XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil

Potamotrygonidae is a representative family of South American freshwater elasmobranchs. Cytogenetic studies were performed in a Potamotrygon species from the middle Negro River, Amazonas, Brazil, here named as Potamotrygon sp. C. Mitotic and meiotic chromosomes were analyzed using conventional staining techniques, C-banding, and detection of the nucleolus organizing regions (NOR) with Silver nitrate (Ag-NOR). The diploid number was distinct between sexes, with males having 2n = 67 chromosomes, karyotype formula 19m + 8sm + 10st + 30a, and fundamental number (FN) = 104, and females having 2n = 68 chromosomes, karyotype formula 20m + 8sm + 10st + 30a, and FN = 106. A large chromosome, corresponding to pair number two in the female karyotype, was missing in the male complement. Male meiotic cells had 33 bivalents plus a large univalent chromosome in metaphase I, and n = 33 and n = 34 chromosomes in metaphase II. These characteristics are consistent with a sex chromosome system of the XX/XO type. Several Ag-NOR sites were identified in both male and female karyotypes. Positive C-banding was located only in the centromeric regions of the chromosomes. This sex chromosome system, which rarely occurs in fish, is now being described for the first time among the freshwater rays of the Amazon basin.     

Karyological analysis of the genus Canna (Cannaceae)

Somatic chromosome numbers and karyological features in 22 taxa of the genus Canna (Cannaceae) were investigated and are presented here for the first time. The chromosome numbers were primarily reported for C. bangii, C. indica var. sanctae-rosae and C. tulianensis. A triploid somatic chromosome number of 2n = 27 was found only in C. discolor, while the remaining taxa were diploids with 2n = 18. Chromosome numbers are considered to be remarkably stable within the Cannaceae. Canna shows a symmetric karyotype that consists mostly of median chromosomes, but with some subterminal or submedian chromosomes, and a karyotype homogenous in variation in chromosome length. The karyotype of some taxa of Canna, furthermore, is characterized by median chromosomes with a secondary constriction in the centromeric region. Cytotaxonomical characteristics of the family Cannaceae and the related groups in the Zingiberales are discussed.     

Molecular and cytogenetic analyses provide evidence of the introgression of chromosomal segments from the wild <Emphasis Type="Italic">Cucumis hystrix</Emphasis> into the cultivated cucumber through the bridge of a synthetic allotetraploid

Cucumis × hytivus (2n = 4× = 38) is a synthetic allotetraploid obtained from interspecific hybridization between the cucumber (2n = 2× = 14) and its wild relative C. hystrix (2n = 2× = 24). The synthesis of this species built a bridge for cucumber improvement through gene introgression. Allotriploid and introgression lines (ILs) have previously been produced and characterized with respect to morphology, cytology, and molecular markers. However, no clear evidence of how the chromosomal segments of C. hystrix were introgressed and inherited was found owing to the small size of chromosomes. In the present study, cucumber-C. hystrix introgression lines were developed by backcrossing the allotriploid to North China cucumber breeding line “P01” followed by self-pollination. The introgressed segments of C. hystrix in the ILs were revealed by meiotic pachytene chromosome analysis. Fluorescence in situ hybridization (FISH) was performed on pachytene chromosomes using fosmid clones from cucumber, which confirmed that introgression occurred in the long arm of chromosome 7. Molecular analysis using a set of 53 simple sequence repeats (SSRs) indicated that the chromosomal segments of C. hystrix were introduced into 4 cucumber chromosomes, the short arms of chromosomes 2 and 6, and long arms of chromosomes 3 and 7. The inheritance of alien sequences in the long arm of chromosome 7 was investigated with 21 SSRs in self-pollinated progenies. C. hystrix-specific bands of several SSRs were still present in some individuals, indicating that the introgressed segment was partially preserved. The first unambiguous identification of alien chromosome segments in cucumber ILs using combined molecular cytogenetics could facilitate the determination of effects of wild alleles and promote cucumber improvement.     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

Considerations on karyotype evolution in the genera Imparfinis Eigenmann and Norris 1900 and Pimelodella Eigenmann and Eigenmann 1888 (Siluriformes: Heptapteridae)

