Physical mapping of 5S rDNA in Eucalyptus dunnii Maiden and Zea mays L. by the PRINS technique

A detecção de sequências de DNA in situ por meio de técnicas de citogenética molecular consiste em uma das metodologias mais utilizadas para o mapeamento físico. Embora seja amplamente aplicada na pesquisa genômica, a Hibridização Fluorescente In Situ (FISH - Fluorescent In Situ Hybrid/zation) tem sido considerada uma técnica relativamente demorada, uma vez que requer a construção de uma sonda marcada. A Reação de Amplificação In Situ (PRINS Primed ln situ Label/ing), por sua vez, consiste em uma alternativa sensível e rápida em relação a FISH. No entanto, a especificidade e sensibilidade da técnica de PRINS podem ser influenciadas por fatores especie-específicos. Desse modo, o presente estudo teve como objetivo adaptar um protocolo de PRINS reprodutível para o mapeamento físico de genes de rRNA 58 em duas espécies com cromossomos de tamanhos relativamente diferentes: Eucalyptus dunnii Maiden e Zea mays L. Inicialmente, meristemas radiculares de ambas as espécies foram sincronizados com hidroxiureia e tratados com o agente anti- tubulínico amiprofos-metil para o acúmulo de metáfases. Em seguida, os meristemas foram submetidos a maceração enzimática e as lâminas confeccionadas pelas técnicas de dissociação celular e secagem ao ar. Após o pré-tratamento das lâminas, um par de primers específicos para a região 58 de E. glóbulus foi utilizado para a reação de PRINS, que consistiu de um único ciclo. A reação foi conduzida em preparações de E. dunnii e Z. mays contendo cromossomos metafásicos morfologicamente preservados, sem resíduos de citoplasma ou sobreposições. A técnica de FISH foi adicionalmente aplicada na espécie Z. mays com o intuito de confirmar os resultados obtidos pela PRINS. O mesmo par de primers foi utilizado para a construção de sondas marcadas com fluorescência, as quais foram posteriormente hibridizadas com o DNA alvo. Os cariótipos de E. dunnii e Z. mays exibiram 2n = 2x = 22 e Zn = 2x = 20 cromossomos, respectivamente. Em ambas as espécies, o cromossomo portador da constrição secundaria foi classificado como o número 6. O protocolo de citogenética clássica associado a reação de PRIN8 resultou em sinais nítidos para a sequência de rDNA 58 tanto em núcleos interfásicos quanto em cromossomos metafásicos de E. dunnii e Z. mays. Em E. dunnii, os genes de rRNA 58 foram mapeados no braço curto do cromossomo 5, em posição pericentromérica. Na espécie Z. mays, o sinal foi localizado na região terminal do braço longo do cromossomo 2. A técnica de FISH confirmou o resultado obtido pela PRIN8 nessa especie. Embora o tamanho dos cromossomos possa influenciar na resolução de técnicas de citogenética molecular, o protocolo descrito resultou em uma elevada razão sinal:background para ambas as espécies estudadas, evidenciando sua reprodutibilidade. Esse estudo consiste no primeiro relato do mapeamento de sequências específicas em E. dunnii e Z. mays pela PRIN8, contribuindo para o desenvolvimento e aperfeiçoamento das técnicas de mapeamento físico em espécies vegetais. Além disso, a localização dos genes de rDNA 58 ainda não se encontra disponível no mapa de ligação ou no genoma sequenciado de Eucalyptus. Dentro desse contexto, a localização física das sequências de rDNA 58 de E. dunnii pode ser integrada aos dados de sequenciamento e auxiliar no alinhamento de sequencias e posicionamento dos genes de rDNA 58 no genoma sequenciado.The in situ detection of DNA sequences by molecular cytogenetic techniques is one of the most used methodologies for physical mapping. Although widely applied in genomic research, the Fluorescent ln situ Hybridization (FISH) has been considered a relatively time-consuming technique, due to the need of constructing a labeled probe. The Primed ln situ Labelling (PRIN8), on the other hand, is a sensitive and fast alternative to FI8H. Nevertheless, the specificity and sensitivity of the PRIN8 technique may be influenced by species-specific factors. Therefore, the present study aimed to adapt a reproducible PRIN8 protocol for the physical mapping of 58 rRNA genes in two species with chromosomes of relatively different sizes: Eucalyptus dunnii Maiden and Zea mays L. Initially, root meristems of both species were synchronized with hydroxyurea (HU) and treated with the anti-tubulin agent amiprophos methyl (APM) for metaphase accumulation. Then, the meristems were submitted to enzymatic maceration and the slides were prepared by the cell dissociation and air drying techniques. After pre-treatment of the slides, a pair of primers specific to the 58 region of E. globulus was used to the PRIN8 reaction, which consisted of a single cycle. The reaction was conducted on preparations of both E. dunnii and Z. mays containing morphologically preserved metaphasic chromosomes, without cytoplasmic debris or overlaps. The FISH technique was additionally conducted in the species Z. mays with the aim to confirm the results obtained by PRIN8. The same pair of primers was used for constructing the fluorescently labeled probes, which were subsequently hybridized to the target DNA. The karyotypes of E. dunnii and Z. mays exhibited 2n = 2x = 22 and 2n = 2x = 20 chromosomes, respectively. In both species, the chromosome carrying the secondary constriction was classified as number 6. The classical cytogenetics protocol associated with PRIN8 resulted in clear signals for the 58 rDNA sequence in both interphase nucleus and metaphase chromosomes of E. dunnii and Z. mays. ln E. dunnii, the 58 rRNA genes were mapped on the short arm of chromosome 5, in pericentromeric position. In the species Z. mays, the signal was located in the terminal region of the chromosome 2 long arm. The FISH technique confirmed the result obtained by the PRINS reaction in this species. Although the chromosome size may influence the resolution of molecular cytogenetic techniques, the described protocol resulted in a high signal:background ratio for both studied species, evidencing its reproducibility. This study comprises the first report on the mapping of specific sequences in E. dunnii and Z. mays by the PRINS technique, contributing to de development and improvement of physical mapping techniques in plant species. In addition, the localization of 58 rDNA genes is not yet available in the linkage map or in the sequenced genome of Eucalyptus. In this context, the physical localization of E. dunnii 58 rDNA may be integrated With sequencing data and assist in sequence alignment and positioning of 58 rDNA genes in the sequenced genome.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Citogenômica em Coffea L.

