Bridging the Gap: Combining Genomics and Transcriptomics Approaches to Understand Stylosanthes scabra, an Orphan Legume from the Brazilian Caatinga.

Stylosanthes scabra is a scientifically orphaned legume found in the Brazilian Caatinga biome (a semi-arid environment). This work utilized omics approaches to investigate some ecophysiological aspects of stress tolerance/resistance in S. scabra, study its genomic landscape, and predict potential metabolic pathways. Considering its high-confidence conceptual proteome, 1694 (~2.6%) proteins were associated with resistance proteins, some of which were found in soybean QTL regions that confer resistance to Asian soybean rust. S. scabra was also found to be a potential source of terpenes, as biosynthetic gene clusters associated with terpene biosynthesis were identified in its genome. The analysis revealed that mobile elements comprised approximately 59% of the sequenced genome. In the remaining 41% of the sections, some of the 22,681 protein-coding gene families were categorized into two informational groups: those that were specific to S. scabra and those that expanded significantly compared to their immediate ancestor. Biological process enrichment analyses indicated that these gene families play fundamental roles in the adaptation of S. scabra to extreme environments. Additionally, phylogenomic analysis indicated a close evolutionary relationship between the genera Stylosanthes and Arachis. Finally, this study found a high number (57) of aquaporin-encoding loci in the S. scabra genome. RNA-Seq and qPCR data suggested that the PIP subfamily may play a key role in the species' adaptation to water deficit conditions. Overall, these results provide valuable insights into S. scabra biology and a wealth of gene/transcript information for future legume omics studies.

1H NMR-based metabolic profile and chemometric analysis for the discrimination of Passiflora species genotypic variations.

The species of the genus Passiflora (Passifloraceae family) have been used as food, cosmetic and traditional herbal. As a result, the Passiflora species are widely cultivated and has an economic, medicinal and ornamental importance. The popular designation as "passion fruit" and chemical profile of several Passiflora species remains unknown. The lack of chemical information contributes to the erroneous classification and adulteration. In recent years, special attention has been paid to the bioactivity and phytochemical profiles of several Passiflora species extracts. In this research, 1H NMR-based metabolic profiling coupled with chemometric tools was used to characterize and distinguish extracts obtained from different wild Passiflora species (P. alata, P. cincinnata, and P. setacea) and genetic varieties (P. alata var. BRS Pérola do Cerrado, P. cincinnata var. BRS Sertão Forte, and P. setacea var. BRS Pérola do Cerrado). Fourteen metabolites were identified by 1D and 2D NMR experiments, highlighting the presence of fatty acids, carbohydrates, saponins, alkaloids, and mainly C-glycosidic flavones. Principal components analysis (PCA) allowed discrimination of Passiflora extracts, which the quadranguloside, oleanolic acid-3-sophoroside, α-glucose, ß-glucose, and vitexin-2-O"-rhamnoside were relevant in the differentiation of P. alata and P. alata var. BRS Pérola do Cerrado, while the flavones isovitexin and isovitexin-2-O"-xyloside were dominant in the grouping of P. setacea and P. setacea var. BRS Pérola do Cerrado, and finally P. cincinnata and P. cincinnata var. BRS Sertão Forte grouped by the influence of the fatty acids, sucrose, flavones (isoorientin and vitexin-2-O"-xyloside), and trigonelline. The varieties of P. setacea, and P. cincinnata are chemically equivalent to the original Passiflora species. However, the PCA analysis showed that the genetic variety of P. alata occupied a different position in the scores plot provoked mainly by the presence of oleanolic acid-3-sophoroside. The 1H NMR metabolic profile can be efficient for quality control evaluation, and can contribute to the investigation of new alternatives for official Passiflora herbal medicines.

Dehydration response in Stylosanthes scabra: Transcriptional, biochemical, and physiological modulations.

