Mapeamento cromossômico de DNA satélite e comportamento meiótico no complexo Poliploide Lippia alba (Mill.) N. E. Br.

Lippia alba (Verbenaceae) is a tropical aromatic shrub with extensive phenotypical and genomic plasticity widely used in traditional medicine. Recently, the species was described as a new natural autopolyploid complex with five distinct chromosome numbers (2x=30, 2x=30+8, 3x=45, 4x=60 and 6x=90). Strides have been done in order to understand the cytotypes origin and species evolution. In this study, a detailed karyotype of L. alba using Fluorescence in situ Hybridization (FISH) with species-specific probes was described. We also report the meiosis behavior and pollen viability in sixty accessions. Using massive parallel sequencing (IIlumina MiSeq platform) new cytogenetic landmarks (CL66 and CL98) were chosen for probing all cytotypes described for the species. For meiotic analysis, the percentage of abnormalities was quantified, evaluating around 100 cells in six stages (metaphase I; anaphase I + telophase I; metaphase II; anaphase II + telophase II). Around 1,000 pollen per accession were used to estimate pollen viability. FISH results revealed that both satDNA arrays are located preferentially on terminal sites of the chromosomes. In general, the CL98 repeat showed a uniform pattern in different accessions. We observed 2, 3, 4, and 6 marked chromosomes respectively in diploid, triploid, tetraploid and hexaploid accessions revealing that the number of depicted chromosomes varied proportionally according to the ploidy level. On the other hand, the CL66 repeat was polymorphic. Great variations were observed among the accessions mainly within the diploids. In general, the meiotic analysis revealed higher index of abnormalities in polyploid cytotypes. However, some 2x accessions also showed considerable irregularities during the microsporogenesis. Desynapsis, unequal segregation, lost chromosomes, triads and polyads were the most common irregularities observed. Pollen viability analysis corroborated the meiosis data. It was possible to conclude that 1) the development of specific landmarks for L. alba was efficient; 2) the karyotypic profiles of both satDNA revealed different behavior; 3) microsporogenesis analysis and pollen viability of 2x accessions suggest that diploids are the key point for the origin of the polyploid complex and 4) independent and multiples events of genome duplication associated to chromosome rearrangements may have generated great karyotypic variation in the species. L. alba karyotype is possibly under stabilization process making the species an important model to study natural polyploids in the tropics.Lippia alba (Verbenaceae), é uma espécie herbácea tropical com grande plasticidade fenotípica e genômica, amplamente utilizada na medicina popular. Recentemente, a espécie foi descrita como um novo complexo autopoliploide contendo cinco números cromossômicos (2x=30, 2x=30+8, 3x=45, 4x=60 e 6x=90), e esforços têm sido feitos a fim de entender sua origem e evolução. No presente trabalho, foram descritos perfis cariotípicos mais detalhados da espécie, por meio de mapeamento cromossômico utilizando sondas espécie-específicas e análises do comportamento meiótico e de viabilidade polínica. A partir do sequenciamento genômico de baixa cobertura (IIlumina MiSeq), foram desenvolvidos novos marcadores citogenéticos (denominados CL66 e CL98) os quais foram utilizados para o mapeamento cromossômico em acessos representando os cinco citótipos do complexo. Para a análise meiótica, seis estágios da divisão (metáfase I; anáfase I + telófase I; metáfase II; anáfase II + telófase II) foram quantificados, e aproximadamente, 100 células foram avaliadas para cada estágio. Os mesmos acessos foram avaliados quanto à viabilidade polínica (1.000 grãos de pólen foram quantificados para cada indivíduo). Os resultados da Hibridização Fluorescente in situ (FISH) revelaram que ambas as repetições satélite estão localizadas na porção terminal dos cromossomos. Em geral, a repetição CL98 mostrou um padrão uniforme nos diferentes acessos. Foram observados dois, três, quatro e seis cromossomos marcados em diploides, triploides, tetraploides e hexaploide, respectivamente, revelando que o número de cromossomos marcados variou proporcionalmente, de acordo com o nível de ploidia do acesso. Por outro lado, a repetição CL66 apresentou-se polimórfica. Variações foram observadas entre os acessos, principalmente, entre os indivíduos diploides. Com relação às análises meióticas, alto percentual de irregularidade foi observado nos citótipos poliploides. Entretanto, alguns acessos 2x também mostraram consideráveis erros durante a microsporogênese. Entre as irregularidades encontradas, destacam-se: pareamento cromossômico anormal; segregação cromossômica desigual; cromossomos perdidos; tríades e políades. Os resultados da viabilidade polínica corroboraram os dados da meiose. A partir do conjunto de dados obtidos foi possível concluir que 1) a metodologia para o desenvolvimento de marcadores cromossômicos específicos para L. alba mostrou-se eficiente; 2) as repetições satélite exibiram diferentes comportamentos (estável e dinâmico) no genoma de L. alba; 3) a ocorrência de microsporogênese irregular em diploides, associada à viabilidade polínica, sugerem que os acessos 2x sejam elementos importantes na formação do complexo poliploide e 4) a ampla variação cariotípica observada na espécie pode ser consequência de múltiplos e independentes eventos de duplicação genômica, aliado a rearranjos cromossômicos. Possivelmente, L. alba encontra-se em processo de estabilização do seu cariótipo tornando a espécie, um importante modelo para estudos de poliploides naturais nos trópicos

