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Corresponding author: Ana Maria Benko-Iseppon ( ana.benko.iseppon@pq.cnpq.br ) Academic editor: Gennady Karlov
© 2016 Karla C. B. Santana, Diego S. B. Pinangé, Santelmo Vasconcelos, Ana Rafaela Oliveira, Ana Christina Brasileiro-Vidal, Marccus V. Alves, Ana Maria Benko-Iseppon.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Santana KCB, Pinangé DSB, Vasconcelos S, Oliveira AR, Brasileiro-Vidal AC, Alves MV, Benko-Iseppon AM (2016) Unraveling the karyotype structure of the spurges Euphorbia hirta Linnaeus, 1753 and E. hyssopifolia Linnaeus, 1753 (Euphorbiaceae) using genome size estimation and heterochromatin differentiation. Comparative Cytogenetics 10(4): 657-669. https://doi.org/10.3897/CompCytogen.v10i4.8193
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Euphorbia Linnaeus, 1753 (Euphorbiaceae) is one of the most diverse and complex genera among the angiosperms, showing a huge diversity in morphologic traits and ecologic patterns. In order to improve the knowledge of the karyotype organization of Euphorbia hirta (2n = 18) and E. hyssopifolia (2n = 12), cytogenetic studies were performed by means of conventional staining with Giemsa, genome size estimations with flow cytometry, heterochromatin differentiation with chromomycin A3 (CMA) and 4’,6-diamidino-2-phenylindole (DAPI) and Giemsa C-banding, fluorescent in situ hybridization (FISH) with 45S and 5S rDNA probes, and impregnation with silver nitrate (AgNO3). Our results revealed small metacentric chromosomes, CMA+/DAPI0 heterochromatin in the pericentromeric regions of all chromosomes and CMA+/DAPI− in the distal part of chromosome arms carriers of nucleolar organizing regions (NORs). The DNA content measurements revealed small genomes for both species: E. hirta with 2C = 0.77 pg and E. hyssopifolia with 2C = 1.41 pg. After FISH procedures, E. hirta, and E. hyssopifolia presented three and four pairs of terminal 45S rDNA sites, respectively, colocalizing with CMA+ heterochromatic blocks, besides only one interstitial pair of 5S rDNA signals. Additionally, the maximum number of active NORs agreed with the total number of observed 45S rDNA sites. This work represents the first analysis using FISH in the subfamily Euphorbioideae, revealing a significant number of chromosomal markers, which may be very helpful to understand evolutionary patterns among Euphorbia species.
Cytotaxonomy, CMA/DAPI, FISH, genome size, rDNA, RONs
The giant genus Euphorbia (spurges), a member of the family Euphorbiaceae, is one of the largest and most diverse groups of the plant kingdom, consisting of more than 2000 species with a very wide geographic distribution (
Similarly to the family as a whole, the genus Euphorbia is an extremely diversified group, not only taking into account morphology and habit (
In groups with such a complex classification, the knowledge about chromosome features, such as the organization of interphase nuclei, diploid number, nuclear DNA content and physical mapping of repetitive DNA, may be critical to support studies on systematics and understanding evolutionary pathways (
Fruits of specimens of E. hirta (vouchers: K.C.B. Santana 04, 05 and 06 – UFP) and E. hyssopifolia (vouchers: K.C.B. Santana 01, 02 and 03 – UFP) were collected in urban fragments of the Atlantic Forest in Recife (Pernambuco, Brazil). Subsequently, they were incubated at 50 °C for 5 h and then transferred to room temperature (ca. 25 °C) for three to four days to release the seeds, which were germinated in Petri dishes under an artificial system of circadian lighting (≥ 1,500 lux) at ~35 °C. Root tips were pre-treated with 2 mM 8-hydroxyquinoline for 90 min at room temperature and 23 h at 8 °C. For the conventional staining, fluorochromes and FISH procedures, the roots were fixated in ethanol:acetic acid (3:1, v:v), for 4–6 h at room temperature and stored at −20 °C.
The preparation of slides followed the methodology used by
To estimate the DNA C-values, approximately 20-30 mg of fresh leaves from E. hirta and E. hyssopifolia were chopped on ice with 1 mL of GPB buffer (
The C-banding methodology followed the procedures described by
The impregnation with silver nitrate followed the protocol described by
The CMA/DAPI banding followed
Images of the best cells were captured with a Leica DMLB epifluorescence microscope with a Leica DFC 340FX camera, using the software Leica CW4000. Images were optimized for best contrast and brightness and the photos of FISH with 5S and 45 rDNA probe were pseudocolored in red and green, respectively (to allow the superposition of images), using Adobe Photoshop CS4 (Adobe Systems Incorporated). Additionally, chromosomes of 10 cells stained with DAPI of each species were measured to obtain the chromosome sizes and the relationship between the chromosome arms according to
The interphase nuclei of both species were predominantly semi-reticulated with a proximal pattern of condensation (Figures
Karyotype analysis of Euphorbia hirta (2n = 18). Standard staining of mitotic interphase nucleus (A); standard staining of mitotic metaphase (B); silver impregnation of mitotic interphase nucleus (C); fluorochrome banding of metaphase chromosomes stained with CMA (D) and DAPI (E) and superposed images (F); C-banding of chromosomes stained with CMA/DAPI (C-CMA/DAPI; G–H); and metaphase chromosomes hybridized with 5S (red) and 45S (green) rDNA probes (I). Arrows and arrowheads indicate 5S and 45S rDNA sites, respectively.