Stylosanthes scabra, popularly known as stylo, is native to the Brazilian Caatinga semiarid region and stands out as a drought-tolerant shrub forage crop. This work provides information about the plant response during the first 48 h of water deficit, followed by a rehydration treatment. Besides root transcriptomics data, 13 physiological or biochemical parameters were scrutinized. Additionally, RNA-Seq annotated transcripts not associated with the "Viridiplantae" clade were taxonomically categorized. It was found that S. scabra quickly perceives and recovers from the oscillations of the imposed water regime. Physiologically, mechanisms that minimize evapotranspiration or protect the photosynthetic apparatus stood out. Biochemically, it was found that the root tissue invests in synthesizing compounds that can act as osmolytes (proline and sugars), emphasizing the importance of osmoregulation to water deficit acclimation. Consistently, transcriptome and qPCR analyses showed that a set of enriched biological processes with upregulated (UR) transcripts were involved in protective functions against reactive oxygen species or encoding enzymes of important metabolic pathways, which might contribute to S. scabra response to water deficit. Additionally, several UR kinases and transcription factors were identified. Finally, in an innovative approach, some naturally occurring microbial groups (such as Schizosaccharomyces, Bradyrhizobium, etc.) were identified in the S. scabra roots. This study reveals insights into the physiological, biochemical, and molecular mechanisms underlying the S. scabra response to water deficit and provides candidate genes that may be useful in developing drought-tolerant crop varieties through biotechnological applications.

Development of the prickly pear cactus Opuntia stricta (Haw.) Haw. (Cactaceae) in vitro in response to the replacement of potassium nitrate for a commercial kno 3 fertilizer / Desenvolvimento da palma Opuntia stricta (Haw.) Haw. (Cactaceae) in vitro em resposta à substituição de nitrato de potássio (P.A.) por fertilizante comercial KNO 3

ABSTRACT: In micropropagation, potassium nitrate (KNO3), an ACS reagent grade chemical, used in the preparation of growing mediums is expensive and its procurement depends on bureaucratic procedures, as it is controlled by the Brazilian Army. This research to assessed the effect of replacing the ACS KNO3 for a commercially available fertilizer (KNO3- based) on the micropropagation of the prickly pear cactus (Opuntia stricta (Haw.) Haw. cv. Elephant Ear. Treatments used six different fertilizer concentrations (0, 0.5, 1, 1.5, 2 and 2.5 g L-1) and a control consisting of 1.9 g L-1 KNO3, as shown in the MS salts. The survival, size and number of sprouts and the value of fresh biomass were evaluated. After seedling acclimation, we assessed the survival, number of sprouts, length, and number of roots, racket formation, average fresh biomass mass, macronutrient absorption and morphological changes of the seedlings. Explants inoculated with fertilizers at concentrations of 0.0; 2.0 and 2.5 g L-¹ did not grow. The response of explants at concentrations of 0.5 and 1.5 g L-1 of the fertilizer were the same as those developed in a KNO3 medium, and at a concentration of 1.0 g L-1, in all variables, the means were higher than those of the control medium. Therefore, it showed the feasibility of using fertilizers in the in vitro cultivation of the prickly pear cactus, which may remove bureaucratic barriers and reduce product costs by 99.12%.

RESUMO: Na micropropagação, o nitrato de potássio (KNO3), reagente puro para análise (P.A.), utilizado no preparo dos meios de cultura, possui custo elevado e a sua aquisição depende de trâmites burocráticos, por se tratar de substância controlada pelo Exército Brasileiro. O objetivo deste trabalho foi avaliar o efeito da substituição do KNO3 P.A. por fertilizante comercial (com fonte de KNO3), encontrado livremente no comércio, na micropropagação de palma (Opuntia stricta (Haw.) Haw. cv Orelha de Elefante. Os tratamentos foram de seis concentrações do fertilizante (0; 0,5; 1; 1,5; 2 e 2,5 g L-1) e um controle constituído de 1,9 g L-1 de reagente KNO3, conforme mostrado nos sais MS. Avaliou-se a sobrevivência, tamanho e número de brotações do explante, e o valor da biomassa fresca. Após a aclimatização das mudas avaliou-se a sobrevivência, número de brotações, comprimento da parte aérea, número de raízes, formação da raquete, massa média da biomassa fresca, absorção de macronutrientes e alterações morfológicas das mudas. Os explantes inoculados em meio com fertilizantes nas concentrações de 0,0; 2,0 e 2,5 g L-¹ não se desenvolveram. A resposta dos explantes nas concentrações de 0,5 e 1,5 g L-1 do fertilizante foram iguais aos desenvolvidos em meio contendo KNO3, e na concentração de 1,0 g L-1, em todas as variáveis, as médias foram superiores em relação as do controle. Dessa forma, constatou-se a viabilidade do uso do fertilizante no cultivo in vitro da palma, o que propiciou a eliminação dos entraves burocráticos e redução no custo de 99,12% na compra do produto.

