Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re‐arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.     

Failure of interspecies androgenesis in salmonids

Androgenetic development of salmonid embryos was induced in recipient oocytes from the same or other species (intra- or interspecies androgenesis). Parameters for induced androgenesis were investigated in brown trout Salmo trutta and brook trout Salvelinus fontinalis . Reciprocal androgenetic and control crosses were conducted among fishes from three genera: Oncorhynchus (rainbow trout, O. mykiss ), Salmo (brown trout) and Salvelinus (brook trout), and within two genera: Salmo (brown trout and Atlantic salmon, S. salar ) and Salvelinus (brook trout and Arctic charr, S. alpinus ). Live hatched androgenetic progenies were obtained in all intraspecies variants, where oocytes and sperm originated from the same species. Interspecies androgenesis resulted in no viable larvae, despite the fact that most hybrid controls and intraspecies androgenetic individuals were viable. When recipient oocytes originated from other genera (interspecific intergeneric androgenesis), embryonic development ceased in early developmental stages, except for haploid controls of brook trout produced in eggs of brown trout. Survival of embryos to the eyed-egg stage was relatively high in the intrageneric androgenesis experiment. Nevertheless, none of these embryos survived to hatching. Some of the presumed Atlantic salmon individuals developing in brown trout eggs contained maternal DNA, questioning the accuracy of enucleation using irradiation. The inability to induce interspecific androgenesis among the examined salmonid species may have been the result of substantial kariotypical and developmental differences between spermatozoal donors and oocyte recipients, causing an incompatibility between maternal cytoplasmic regulatory factors and the paternal nuclear genome.     

Distribution of Telomeric DNA Sequences on the X-Radiation-Induced Chromosome Fragments Observed in the Genome of Androgenetic Brook Trout ( Salvelinus fontinalis , Mitchill 1814)

Cytogenetic screening of the androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814) offspring hatched from eggs exposed to 420 Gy of X-radiation before insemination exhibited residues of the irradiated maternal nuclear genome in the form of small chromosome fragments. Remnants of the irradiated chromosomes had different sizes, and their number varied intraindividually from 1 to 15. To efficiently pass through the series of the cell divisions, such chromosome fragments must have had functional kinetochores. Distribution patterns of the telomeric hybridization signals on the chromosome fragments enabled us to distinguish their 3 groups: (i) telomere-less ring chromosomes with fused broken chromosome arms, (ii) rings formed in the course of fusion of the radiation-broken chromosome arm with the opposite telomeric region and exhibiting interstitial telomeric signals at the fusion point, and (iii) chromosome fragments with fused unprotected sister chromatids of 1 broken arm and intact telomeres from the other arm. Disturbances during segregation of such fragments, mainly breakages during anaphase, may partially explain intraindividual variation in the number and size of the chromosome fragments observed in the androgenetic brook trout.     

Uniparental chromosome elimination in the early embryogenesis of the inviable salmonid hybrids between masu salmon female and rainbow trout male

Chromosome elimination through chromosome loss and partial deletion is known to be one of the causes of embryonic inviability in some salmonid interspecific hybrids. Using fluorescence in situ hybridization and related techniques, including whole chromosome painting and comparative genomic hybridization, parental origin of eliminated chromosomes was identified in the inviable hybrids between masu salmon (Ms, Oncorhynchus masou) female and rainbow trout (Rb, O. mykiss) male at the early embryonic stage prior to death. In these hybrids, the haploid Rb chromosome number decreased to nearly half, whereas the Ms chromosomes were retained as one or occasionally two full haploid complements. The Rb chromosomes were also involved in the frequently observed fragments and micronuclei. Whereas the occurrence of fragments was constant throughout the observed period, chromosome loss occurred mainly from just after fertilization to the blastulae stage. In tissue sections and cell spreads of late blastula, some Rb chromosomes were trapped in the midzone from ana- to telophase, resulting in micronuclei at the subsequent interphase. Micronuclei and mitotic abnormalities were also observed in the androgenetic haploid hybrids. However, such abnormalities were seldom or never observed in the viable reciprocal hybrids. The present findings suggest that the paternal Rb chromosomes in the inviable hybrids are preferentially eliminated through mitotic abnormalities during early embryogenesis, owing to a possible incompatibility between the maternal Ms cytoplasm and paternal Rb genome. Received: 22 August 1996; in revised form: 14 November 1996 / Accepted: 20 November 1996     

