Flow cytometric determination of genome size in European sunbleak Leucaspius delineatus (Heckel, 1843)

The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak’s erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 μm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 μm2, respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

<p>Abstract</p> <p>Background</p> <p>Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution.</p> <p>Methods</p> <p>Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone.</p> <p>Results</p> <p>Chromosome number, <it>Cot</it>-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A.</p> <p>Conclusions</p> <p>The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability. Based on the present and previous findings, we discuss the association of rapid genetic changes and high genetic diversity with various ploidy levels and multiple reproduction modes in several unisexual and sexual complexes of vertebrates and even other invertebrates.</p

Quantifying the threat of extinction from Muller's ratchet in the diploid Amazon molly (Poecilia formosa)

BACKGROUND: The Amazon molly (Poecilia formosa) is a small unisexual fish that has been suspected of being threatened by extinction from the stochastic accumulation of slightly deleterious mutations that is caused by Muller's ratchet in non-recombining populations. However, no detailed quantification of the extent of this threat is available. RESULTS: Here we quantify genomic decay in this fish by using a simple model of Muller's ratchet with the most realistic parameter combinations available employing the evolution@home global computing system. We also describe simple extensions of the standard model of Muller's ratchet that allow us to deal with selfing diploids, triploids and mitotic recombination. We show that Muller's ratchet creates a threat of extinction for the Amazon molly for many biologically realistic parameter combinations. In most cases, extinction is expected to occur within a time frame that is less than previous estimates of the age of the species, leading to a genomic decay paradox. CONCLUSION: How then does the Amazon molly survive? Several biological processes could individually or in combination solve this genomic decay paradox, including paternal leakage of undamaged DNA from sexual sister species, compensatory mutations and many others. More research is needed to quantify the contribution of these potential solutions towards the survival of the Amazon molly and other (ancient) asexual species

A vertebrate reproductive system involving three ploidy levels : hybrid origin of triploids in a contact zone of diploid and tetraploid green toads (Bufo viridis subgroup)

The rise and consequences of polyploidy in vertebrates, whose origin was associated with genome duplications, may be best studied in natural diploid and polyploid populations. In a diploid/tetraploid (2n/4n) geographic contact zone of Palearctic green toads in northern Kyrgyzstan, we examine 4ns and triploids (3n) of unknown genetic composition and origins. Using mitochondrial and nuclear sequence, and nuclear microsatellite markers in 84 individuals, we show that 4n (Bufo pewzowi) are allopolyploids, with a geographically proximate 2n species (B. turanensis) being their maternal ancestor and their paternal ancestor as yet unidentified. Local 3n forms arise through hybridization. Adult 3n mature males (B. turanensis mtDNA) have 2n mothers and 4n fathers, but seem distinguishable by nuclear profiles from partly aneuploid 3n tadpoles (with B. pewzowi mtDNA). These observations suggest multiple pathways to the formation of triploids in the contact zone, involving both reciprocal origins. To explain the phenomena in the system, we favor a hypothesis where 3n males (with B. turanensis mtDNA) backcross with 4n and 2n females. Together with previous studies of a separately evolved, sexually reproducing 3n lineage, these observations reveal complex reproductive interactions among toads of different ploidy levels and multiple pathways to the evolution of polyploid lineages

Distribution and stability of supernumerary microchromosomes in natural populations of the Amazon molly, Poecilia formosa.

In animals, supernumerary chromosomes and their evolution have mostly been studied in sexual reproducing species. In the present study, for the first time, the natural distribution and stability of supernumerary microchromosomes were investigated in the unisexual fish species Poecilia formosa. Natural habitats throughout the range of P. formosa were screened for the presence of microchromosomes over several years. A high frequency of microchromosomes was found in the Río Purificación river system. Evidence points to the presence of the same microchromosome lineage over many generations. No supernumerary chromosomes were found elsewhere than in the Río Purificación representing a significant difference in the distribution of microchromosome-bearing individuals between the Río Purificación and all other collection sites

Isolation of a Cancer-Associated Microchromosome in the Sperm-Dependent Parthenogen Poecilia formosa

In the asexual all-female fish species Poecilia formosa, the Amazon molly, supernumerary chromosomes have frequently been found in both laboratory-reared and wild-caught individuals. While wild-caught individuals with B chromosomes are phenotypically indifferent from conspecifics, individuals carrying B chromosomes from recent introgression events in the laboratory show phenotypic changes. Former analyses showed that the expression of a pigment cell locus is associated with the presence of these B chromosomes. In addition, they contain a so far unidentified locus that confers a higher susceptibility to tumor formation in the presence of pigmentation pattern. Isolation by microdissection and hybridization to metaphase chromosomes revealed that they contain one or several sequences with similarity to a highly repetitive pericentromeric and subtelomeric sequence in A chromosomes. Isolation of one particular sequence by AFLP showed that the B chromosomes contain at least 1 copy of an A-chromosomal region which is highly conserved in the whole genus Poecilia, i.e. more than 5 million years old. We propose it to be a single copy sequence