Rapid plant DNA and RNA extraction protocol using a bench drill

Plant DNA and RNA extraction methods are well established, with a wide range of protocols, depending on the purposes of each laboratory/research. Nowadays, quick, inexpensive and easy plant DNA and RNA extraction methods are highly sought after. We developed an optimized protocol for plant DNA and RNA extraction that uses an inexpensive bench drill and plastic bags and does not require liquid nitrogen. DNA from leaves and RNA from leaves and roots of banana, pineapple, citrus, papaya, passion fruit and cassava, were extracted using a basic cetyltrimethylammonium bromide method. Both nucleic acids were quantified and evaluated for quality based on agarose gel electrophoresis. The DNA and RNA extractions were successful for all species, and RNA quality in pellets was maintained after storage at room temperature for three weeks. This protocol can reduce costs considerably in laboratories with ongoing routine activities of DNA and RNA extraction for genetic diversity and gene expression analyses, where other conventional methods have not been successful due to explant, condition of samples and quantity and quality of nucleic acids. This is especially relevant for many laboratories in developing countries where the cost and availability of liquid nitrogen may be a constraint

Diversidade genética de populações de Xanthomonas phaseoli pv. manihotis em mandioca por meio de marcadores rep-PCR e VNTRs

The objective of this work was to evaluate the genetic diversity of Xanthomonas phaseoli pv. manihotis (Xpm) from eight populations from five cassava producing states in Brazil, through the rep-PCR (BOX-PCR and ERIC-PCR) and variable number of tandem repeat (VNTR) markers. Cassava leaves with symptoms of cassava bacterial blight were collected in eight municipalities, and the Xpm isolates were identified by amplification with primers specific for these isolates. The identity of the Xpm isolates was confirmed with the BOX-PCR, ERIC-PCR, and VNTR markers. The observed selection pressure, together with the mode of reproduction and the mechanisms that increase genetic variability, allows of the pathogen populations to adapt according to microclimate variation, contributing to a differentiated reproductive success. ERIC-PCR and VNTRs are the best markers for evaluating the genetic variability in the eight studied Xpm populations. However, ERIC-PCR is the marker that best separated the groups by population and presented a higher similarity between the isolates of the same population. The study of the genetic diversity of Xpm is key to improve disease monitoring and management strategies in cassava crops.O objetivo deste trabalho foi avaliar a diversidade genética de Xanthomonas phaseoli pv. manihotis (Xpm) de oito populações de cinco estados produtores de mandioca no Brasil, por meio de marcadores rep-PCR (BOX-PCR e ERIC-PCR) e variable number of tandem repeats (VNTRs). Folhas de mandioca com sintomas de crestamento bacteriano foram coletadas em oito municípios, e os isolados Xpm foram identificados por amplificação com iniciadores específicos para esses isolados. A identidade dos isolados Xmp foi confirmada com os marcadores BOX-PCR, ERIC-PCR e VNTRs. A pressão de seleção observada, junto com o modo de reprodução e os mecanismos que aumentam a variabilidade genética, permite que as populações do patógeno se adaptem de acordo com a variação dos microclimas, o que contribui para o sucesso reprodutivo diferenciado. ERIC-PCR e VNTRs são os melhores marcadores para avaliar a variabilidade genética das oito populações Xpm estudadas. No entanto, ERIC-PCR é o marcador que melhor separou os grupos por população e apresentou maior similaridade entre os isolados de uma mesma população. O estudo da diversidade genética de Xpm é fundamental para delinear estratégias de manejo e monitoramento de doenças na cultura da mandioca

Recurrent water deficit causes epigenetic and hormonal changes in citrus plants

The present study evaluated the physiological, molecular and hormonal parameters from scion/rootstock interaction of citrus plants during recurrent water deficit. Responses of the Valencia (VO) scion variety grafted on two rootstocks with different soil water extraction capacities, Rangpur Lime (RL) and Sunki Maravilha (SM), during three successive periods of water deficit: plants exposed to a single episode of water deficit (WD1) and plants exposed to two (WD2) and three (WD3) recurrent periods of WD were compared. The combinations VO/RL and VO/SM presented polymorphic alterations of epigenetic marks and hormonal (i.e. abscisic acid, auxins and salicylicacid) profiles, which were particularly prominent when VO/SM plantswere exposed toWD3 treatment. Upon successive drought events, the VO/SM combination presented acclimatization characteristics that enable higher tolerance to water deficit by increasing transpiration (E), stomatal conductance (g s ) and photosynthetic rate (A), which in turn may have facilitated the whole plant survival. Besides providing comprehensive data on the scion/rootstock interactions upon successive stress events, this study brings the first dataset suggesting that epigenetic alterations in citrus plants triggered by recurrent water deficit lead to improved drought tolerance in this crop species. (Résumé d'auteur