Cadaveric sperm induces intergeneric androgenesis in the fish, Hemigrammus caudovittatus

Intergeneric androgenetic golden Buenos Aires tetra (BT), Hemigrammus caudovittatus was generated using sperm drawn from post-mortem males preserved at -20 degrees C for 10, 20, 30 and 40 days or fresh sperm to activate the UV-irradiated oocytes of black widow tetra (WT), Gymnocorymbus ternetzi. UV-irradiation (4.2 W/m(2)) of the oocytes for 3 min inactivated their nuclear genome. Fry hatched out from these activated oocytes were haploids; suffering haploid syndrome, they died before or within 48 h after hatching. Fresh BT sperm activated 95% oocytes; however, the sperm drawn from post-mortem males preserved at -20 degrees C for 60 (within glycerol packing) and 30 days (without glycerol packing) activated only 24 and 19% oocytes, respectively. Following activation, diploidy was restored by shocking the 25-min-old embryos at 41 degrees C for 2 min. Nuclear genomic inactivation of the oocytes was confirmed by (i) production of 100% haploids, (ii) karyotype and erythrocyte measurements, (iii) phenotypic markers, (iv) progeny testing and (v) species-specific marker. At hatching, survival of androgenotes decreased from 11% for those induced with fresh sperm to 4% for those generated using sperm from 30-day-old post-mortem males. Reproductive performance of the 'fresh' and 'cadaveric' F(0) and F(1) androgenetic males (Y(2)Y(2)) was superior to the control (X(1)Y(2)). Crosses involving homozygous (Y(2)Y(2)) 'fresh' F(0) androgenetic males with heterozygous females (X(1)X(2)) and F(0) homozygous males (Y(2)Y(2)) with females (X(2)X(2)) produced 2-4% unexpected female progenies. Paternal autosomes, inherited by the homozygous androgenetic female (X(2)X(2)), induced the production of female progenies in significantly less number of crosses than the crosses with heterozygous females (X(1)X(2)), which carried equal number of paternal and maternal autosomes. PCR analyses of the genomic DNA of normal male and unexpected F(1) and F(2) female progenies amplified by DMRT 1 specific primer produced bands of 237 and 300 bp length, and thereby confirmed that these unexpected females were genetic males. RAPD analyses of the androgenetic progenies showed that their genome was not contaminated with maternal genome.     

Production of androgenetic diploid rainbow trout

Haploid androgenesis was induced in rainbow trout (Salmo gairdneri) when eggs were irradiated with 60Co gamma radiation prior to fertilization. Diploidy was restored to the androgenetic haploid zygotes by suppression of first cleavage division using hydrostatic pressure. Peak survival in the androgenetic diploid lots (32.5-38.9 percent of control) occurred when a pressure shock of 9000 pounds per square inch lasting from one to three minutes was applied to the eggs 345 minutes post-fertilization. Chromosomal analysis confirmed diploidy in the androgenetic individuals and suggested that YY rainbow trout are viable to at least the "eyed stage" of development. Inheritance patterns at two loci confirmed all-paternal inheritance. The relatively high yields of completely homozygous androgenetic rainbow trout and the potential for the use of androgenesis in the production of inbred lines and in genetic studies indicate that androgenesis may become a valuable tool in fish research and breeding.     

Fate of maternal mtDNA following 60Co inactivation of maternal nuclear DNA in unfertilized salmonid eggs.

