Chromosomal differentiation betweenPinus heldreichiiandPinus nigra

Abstract

-Two tertiary relict pines, Pinus heldreichii and P. nigra, have been considered as taxonomically very close species. Both species have very discontinuous geographical distributions which overlap in some localities in Balkan Peninsula. The pattern of heterochromatin regions distribution (AT-, GC-rich and DAPI positive heterochromatin) and activity of nucleolar organizing regions (NORs) were analyzed for these two pines by fluorochrome banding and silver staining respectively. Morphometric data for karyotype of P. heldreichii were presented here for the first time. Chromomycin (CMA) banding pattern was particularly species specific, P. heldreichii possessed 12 and P. nigra 24 bands. In comparison with other species from subsection Pinus, P. heldreichii displayed particular CMA-banding pattern. Hoechst banding was less specific and similar to other pines. DAPI staining was applied after DNA denaturation/renaturation and revealed important differences in number and position of bands between the two species. Number of secondary constrictions (SCs), of NORs and of nucleoli also differed between P. heldreichii (10) and P. nigra (12). Our results proved a clear interspecific differentiation at the chromosomal level between the two species and add some data to discuss about the possible new subsectional placement of P. heldreichii. Pinus / heterochromatin / fluorochrome banding / G-C and A-T rich DNA / interspecific differentiationRésumé -Différentiation chromosomique entre Pinus heldreichii et Pinus nigra. Deux reliques tertiaires, Pinus heldreichii et P. nigra, sont considérées comme espèces taxonomiquement très proches. Leur distribution géographique est très discontinue, se chevauchant dans quelques localités des Balkans. La répartition des régions hétérochromatiques (AT-, GC-riche et hétérochromatine constitutive nonspécifique) et l'activité des organisateurs nucléolaires (NORs) sont analysées par fluorochrome banding et coloration au nitrate d'argent respectivement. Les données morphometriques du caryotype de P. heldreichii sont présentées ici pour la première fois. La distribution des bandes GC-riches est particulièrement spécifique, P. heldreichii possède 12 et P. nigra 24 bandes. En comparaison avec d'autres espèces de la subsection Pinus, P. heldreichii présente un chromomycin banding particulier. Les bandes AT-riches sont moins spécifiques et leur localisation est similaire aux autres pins. La coloration au DAPI, effectuée après la dénaturation/renaturation de l'ADN, révèle des différences importantes en nombre et position des bandes entre deux espèces. Le nombre des constrictions secondaires (SCs), des NORs et des nucléoles diffère également entre P. heldreichii (10) et P. nigra (12). Nos résultats montrent une différentiation nette au niveau chromosomique entre ces deux espèces et ouvrent la discussion sur un changement éventuel de subsection pour P. heldreichii. Pinus / hétérochromatine / fluorochrome banding / ADN riche en G-C et A-T / différentiation interspécifique

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

“…DAPI-bands appeared at proximal region and thin DAPI-bands did at interstitial region. DAPI-dots were observed at centromeric region as previous reports (Hizume et al 1983(Hizume et al , 1989(Hizume et al , 1990Bogunić et al 2006). Number of interstitial CMA-bands, and proximal bands of CMA and DAPI varied among species.…”

“…The fluorescent banding pattern of each species showed some variation of mainly in interstitial CMA-bands and fluorescence of proximal bands as previous reports (Hizume et al 1989;Bogunić et al 2006). Intraspecific variations of fluorescent bands in each species were not described this time.…”

“…Interstitial CMA-bands locating at secondary constriction containing rDNA are six to eight in karyotypes of 16 Pinus species examined as previous study (Hizume et al 1983, 1989, 1990, 1992, Jacobs et al 2000, Bogunić et al 2006) and the number of CMA-bands is not found relation with any taxonomic ranks. Six species of subsection Pinus had six to ten proximal CMA-bands and other species had few (four or six) proximal CMA-bands.…”

“…In addition to interstitial CMA-bands at secondary constrictions, CMA-bands appear at the proximal region of some chromosomes, and DAPI-bands also appeared at the proximal and interstitial regions of most chromosomes. The fluorescent bandings applied sequentially to supply many fluorescent bands which contribute to identification of homologous chromosomes and then comparative karyotype analysis among populations and species (Hizume et al 1983(Hizume et al , 1989(Hizume et al , 1990Bogunić et al 2006).…”

“…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. Fluorescent band patterns were reported in four and eight taxa of subgenera Pinus and Strobus, respectively (Hizume et al 1983(Hizume et al , 1989(Hizume et al , 1990Bogunić et al 2006). In more species chromosomal information is need for understand of species relationships in respect to chromosomes in a large genus Pinus.…”