Diversidade genética entre diploides melhorados de bananeira por meio de variáveis canônicas e do método Ward‑MLM

The objective of this work was to estimate the genetic diversity of improved banana diploids using data from quantitative analysis and from simple sequence repeats (SSR) marker, simultaneously. The experiment was carried out with 33 diploids, in an augmented block design with 30 regular treatments and three common ones. Eighteen agronomic characteristics and 20 SSR primers were used. The agronomic characteristics and the SSR were analyzed simultaneously by the Ward‑MLM, cluster, and IML procedures. The Ward clustering method considered the combined matrix obtained by the Gower algorithm. The Ward‑MLM procedure identified three ideal groups (G1, G2, and G3) based on pseudo‑F and pseudo‑t2 statistics. The dendrogram showed relative similarity between the G1 genotypes, justified by genealogy. In G2, 'Calcutta 4' appears in 62% of the genealogies. Similar behavior was observed in G3, in which the 028003‑01 diploid is the male parent of the 086079‑10 and 042079‑06 genotypes. The method with canonical variables had greater discriminatory power than Ward‑MLM. Although reduced, the genetic variability available is sufficient to be used in the development of new hybrids.O objetivo deste trabalho foi estimar a diversidade genética entre diploides melhorados de bananeira por meio de dados quantitativos e de marcadores de sequências simples repetidas (SSR), simultaneamente. O experimento foi conduzido com 33 diploides, em blocos aumentados com 30 tratamentos regulares e três comuns. Dezoito características agronômicas e 20 iniciadores SSR foram usados. Os dados agronômicos e de SSR foram analisados, simultaneamente, via os procedimentos Ward‑MLM, de agrupamento e IML. O método de agrupamento de Ward considerou matriz híbrida obtida pelo algoritmo de Gower. O procedimento Ward‑MLM identificou três grupos (G1, G2 e G3) baseados nas estatísticas de pseudo‑F and pseudo‑t2. O dendrograma mostrou similaridade relativa entre os genótipos do G1, justificada pela genealogia. No G2, 'Calcutta 4' aparece em 62% das genealogias. Comportamento similar foi observado no grupo G3, em que o diploide 028003‑01 é o parental masculino dos genótipos 086079‑10 e 042079‑06. O método com uso de variáveis canônicas teve maior poder discriminatório que o Ward‑MLM. Embora reduzida, a variabilidade genética disponível é suficiente para ser usada no desenvolvimento de novos híbridos

Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract\ud \ud \ud \ud Background\ud \ud Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa.\ud \ud \ud \ud Results\ud \ud From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis.\ud \ud \ud \ud Conclusions\ud \ud The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.This work was funded by FAPESP [2008/03470-0] and CNPq. Technical assistance by Luis Eduardo Fonseca was greatly appreciated. The authors (ONJ, SSO, EP, AF) are grateful for the fellowships provided by CNPq and GGS to FAPESP [2010/01398-0]

Factors influencing terrestriality in primates of the Americas and Madagascar

Among mammals, the order Primates is exceptional in having a high taxonomic richness in which the taxa are arboreal, semiterrestrial, or terrestrial. Although habitual terrestriality is pervasive among the apes and African and Asian monkeys (catarrhines), it is largely absent among monkeys of the Americas (platyrrhines), as well as galagos, lemurs, and lorises (strepsirrhines), which are mostly arboreal. Numerous ecological drivers and species-specific factors are suggested to set the conditions for an evolutionary shift from arboreality to terrestriality, and current environmental conditions may provide analogous scenarios to those transitional periods. Therefore, we investigated predominantly arboreal, diurnal primate genera from the Americas and Madagascar that lack fully terrestrial taxa, to determine whether ecological drivers (habitat canopy cover, predation risk, maximum temperature, precipitation, primate species richness, human population density, and distance to roads) or species-specific traits (bodymass, group size, and degree of frugivory) associate with increased terrestriality. We collated 150,961 observation hours across 2,227 months from 47 species at 20 sites in Madagascar and 48 sites in the Americas. Multiple factors were associated with ground use in these otherwise arboreal species, including increased temperature, a decrease in canopy cover, a dietary shift away from frugivory, and larger group size. These factors mostly explain intraspecific differences in terrestriality. As humanity modifies habitats and causes climate change, our results suggest that species already inhabiting hot, sparsely canopied sites, and exhibiting more generalized diets, are more likely to shift toward greater ground use