Heptapteridae is one of the fish families of the order Siluriformes with a wide distribution throughout the basins of the Neotropical region. The genera Imparfinis and Pimelodella comprise few species and/or populations with some chromosome information. Specimens of Imparfinis schubarti, Imparfinis mirini, and Pimelodella meeki from different sites located in the Paranapanema River Basin/PR/Brazil were cytogenetically analyzed. The two species of the genus Imparfinis exhibited 2n = 58 and FN = 116: I. schubarti, with a karyotypic formula of 30m + 28sm and I. mirini with a karyotype formula of 36m + 22sm. P. meeki presented a karyotype of 2n = 46 characterized by 26m + 14sm + 6st and FN = 92, confirming a variability in the 2n of the Heptapteridae family. Both Imparfinis species exhibited interstitial NORs in pair 1, coincident with a secondary constriction; P. meeki presented NORs located in the terminal position on the short arm of pair 17. All AgNORs were coincident with 18S rDNA probe and CMA₃ positive. P. meeki showed a small amount of heterochromatin rich in AT and GC bases. The heterochromatin in I. schubarti was CMA₃ positive. In I. mirini the heterochromatin was DAPI-positive. Furthermore, the long arm of one of the chromosomes of pair 19 revealed the presence of heterochromatic heteromorphism only in male individuals. After meiotic analyses, this heteromorphism could be easily identified in the pachytene and metaphase I stages, and was heteropyknotic and DAPI positive. This feature may be an indication of initial differentiation of sex chromosomes in I. mirini, increasing the great karyotypic variability within this family of fish.     

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit’s performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.     

Chromosome numbers of the Australian Cymodoceaceae

The somatic chromosome numbers of the eleven Australian seagrass species belonging to five genera in the Family Cymodoceaceae were determined. The chromosome numbers in Amphibolis and Thalassodendron are reported for the first time. Cymodocea and Halodule species have the following chromosome numbers: Cymodocea angustata Ostenf., 2n = 14, 28; C. rotundata Ehrenb. & Hempr. ex Asch., 2n = 14; C. serrulata (R. Br.) Asch. & Magnus, 2n = 14, 28; Halodule pinifolia (Miki) den Hartog, 2n = 32; H. uninervis (Forsk.) Asch., 2n = 32; H. tridentata (Steinh.) Endl. ex Unger, 2n = 14. Halodule has the highest chromosome numbers among the seagrasses and they are the largest in sizes with a distinct bimodal type in the family. Syringodium isoetifolium (Asch.) Dandy has 2n = 20. Both endemic Amphibolis antarctica (Labill.) Sonder ex Asch. and A. griffithii (J. Black) den Hartog have 2n = ca. 36 and have the smallest chromosomes in the family appearing as small dots. Thalassodendron pachyrhizum den Hartog has 2n = 28. Chromosome numbers appear to be identical or closely related among different species in the same genus but they vary in the five genera in the Cymodoceaceae suggesting that these five genera may have evolved independently in the past.     

Nucleolar activity in Triticum x Thinopyrum hybrids with different ploidy level

The activity of nucleolar organizer regions (NORs) and the presence of ribosomal DNA (rDNA) zones were studied in two Triticum x Thinopyrum hybrids: a hexaploid hybrid with 2n = 42 chromosomes, named trigopiro SH16 INTA, and a decaploid hybrid with 2n = 56 chromosomes, named trigopiro Don Noé INTA. The use of the pTa71 probe revealed the presence of 10 rDNA signals in both hybrids, whereas the Ag-NORs technique showed 10 signals in SH16 and 8 in Don Noé. We concluded that all trigopiro SH16 INTA NORs are active and that the activity of one NOR pair of trigopiro Don Noé INTA is suppressed. Therefore, the amphiplasty phenomenon is present in trigopiro Don Noé INTA but not in trigopiro SH16 INTA.     

Karyological evidence for interspecific hybridization between Cichla monoculus and C. temensis (Perciformes, Cichlidae) in the Amazon

Cichla monoculus, Cichla temensis (peacock bass or tucunare), and its presumed hybrids, were cytogenetically analyzed. The fish were collected at three distinct sites in the central Amazon basin, namely in the Uatuma (C. monoculus, C. temensis and their natural hybrid), Jau (C. temensis), and Solimoes rivers (C. monoculus). The two species and the natural hybrid showed the same diploid number, 2n=48 acrocentric chromosomes. Single NORs were detected in the distal region of the long arm in all three species. However, in C. monoculus, the NOR was found on the second pair of the complement, in C. temensis, on the third pair and in the hybrid two NOR patterns were found, one on the second pair and the other on the third pair of chromosomes. The two species and the hybrid have their constitutive heterochromatin located in the pericentromeric region of all chromosomes and an interstitial C-band located on the largest chromosome pair. The great similarity in the chromosome number and morphology, chromosome size class differences, the NOR patterns and C-banding suggested chromosomal stasis during speciation and hybridization of Cichla.     

Meiotic behavior and pollen fertility in triploid and tetraploid natural populations of Campuloclinium macrocephalum (Eupatorieae,Asteraceae)

Campuloclinium macrocephalum DC. is a perennial herb widely distributed in the New World and introduced in South Africa, where it is commonly called “pompom weed”. This species is considered one of the most important weeds of Brazil and one of the problematic invasive plants of South Africa. The meiotic system can be studied to assess the ability of a weed to spread, but only few studies on C. macrocephalum have been realized. In this study, we examined the meiotic behavior and pollen fertility of 14 natural populations of C. macrocephalum from Argentina and Uruguay. Meiotic analysis revealed 2 triploid (2n = 3x = 30), 11 tetraploid (2n = 4x = 40) and 1 mixed population (2n = 2x = 20, 2n = 4x = 40). Both, triploid and tetraploid specimens showed a widely variable meiotic behavior with irregular chromosome pairing showing univalents, bivalents, trivalents (in triploids) and tetravalents (in tetraploids) at diacinesis of first meiotic division. Different abnormalities were observed, such as: laggard chromosomes, chromatin bridges, and out of plate chromosomes at metaphase I. During meiosis I (prophase), some cells showed the phenomenon of cytomixis or chromatin transfer between pollen mother cells. The meiotic indexes suggest that only four populations were normally fertile (over 90 % of fertile pollen), indicating meiotically stable plants. The remaining populations share variable pollen fertility, with triploids ranging from 46.64 to 54.83 % and tetraploids varying from 3.54 to 45.30 %. We suggest that polyploidy seems to be recurrent in C. macrocephalum, promoting partial sterility of pollen grains, generating large numbers of individuals by apomixis promoting invasion of crop fields. This study presents the meiotic behavior of this weed, these could be useful for future studies of biological control in areas with no natural enemies.     

Physical localization of rRNA genes by fluorescence in situ hybridization (FISH) and analysis of spacer length variants of 45S rRNA (slvs) genes in some species of genus Sesbania

The somatic chromosome numbers 2n = 12 in S. macrantha, S. coerulescens and 2n = 24 in S. simplicuscula were determined, additionally the contradictory chromosome numbers of S. bispinosa (2n = 12, 13, 14, 24) and S. pachycarpa (2n 12, 14) were determined as 2n = 24 and 2n = 14, respectively. The number of 5S rDNA sites in chromosome pair 1 was highly conserved in all the diploid and tetraploid species studied irrespective of their geographic distribution, suggesting that all diploid and tetraploid species/cytotypes of Sesbania analyzed in the present study are in close proximity. Cytogenetic mapping of the 45S multi-gene family was also carried out using fluorescence in situ hybridization. 45S rDNA was consistently located on short or long arms of two sub-metacentric chromosome pairs except on one chromosome pair in S. macrantha and on three chromosome pairs in S. bispinosa and S. cannabina. Out of these nine species, we observed the homogenization of intergenic spacer in six species and find only one spacer length variant (slv) located on one to three chromosome loci. However, three of the species were observed to have two slvs located on two different chromosomes. The species were grouped as per their evolutionary relationship on the basis of the results of the present study.     

Ploidy variation in Trigonobalanus verticillata (Fagaceae)

Cytological studies were carried out on two populations of Trigonobalanus verticillata in Yinggeling, Hainan Province, China (YGL), and Fraser’s Hill, Malaysia (FH). In the two populations, the pattern of interphase nuclei was of the simple chromocentre type, the mitotic prophases were of the proximal interstitial type and chromosome numbers were 2n = 2x = 14 (YGL) and 2n = 6x = 42 (FH), representing diploid and hexaploid respectively. The basic chromosome number of the species was x = 7; this is a unique number in the Fagaceae and in the Fagales as well. The characteristic karyotype of the diploid population in China was 2n = 14 = 8m + 4sm + 2st. Based on the new finding of the chromosome number in this study, we clarify the previous dispute about the basic chromosome number and propose that the two Asian Trigonobalanus species have closer affinity.     

Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes

Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA₃-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a “leuciscine” cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.     

Ploidy stability in embryogenic cultures and regenerated plantlets of tamarillo

Ploidy levels of short-term (1 and 2 years) and long-term (7 and 10 years) embryogenic cultures as well as of regenerated plantlets of tamarillo were analyzed by flow cytometry and chromosome counts. Embryogenic cultures were induced from expanding leaves cultured in the presence of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) and monthly subcultured on the same media. Embryo development and plantlets were obtained following subculture of the embryogenic tissue in auxin free medium containing gibberellic acid (GA₃). Seedlings and rooted shoots from axillary shoot proliferation were used as controls. The results showed that in long-term embryogenic cultures the ability to develop somatic embryos and plantlets was reduced. Embryogenic tissues maintained for 10 years were mostly aneuploids of the tetraploid (2n = 4x = 48) level whereas those kept in culture for 7 years or less were also mostly aneuploids but of the diploid (2n = 2x = 24) level. The results obtained by flow cytometry were, in general, consistent with those obtained by chromosome counts. The chromosome alteration observed in the embryogenic tissues was already present after 1 year of culture and increased with culture age, hence impairing the maintenance of these tissues for long periods without affecting chromosome stability of the regenerated plantlets. However, the occurrence of triploids and tetraploids as well as aneuploids can be useful for breeding purposes. A value around 23 pg/2C was found for the genome size of tamarillo largely exceeding the value previously published (15.50 pg/2C).     

Tetraploid induction of Mitracarpus hirtus L. by colchicine and its characterization including antibacterial activity

Tetraploid plants were successfully induced for the first time in Mitracarpus hirtus L., by overnight immersion of shoot meristems in 0.1 % colchicine solution, followed by in vitro culture leading to plant regeneration. Examination of ploidy level by flow cytometric analysis and counting chromosome number at metaphase confirmed that original diploid plant (WT1) contained chromosome number as 2n = 2x = 28, whereas 2n = 4x = 56 was observed in the tetraploids induced with colchicine treatment (CC102 and CC110). Thicker root formation, larger stomata (1.3–2 times), and lower density of stomata (1.7–4 times) were observed in these tetraploid plants. After transplantation to the pot, tetraploid plant (CC110) showed higher fresh weights of aerial part and leaves (1.5 and 1.4 times respectively) than diploid. However, the methanolic extracts from leaves of tetraploid line CC102 showed inhibition against human pathogenic bacterium, S. aureus while WT1 and CC110 showed no activity. GC–MS revealed 40 unique compounds present in CC102, but absent in WT1 and CC110. Through hierarchical clustering analysis the 40 unique compounds in CC102 formed a cluster group found to correlate with anti-S. aurens activity. These results suggested that tetraploid M. hirtus CC102 created in this study provides a novel source of compounds useful in fighting infectious disease.     