Evolução cromossômica no complexo poliploide Lippia alba (Mill.) N. E. Br.

The polyploidy is considered one of the most important factors in plant speciation, Lippia alba is an important medicinal herb being popularly used mainly in the treatment of gastric and respiratory problems. Once your popular use is employed as palliative for various human diseases. The species, of widely distribution possess extensive morphological plasticity and different chemotypes in addition to great karyotype variation. The present study aimed to characterize by means of cytogenetic techniques different cytotypes collected in natural environment and help to understand the dynamic of possible polyploid complex formation of the in L. alba. By means of chromosome banding and fluorescence in situ hibridization techniques in addition to meiotic and pollen viability analyses accomplished in five cytotypes of polyploid complex, it was possible to observe numerical and structural variations. The results suggest that intraspecific crosses autopolyploidization events caused the wide karyological variation in L. alba. The FISH technique, specially the rDNA 5S region, revealed important indications of autopolyploidy, once the number of marks of monoploid complement remained constant regardless of chromosome number. Moreover, the high rates of meiotic irregularities observed in triploid, aneuploid, tetraploid and hexaploid accessions also indicates abnormal paring and chromosomal segregation , a typical phenomenon of newly formed polypoids. In addition, the results of pollen viability by means of FDA test corroborate the meiotic data and support the hypothesis of of a polyploid complex formation in L. alba.A poliploidia é considerada um dos principais fatores envolvidos na formação de novas espécies. Lippia alba é uma espécie herbácea de grande importância medicinal, sendo empregada popularmente no tratamento de problemas gástricos e respiratórios. A espécie, de ampla distribuição caracteriza-se por apresentar vasta plasticidade morfológica e variados quimiotipos além de grande variação cariotípica. O presente trabalho teve como objetivo caracterizar por meio de técnicas citogenéticas, diferentes citótipos coletados em ambientes naturais e contribuir para entender a dinâmica da formação de um possível complexo poliploide em L. alba. A partir de técnicas de bandeamento cromossômico e hibridização fluorescente in situ somadas às análises meióticas e de viabilidade polínica realizadas em cinco citótipos do complexo poliploide, foi possível verificar variações cromossômicas numéricas e estruturais. Os resultados sugerem que cruzamentos intraespecíficos e eventos de autopoliploidização originaram esta ampla variação cariológica em L. alba. A técnica de FISH, em especial a região de DNAr 5S, revelou indícios fortes de autopoliploidia, já que o número de sítios do complemento monoploide apresentou-se constante independente do nível de ploidia. Além disso, os altos índices de irregularidades meióticas encontrados nos acessos triploides, aneuploide, tetraploide e hexaploide também são indícios de pareamento e segregação cromossômica anormal, fenômeno típico de poliploides recém-formados. Em adição, os resultados de viabilidade polínica com o corante FDA corroboram os dados meióticos e incrementam a hipótese de formação de um complexo poliploide em L. alba.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Contributions to cytogenetics of Plectranthus barbatus Andr. (Lamiaceae): a medicinal plant

Accessions of Plectranthus barbatus (Lamiaceae), a medicinal plant, were investigated using a cytogenetic approach and flow cytometry (FCM). Here, we describe for the first time details of the karyotype including chromosome morphology, physical mapping of GC rich bands (CMA3 banding), as well as the mapping of 45S and 5S rDNA sites. All accessions studied showed karyotypes with 2n = 30 small metacentric and submetacentric chromosomes. The CMA3 banding and fluorescent in situ hybridization techniques revealed coincidence between CMA3 bands and 45S rDNA sites (6 terminal marks) while for the 5S rDNA were observed 4 subterminal marks no coincident with CMA3 marks. For nuclear genome size measurement, the FCM procedure provided histograms with G0/G1 peaks exhibiting CV between 2.0–4.9 and the mean values obtained for the species was 2C = 2.78 pg, with AT% = 61.08 and GC% = 38.92. The cytogenetic data obtained here present new and important information which enables the characterization of P. barbatus

rDNA mapping, heterochromatin characterization and AT/GC content of Agapanthus africanus (L.) Hoffmanns (Agapanthaceae)