The genus Coffea comprises ~124 species, including C. arabica and C. canephora, which are responsible for the commercial production of coffee beans. Although most cytogenomic researches are focused on commercial species and its relatives, efforts have been made to expand the scope to wild species. However, molecular cytogenetic data are still limited to karyotype organization, especially due to low quality of the cytogenetic preparations. Considering this, the present work is focused on the cytogenomics of Coffea species. The first chapter provides an up to date review on the history of Coffea cytogenomics, initiating with the first classical cytogenetic studies, encompassing the main challenges and landmarks in cytogenetics and genomics, as well as their integration. These cytogenomic data allowed us to understand the phylogenetic relationships in Coffea, as well as their genomic origins, highlighting the relatively recent events of allopolyploidy. These events include the origin of the allotetraploid C. arabica (2n = 4x = 44, C. canephora x C. eugenioides) and the allotriploid hybrid ‘Hibrido de Timor (2n =3 x = 33, C. arabica x C. canephora). The second chapter is devoted to the study of repetitive sequences (repeatome) of the C. eugenioides genome, by integrating bioinformatic tools with cytogenetic mapping. We showed that repetitive sequences comprise ~47% of C. eugenioides genome, with mobile elements representing 45%. The Ty3/Gypsy to Ty1/Copia ratio was high (32:4), as also observed for other Coffea species. We mapped three Ty1/Copia (Bianca, TAR and Tork) and one Ty3/Gypsy (Athila) LTR-retrotransposon in metaphase chromosomes and interphase nuclei of C. eugenioides. Their distribution exhibited a clustered pattern throughout different chromosomes and regions of the chromosomes. The results obtained here are unprecedented for Coffea. Thus, we hope they lay the background for further investigations regarding Coffea cytogenomics. Keywords: Coffee. Molecular Cytogenetics. Polyploidy. Genomics. Repeatome. Mobile Elements.O gênero Coffea compreende ~124 espécies, incluindo C. arabica e C. canephora, as quais são responsáveis pela produção comercial de grãos de café. Embora a maioria das pesquisas citogenômicas foquem nas espécies comerciais ou espécies relacionadas, esforços têm sido feitos para incluir espécies selvagens. No entanto, os dados de citogenética molecular ainda se limitam à organização do cariótipo, especialmente devido à baixa qualidade das preparações citogenéticas. Considerando o exposto, o presente trabalho tem como foco a citogenômica de espécies de Coffea. O primeiro capítulo apresenta uma revisão atualizada acerca da história da citogenômica em Coffea, iniciando com os primeiros estudos de citogenética clássica, abrangendo os principais desafios e marcos na citogenética e na genômica, assim como sua integração. Esses dados citogenômicos nos permitiram compreender as relações filogenéticas em Coffea, bem como suas origens genômicas, destacando os eventos relativamente recentes de alopoliploidia. Esses eventos incluem a origem do alotetraploide C. arabica (2n = 4x = 44, C. canephora x C. eugenioides) e do hibrido alotriploide 'Hibrido de Timor' (2n = 3 x = 33, C. arabica x C. canephora). O segundo capitulo é dedicado ao estudo de sequências repetitivas (repetoma) do genoma de C. eugenioides, por meio da integração de ferramentas bioinformáticas com mapeamento citogenético. Mostramos que as sequências repetitivas compreendem ~47% do genoma de C. eugenioides, com os elementos móveis representando 45%. A relação de Ty3/Gypsy para Ty1/Copia foi alta (32:4), assim como observado para outras espécies de Coffea. Nós também mapeamos três LTR-retrotransposons Ty1/Copia (Bianca, TAR e Tork) e um elemento Ty3/Gypsy (Athila) em cromossomos metafasicos e nucleos interfasicos de C. eugenioides. Sua distribuição exibiu um padrão agrupado, ao longo diferentes cromossomos e regiões dos cromossomos. Os resultados obtidos aqui são inéditos para Coffea. Portanto, esperamos que eles sirvam como base para futuras investigações a respeito da citogenômica de Coffea. Palavras-chave: Café. Citogenética Molecular. Poliploidia. Genômica. Repetoma. Elementos Móveis

