Cytogenomic Characterization of Transposable Elements and Satellite DNA in Passiflora L. Species.

The species Passiflora alata, P. cincinnata, and P. edulis have great economic value due to the use of their fruits for human consumption. In this study, we compared the repetitive genome fractions of these three species. The compositions of the repetitive DNA of these three species' genomes were analyzed using clustering and identification of the repetitive sequences with RepeatExplorer. It was found that repetitive DNA content represents 74.70%, 66.86%, and 62.24% of the genome of P. alata, P. edulis, and P. cincinnata, respectively. LTR Ty3/Gypsy retrotransposons represent the highest genome proportions in P. alata and P. edulis, while Ty1/Copia comprises the largest proportion of P. cincinnata genome. Chromosomal mapping by Fluorescent In Situ Hybridization (FISH) showed that LTR retrotransposons have a dispersed distribution along chromosomes. The subtelomeric region of chromosomes is where 145 bp satellite DNA is located, suggesting that these elements may play important roles in genome structure and organization in these species. In this work, we obtained the first global characterization of the composition of repetitive DNA in Passiflora, showing that an increase in genome size is related to an increase in repetitive DNA, which represents an important evolutionary route for these species.

Karyotyping of commercial cultivars of melon (Cucumis melo L.).

BACKGROUND: This study on cultivars of melon (Cucumis melo L.) marketed in Brazil was conducted to obtain information to be used in breeding programs of this species. Little is known about the karyotype variability among C. melo L. cultivars targeted at the consumer market. The objective of the present study was to verify the karyotype variability in eight commercial melon cultivars used in the Brazilian market. METHODS AND RESULTS: Slides were stained with 2% Giemsa and assembled with Neomount to perform chromosomal morphometry. GC-rich heterochromatin was observed by CMA3/DAPI staining. 5 S rDNA, centromeric satellite DNA (SatDNA), and telomeric sites were visualized using fluorescence in situ hybridization. All images were captured on an Olympus BX41 microscope equipped with a 5 M Olympus DP25 digital camera and DP2-BSW software. The cultivars showed symmetrical karyotypes with significant differences in total chromosome length and average chromosome size. Heterochromatic CMA3+ blocks were observed in terminal regions related to satellites (secondary constrictions), as well as in centromeric and pericentromeric regions. A single chromosomal pair of 5 S rDNA sites was observed in all cultivars, but at distinct locations. Centromeric satellite sequences, tested for the first time in melon, revealed only centromeric sites. Telomeric sites were observed in all the chromosomes of the cultivars. CONCLUSIONS: Karyotype variation was observed in cultivars of melon, which were analyzed for chromosomal morphology and localization of GC-rich heterochromatin, as well centromeric SatDNA, rDNA, and telomeric chromosomal markers. Hence, these cultivars can be used in future breeding programs.

Identification of 45S rDNA in Passiflora using low coverage sequencing: analysis of GC content and chromosomal localization.

BACKGROUND: The 45S rDNA is considered the most useful chromosomal marker for cytogenetic analysis of Passiflora. Amplification of 45S rDNA sequence via PCR are more advantageous than sequence maintenance in vectors for chromosomal hybridization via FISH. We aimed both to identify 45S rDNA by sequencing data for chromosomal localization and to verify the relationship between GC content and CMA3/DAPI banding. METHODS AND RESULTS: Low-coverage sequencing of Passiflora alata, P. cincinnata, and P. edulis was performed, and 45S rDNA units were identified using RepeatExplorer. The 45S rDNA units were used to construct a neighbor-joining tree to verify the similarities between the three species' 18S and 26S rDNA sequences. Clusters (CL)116 (P. alata), CL71 (P. cincinnata), and CL116 (P. edulis) were remarkably similar among the three species, and the 26S rDNA sequences of the clusters were similar to those of Populus tremuloides, Salix interior, and Averrhoa carambola (98% identity). The 26S rDNA was cytologically localized in the chromosomes of P. edulis, P. bahiensis, and the backcrossed hybrid (P. sublanceolata vs. HD13). The hybridization transfer capacity was evaluated in Citrus sunki and Cucumis melo. Finally, a chromosomal pair with a heteromorphic 26S rDNA site was observed in P. edulis, which was the same to that observed for CMA3. CONCLUSION: The amplification of the 26S rDNA in Passiflora via PCR and the chromosomal localization in Passiflora and other plant species was successfully achieved. The CMA3 bands were found to be related not only to the amount of GC but also to its structure and the number of repetitions.

Low coverage sequencing for repetitive DNA analysis in Passiflora edulis Sims: citogenomic characterization of transposable elements and satellite DNA.

BACKGROUND: The cytogenomic study of repetitive regions is fundamental for the understanding of morphofunctional mechanisms and genome evolution. Passiflora edulis a species of relevant agronomic value, this work had its genome sequenced by next generation sequencing and bioinformatics analysis performed by RepeatExplorer pipeline. The clusters allowed the identification and characterization of repetitive elements (predominant contributors to most plant genomes). The aim of this study was to identify, characterize and map the repetitive DNA of P. edulis, providing important cytogenomic markers, especially sequences associated with the centromere. RESULTS: Three clusters of satellite DNAs (69, 118 and 207) and seven clusters of Long Terminal Repeat (LTR) retrotransposons of the superfamilies Ty1/Copy and Ty3/Gypsy and families Angela, Athila, Chromovirus and Maximus-Sire (6, 11, 36, 43, 86, 94 and 135) were characterized and analyzed. The chromosome mapping of satellite DNAs showed two hybridization sites co-located in the 5S rDNA region (PeSat_1), subterminal hybridizations (PeSat_3) and hybridization in four sites, co-located in the 45S rDNA region (PeSat_2). Most of the retroelements hybridizations showed signals scattered in the chromosomes, diverging in abundance, and only the cluster 6 presented pericentromeric regions marking. No satellite DNAs and retroelement associated with centromere was observed. CONCLUSION: P. edulis has a highly repetitive genome, with the predominance of Ty3/Gypsy LTR retrotransposon. The satellite DNAs and LTR retrotransposon characterized are promising markers for investigation of the evolutionary patterns and genetic distinction of species and hybrids of Passiflora.

Correction to: low coverage sequencing for repetitive DNA analysis in Passiflora edulis Sims: citogenomic characterization of transposable elements and satellite DNA.

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Karyotype diversity and 2C DNA content in species of the Caesalpinia group.

BACKGROUND: The Leguminosae family is the third-largest family of angiosperms, and Caesalpinioideae is its second-largest subfamily. A great number of species (approximately 205) are found in the Caesalpinia group within this subfamily; together with these species' phenotypic plasticity and the similarities in their morphological descriptors, make this a complex group for taxonomic and phylogenetic studies. The objective of the present work was to evaluate the karyotypic diversity and the 2C DNA content variation in 10 species of the Caesalpinia group, representing six genera: Paubrasilia, Caesalpinia, Cenostigma, Poincianella, Erythrostemon and Libidibia. The GC-rich heterochromatin and 45S rDNA sites (which are used as chromosome markers) were located to evaluate the karyotype diversity in the clade. The variation in the 2C DNA content was determined through flow cytometry. RESULTS: The fluorochrome banding indicated that the chromomycin A3+/4',6-diamidino-2-phenylindole- blocks were exclusively in the terminal regions of the chromosomes, coinciding with 45S rDNA sites in all analyzed species. Physical mapping of the species (through fluorescence in situ hybridization) revealed variation in the size of the hybridization signals and in the number and distribution of the 45S rDNA sites. All hybridization sites were in the terminal regions of the chromosomes. In addition, all species had a hybridization site in the fourth chromosome pair. The 2C DNA content ranged from 1.54 pg in Erythrostemon calycina to 2.82 pg in the Paubrasilia echinata large-leaf variant. The Pa. echinata small-leaf variant was isolated from the other leaf variants through Scoot-Knott clustering. CONCLUSIONS: The chromosome diversity and the variation in the 2C DNA content reinforce that the actual taxonomy and clustering of the analyzed taxa requires more genera that were previously proposed. This fact indicates that taxonomy, phylogeny and cytoevolutionary inference related to the complex Caesalpinia group have to be done through integrative evaluation.

Identification and characterization of karyotype in Passiflora hybrids using FISH and GISH.

BACKGROUND: A great interest exists in the production of hybrid plants of the genus Passiflora given the beauty and exotic features of its flowers which have ornamental value. Hybrid paternity confirmation is therefore important for assuring germplasm origin, and is typically carried out by molecular marker segregation. The aim of this study was to karyotypically characterize the chromosome heritance patterns of the progeny resultant from a cross of P. gardneri and P. gibertii using classical cytogenetics, chromosome banding, and molecular cytogenetics. RESULTS: All analyzed genotypes showed the same diploid chromosome number as the genitor species: 2n = 18. Classical and CMA3 and DAPI staining allowed for chromosome counting and satellite identification (secondary constrictions). Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used to characterize subgenomes by either identifying rDNA-specific genome patterns or parental genomes, respectively. CONCLUSIONS: The heritance of chromosomal markers presenting rDNA sites from each parent for genome identification confirmed that all obtained plants were hybrids. These results will improve breeding programs involving the species of this genus. Apart from confirming hybridization, GISH allowed the visualization of recombination between the homeologous chromosome and the introgression of sequences of interest.

Anatomical and histochemical aspects of zigotic embryo and leaves in Coqueiro Anão / Aspectos anatômicos e histoquímicos de embrião zigótico e folhas em Coqueiro Anão

The paper provides information about histochemical staining reactions in leaves and embryos of 'Coqueiro Anão' (Cocos nucifera). It was compared in vitro coconut and autotrophic palm leaves. Reactions for insoluble polysaccharides and acidic compounds, protein, extractable lipids, lignin and other classes of compounds were tested using histochemical tests. None sample gave positive reaction for lignin and phenolic compounds. All the samples gave positive reaction for protein, starch and insoluble polysaccharides while acidic compounds were positive only in in vitro leaves. Both in vitro and autotrophic leaves gave positive reaction for lipids showing presence of cuticle even in in vitro leaves. Only autotrophic palm leaves showed idioblasts containing calcium oxalate crystals.

Este trabalho fornece informações sobre resultados de testes histoquímicos em folhas e embriões zigóticos de 'coqueiro anão' (Cocos nucifera). Foram comparadas folhas de palmeiras de cultura in vitro e autotróficas. Reações para polissacarídeos insolúveis e compostos acídicos, proteínas, lipídios, lignina e outras classes de compostos foram testadas por meio de testes histoquímicos. Nenhuma amostra foi positiva para lignina e compostos fenólicos. Todas as amostras foram positivas para proteína, amido e polissacarídeos insolúveis, enquanto compostos acídicos foram observados apenas em folhas cultivadas in vitro. Amostras de folhas in vitro e autotróficas apresentaram reação positiva para lipídios, demonstrando a presença de cutícula mesmo em plântulas in vitro. Somente folhas de palmeiras autotróficas apresentaram ideoblastos contendo cristais de oxalato de cálcio.

Reproductive studies in ipecac (Psychotria ipecacuanha (Brot. ) stockes; Rubiaceae): pollen development and morphology

The aim of this work was to carry out the reproductive studies on Brazilian accessions of ipecac, Psychotria ipecacuanha. It presented heterostyly, with brevistylous and longistylous flowers. The pollen development was observed from the sections of the anthers embedded in resin. Anther development was normal as usually observed in dicotyledones, displaying four layers: outer epidermis, endothecium, middle layer and inner tapetum. The pollen was bicellular and filled with starch at the microspore stage. Pollen morphology was studied using SEM, which showed pollen polymorphism within and between the two floral morphs. Five types of pollen with reticulate or perforate exine were identified. The characteristics showed that the sexual process was as important as the vegetative propagation for the reproduction of this species.

Foram realizados estudos reprodutivos em acessos brasileiros de poaia, Psychotria ipecacuanha. Poaia apresenta heterostilia, com flores brevistilas e longistilas. O desenvolvimento do pólen foi estudado em cortes de anteras embebidas em resina. O desenvolvimento da antera seguiu o padrão normal para as dicotiledôneas, a qual apresentou quatro camadas: epiderme, endotécio, camada média, e tapete, a mais interna. O pólen apresentou-se bicelular e preenchido com amido no estágio de micrósporo. A morfologia do pólen foi estudada utilizando-se MEV. Foi observado polimorfismo polínico dentro e entre as duas formas florais. Foram identificados cinco tipos de grãos de pólen, com exina reticulada ou perfurada. Em seu hábitat natural, sabe-se que essa espécie apresenta propagação por multiplicação vegetativa, mas as características estudadas demonstraram que o mecanismo sexuado é tão importante para a reprodução dessa espécie quanto à propagação vegetativa.

Optical and ultrastructural study of the pollen grain development in hermaphrodite papaya tree (Carica papaya L.)

The objective of this study was to describe the pollen grain development in hermaphrodite papaya tree. The flower buds were collected at different stages of the development and the anthers were treated chemically for observation under optical and electronic transmission microscopes. The pollen grain development followed the normal pattern described for the Angiosperms. The pollen grain development was described from meiocyte to the mature pollen grain. In the microsporogenesis, the microspore mother cells or the meiocytes underwent meiosis giving rise to the tetrads that were enclosed by the calose. Later, the tetrads were released by the dissolution of the calose by calase activity and microspores underwent mitosis. Microgametogenesis was characterized by asymmetrical mitotic division of each microspore giving rise to bi-nucleate pollen grains. The structures similar to the plastids were found in the cytoplasm and close to the nucleus of the generative cell. Gradual degeneration was observed in the tapetum during the male gamete development.

Esta pesquisa descreve os estádios de formação do gameta masculino (grão de pólen) em mamoeiro. Para isto foram coletados botões florais em diferentes etapas de desenvolvimento e as anteras foram tratadas quimicamente para serem observadas ao microscópio óptico e eletrônico de transmissão. O desenvolvimento do grão de pólen seguiu o padrão normal para Angiospermas. Foram descritos os estádios de desenvolvimento do grão de pólen desde meiócitos ou células mães de micrósporos até o grão de pólen binucleado. Na microesporogênese, as células esporogênicas sofreram meiose dando origem as tétrades, com posterior liberação dos micrósporos da tétrade. A microgametogênese caracterizou-se, principalmente, pela divisão mitótica assimétrica de cada micrósporo originando grãos de pólen binucleados. Estruturas semelhantes a plastídios foram encontradas no citoplasma da célula generativa e agregados próximos ao núcleo da célula generativa. Observou-se degeneração gradativa das células do tapete no decorrer da formação do gameta.

Cytogenetic studies in some species of Passiflora L. (Passifloraceae): a review emphasizing Brazilian species

The Passifloraceae is represented by species of tropical and subtropical origin. The Passiflora is the richest genus with approximately 450 species, 200 of them being native to Brazil. Recent karyological studies have reported the basic chromosome number for the Passiflora genus as x = 6, whereas x = 9, x = 10 and x = 12 were established as secondary basic numbers. High rates of fertility occur in most Passiflora species, since both meiotic index and pollen viability are above 90 percent. Unusual meiotic behavior has been described in some taxa. Unviable pollen were observed in some diploids species. The genome size varies from 1.83 to 5.36 pg, and significant interspecific variance has been observed. Studies using the FISH methodology have shown that there are two to three rDNA 45S sites and one 5S site in the species analyzed. In this review, information about the above-mentioned studies is presented and discussed in detail.

A família Passifloraceae é representada por espécies de origem tropical e subtropical. Passiflora é o gênero mais rico, com aproximadamente 450 espécies, cerca de 200 delas nativas do Brasil. Recentes estudos cariológicos têm relatado o número básico de cromossomos para o gênero Passiflora como sendo x = 6, enquanto x = 9, x = 10 e x = 12 foram considerados números básicos secundários. Altas taxas de fertilidade são observadas na maioria das espécies de Passiflora, uma vez que o índice meiótico e a viabilidade polínica apresentam-se acima de 90 por cento.Comportamento meiótico irregular tem sido descrito para alguns taxas. Grãos de pólen inviáveis foram observados em espécies diplóides. O tamanho do genoma varia de 1,83 a 5,36 pg, e variação interespecífica significativa tem sido observada. Estudos usando a metodologia de hibridização in situ (FISH) tem demonstrado haver de dois a três sites de DNAr 45S e um site de DNAr 5S nas espécies analisadas. Nesta revisão, informações sobre os estudos acima mencionados são apresentados e discutidos em detalhes.