The karyotype of Adenia and the origin of the base number x = 12 in Passifloroideae (Passifloraceae).

Adenia is an Old World genus of Passifloroideae closely related to Passiflora. The two genera comprise the large majority of Passifloroideae species, although most studies are concentrated on Passiflora. Cytological analyses reveal that changes in chromosome numbers played an important role in the evolution of Passiflora, whereas in the remaining genera little is known, hindering the identification of the base number of the family. Here we analyzed the chromosome number and the 35S rDNA sites of three species of Adenia and reevaluated the base number (x) of the subfamily Passifloroideae and the family Passifloraceae, including chromosome data for Turneroideae and Malesherbioideae. The chromosome number of Adenia species seemed to be stable with 2n = 24 or 48 and one or two pairs of rDNA sites, very similar to Passiflora subgenus Astrophea, suggesting a common ancestral karyotype with x = 12. Differently, Turneroideae and Malesherbioideae present x = 7. A whole genomic duplication detected after the separation of Passifloroideae and Malesherbioideae suggests that the base number of Passifloraceae most probably was x = 7, which by dysploidy and polyploidy generated x = 12 for the subfamily Passifloroideae.

Large vs small genomes in Passiflora: the influence of the mobilome and the satellitome.

MAIN CONCLUSIONS: While two lineages of retrotransposons were more abundant in larger Passiflora genomes, the satellitome was more diverse and abundant in the smallest genome analysed. Repetitive sequences are ubiquitous and fast-evolving elements responsible for size variation and large-scale organization of plant genomes. Within Passiflora genus, a tenfold variation in genome size, not attributed to polyploidy, is known. Here, we applied a combined in silico and cytological approach to study the organization and diversification of repetitive elements in three species of this genus representing its known range in genome size variation. Sequences were classified in terms of type and repetitiveness and the most abundant were mapped to chromosomes. We identified long terminal repeat (LTR) retrotransposons as the most abundant elements in the three genomes, showing a considerable variation among species. Satellite DNAs (satDNAs) were less representative, but highly diverse between subgenera. Our results clearly confirm that the largest genome species (Passiflora quadrangularis) presents a higher accumulation of repetitive DNA sequences, specially Angela and Tekay elements, making up most of its genome. Passiflora cincinnata, with intermediate genome and from the same subgenus, showed similarity with P. quadrangularis regarding the families of repetitive DNA sequences, but in different proportions. On the other hand, Passiflora organensis, the smallest genome, from a different subgenus, presented greater diversity and the highest proportion of satDNA. Altogether, our data indicates that while large genomes evolved by an accumulation of retrotransposons, the smallest genome known for the genus has evolved by diversification of different repeat types, particularly satDNAs.

Selection of cowpea cultivars for high temperature tolerance: physiological, biochemical and yield aspects.