Karyotype analysis of Euphorbia hyssopifolia (2n = 12). Standard staining of mitotic interphase nucleus (A); standard staining of mitotic metaphase (B); silver impregnation of mitotic interphase nucleus (C); fluorochrome banding of metaphase chromosomes stained with CMA (D) and DAPI (E) and superposed images (F); C-banding of chromosomes stained with CMA/DAPI (C-CMA/DAPI; G–H); and metaphase chromosomes hybridized with 5S (red) and 45S (green) rDNA probes (I). Arrows and arrowheads indicate 5S and 45S rDNA sites, respectively.
Representative idiograms of Euphorbia hirta and E. hyssopifolia chromosomes. The black dots in the chart in the inferior right corner associate the chromosome marks (rows) with their respective colors (columns) in the chromosomes.
The karyotype presented metacentric and submetacentric chromosomes with gradual decreasing sizes, ranging in average from 1.21 µm to 2.58 µm, for E. hirta, and from 1.43 µm to 2.04 µm, for E. hyssopifolia. In general, Euphorbiaceae species exhibit small chromosomes (see
Euphorbia hirta presented a smaller genome size (2C = 0.77 ± 0.02 pg) than E. hyssopifolia (2C = 1.41 ± 0.04 pg). These results fit in the known range of DNA content of species of the genus, which varies from 2C = 0.70 pg to 2C = 18.80 pg (see Bennett and Leitch 2012). According to the most comprehensive phylogenetic reconstruction based on nuclear and plastid sequences for Euphorbia subgenus Chamaesyce Gray, 1821, provided by
However, despite accounting for more than half of the known genome sizes of members of Euphorbiaceae (19 out of 33 analyzed species), the proportion of Euphorbia analyzed species is considerably low, being less than 1% of the genus. Thus, the noteworthy range of variation of 41× among the species of the genus analyzed so far, between the diploid species E. peplus Linnaeus, 1758 (2n = 22; 2C = 0.70 pg) and E. polygona Haworth, 1803 (2n = 20; 2C = 28.70), although quite high, may be an underestimation (see Bennet and Leitch 2016). Also, while this is the first report of the genome size of E. hyssopifolia,
In the CMA/DAPI banding, the pericentromeric region of all chromosomes showed positive bands for CMA and were negative for DAPI (CMA+/DAPI−) for both species (Figures
The FISH procedures revealed 45S rDNA terminal sites for both E. hirta (short arm of chromosome pairs 1, 3 and 5) and E. hyssopifolia (short arm of chromosome pairs 1, 2, 3 and 4) (Figures
The maximum number of nucleoli per interphase nuclei visualized through impregnation with silver nitrate in E. hirta and E. hyssopifolia were six and eight, respectively (Figure
Nucleolar frequency by interphase nucleus in mitotic cells of Euphorbia hirta and E. hyssopifolia.
Species | Nucleoli per cell | Number of cells | |||||
---|---|---|---|---|---|---|---|
1 (%) | 2 (%) | 3 (%) | 4 (%) | 5–6 (%) | 7–8 (%) | ||
Euphorbia hirta | 54.04 | 34.25 | 9.89 | 1.31 | 0.52 | - | 5054 |
Euphorbia hyssopifolia | 34.72 | 40.19 | 19.35 | 4.91 | 0.72 | 0.12 | 4280 |
It is interesting to note that despite similarities in the morphology, habit and occurrence of both here studied species in the sampled area, no evidence of hybridization was detected during field work. This was confirmed by a recent report using phytochemical profiling and ISSR (Inter-Simple Sequence Repeat) markers, positioning both taxa in distinct branches (
The present analysis characterized the chromosomes of two Euphorbia species, being a pioneer in the application of the FISH methodology with members of the subfamily Euphorbioideae. The physical mapping of repetitive DNA played a complementary role between the different methodologies employed, generating markers that showed a relatively high conservation of the distribution pattern of heterochromatin between E. hirta and E. hyssopifolia. These findings indicated the high potential of the employed approaches in describing chromosome markers that may be very helpful differentiate species and understand karyotype evolution within such a diverse genus.
The authors thank Dr. Nelson Carvalho (Department of Sustainable Development, Vale Institute of Technology, Belém, Brazil) for providing use of the flow cytometer and Prof. Dr. João Loureiro (Department of Life Sciences, University of Coimbra, Coimbra, Portugal) and Dr. Marcelo Guerra (Department of Botany, Federal University of Pernambuco, Recife, Brazil) with the valuable support for the realization of the procedures of C-DNA measurement. This work was carried out with the financial support of the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE).