Anatomy,pollen, and chromosomes of <Emphasis Type="Italic">Adenoa</Emphasis> (Turneraceae), a monotypic genus endemic to Cuba

The monotypic genus Adenoa is endemic to Cuba. Its name alludes to the presence of minute glands on the petal margin, identified in the present study as lachrymiform colleters. Here we describe the morphological, anatomical, palynological, and chromosome features that characterize Adenoa cubensis. The indumentum of Adenoa consists only of stellate trichomes. Unlike many species of the new world genera Piriqueta and Turnera, Adenoa lacks glandular hairs and extrafloral nectaries. Adenoa, Piriqueta, and Turnera share the presence of standard, sessile, and lachrymiform colleters. The leaves of Adenoa have xeromorphic features, which include entire, revolute blade margins, an adaxial hypodermis, and stomata restricted to the abaxial surface. The chromosome number is 2n?=?14, which is probably the ancestral number of the family. Adenoa chromosomes are similar in size to those of Turnera, and are larger than those of Piriqueta. Using the available data, we discuss relationships among the new world genera of Turneraceae.     

More sex chromosomes than autosomes in the Amazonian frog <Emphasis Type="Italic">Leptodactylus pentadactylus</Emphasis>

Heteromorphic sex chromosomes are common in eukaryotes and largely ubiquitous in birds and mammals. The largest number of multiple sex chromosomes in vertebrates known today is found in the monotreme platypus (Ornithorhynchus anatinus, 2n?=?52) which exhibits precisely 10 sex chromosomes. Interestingly, fish, amphibians, and reptiles have sex determination mechanisms that do or do not involve morphologically differentiated sex chromosomes. Relatively few amphibian species carry heteromorphic sex chromosomes, and when present, they are frequently represented by only one pair, either XX:XY or ZZ:ZW types. Here, in contrast, with several evidences, from classical and molecular cytogenetic analyses, we found 12 sex chromosomes in a Brazilian population of the smoky jungle frog, designated as Leptodactylus pentadactylus Laurenti, 1768 (Leptodactylinae), which has a karyotype with 2n?=?22 chromosomes. Males exhibited an astonishing stable ring-shaped meiotic chain composed of six X and six Y chromosomes. The number of sex chromosomes is larger than the number of autosomes found, and these data represent the largest number of multiple sex chromosomes ever found among vertebrate species. Additionally, sequence and karyotype variation data suggest that this species may represent a complex of species, in which the chromosomal rearrangements may possibly have played an important role in the evolution process.     

<Emphasis Type="Italic">Oswaldocruzia lanfrediae</Emphasis> n. sp. (Strongylida: Molineidae), a parasite of <Emphasis Type="Italic">Leptodactylus paraensis</Emphasis> Heyer (Anura: Leptodactylidae) in Brazil

Leptodactylus paraensis Heyer, is a Neotropical anuran species that inhabits Rainforest habitats in the eastern Amazon, but because it has only been recently separated from the Leptodactylus pentadactylus (Laurenti) species group, little is known about its helminth fauna. This study describes a new species of Oswaldocruzia Travassos, 1917 and records the first occurrence of this genus parasitising L. paraensis and the second species for the Caxiuanã National Forest in the eastern Amazon, Brazil. Oswaldocruzia lanfrediae n. sp. is characterised by having an anterior extremity with a smooth cephalic vesicle divided into two portions, a claviform oesophagus, well-developed cuticular longitudinal ridges and lateral alae. Females have a well-developed ovojector, with didelphic and amphidelphic uteri. Males show complex robust spicules divided into a slightly curved shoe, a bifurcated fork and a blade terminating in 2–3 processes. The new species differs from its congeners especially regarding the lateral alae and the morphology of the spicules, in addition to morphometric characters such as body size, oesophagus length, deirid position, nerve-ring position and relative position of the vulva in females.     

Molecular cytogenetic analysis of DNA from pericentric heterochromatin of chromosome 2L of malaria mosquito <Emphasis Type="Italic">Anopheles beklemishevi</Emphasis> (culicidae,Diptera)

Using the method of microdissection of polytene chromosomes, followed by in situ hybridization, chromosomal localization of region-specific DNA probe from pericentic heterochromatin of chromosome 2L of Anopheles beklemishevi Stegnii et Kabanova was examined on polytene chromosomes of Anopheles atroparvus van Thiel, An. messeae Fall, and An. beklemishevi. DNA sequences homologous to the probe used were found in all species examined on chromosomes 2 and 3 in pericentric regions and in attachment regions. The exclusion were the attachment regions of chromosome XL in An. beklemishevi and An. messeae, and pericentric region of arm 2R in An. messeae. Pericentric α -heterochromatin of arm 2L in An. messeae and arm 3R in An. atroparvus also contained no sequences homologous to the DNA probe. The data obtained were compared with the earlier obtained data on localization of species-specific probe from the segment of chromosome 2R of An. atroparvus on chromosomes of An. artoparvus, An. messeae, and An. beklemishevi. The differences between the species in the sites of probes localization and fluorescence intensity revealed pointed to the existence of individual sequence associations in the regions of chromosomes attachment.     

Integrated gene mapping and synteny studies give insights into the evolution of a sex proto-chromosome in <Emphasis Type="Italic">Solea senegalensis</Emphasis>

The evolution of genes related to sex and reproduction in fish shows high plasticity and, to date, the sex determination system has only been identified in a few species. Solea senegalensis has 42 chromosomes and an XX/XY chromosome system for sex determination, while related species show the ZZ/ZW system. Next-generation sequencing (NGS), multi-color fluorescence in situ hybridization (mFISH) techniques, and bioinformatics analysis have been carried out, with the objective of revealing new information about sex determination and reproduction in S. senegalensis. To that end, several bacterial artificial chromosome (BAC) clones that contain candidate genes involved in such processes (dmrt1, dmrt2, dmrt3, dmrt4, sox3, sox6, sox8, sox9, lh, cyp19a1a, amh, vasa, aqp3, and nanos3) were analyzed and compared with the same region in other related species. Synteny studies showed that the co-localization of dmrt1-dmrt2-drmt3 in the largest metacentric chromosome of S. senegalensis is coincident with that found in the Z chromosome of Cynoglossus semilaevis, which would potentially make this a sex proto-chromosome. Phylogenetic studies show the close proximity of S. senegalensis to Oryzias latipes, a species with an XX/XY system and a sex master gene. Comparative mapping provides evidence of the preferential association of these candidate genes in particular chromosome pairs. By using the NGS and mFISH techniques, it has been possible to obtain an integrated genetic map, which shows that 15 out of 21 chromosome pairs of S. senegalensis have at least one BAC clone. This result is important for distinguishing those chromosome pairs of S. senegalensis that are similar in shape and size. The mFISH analysis shows the following co-localizations in the same chromosomes: dmrt1-dmrt2-dmrt3, dmrt4-sox9-thrb, aqp3-sox8, cyp19a1a-fshb, igsf9b-sox3, and lysg-sox6.     

Behavior of sex chromosomes at early meiosis stages in three wood mice species of the genus <Emphasis Type="Italic">Apodemus</Emphasis> (Rodentia,Muridae)

The prophase of the first meiotic division was studied in field mice of the species Apodemus (Sylvaemus) flavicollis, A. (S.) ponticus, and A. (S.) uralensis by light and electron microscopy. The karyotypes of three species were described on the base of electron microscopy of synaptonemal complexes in spermatocytes I. The axial elements of the sex chromosomes at early-middle pachytene synapse along the major portion of the Y axis; at late pachytene-early diplotene, the synapsis region shrinks; and at diakinesis-metaphase I, X and Y chromosomes associate end-to-end in all species studied. The behavior of sex chromosomes in the synapsis in the species studied was quite uniform. The results are discussed in the context of earlier data on the behavior of sex chromosomes in various rodent species in meiosis prophase I and their banding.     

Genetic Regulation of Meiotic Chromosome Pairing by Chromosome 3<Emphasis Type="Italic">D</Emphasis> of <Emphasis Type="Italic">Triticum aestivum</Emphasis>

IN hexaploid wheat (Triticum aestivum, 2n = 6x = 42) the constituent genomes A, B and D derive from closely related diploid species (2n = 2x = 14) within the sub-tribe Triticinae^1–4. The seven different chromosomes of each genome have genetically equivalent (homoeologous) chromosomes in the other two genomes⁵. Homoeologous chromosomes generally compensate each other in nullisomic-tetrasomic combinations⁵.     