Molecular cytogenetic characterization of the Amazon River dolphin Inia geoffrensis

Classical and molecular cytogenetic (18S rDNA, telomeric sequence, and LINE-1 retrotransposon probes) studies were carried out to contribute to an understanding of the organization of repeated DNA elements in the Amazon River dolphin (boto, Inia geoffrensis). Twenty-seven specimens were examined, each presenting 2n?=?44 chromosomes, the karyotype formula?12m?+?14sm?+?6st?+?10t?+?XX/XY, and fundamental number (FN)?=?74. C-positive heterochromatin was observed in terminal and interstitial positions, with the occurrence of polymorphism. Interstitial telomeric sequences were not observed. The nucleolar organizer region (NOR) was located at a single site on a smallest autosomal pair. LINE-1 was preferentially distributed in the euchromatin regions, with the greatest accumulation on the X chromosome. Although the karyotype structure in cetaceans is considered to be conserved, the boto karyotype demonstrated significant variations in its formula, heterochromatin distribution, and the location of the NOR compared to other cetacean species. These results contribute to knowledge of the chromosome organization in boto and to a better understanding of karyoevolution in cetaceans.     

Characterization of methicillin-resistant coagulase-negative staphylococci in milk from cows with mastitis in Brazil

Staphylococci are one of the most prevalent microorganisms in bovine mastitis. Staphylococcus spp. are widespread in the environment, and can infect animals and humans as opportunistic pathogens. The objective of this study was to determine the frequency of methicillin-resistance (MR) among coagulase-negative staphylococci (CoNS) previously obtained from milk of mastitic cows in Brazil and to characterize the antimicrobial resistance phenotype/genotype and the SCCmec type of MRCoNS isolates. Identification of MRCoNS was based on both biochemical and molecular methods. Susceptibility testing for eleven antimicrobials was performed by disk-diffusion agar. Antimicrobial resistance genes and SCCmec were investigated by specific PCRs. Twenty-six MRCoNS were detected (20 % of total CoNS), obtained from 24 animals, and were identified as follows: S. epidermidis (7 isolates), S. chromogenes (7), S. warneri (6), S. hyicus (5) and S. simulans (1). All MRCoNS isolates carried mecA while the mecC gene was not detected in any CoNS. The SCCmec IVa was demonstrated in nine MRCoNS, while the remaining 17 isolates harbored non-typeable SCCmec cassettes. In addition to oxacillin and cefoxitin resistance, MRCoNS showed resistance to tetracycline (n = 7), streptomycin (n = 6), tobramycin (n = 6), and gentamicin (n = 4), and harbored the genes tet(K) (n = 7), str (n = 3), ant(4′) (n = 6) and aac(6′)-aph(2″) (n = 4), respectively. In addition, seven strains showed intermediate resistance to clindamycin and two to streptomycin, of which two harboured the lnu(B) and lsa(E) genes and two the aad(E) gene, respectively. One isolate presented intermediate erythromycin and clindamycin resistance and harbored an erm(C) gene with an uncommon 89-bp deletion rendering a premature stop codon. MRCoNS can be implicated in mastitis of cows and they constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria.     

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): comparative chromosome mapping of repetitive sequences

Erythrinus erythrinus presents extensive karyotypic diversity, with four karyomorphs (A–D) differing in the number of chromosomes, karyotype structure or sex chromosomes systems. Karyomorph A has 2n = 54 chromosomes in males and females without heteromorphic sex chromosomes, while karyomorph C has 2n = 52 chromosomes in females and 2n = 51 chromosomes in males, due a X₁X₁X₂X₂/X₁X₂Y sex chromosome system. Three allopatric populations of the karyomorph A and one population of the karyomorph C were now in deep investigated by molecular cytogenetic analyses, using repetitive DNAs as probes. The results reinforced the relatedness among populations of the karyomorph A, despite their large geographic distribution. Karyomorph C, however, showed a remarkably difference in the genomic constitution, especially concerning the amount and distribution of the 5S rDNA and Rex3 sequences on chromosomes. In addition, although karyomorphs C and D share several features, exclusive chromosomal markers show the derivative evolutionary pathway between them. Thus, besides the classical chromosomal rearrangements, the repetitive DNAs were useful tools to reveal the biodiversity, relatedness and differentiation of this fish group. The chromosomal set strongly corroborates that E. erythrinus corresponds to a species complex instead of a single biological entity.