High temperature stress can hinder the development of cowpea resulting in several damages including vegetative and reproductive phases of the crop. In this context, the objective of this study was to select cowpea cultivars tolerant to high temperature stress using various parameters related to physiological, biochemical, and yield aspects. For this, the cultivars Carijó, Itaim, Pujante, Rouxinol, and Tapahium were used, maintained in two temperature regimes: 20-26-33 °C and 24.8-30.8-37.8 °C. The experiment was carried out in growth chambers, in a 5 × 2 factorial arrangement (cultivars × temperature regimes). Responses differentiated among the cultivars Carijó, Itaim, Pujante, Rouxinol, and Tapahium with the increase of 4.8 °C in air temperature. The high temperature promoted a greater quantity of aborted flowers, leading to a reduction in the yield of the cultivars Carijó, Pujante, Rouxinol, and Tapahium. The photosynthesis, stomatal conductance, leaf transpiration and enzymatic activities were significantly influenced by high temperature. From the combination of the responses of biometric, physiological and productive variables, the cultivar Itaim can be considered as tolerant to an increase of 4.8 °C in air temperature.

Origin and parental genome characterization of the allotetraploid Stylosanthes scabra Vogel (Papilionoideae, Leguminosae), an important legume pasture crop.

Backgrounds and Aims: The genus Stylosanthes includes nitrogen-fixing and drought-tolerant species of considerable economic importance for perennial pasture, green manure and land recovery. Stylosanthes scabra is adapted to variable soil conditions, being cultivated to improve pastures and soils worldwide. Previous studies have proposed S. scabra as an allotetraploid species (2n = 40) with a putative diploid A genome progenitor S. hamata or S. seabrana (2n = 20) and the B genome progenitor S. viscosa (2n = 20). We aimed to provide conclusive evidence for the origin of S. scabra. Methods: We performed fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) experiments and Illumina paired-end sequencing of S. scabra, S. hamata and S. viscosa genomic DNA, to assemble and compare complete ribosomal DNA (rDNA) units and chloroplast genomes. Plastome- and genome-wide single nucleotide variation detection was also performed. Key Results: GISH and phylogenetic analyses of plastid DNA and rDNA sequences support that S. scabra is an allotetraploid formed from 0.63 to 0.52 million years ago (Mya), from progenitors with a similar genome structure to the maternal donor S. hamata and the paternal donor S. viscosa. FISH revealed a non-additive number of 35S rDNA sites in S. scabra compared with its progenitors, indicating the loss of one locus from A genome origin. In S. scabra, most 5S rDNA units were similar to S. viscosa, while one 5S rDNA site of reduced size most probably came from an A genome species as revealed by GISH and in silico analysis. Conclusions: Our approach combined whole-plastome and rDNA assembly with additional cytogenetic analysis to shed light successfully on the allotetraploid origin of S. scabra. We propose a Middle Pleistocene origin for S. scabra involving species with maternal A and paternal B genomes. Our data also suggest that variation found in rDNA units in S. scabra and its progenitors reveals differences that can be explained by homogenization, deletion and amplification processes that have occurred since its origin.

Application of Arbuscular Mycorrhizal Fungi during the Acclimatization of Alpinia purpurata to Induce Tolerance to Meloidogyne arenaria.

An experiment was conducted to evaluate the tolerance of micropropagated and mycorrhized alpinia plants to the parasite Meloidogyne arenaria. The experimental design was completely randomized with a factorial arrangement of four inoculation treatments with arbuscular mycorrhizal fungi (AMF) (Gigaspora albida, Claroideoglomus etunicatum, Acaulospora longula, and a non-inoculated control) in the presence or absence of M. arenaria with five replicates. The following characteristics were evaluated after 270 days of mycorrhization and 170 days of M. arenaria inoculation: height, number of leaves and tillers, fresh mass of aerial and subterranean parts, dry mass of aerial parts, foliar area, nutritional content, mycorrhizal colonization, AMF sporulation, and the number of galls, egg masses, and eggs. The results indicated a significant interaction between the treatments for AMF spore density, total mycorrhizal colonization, and nutrient content (Zn, Na, and N), while the remaining parameters were influenced by either AMF or nematodes. Plants inoculated with A. longula or C. etunicatum exhibited greater growth than the control. Lower N content was observed in plants inoculated with AMF, while Zn and Na were found in larger quantities in plants inoculated with C. etunicatum. Fewer galls were observed on mycorrhized plants, and egg mass production and the number of eggs were lower in plants inoculated with G. albida. Plants inoculated with A. longula showed a higher percentage of total mycorrhizal colonization in the presence of the nematode. Therefore, the association of micropropagated alpinia plants and A. longula enhanced tolerance to parasitism by M. arenaria.