Two new cytotypes reinforce that <Emphasis Type="Italic">Micronycteris hirsuta</Emphasis> Peters, 1869 does not represent a monotypic taxon

Background

The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n?=?26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data.

Results

We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n?=?26 and 25, NF?=?32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA.

Conclusions

The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

     

The karyotype of the great sculpin, <Emphasis Type="Italic">Myoxocephalus polyacanthocephalus</Emphasis> (Pallas, 1814) (Pisces: Cottidae), from the Russian part of the species range

The karyotype of the great sculpin, Myoxocephalus polyacanthocephalus (Pallas, 1814) (Pisces: Cottidae) from the Sea of Okhotsk and the Sea of Japan has been studied for the first time. The karyotype is stable; it consists of 40 chromosomes (4 metacentric, 2 submeta-subtelocentric, 20 subtelocentric, and 14 acrocentric chromosomes); the number of chromosomal arms is 44 + 2. Nucleolar organizer regions (NOR) are found in the telomeric region of the arm in one homologue of a pair of small metacentric chromosomes, using the Ag-NOR banding technique. A comparative analysis of the karyotype of M. polyacanthocephalus and the karyotypes of other Myoxocephalus species (M. stelleri, M. brandtii, M. jaok, M. ochotensis, and M. scorpius) has been carried out based on the main karyotype characters, as well as on the number and localization of NORs. The identified differences make it possible to differentiate the studied species, whereas the general traits indicate their taxonomic proximity.     

Chromosomal features of nucleolar dominance in hybrids between the Neotropical fish <Emphasis Type="Italic">Leporinus macrocephalus</Emphasis> and <Emphasis Type="Italic">Leporinus elongatus</Emphasis> (Characiformes,Anostomidae)

In the present study, the chromosomal mechanisms of nucleolar dominance were analyzed in the hybrid lineage “Piaupara,” which resulted from crossing the Leporinus macrocephalus female (Piauçu) and L. elongatus male (Piapara) fish. The analyses demonstrated that, in the hybrid, the nucleolar region inherited from L. elongatus presented higher activity, with expression in 100% of the cells, whereas the nucleolar region from L. macrocephalus appeared active at a frequency of 11.6%. The FISH technique with an 18S probe showed that the ribosomal DNA of the nucleolar region was not lost in the hybrid, and the results therefore demonstrated invariable marks in two chromosomes, each originating from one parent. An interesting difference between the nucleolar regions of the parental species was the association of the NOR with heterochromatic blocks (repetitive DNA) in L. elongatus, which could act as a determinative element in the establishment of this process.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

A comparative cytogenetic study of the tetraploid oat species with the A and C genomes: <Emphasis Type="Italic">Avena insularis,A. magna</Emphasis>, and <Emphasis Type="Italic">A. murphyi</Emphasis>

C-banding of chromosomes and in situ hybridization with the probes pTa71 and pTa794 were used for a comparative cytogenetic study of the three tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi. These species were similar in the structure and C-banding patterns of several chromosomes as well as in the location of the loci 5S rRNA genes and major NOR sites; however, they differed in the number and localization of minor 45S rDNA loci as well as in the morphology and distribution of heterochromatin in some chromosomes. According to the data obtained, A. insularis is closer to A. magna, whereas A. murphyi is somewhat separated from these two species. Presumably, all the three studied species originated from the same tetraploid ancestor, and their divergence is connected with various species-specific chromosome rearrangements. The evolution of A. murphyi is likely to have occurred independently of the other two species.     

Chromosome number variability in central european members of the<Emphasis Type="Italic">Festuca ovina</Emphasis> and<Emphasis Type="Italic">F. pallens</Emphasis> groups (sect.<Emphasis Type="Italic">Festuca</Emphasis>)

