed exact karyotyping of 26 Pinus congeners. We used these new data to examine species relationships. The 35S rDNA and 5S rDNA, Arabidopsis-type telomere repeat sequences, and the proximal CMA band-specific repeat (PCSR) of P. densiflora were used as FISH probes for our analysis of chromosomes in 26 Pinus species. Each species had a unique FISH karyotype and most homologous chromosome pairs were identified. The FISH karyotypes were used to compare corresponding or homologous chromosomes among the species. Common or similar FISH signal patterns appeared in closely related species. Species that had inherited common FISH signal patterns were classified into four karyotype groups. We used cluster analyses to compare quantitative differences in FISH signals within these groups. The results of these analyses were consistent with recent systematic interpretations and resolved differences among existing taxonomic systems based on diverse methodologies. Our results indicate that FISH signal patterns reflect the history of species differentiation and that comparative FISH karyotyping has potential as an important tool for studying the taxonomy or phylogeny of Pinus.

Chromosome banding in the genus <i>Pinus</i> V. Fluorescent banding patterns in 16 diploxylon pines

“…The multi-probe FISH seems the most successful technique for identification of homologous chromosomes and comparative analysis in Pinus species (Hizume et al 2002;Shibata et al 2016). The probes used in the FISH are repetitive sequences and their FISH signals appear at fluorescent bands, and there is a limit in usable number of probes.…”

“…For more detail understands of chromosomes excellent techniques need to be used. In Pinus several techniques have been applied to chromosome analyses such as observation of small constrictions, C-banding, G-banding, fluorescent banding and in situ hybridization (Tanaka and Hizume 1980;Hizume et al 1983Hizume et al , 1989Hizume et al , 1990Hizume et al , 2002Doudrick et al 1995;Lubaretz et al 1996;Jacobs et al 2000;Shibata et al 2016;Bogunić et al 2011a,b). An effectiveness of fluorescent banding method and fluorescent in situ hybridization (FISH) using several proves is demonstrated in several pine species (Hizume et al 1983(Hizume et al , 1989(Hizume et al , 1990(Hizume et al , 2002Bogunić et al 2011a, b).…”

“…An effectiveness of fluorescent banding method and fluorescent in situ hybridization (FISH) using several proves is demonstrated in several pine species (Hizume et al 1983(Hizume et al , 1989(Hizume et al , 1990(Hizume et al , 2002Bogunić et al 2011a, b). The multi-probe FISH supplies the most information of chromosome structure in large scale and of comparative analysis of species among techniques (Hizume et al 2002;Islam-Faridi et al 2007;Shibata et al 2016), but need expensive reagents and several instruments. The fluorescent band method sequentially using two base-specific fluorochromes is more easy and simple technique by just staining and supplies comparable results for FISH.…”

Chromosome banding in the genus <i>Pinus</i> IV. Fluorescent banding patterns of chromosomes in eight taxa of haploxylone pines

“…Conserved karyotype is suitable for precise comparative karyotype analysis after an identification of each homoeologous chromosome and expected to supply valuable phylogenetic information. For chromosome identification of each homologous pair in chromosome complement, several techniques such as C-banding (Borzan and Papeš 1978;Tanaka and Hizume 1980;MacPherson and Filion 1981), G-banding (Drewry 1982), fluorescent banding (Hizume et al 1983(Hizume et al , 1989b(Hizume et al , 1990Jacobs et al 2000;Islam-Faridi et al 2007) and in situ hybridization (ISH) including fluorescent in situ hybridization (FISH) of rDNA probes Doudrick et al 1995;Lubaretz et al 1996;Jacobs et al 2000;Liu et al 2003;Cai et al 2006;Islam-Faridi et al 2007;Shibata et al 2016) and, other probes such as PCSR and telomere sequences (Hizume et al 2002b;Islam-Faridi et al 2007;Shibata et al 2016) were used for chromosome analysis in Pinus species. The fluorescent banding using CMA and DAPI displayed many fluorescent bands at interstitial and/or centromeric regions of most chromosomes in Pinus.…”

“…Their band patterns were useful for chromosome identification and comparative karyotype analysis which revealed relationships among three Japanese species in subgenus Pinus (Hizume et al 1983(Hizume et al , 1989b(Hizume et al , 1990. Recently the FISH with two or more probes was adapted to species of subgenera Pinus (Hizume et al 2002b;Liu et al 2003;Islam-Faridi et al 2007;Shibata et al 2016) and Strobus (Cai et al 2006;Shibata et al 2016). Shibata et al (2016) showed a usefulness of multi FISH for chromosome identification and comparative karyotype analysis in many Pinus species.…”