Symbiotic compatibility between arbuscular mycorrhizal fungi (autoctone or exotic) and three native species of the Caatinga in different phosphorus levels / Compatibilidade simbiótica entre fungos micorrízicos arbusculares (autóctone ou exótico) e três espécies nativas da Caatinga em diferentes níveis de fósforo

The aim of this study was to evaluate three plant species native to Caatinga (Aspidosperma pyrifolium, Ziziphus joazeiro and Pseudobombax simplicifolium) in response to inoculation with two arbuscular mycorrhizal fungi (autoctone: Claroideoglomus etunicatum or exotic: Acaulospora longula) and phosphate fertilization. The experiment was conducted in a greenhouse with a completely randomized design involving a factorial arrangement of three inoculation treatments (control, C. etunicatum or A. longula) and 4 levels of single superphosphate (0 - no added P2O5, 12, 24 and 48 mg dm-3) in 10 repetitions. Under the conditions of this study, the inoculum of C. etunicatum, autoctone to the semi-arid region, showed great compatibility and efficiency in promoting plant growth and nutrient intake when compared to the exotic inoculum of A. longula. Plants of A. pyrifolium were not responsive to mycorrhization and phosphate fertilization. In soil with low phosphorus content, mycorrhized plants of Z. joazeiro and P. simplicifolium presented better vegetative development with high shoot dry weight. Mycorrhized plants of Z. joazeiro had a proportional increase in the levels of macro- and micronutrients at the phosphorus doses applied. Mycorrhizal symbiosis may have a fundamental role in the development of these species from the Caatinga.

O objetivo do trabalho foi avaliar o desenvolvimento de três espécies vegetais nativas da Caatinga (Aspidosperma pyrifolium, Ziziphus joazeiro e Pseudobombax simplicifolium) em resposta à inoculação com dois fungos micorrízicos arbusculares (autóctone: Claroideoglomus etunicatum ou exótico: Acaulospora longula) e adubação fosfatada. O experimento foi realizado em casa de vegetação em delineamento inteiramente casualizado em arranjo fatorial com três tratamentos de inoculação (Controle, C. etunicatum ou A. longula) e quatro doses de superfosfato simples (0 - sem adição de P2O5, 12, 24 e 48 mg dm -3), em dez repetições. Nas condições deste estudo, o inóculo de C. etunicatum, autóctone da região semiárida, demonstrou maior compatibilidade e eficiência na promoção do crescimento e nutrição das plantas quando comparado ao inóculo exótico de A. longula. Plantas de A. pyrifolium não foram responsivas à micorrização e adubação fosfatada. Em solo com menor teor de fósforo as plantas de Z. joazeiro e P. simplicifolium micorrizadas apresentaram melhor desenvolvimento vegetativo, com elevada produção de biomassa seca da parte aérea. Plantas de Z. joazeiro micorrizadas tiveram aumento nos teores de macro e micronutrientes proporcional às doses de fósforo aplicadas. Conclui-se que simbiose micorrízica pode ter papel fundamental no desenvolvimento destas espécies nativas da Caatinga

Responses of Guava Plants to Inoculation with Arbuscular Mycorrhizal Fungi in Soil Infested with Meloidogyne enterolobii.