Polinização seletiva em maracujazeiro amarelo (Passiflora edulis f. flavicarpa) monitorada por vetores canônicos / Seletive pollination in yellow passion fruit (Passiflora edulis f. flavicarpa) by canonic vectors

Objetivou-se neste trabalho avaliar a influência da planta doadora de pólen sobre as características dos frutos produzidos na cultura do maracujazeiro-amarelo (Passiflora edulis f. falvicarpa). Neste contexto, um experimento foi conduzido, segundo o esquema de cruzamentos controlados. Empregaram-se 23 plantas "genitor masculino" e 103 plantas receptoras de pólen, perfazendo o total de 185 frutos avaliados segundo as características: peso, largura, comprimento, número de sementes e peso relativo de 100 sementes. A avaliação dessas características, segundo análise de variância multivariada (MANOVA), permitiu a verificação de efeitos significativos ("genitor masculino" e "fêmea hierarquizada a genitor masculino"). A partir das informações da MANOVA, construiu-se uma função discriminante, constituída pela primeira variável canônica baseada na matriz de soma de quadrados para a fonte de variação "genitor masculino" (Importância relativa = 58,10 por cento). A análise desta permitiu verificar elevado efeito da fonte de variação "genitor masculino" sobre as características dos frutos, cujos indivíduos foram agrupados univariamente em três grupos distintos. Tais resultados permitem concluir que tanto progenitores femininos como masculinos podem influenciar os resultados obtidos em esquemas de seleção massal.

This research was aiemd at evaluating the influence of the relactive pollination of pollen donor plant in obtaition higher fruits of yellow passion (Passiflora edulis f. falvicarpa). To do so, an experiment was conduced according to the controlled crosses, using 23 male plants and 103 pollen receptor plants. The total of fruits obtained was 185 units, that were evaluated according to the following traits: fruit weight, fruit diameter, fruit length, fruit seed number and relative weight of 100 seeds. The data were analyzed by using multivariate analysis (MANOVA). The discriminant function was constituted by the first canonic variable based on matrix of square sum of male (relative importance = 58.1). The discriminant function analysis permitted to verify high effect of male in relation to fruit traits, whose individuals were grouped in three distinct classes. The results indicate that female as well as male can influence the results obtained in mass selection.

Structural, hystochemical and cytochemical characteristics of the stigma and style in Passiflora edulis f. flavicarpa ( Passifloraceae )

Foram realizadas observações sobre aspectos anatômicos do estigma e estilo de maracujazeiro amarelo, uma vez que estudos detalhados não têm sido encontrados em literatura recente. O estigma é seco, não-ramificado com papilas multiseriadas e multicelulares, com células altamente vacuoladas e parede celular relativamente fina. O estilo é sólido apresentando no centro o tecido de transmissão com espaços intercelulares contendo uma matriz que inclui polissacarídeos e substâncias lipídicas. Durante o desenvolvimento, células jovens do tecido de transmissão não apresentam espaços intercelulares, ao contrário do pistilo maduro. O estigma e estilo contêm citoplasma periférico apresentando um perfil citoplasmático com núcleo e um nucléolo, mitocôndrias distribuídas espaçadamente, extenso retículo endoplasmático rugoso, ribossomos e amiloplastos.

Flow cytometric analysis of genome size variation in some Passiflora species.

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.

Embryo sac development in yellow passion fruit Passiflora edulis f. flavicarpa (Passifloraceae)

The yellow passion fruit, Passiflora edulis f. flavicarpa, is one of the most important Brazilian fruit crops. It is an allogamous, diploid, and self-incompatible species. It has hermaphrodite, solitary flowers, located in the leaf axils and protected by leaf bracts. The flower has an androgynophore, which is a straight stalk supporting its reproductive parts. There are usually five anthers, located at the tip of each of the five filaments. The ovary is borne just above the filaments, at the top of the androgynophore; there are three styles that are united at their base, and at the top there are three stigmas. The objective of this research was to observe embryo sac development in yellow passion flowers. Ovaries at different stages of development were fixed in FAA (formalin, acetic acid and alcohol solution), hydrated, stained with Mayer's hemalum, and dehydrated. Ovules were cleared by using methyl salicylate, mounted on slides, and observed through a confocal scanning laser microscope. The yellow passion fruit ovule is bitegmic, crassinucellate, and anatropous, and its gametophyte development is of the Polygonum type. After meiosis, functional megaspores under go three successive mitotic divisions, resulting in an eight-nucleate megagametophyte: the egg apparatus at the micropylar end, two polar nuclei at the cell center, and three antipodals at the chalazal end. The egg apparatus is formed by an egg cell and two synergids, each with a filiform apparatus. The mature embryo sac has an egg cell, two synergids, two polar nuclei, and three antipodes, as has been described for most angiosperms