The polyploidy and its key role in plant breeding

Polyploidy is a major force in the evolution of both wild and cultivated plants. Polyploid organisms often exhibit increased vigor and, in some cases, outperform their diploid relatives in several aspects. This remarkable superiority of polyploids has been the target of many plant breeders in the last century, who have induced polyploidy and/or used natural polyploids in many ways to obtain increasingly improved plant cultivars. Some of the most important consequences of polyploidy for plant breeding are the increment in plant organs (“gigas” effect), buffering of deleterious mutations, increased heterozygosity, and heterosis (hybrid vigor). Regarding such features as tools, cultivars have been generated with higher yield levels, improving the product quality and increasing the tolerance to both biotic and abiotic stresses. In some cases, when the crossing between two species is not possible because of differences in ploidy level, polyploids can be used as a bridge for gene transferring between them. In addition, polyploidy often results in reduced fertility due to meiotic errors, allowing the production of seedless varieties. On the other hand, the genome doubling in a newly formed sterile hybrid allows the restoration of its fertility. Based on these aspects, the present review initially concerns the origin, frequency and classification of the polyploids, progressing to show the revolution promoted by the discovery of natural polyploids and polyploidization induction in the breeding program status of distinct crops

Embryogenic potential of immature zygotic embryos of Passiflora: a new advance for in vitro propagation without plant growth regulators

In vitro strategies for Passiflora have been developed owing to its economic and ecological importance. However, plantlet regeneration through somatic embryogenesis has presented some problems, such as the reproducibility of the protocol and formation of abnormal embryos and plantlets. Thus, this study aimed to establish a protocol exploring the embryogenic potential of immature zygotic embryos (IZE) of the wild species Passiflora miniata Vanderpl. and Passiflora speciosa Gardn. Friable calli, which formed on the abaxial surface of the cotyledons, yielded globular, heart-shaped, torpedo and cotyledonary somatic embryos, characterising the embryogenic response as asynchronous. A high percentage of normal regenerants (90 %) was obtained from IZE in media lacking 2,4-dichlorophenoxyacetic acid (2,4-D) in comparison to the value of normal plantlets (60 %) regenerated from mature zygotic embryos inoculated in media with 2,4-D. This result demonstrates that IZE of P. miniata and P. speciosa possess sufficient levels of endogenous phytohormones to trigger a high rate of indirect somatic embryogenesis. All regenerated plantlets had the same genome size and chromosome number as the explant donor plants. Therefore, the indirect embryogenic pathway, employing IZE inoculated into media free of growth regulators, did not cause changes in the karyotype and morphology. Based on these results, IZE should be considered as explant for the establishment of somatic embryogenesis in other species. Besides, a new, reliable and relatively rapid protocol to recover plantlets of P. miniata and P. speciosa yielded several plants, which were acclimatised and used for ornamental purposes and breeding programs, and for reintroduction into biological reserves

Indirect somatic embryogenesis in Coffea with different ploidy levels: a revisiting and updating study