ABSTRACT Agapanthus (Agapanthaceae) has 10 species described. However, most taxonomists differ respect to this number because the great phenotypic plasticity of the species. The cytogenetic has been an important tool to aid the plant taxon identification, and to date, all taxa of Agapanthus L'Héritier studied cytologically, presented 2n = 30. Although the species possess large chromosomes, the group is karyologically little explored. This work aimed to increase the cytogenetic knowledge of Agapanthus africanus (L.) Hoffmanns by utilization of chromosome banding techniques with DAPI / CMA3 and Fluorescent in situ Hybridization (FISH). In addition, flow cytometry was used for determination of DNA content and the percentage of AT / GC nitrogenous bases. Plants studied showed 2n = 30 chromosomes, ranging from 4.34 - 8.55 µm, with the karyotype formulae (KF) = 10m + 5sm. Through FISH, one 45S rDNA signal was observed proximally to centromere of the chromosome 7, while for 5S rDNA sites we observed one signal proximally to centromere of chromosome 9. The 2C DNA content estimated for the species was 2C = 24.4 with 59% of AT and 41% of GC. Our data allowed important upgrade for biology and cytotaxonomy of Agapanthus africanus (L.) Hoffmanns

In vitro regeneration of triploid plants from mature endosperm culture of commercial passionfruit (Passiflora edulis Sims)

Due to the triploid nature of endosperm, an embryonic reserve tissue, in vitro culture of endosperm tissues has been considered a direct method for production of polyploids. In the present study, we report the establishment of an in vitro regeneration system from endosperm culture for production of triploid Passiflora edulis plants, the main commercial species of passionfruit. Surface-sterilized endosperms were cultured in MS medium with different concentrations of benzyladenine (BA), thidiazuron (TDZ), and kinetin (KIN). The cultures were maintained in a growth chamber under controlled conditions, for 60 days. Thidiazuron was the only type of cytokinin that induced shoot production in the endosperm tissues; the highest number of shoots was produced in the presence of 4.5 and 9.0 μM TDZ. Flow cytometry and chromosomal analysis confirmed that endosperm-derived plants were triploid. The internal standard, Pisum sativum, and the diploid control, seed-derived Passiflora edulis plants (2n = 2× = 18), showed average DNA quantities of 9.09 and 3.35 pg respectively. Endosperm-derived P. edulis plants showed an average DNA content of 5.10 pg and a chromosome count of 27 (3n = 3× = 27), the same ploidy level as the endosperm (triploid). Our data open new prospects for breeding of passionfruit by means of a stable and reproducible regeneration system from endosperm culture leading to generation of triploid plants

In vitro regeneration of triploid plants from mature endosperm culture of commercial passionfruit ( Passiflora edulis Sims)

Due to the triploid nature of endosperm, an embryonic reserve tissue, in vitro culture of endosperm tissues has been considered a direct method for production of polyploids. In the present study, we report the establishment of an in vitro regeneration system from endosperm culture for production of triploid Passiflora edulis plants, the main commercial species of passionfruit. Surface-sterilized endosperms were cultured in MS medium with different concentrations of benzyladenine (BA), thidiazuron (TDZ), and kinetin (KIN). The cultures were maintained in a growth chamber under controlled conditions, for 60 days. Thidiazuron was the only type of cytokinin that induced shoot production in the endosperm tissues; the highest number of shoots was produced in the presence of 4.5 and 9.0 μM TDZ. Flow cytometry and chromosomal analysis confirmed that endosperm-derived plants were triploid. The internal standard, Pisum sativum, and the diploid control, seed-derived Passiflora edulis plants (2n = 2× = 18), showed average DNA quantities of 9.09 and 3.35 pg respectively. Endosperm-derived P. edulis plants showed an average DNA content of 5.10 pg and a chromosome count of 27 (3n = 3× = 27), the same ploidy level as the endosperm (triploid). Our data open new prospects for breeding of passionfruit by means of a stable and reproducible regeneration system from endosperm culture leading to generation of triploid plants

ATLANTIC ANTS: a data set of ants in Atlantic Forests of South America

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