In the Northeast of Brazil, expansion of guava crops has been impaired by Meloidogyne enterolobii that causes root galls, leaf fall and plant death. Considering the fact that arbuscular mycorrhizal Fungi (AMF) improve plant growth giving protection against damages by plant pathogens, this work was carried out to select AMF efficient to increase production of guava seedlings and their tolerance to M. enterolobii. Seedlings of guava were inoculated with 200 spores of Gigaspora albida, Glomus etunicatum or Acaulospora longula and 55 days later with 4,000 eggs of M. enterolobii. The interactions between the AMF and M. enterolobii were assessed by measuring leaf number, aerial dry biomass, CO2 evolution and arbuscular and total mycorrhizal colonization. In general, plant growth was improved by the treatments with A. longula or with G. albida. The presence of the nematode decreased arbuscular colonization and increased general enzymatic activity. Higher dehydrogenase activity occurred with the A. longula treatment and CO2 evolution was higher in the control with the nematode. More spores and higher production of glomalin-related soil proteins were observed in the treatment with G. albida. The numbers of galls, egg masses and eggs were reduced in the presence of A. longula. Inoculation with this fungus benefitted plant growth and decreased nematode reproduction.

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil / Resistência antimicrobiana de Staphylococcus spp. de mastite de pequenos ruminantes no Brasil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.

O presente trabalho teve como objetivo determinar os padrões de resistência a agentes antimicrobianos e identificar marcadores moleculares de resistência em Staphylococcus spp. (n=210) isolados de mastite de pequenos ruminantes no Brasil. Os padrões de resistência a agentes antimicrobianos foram avaliados pelo teste de difusão em disco e pela detecção da presença dos genes mecA, blaZ, ermA, ermB, ermC e msrA via PCR. O teste da bomba de efluxo foi realizado utilizando brometo de etídio e a produção de biofime foi determinada pelo teste do vermelho congo em paralelo com o PCR para detecção do gene icaD. Os isolados foram mais resistentes a amoxicilina (50,0%), estreptomicina (42,8%), tetraciclina (40,4%), lincomicina (39,0%) e eritromicina (33,8%). Setenta e um (33,8%) isolados foram sensíveis a todas as drogas testadas e 41 foram positivos para a bomba de efluxo. Embora a resistência fenotípica a oxacilina tenha sido observada observada em 12,8% dos isolados, nenhum possuiu o gene mecA. Entretanto, 45,7% dos isolados continham a gene blaZ, indicando que a produção de beta-lactamases foi o principal mecanismo associado com a resistência dos Staphylococcus aos beta-lactâmicos. Os outros determinantes de resistência a agentes antimicrobianos ermA, ermb, ermC e msrA foram observados em 1,4%, 10,4%, 16,2% e 0,9% dos isolados respectivamente. Além disso, o gene icaD foi detectado em 32,9% dos isolados. Setenta e três isolados (54 de cabras e 19 de ovelhas) foram negativos para todos os genes de resistência testados e 69 isolados apresentaram dois ou mais genes de resistência. A associação entre blaZ, ermA, ermB, ermC e bomba de efluxo foi observada em 17 isolados dos quais 14 eram oriundos de cabras e três de ovelhas. Os dados obtidos no presente estudo indicam a resistência dos isolados aos beta-lactâmicos, o que pode estar associado ao uso sem controle veterinário destas drogas nos animais.

Comparative cytogenetic analysis of two grasshopper species of the tribe Abracrini (Ommatolampinae, Acrididae).

The grasshopper species Orthoscapheus rufipes and Eujivarus fusiformis were analyzed using several cytogenetic techniques. The karyotype of O. rufipes, described here for the first time, had a diploid number of 2n = 23, whereas E. fusiformis had a karyotype with 2n = 21. The two species showed the same mechanism of sex determination (XO type) but differed in chromosome morphology. Pericentromeric blocks of constitutive heterochromatin (CH) were detected in the chromosome complement of both species. CMA(3)/DA/DAPI staining revealed CMA(3)-positive blocks in CH regions in four autosomal bivalents of O. rufipes and in two of E. fusiformis. The location of active NORs differed between the two species, occurring in bivalents M(6) and S(9) of O. rufipes and M(6) and M(7) of E. fusiformsi. The rDNA sites revealed by FISH coincided with the number and position of the active NORs detected by AgNO(3) staining. The variability in chromosomal markers accounted for the karyotype differentiation observed in the tribe Abracrini.