The effects of gamma irradiation on nuclear DNA and mitochondrial DNA (mtDNA) were examined by exposing unfertilized salmonid eggs to a 60Co source. Brown trout (Salmo trutta) eggs exposed to 60Co were fertilized with sperm from brook trout (Salvelinus fontinalis), and brook trout eggs exposed to 60Co were fertilized with sperm from splake males (S. namaycush x S. fontinalis). In both types of matings only paternal allozymes were found in embryos, confirming the inactivation of the nuclear genome in the eggs. Analysis of mtDNA in these same embryos showed exclusively maternal mtDNA. The absence of paternal mtDNA among any of the embryos supports the predominance of maternal inheritance of mtDNA in vertebrates and suggests that mtDNAs are more resistant to cobalt inactivation than nuclear DNAs based on structure or numerical superiority to maternal nuclear DNA. Inactivation of maternal nuclear DNA, fertilization, and an induced return to the diploid state provide a means for producing an inbred organism having the nuclear genome of the paternal parent (androgenetic) and the mitochondrial genome of the female.     

Genetic inactivation of Leuciscus idus L. (ide) oocytes using UV irradiation

Oocytes of Leuciscus idus were genetically inactivated using ultraviolet (UV) irradiation. Eggs for the experiment were obtained from dark-coloured females, whereas milt was taken from yellow-coloured (recessive marker) males. The survival at the eleutheroembryo stage (free embryo) in all experimental groups fertilized with genetically inactivated spermatozoa was much lower than in control groups. All haploid embryos showed morphological abnormalities, such as a stunted body and a poorly formed retina, and the condition was referred to as the haploid syndrome. The androgenetic origin (haploid or diploid embryos) was checked using a recessive colour marker ('blond'). The optimal doses of UV irradiation were 3,456-4,608 Jm(-2) at which almost 100% haploid embryos were produced at a hatching rate of >15%. Lower UV-ray doses influenced abnormal embryo development. Ploidy level recognition showed a typical value of mean active nucleoli per cell in haploid and diploid (control fish and spontaneous androgenotes) specimens. Abnormal dark embryos were classified as aneuploids.     

Techniques of chromosome manipulation in rainbow trout: a new evaluation with karyology

Summary Experiments, supported by extensive karyology, were carried out to evaluate the different techniques used for chromosome manipulation in rainbow trout. Eggs, when subjected to early heat shocks, changed from haploidy to diploidy and from diploidy to triploidy. In this respect heat shocks differ from pressure shocks which induce gradual transitions between successive ploidy levels. Sperm treatment with dimethylsulphate yields haploid embryos containing residual sperm chromatin fragments, in contrast to treatment with ultraviolet rays.     

Evolution of karyotype in haploid cell lines of Drosophila melanogaster

Seven continuous cell lines have been established in vitro from lethal embryos produced by the female sterile mutant mh 1182 of Drosophila melanogaster. Six lines show haploid metaphases. Karyotype analysis revealed a high level of aneuploid cells with frequent chromosome fragments. In three lines, haploid cells were quickly overgrown by diploid cells. Two lines were more stable but the proportion of haploid cells decreased with time. One line was stable, showing 80-90% of haploid cells for over 1 000 cell generations. Stable haploid clones have been isolated from two lines. Crossing of mh 1182/mh 1182 females with males bearing a ring X chromosome shows that the haploid genome retained in the cells is of maternal origin and that the diploid cells derive from pre-existing haploid cells. The appearance of the diploid cells and the conditions of karyotypic stability are analysed.     

Robertsonian polymorphism and constitutive heterochromatin distribution in chromosomes of the rainbow trout (Salmo gairdneri).