Chromosome numbers for 98 plants ofF. pallens, 19 ofF. psammophila, F. belensis andF. vaginata, and 44 ofF. ovina (originating from Austria, the Czech Republic, Germany, Slovakia and Latvia) are given. In addition to theF. ovina andF. pallens groups, chromosome counts for the following taxa are also reported:F. alpestris (2n=14) reported for the first time in this work,F. amethystina subsp.amethystina (2n=28),F. brevipila (2n=42),F. cinerea (2n=28),F. rupicola subsp.rupicola (2n=42) andF. versicolor subsp.versicolor (2n=14).InF. pallens, two ploidy levels (2n=2x=14+0-1B, 2n=4x=28+0-1B) as well as two natural triploid plants (2n=21+0-1B), were found. In addition to the fourF. pallens types that have been distinguished in Austria, one new tetraploid type (F. pallens “scabrifolia”) from the Czech Republic and Germany is reported and its taxonomy is discussed. The distributions of the Oberösterreich-Niederösterreich and Pannonisches-HügellandF. pallens types outside of Austria are documented.Only the diploid chromosome number (2n=14) was found inF. psammophila andF. vaginata. Chromosome numbers forF. psammophila subsp.muellerstollii andF. belensis (both 2n=14) were determined here for the first time. Two ploidy levels, 2n=14+0-5B corresponding toF. ovina subsp.ovina and 2n=28 corresponding toF. ovina subsp.guestphalica andF. cf.duernsteinensis were confirmed inF. ovina. Differences in chromosome structure (simple and multiple secondary constrictions) betweenF. pallens as opposed toF. psammophila andF. vaginata are discussed. A complete survey of published chromosome counts for Central European species from theF. ovina andF. pallens groups is included.     

A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells

Background

In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-L ike K inase (TLK1B) and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line.

Results

Expression of a kinase-dead mutant resulted in reduction of phosphorylated histone H3, which could have consequences in mitotic segregation of chromosomes. When analyzed by FACS and microscopy, these cells displayed high chromosome number instability and aneuploidy. This phenomenon was accompanied by less condensed chromosomes at mitosis; failure of a number of chromosomes to align properly on the metaphase plate; failure of some chromosomes to attach to microtubules; and the occasional presentation of two bipolar spindles. We also used a different method (siRNA) to reduce the level of endogenous TLK1, but in this case, the main result was a strong block of cell cycle progression suggesting that TLK1 may also play a role in progression from G1. This block in S phase progression could also offer a different explanation of some of the later mitotic defects.

Conclusions

TLK1 has a function important for proper chromosome segregation and maintenance of diploid cells at mitosis in mammalian cells that could be mediated by reduced phosphorylation of histone H3 and condensation of chromosomes, although other explanations to the phenotype are possible.

     

Banding and FISH in three species of <Emphasis Type="Italic">Vernonia</Emphasis>, subsection <Emphasis Type="Italic">Macrocephalae</Emphasis> (Asteraceae,Vernonieae)

To complement our knowledge about the karyotypes of the genus Vernonia Schreb., different techniques of chromosome banding, including AgNOR, triple staining with fluorochromes CMA/DA/DAPI (CDD), and fluorescence in situ hybridization (FISH) for the 45S rDNA probe, were applied to three species of subsection Macrocephalae. Vernonia bardanoides was collected from an area of cerrado (savanna) vegetation in Itirapina, São Paulo State, Brazil, and V. linearifolia and V. tomentella were collected from areas of rocky, open altitudinal vegetation in Joaquim Felicio and Diamantina, respectively, in Minas Gerais State. All species showed two terminal CMA⁺ and NOR bands. FISH indicated two terminal 45S rDNA sites in V. linearifolia and V. tomentella, and six in V. bardanoides.     

<Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> and <Emphasis Type="Italic">Adalia bipunctata</Emphasis> combination for the control of black bean aphid<Emphasis Type="Italic">, Aphis fabae</Emphasis>

The predator Adalia bipunctata (Coleoptera: Coccinellidae) and the entomopathogenic fungus Lecanicillium muscarium, have been considered as potential biological control against aphids. While it can be difficult to achieve a high control level of Aphis fabae Scopoli (Hemiptera: Aphididae) using only a single beneficial agent, the research presented here aimed to determine the interaction between L. muscarium and A. bipunctata potential for control against A. fabae. Lecanicillium muscarium was found to cause about 30% mortality in A. bipunctata and with a reduction in feeding by about 15%. However, co-application of A. bipunctata and L. muscarium can cause an addititive effect in reducing aphid populations, resulting in about 90% reduction in aphid populations compared with control treatment. Thus, these two biocontrol agents have the potential to be complementary. This research study demonstrates that it is possible to combine A. bipunctata with L. muscarium to provide a sustainable method for management of A. fabae on broad bean cropping system and that field studies are required.     

