Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes.

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.

Laser microdissection was used for the preparation of whole chromosome painting probes in Silurana (Xenopus) tropicalis. Subsequent cross-species fluorescence in situ hybridization (Zoo-FISH) on its tetraploid relative Xenopus laevis revealed persistence of chromosomal quartets even after 50-65 million years of separate evolution. Their arrangement is in a partial concordance with previous experiments based on similarity of a high-resolution replication banding pattern. Further support for an allotetraploid origin of X. laevis was given by hybridization with a probe derived from the smallest X. tropicalis chromosome (Xt10). Here, pericentric areas of both arms of Xl 14 and 18 were stained, indicating intrachromosomal rearrangements. The positions of signals were not in agreement with the chromosomal quartets revealed by painting probes Xt 8 and 9 (Xl 11 + 14 and Xl 15 + 18, respectively). This suggests that both X. tropicalis chromosomes underwent non-reciprocal translocation of Xt10 separately in at least two different ancient ancestors. In addition, the observed translocation events could explain the origin of individuals with 18 chromosomes in diploid karyotypes, probably extinct after the genesis of the allotetraploid X. laevis (2n = 36).

Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged (“homomorphic”) sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used “RAD-tags” and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.

A single copy gene, mitochondrial malate dehydrogenase 2 (Mdh2), was localized on Xenopus tropicalis chromosomes by fluorescence in situ hybridization coupled with tyramide signal amplification (FISH-TSA). The respective cDNA was cloned and sequenced. The labeled probe hybridized with a subcentromeric region of the long arms of homologous chromosomes 3. Results of comparison of the gene localization with previously mapped X. laevis paralogs strongly suggest a common evolutionary origin of chromosomes 3 and 8 in X. laevis and chromosome 3 in X. tropicalis. This is the first time that a single copy gene has been visualized on X. tropicalis chromosomes. The FISH-TSA method gives a strong signal with a 1-kb labeled probe.

BackgroundGenomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly.ResultsWe laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly.ConclusionWe present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds.

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70-160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0-1.4 micrograms/10(7) sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25-30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed.

Growth is a priority trait from the point of view of genetic improvement. Molecular markers linked to quantitative trait loci (QTL) have been regarded as useful for marker-assisted selection in complex traits as growth. Polymorphisms have been studied in five candidate genes influencing growth in gilthead seabream (Sparus aurata): the growth hormone (GH), insulin-like growth factor-1 (IGF-1), myostatin (MSTN-1), prolactin (PRL), and somatolactin (SL) genes. Specimens evaluated were from a commercial broodstock comprising 131 breeders (from which 36 males and 44 females contributed to the progeny). In all samples eleven gene fragments, covering more than 13,000 bp, generated by PCR-RFLP, were analyzed; tests were made for significant associations between these markers and growth traits. ANOVA results showed a significant association betweenMSTN-1gene polymorphism and growth traits. Pairwise tests revealed several RFLPs in theMSTN-1gene with significant heterogeneity of genotypes among size groups.PRLandMSTN-1genes presented linkage disequilibrium. TheMSTN-1gene was mapped in the centromeric region of a medium-size acrocentric chromosome pair.