White-blood-cell culture was used to examine the chromosomes of 53 rainbow trout (Salmo gairdneri) from three locations in the Pacific Northwest of the United States. A Robertsonian chromosome polymorphism is present, resulting in diploid numbers of 60, 59, or 58 in different individuals with 104 chromosome arms. The low level of intraindividual Robertsonian variation, differences in the number of subtelocentric chromosomes between individuals with different chromosome numbers, and frequencies of fish with different chromosome numbers in one population suggest that the interindividual differences are inherited and not somatic. C-banding shows that constitutive heterochromatin is localized near the centromeres and near the secondary constriction one chromosome pair.     

Quantitative trait loci x maternal cytoplasmic environment interaction for development rate in Oncorhynchus mykiss

Effects of maternal cytoplasmic environment (MCE) on development rate in rainbow trout were evaluated within a quantitative trait loci (QTL) analysis framework. Previous research had identified QTL for development rate in doubled haploid (DH) progeny produced from a cross between the Oregon State University (OSU) and the Swanson (SW) River rainbow trout clonal lines. In this study, progeny for QTL mapping were produced from a cross between the OSU and Clearwater (CW) River clonal lines. Doubled haploids were produced from the OSU x CW F1 by androgenesis using eggs from different females (or MCEs); with androgenesis, the maternal nuclear genome was destroyed by irradiation and diploidy was restored by blocking the first embryonic cleavage by heat shock. All embryos were incubated at the same temperature and development rate quantified as time to hatch. Using a linkage map constructed primarily with AFLP markers, QTL mapping was performed, including MCE covariates and QTL x MCE effects in models for testing. The major QTL for development rate in the OSU x SW cross overlaps with the major QTL found in this OSU x CW cross; effects at this locus were the same across MCEs. Both MCE and QTL x MCE effects contribute to variability in development rate, but QTL x MCE were minor and detected only at small-effect QTL.     

Parthenogenetic haploid plants using gamma irradiated pollen in snapmelon (<Emphasis Type="Italic">Cucumis melo</Emphasis> var. <Emphasis Type="Italic">momordica</Emphasis>)

Anthers of snapmelon (Cucumis melo L. var. momordica) were irradiated with varied doses of gamma rays (150, 200, 250, 300 and 350 Gray). Then pollen from irradiated anthers was used for pollinating female flowers. Results revealed that 250 Gray of gamma-irradiation was successful in inducing parthenogenesis and fruit development, whereas, low (150 and 200 Gray) or high (300 and 350 Gray) irradiation doses were not effective in inducing haploid embryos. Embryos at a range of developmental stages were dissected from fruits harvested after 21 days of pollination and cultured on E20A medium. Among these embryos cultured, only cotyledonary embryos germinated into plantlets. Chromosome counting, performed on the roots of regenerated plants, showed the haploid level (n = 12). Ploidy analysis using flow cytometer, measurement of stomatal cells and counting of chloroplast in the guard cells also corroborated the haploid nature of regenerated plants.     

Evaluation of different doses of UV irradiation to loach eggs for genetic inactivation of the maternal genome

Genetic inactivation of the egg nucleus is an indispensable step in the production of androgenetic embryos in teleosts. However, few experimental studies have focused on determining the most effective means of achieving complete inactivation of the maternal genome. Here, we sought to identify the optimum conditions of ultraviolet (UV) irradiation for complete inactivation of the loach egg nucleus. Unfertilized eggs were UV irradiated from above with a dose in the range 0-200 mJ/cm2. Successful inactivation of the maternal genome was evaluated by the exclusive expression of a paternally inherited color phenotype. The presence or absence of putative maternal chromosome fragments was screened by flow cytometry of DNA content and by cytogenetic analysis. The majority of the larvae derived from irradiated eggs had an abnormal appearance. Haploid individuals were detected by measurement of DNA content flow cytometry and by chromosome counting in the groups that received more than 75 mJ/cm2 groups. Although the coefficient of variation of DNA content was apparently reduced in the 125-200 mJ/cm2 groups, chromosome fragments were still detected in all the groups from irradiated eggs. Inactivation of the egg nucleus was also histologically elucidated by the presence or absence of residual nuclear material in anuclear embryos that developed from UV-irradiated eggs fertilized with UV-irradiated sperm. Embryos that were completely or near-completely anuclear were found in the 150 and 200 mJ/cm2 groups. We conclude that the optimum UV dose for complete genetic inactivation of the egg nucleus is more than 150 mJ/cm2.     