Karyologic diversification and phylogenetic relationships of the genus<Emphasis Type="Italic">Thalpomys</Emphasis> (Rodentia,Sigmodontinae)

We describe the karyotype ofThalpomys species, from different Brazilian localities of the Cerrado.Thalpomys cerradensis Herskovitz, 1990 showed 2n=36, FN=34 andT. lasiotis Thomas, 1916 2n=38, FN=38. Comparisons of G-band karyotypes showed evident inter-specific homologies indicating that their chromosome complements could be derived from one another by two presumed rearrangements. Both species showed pericentromeric C-band regions in almost all chromosomes but a comparison with CMA3/DA/DAPI staining indicated that the molecular content of heterochromatic regions was different.T. lasiotis specimens from two different localities differed in the morphology of the X chromosome due to the presence of a short heterochromatic arm. These chromosome types are apparently fixed in each population rather than maintained as a polymorphic variation. Phylogenetic analyses supported the monophyly of the genusThalpomys but was not capable of elucidating its phylogenetic relationship to other Akodontini rodents. These analyses also showed inter-individual variation inT. lasiotis, even within a given population. Phylogenetic analyses placedT. lasiotis specimens with different karyotypes in different monophyletic branches. Molecular and karyologic data confirmed the identity of the genusThalpomys.     

Precise transfers of genes for high grain iron and zinc from wheat-<Emphasis Type="Italic">Aegilops</Emphasis> substitution lines into wheat through pollen irradiation

Nearly 2 billion people worldwide are suffering from iron (Fe) deficiency anemia and zinc (Zn) deficiency. The available elite bread wheat cultivars have inherently low grain micronutrient content. Biofortification for grain Fe and Zn content is one of the most feasible and cost-effective approach for combating widespread deficiency of the micronutrients. QTL controlling high grain Fe and Zn have been mapped on groups 2 and 7 chromosomes of Triticeae. The present study was initiated for precise transfers of genes for high grain Fe and Zn on group 2 and 7 chromosomes of wheat-Aegilops substitution lines to wheat cultivars using pollen radiation hybridization. The pollen radiation hybrids (PRH₁) derived from 1.75 krad irradiated spikes showed the presence of univalents and multivalents in meiotic metaphase-I indicating the effectiveness of radiation dose. In the advanced generation PRH₅, the plants selected with stable chromosome number and high grain Fe and Zn content were analyzed with wheat groups 2 and 7 chromosome specific intron targeted amplified polymorphism (ITAP) markers of the metal homeostasis genes to monitor the transfers of alien genes from the substituted Aegilops chromosomes. The group 2 chromosome derivatives showed the presence of NAS2, FRO2, VIT1, and ZIP2 Aegilops genes whereas the group 7 derivatives had YSL15, NAM, NRAMP5, IRO3, and IRT2 Aegilops genes. The pollen radiation hybrids of both the groups 2 and 7 chromosomes showed more than 30% increase in grain Fe and Zn content with improved yield than the elite wheat cultivar PBW343 LrP indicating small and compensating transfers of metal homeostasis genes of Aegilops into wheat.     

Mutational spectrum of the gene for 21-hydroxylase in the patients with congenital adrenal hyperplasia from Bashkortostan

Molecular genetic analysis of congenital adrenal hyperplasia (CAH) was carried out in 59 patients from the Republic of Baskortostan, which belonged to two main groups. The first group was represented by 35 patients with salt wasting form of the disease, and the second group was comprised of 24 patients with simple virilizing form. Analysis of the CYP21A2 gene in the patients with congenital adrenal hyperplasia from the Republic of Bashkortostan revealed seven different mutations on 81.58% chromosomes, including deletion/conversion delA2 or LGC gene, R356W, I2splice, I172N, Q318X, V281L, and P30L. The mutations were present on 89.71% of chromosomes from the patients with salt wasting form, and in 69.5% of chromosomes from the patients with simple virilizing form. The most frequent mutation was gene deletion/conversion, delA2 or LGC, which was found with the frequency of 30.83%. In six CAH patients the presence of three different mutation clusters on one chromosome was demonstrated: Q318X + R356W, I172N + Q318X, and delA2 or LGC + V281L. For the mutations leading to partial loss of 21-hydroxylase activity and simple virilizing form, 100% conformity of the phenotype to genotype was established.