Comparative cytogenetic analysis of two grasshopper species of the tribe Abracrini (Ommatolampinae, Acrididae)

The grasshopper species Orthoscapheus rufipes and Eujivarus fusiformis were analyzed using several cytogenetic techniques. The karyotype of O. rufipes, described here for the first time, had a diploid number of 2n = 23, whereas E. fusiformis had a karyotype with 2n = 21. The two species showed the same mechanism of sex determination (XO type) but differed in chromosome morphology. Pericentromeric blocks of constitutive heterochromatin (CH) were detected in the chromosome complement of both species. CMA3/DA/DAPI staining revealed CMA3-positive blocks in CH regions in four autosomal bivalents of O. rufipes and in two of E. fusiformis. The location of active NORs differed between the two species, occurring in bivalents M6 and S9 of O. rufipes and M6 and M7 of E. fusiformsi. The rDNA sites revealed by FISH coincided with the number and position of the active NORs detected by AgNO3 staining. The variability in chromosomal markers accounted for the karyotype differentiation observed in the tribe Abracrini.

High levels of chromosomal differentiation in Euchroma gigantea L. 1735 (Coleoptera, Buprestidae)

Euchroma gigantea was karyotypically studied using conventional staining, C-banding, silver nitrate staining and ribosomal fluorescent in situ hybridization (rDNA FISH). Broad wide autosomal polymorphism and a complex sex determination system were found in this beetle. Karyotype complements ranging from 2n = 32, X1X2X3Y1Y2Y3 to 2n = 36,X1X2X3Y1Y2Y3 were detected in the sample analyzed. Punctiform supernumerary chromosomes were present in the different karyotypes. The karyotypic evolution of Brazilian E. gigantea may have taken two directions, reduction in the diploid number of 2n = 36 to 24 through centric fusions or 2n = 24 to 36 due to chromosomal fissions. In addition, pericentric inversions were also involved. The complex multiple sex mechanism of this species seems to be old and well established since it is found in specimens from different populations. Small pericentromeric blocks of constitutive heterochromatin were located on the autosomes and terminal blocks were also found on some small pairs. The sex chromosomes showed larger constitutive heterochromatin blocks. Silver nitrate staining during prophase I of meiosis showed labeling of the sex chromosome chain. However, the rDNA sites could only be precisely determined by FISH, which permitted the identification of these ribosomal sites on chromosomes X1 and X2 of this species.

Chromosome study in Schistocerca (Orthoptera-Acrididae-Cyrtacanthacridinae): karyotypes and distribution patterns of constitutive heterochromatin and nucleolus organizer regions (NORs)

Chromosome analyses were performed in two grasshopper species of the genus Schistocerca, S. pallens and S. flavofasciata. Both species shared the same diploid number (2n = 23, X in males; 2n = 24, XX in females);and a conserved karyotype composed exclusively of acrocentric chromosomes, but differed in their distribution patterns of constitutive heterochromatin and nucleolus organizer regions (NORs). Constitutive heterochromatin was located in the pericentromeric region of all chromosomes in both species. S. flavofasciata presented an additional C-band on the distal region of the long arm of a small autosome pair (S9). Nucleolus organizer regions (NORs), revealed by silver nitrate staining (Ag-NORs), were observed on a medium autosome pair (M5) in both species. S. pallens presented an additional NOR-bearing autosome (M6). The same sites were labeled after FISH with an rDNA probe in S. pallens cells.

A comparative cytogenetic analysis between the grasshopper species Chromacris nuptialis and C. speciosa (Romaleidae): constitutive heterochromatin variability and rDNA sites.