Phylogenomics of several deer species revealed by comparative chromosome painting with Chinese muntjac paints

A set of Chinese muntjac (Muntiacus reevesi) chromosome-specific paints has been hybridized onto the metaphases of sika deer (Cervus nippon, CNI, 2n = 66), red deer (Cervus elaphus, CEL, 2n = 62) and tufted deer (Elaphodus cephalophus, ECE, 2n = 47). Thirty-three homologous autosomal segments were detected in genomes of sika deer and red deer, while 31 autosomal homologous segments were delineated in genome of tufted deer. The Chinese muntjac chromosome X probe painted to the whole X chromosome, and the chromosome Y probe gave signals on the Y chromosome as well as distal region of the X chromosome of each species. Our results confirmed that exclusive Robertsonian translocations have contributed to the karyotypic evolution of sika deer and red deer. In addition to Robertsonian translocation, tandem fusions have played a more important role in the karyotypic evolution of tufted deer. Different types of chromosomal rearrangements have led to great differences in the genome organization between cervinae and muntiacinae species. Our analysis testified that six chromosomal fissions in the proposed 2n = 58 ancestral pecoran karyotype led to the formation of 2n = 70 ancestral cervid karyotype and the deer karyotypes is more derived compare with those of bovid species. Combining previous cytogenetic and molecular systematic studies, we analyzed the genome phylogeny for 11 cervid species.     

Does differential selection on the 5S rDNA explain why the rainbow trout sex chromosome heteromorphism is not linked to the SEX locus?

Many but not all rainbow trout strains have morphologically distinguishable sex chromosomes. In these strains, the short arm of the X has multiple copies of 5S rDNA and a bright DAPI band near the centromere, both of which are missing from the Y chromosome, which has a very small short arm. We examined the presence of these markers using fluorescence in situ hybridization (FISH) in four different YY clonal lines derived from different strains and compared the results with sexed fish of the Donaldson strain with the normal X/Y heteromorphism. The Y chromosome in two of the YY clonal lines (Arlee and Swanson) is indistinguishable from the X chromosome and it is positive for 5S rDNA and the DAPI bright band. On the other hand, both 5S rDNA sequences and the DAPI band were not found on the Y chromosome in Hot Creek and Clearwater which have the normal Y. Thus the presence of these two cytogenetic markers may account for the size difference between the short arm of the X and Y chromosome found in most rainbow trout strains. In fishes the expression of one type of 5S rRNA is restricted to oocytes and previous work suggests that although XX males are fairly common, XY females are rare, implying a selective disadvantage for XY females. A hypothesis is presented to explain why this sex chromosome heteromorphism is not closely linked to the SEX locus, which is found on the long arm of the Y chromosome in rainbow trout.     

Recombination is suppressed over a large region of the rainbow trout Y chromosome

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus ( SEX ), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX , was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.     

Induced androgenesis in acipenserids may be obtained by ultraviolet radiation

The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.     

The cutthroat trout Y chromosome is conserved with that of rainbow trout

Five genetic markers previously shown to be located on the sex chromosomes of rainbow trout (Oncorhynchus mykiss) were tested for linkage with the sex locus of Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) in a genetic cross created from a rainbow x cutthroat male hybrid. We show that the sex locus of both rainbow and cutthroat trout is on the same homologous linkage group. Fluorescence in situ hybridization (FISH) using a probe for the microsatellite marker Omm1665, which maps close to the sex locus of Yellowstone cutthroat trout, was used to identify the Y chromosome of cutthroat trout in the hybrid. The Y chromosome of cutthroat trout is sub-telocentric and lacks a DAPI band found on the short arm of the Y chromosome of some rainbow trout males.