Indirect somatic embryogenesis (ISE) is required for plant propagation and a prerequisite for applications that may provide new germplasms. Genetic, epigenetic and physiological features of the explant donor are barriers for ISE establishment, hindering its wide use. Despite the identification and/or expression analysis of genes during ISE, no approach to establish the karyotype aspects has been performed so far. So, this study aims to establish the ISE and compare the in vitro responses between diploid (Coffea canephora and Coffea eugenioides), allotriploid (“Híbrido de Timor”—HT) and true allotetraploid (Coffea arabica) Coffea in a taxonomic and evolutive scenario. Under the same in vitro conditions, the four Coffea differed from each other during ISE. Leaf explants of the true allopolyploids yielded the highest mean number of friable calli (FC) in relative short time and visually exhibiting more pronounced length. FC of the allotetraploid C. arabica presented the highest mean number of mature cotyledonary somatic embryos (MCSE), which were also recovered faster in this species. However, MCSE mean number in HT was the same or lower than diploid Coffea. Besides, intraspecific variation related to the ISE responses was observed in each Coffea, mainly the mean number of FC obtained from ex vitro and in vitro C. arabica and C. eugenioides explants. So, epigenetic and physiologic features may also have influenced the ISE responses. The findings provide the basis for performing other approaches considering the ploidy level, epigenetic and physiological backgrounds. Besides, the data also contributed for understanding about the consequences of polyploidy

Indirect somatic embryogenesis in Coffea with different ploidy levels: a revisiting and updating study

Indirect somatic embryogenesis (ISE) is required for plant propagation and a prerequisite for applications that may provide new germplasms. Genetic, epigenetic and physiological features of the explant donor are barriers for ISE establishment, hindering its wide use. Despite the identification and/or expression analysis of genes during ISE, no approach to establish the karyotype aspects has been performed so far. So, this study aims to establish the ISE and compare the in vitro responses between diploid (Coffea canephora and Coffea eugenioides), allotriploid (“Híbrido de Timor”—HT) and true allotetraploid (Coffea arabica) Coffea in a taxonomic and evolutive scenario. Under the same in vitro conditions, the four Coffea differed from each other during ISE. Leaf explants of the true allopolyploids yielded the highest mean number of friable calli (FC) in relative short time and visually exhibiting more pronounced length. FC of the allotetraploid C. arabica presented the highest mean number of mature cotyledonary somatic embryos (MCSE), which were also recovered faster in this species. However, MCSE mean number in HT was the same or lower than diploid Coffea. Besides, intraspecific variation related to the ISE responses was observed in each Coffea, mainly the mean number of FC obtained from ex vitro and in vitro C. arabica and C. eugenioides explants. So, epigenetic and physiologic features may also have influenced the ISE responses. The findings provide the basis for performing other approaches considering the ploidy level, epigenetic and physiological backgrounds. Besides, the data also contributed for understanding about the consequences of polyploidy

From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure developed for allotriploid “Híbrido de Timor” (Coffea arabica L. × Coffea canephora Pierre ex A. Froehner)

Since 1966, chromosome doubling has been performed mainly in vitro, associating anti-tubulin treatment and different plant tissues showing proliferative cells. Despite the achieved improvements, some bottlenecks have been pointed out, such as the low rate of polyploids and high rate of mixoploid plantlets. To overcome these hurdles, some approaches have indicated that indirect somatic embryogenesis (ISE) constitutes an alternative trigger for chromosome doubling, especially for homoploid and anorthoploid germplasms. In this way, a guideline has been developed for hexaploidization of the Coffea line “Híbrido de Timor” (HT) ‘CIFC 4106’ (anorthoploid, 3x = 33 chromosomes, 1C = 2.10 pg, Coffea canephora × Coffea arabica) from friable embryogenic calli (FEC) treated with colchicine. From this, a relatively high percentage (49.3%) of HT hexaploids (6x = 66 chromosomes, 2C = 4.20 pg) was obtained, without recovery of mixoploids. Besides confirmation of endomitosis induction through the obtained hexaploids, SSR markers revealed that the FEC/colchicine strategy also resulted in loss of allelic diversity in 39 regenerated HT plantlets, demonstrating its genotoxic effect. Considering these results, the present procedure resolved the main bottlenecks for chromosome doubling, which have been reported since the discovery and isolation of the anti-tubulin colchicine in 1930. Hexaploid HT plantlets have enriched Coffea germplasm banks as a new genetic resource since the resolution of their karyotype and DNA sequence. Just as the true allotetraploid C. arabica and the allotriploid HT ‘CIFC 4106’, the hexaploid HT is relevant to investigate the genomic and phenotypic changes arising from polyploidization events