The chromosomes of Chromacris nuptialis and C. speciosa were comparatively analyzed using different cytogenetic techniques, in order to determine the level of karyotypic similarities and differences between the species. The results show similarities in chromosome number (2n=23,X0) and acrocentric morphology. In some C. nuptialis individuals meiotic irregularities were detected involving the L(2) bivalent. This bivalent was delayed and presented anaphasic bridges and other aberrations. Differences in constitutive heterochromatin (CH) patterns and composition were observed through C-banding and fluorochromes staining. Silver nitrate staining revealed a single medium nucleolar organizer regions (NORs) pair, per species. Differences were also observed in NORs location, which was pericentromeric in C. nuptialis and proximal in C. speciosa. FISH using an rDNA probe confirmed the existence of ribosomal sites coinciding with active regions visualized by silver nitrate. The possible implications of the karyotype differences observed between both species are discussed.

Karyotypic characterization of representatives from Melolonthinae (Coleoptera: Scarabaeidae): karyotypic analysis, banding and fluorescent in situ hybridization (FISH).

Meiotic chromosomes of Phyllophaga (Phytalus) vestita, Phyllophaga (Phyllophaga) aff capillata and Lyogenys fuscus (Melolonthinae) were analyzed by conventional staining, C-banding, fluorochromes, silver nitrate and FISH. The three species had a diploid number of 2n=20 and a sex mechanism of the (Xyp; XYp) parachute type. P. (Phytalus) vestita,P. (Phyllophaga) aff capillata and Lyogenys fuscus showed pericentromeric constitutive heterochromatin (CH) in all autosomal bivalents and on X chromosomes. Staining with CMA3 and DAPI fluorochromes showed that the CH of P. (Phytalus) vestita is not specifically rich in AT and GC-base pairs, whereas in P. (Phyllophaga) aff capillata the sex bivalent and one autosomal pair were found to be enriched in GC base pairs with CMA3, and in Lyogenys fuscus CH was positive for DAPI. Silver nitrate staining revealed nucleolar remnants in all three species. However, FISH obtained a precise identification of nucleolar organizing regions with an rDNA 18S and 25S probe. A signal of hybridization was seen in each species, being detected in the X chromosome of P. (Phytalus) vestita and Lyogenys fuscus, and in a small autosomal bivalent of P. (Phyllophaga) aff capillata.

Variability of the 5S and 45S rDNA sites in Passiflora L. species with distinct base chromosome numbers.

Cytologically, the species of Passiflora with known chromosome number can be divided into four groups: (1) 2n = 12, 24, 36; (2) 2n = 24; (3) 2n = 18, 72; and (4) 2n = 20. The base chromosome number proposed for the genus is x = 6, with x = 9, x = 10 and x = 12 being considered secondary base numbers. In the present study, variability of 5S and 45S rDNA sites was investigated in 20 species of these four groups to check the reliability of this hypothesis. In the group with x = 6, five diploid species (2n = 12) exhibit two 5S rDNA sites and two (P. capsularis, P. morifolia and P. rubra) or four (P. misera 2x and P. tricuspis) 45S rDNA sites. The hexaploid cytotype of P. misera had 12 45S rDNA sites and six 5S rDNA. A tetraploid species, P. suberosa, had ten 45S rDNA sites and four 5S rDNA sites, both in the same chromosomes as the 45S rDNA sites. In the group with x = 9, P. actinia, P. amethystina, P. edmundoi, P. elegans, P. galbana, P. glandulosa and P. mucronata displayed six 45S rDNA sites, whereas P. alata, P. cincinnata, P. edulis f. flavicarpa, P. edulis var. roxo and P. laurifolia had four sites. In this group, all species were diploid (2n = 18) and had only two 5S rDNA sites. Passiflora foetida, the only species with 2n = 20, had six 45S rDNA sites and four 5S rDNA sites. The species with x = 12 (2n = 24), P. haematostigma and P. pentagona, showed four 45S rDNA sites and two 5S rDNA. In general, the number and location of 5S and 45S rDNA sites were consistent with the hypothesis of x = 6 as the probable ancestral genome for the genus, while the groups of species with x = 9, x = 10 and x = 12 were considered to be of tetraploid origin with descending dysploidy and gene silencing of some redundant gene sites, mainly those of 5S rDNA.