Sex-linked markers in the North American green frog (Rana clamitans) developed using DArTseq provide early insight into sex chromosome evolution.

BACKGROUND: The extent to which sex reversal is associated with transitions in sex determining systems (XX-XY, ZZ-ZW, etc.) or abnormal sexual differentiation is predominantly unexplored in amphibians. This is in large part because most amphibian taxa have homomorphic sex chromosomes, which has traditionally made it challenging to identify discordance between phenotypic and genetic sex in amphibians, despite all amphibians having a genetic component to sex determination. Recent advances in molecular techniques such as genome complexity reduction and high throughput sequencing present a valuable avenue for furthering our understanding of sex determination in amphibians and other taxa with homomorphic sex chromosomes like many fish and reptiles. RESULTS: We use DArTseq as a novel approach to identify sex-linked markers in the North American green frog (Rana clamitans melanota) using lab-reared tadpoles as well as wild-caught adults from seven ponds either in undeveloped, forested habitats or suburban ponds known to be subject to contamination by anthropogenic chemicals. The DArTseq methodology identified 13 sex-linked SNP loci and eight presence-absence loci associated with males, indicating an XX-XY system. Both alleles from a single locus show partial high sequence homology to Dmrt1, a gene linked to sex determination and differentiation throughout Metazoa. Two other loci have sequence similarities to regions of the chimpanzee and human X-chromosome as well as the chicken Z-chromosome. Several loci also show geographic variation in sex-linkage, possibly indicating sex chromosome recombination. While all loci are statistically sex-linked, they show varying degrees of female heterozygosity and male homozygosity, providing further evidence that some markers are on regions of the sex chromosomes undergoing higher rates of recombination and therefore further apart from the putative sex determining locus. CONCLUSION: The ease of the DArTseq platform provides a useful avenue for future research on sex reversal and sex chromosome evolution in vertebrates, particularly for non-model species with homomorphic or cryptic or nascent sex chromosomes.

Differential gene expression in Iberian green frogs (Pelophylax perezi) inhabiting a deactivated uranium mine.

Iberian green frogs (Pelophylax perezi) were found inhabiting a deactivated uranium mine, especially an effluent pond, seriously contaminated with metals and radionuclides. These animals were previously assessed for oxidative stress parameters and did not revealed significant alterations. In order to better understand which mechanisms may be involved in the ability to withstand permanent contamination gene expression analysis was performed in the liver, through suppression subtractive hybridization (SSH). The SSH outcome in the liver revealed the up-regulation of genes coding for the ribosomal protein L7a and for several proteins typical from blood plasma: fibrinogen, hemoglobin and albumin. Besides their normal function, some of these proteins can play an important role as protective agents against oxidative stress. This work provides new insights on possible basal protection mechanisms that may act in organisms exposed chronically to contamination.

Construction and Validation of a Dual-Transgene Vector System for Stable Transformation in Plants.

In this study, we constructed dual-transgene vectors (pDT1, pDT7, and pDT7G) that simultaneously co-expressed two genes in plants. ACTIN2 and UBQ10 promoters were used to control the expression of these two genes. The 4×Myc, 3×HA, and 3×Flag reporter genes allowed for the convenient identification of a tunable co-expression system in plants, whereas the dexamethasone (Dex) inducible reporter gene C-terminus of the glucocorticoid receptor (cGR) provided Dex-dependent translocation of the fusion gene between the nucleus and cytoplasm. The function of pDT vectors was validated using four pairwise genes in Nicotiana benthamiana or Arabidopsis thaliana. The co-expression efficiency of two genes from the pDT1 and pDT7G vectors was 35% and 42%, respectively, which ensured the generation of sufficient transgenic materials. These pDT vectors are simple, reliable, efficient, and time-saving tools for the co-expression of two genes through a single transformation event and can be used in the study of protein-protein interactions or multi-component complexes.

Complete mitochondrial genome of the green odorous frog Odorrana margaretae (Anura: Ranidae).

The complete mitochondrial genome (mitogenome) of the green odorous frog Odorrana margaretae (Anura: Ranidae) has been studied. The 17,903 bp circular genome contains the typical complement of 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. The AT content of the overall base compositon of H-strand is 56% and the length of control region is 2501 bp with 63.8% AT content. The arrangement of the protein-coding and ribosomal RNA genes was the same as that found in other anurans. The cluster of rearranged LTPF tRNA genes and the translocation of tRNA(His) gene into the D-loop region are observed.

Transcriptome profile of the green odorous frog (Odorrana margaretae).

Transcriptome profiles provide a practical and inexpensive alternative to explore genomic data in non-model organisms, particularly in amphibians where the genomes are very large and complex. The odorous frog Odorranamargaretae (Anura: Ranidae) is a dominant species in the mountain stream ecosystem of western China. Limited knowledge of its genetic background has hindered research on this species, despite its importance in the ecosystem and as biological resources. Here we report the transcriptome of O. margaretae in order to establish the foundation for genetic research. Using an Illumina sequencing platform, 62,321,166 raw reads were acquired. After a de novo assembly, 37,906 transcripts were obtained, and 18,933 transcripts were annotated to 14,628 genes. We functionally classified these transcripts by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 11,457 unique transcripts were assigned to 52 GO terms, and 1,438 transcripts were assigned to 128 KEGG pathways. Furthermore, we identified 27 potential antimicrobial peptides (AMPs), 50,351 single nucleotide polymorphism (SNP) sites, and 2,574 microsatellite DNA loci. The transcriptome profile of this species will shed more light on its genetic background and provide useful tools for future studies of this species, as well as other species in the genus Odorrana. It will also contribute to the accumulation of amphibian genomic data.

Specific time of exposure during tadpole development influences biological effects of the insecticide carbaryl in green frogs (Lithobates clamitans).

The orchestration of anuran metamorphosis is initiated and integrated by thyroid hormones, which change dynamically during larval development and which may represent a target of disruption by environmental contaminants. Studies have found that some anurans experience increased rates of development when exposed to the insecticide carbaryl later in larval development, suggesting that this insecticide could affect thyroid hormone-associated biological pathways. However, the time in development when tadpoles are sensitive to insecticide exposure has not been clearly defined nor has the mechanism been tested. In two separate studies, we exposed recently hatched green frog (Lithobates clamitans) tadpoles to a single, three day carbaryl exposure in the laboratory at either 2, 4, 8, or 16 weeks post-hatching. We examined the impact of carbaryl exposure on mRNA abundance patterns in the brains of frogs following metamorphosis months after a single three day exposure (experiment 1) and in tadpole tails three days after exposure (experiment 2) using cDNA microarrays and quantitative real time polymerase chain reaction (QPCR) analyses. For tadpoles reared through metamorphosis, we measured tadpole growth and development, as well as time to, mass at, and survival to metamorphosis. Although carbaryl did not significantly impact tadpole development, metamorphosis, or survival, clear exposure-related alterations in both tail and brain transcript levels were evident when tadpoles were exposed to carbaryl, particularly in tadpoles exposed at weeks 8 and 16 post-hatching, indicating both short-term and long-term alterations in mRNA expression. These results indicate that carbaryl can have long-lasting effects on brain development when exposure occurs at sensitive developmental stages, which may have implications for animal fitness and function later in the life cycle.