Chris Murdock

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

BackgroundThrough the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energys Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.ResultsA total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.ConclusionsThis project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions

Real-time PCR is a highly sensitive, relatively easy to perform assay for quantifying mRNA abundance. However, there are several complexities built into the assay that can affect data interpretation. Most notably, the selection of an appropriate internal control for normalization is essential for expression data interpretation. In this study we investigated the suitability of seven commonly used genes [18S ribosomal RNA (18S), alpha tubulin (TUBA), beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), and RNA polymerase II polypeptide B (POLR2B)] as potential quantitative references for normalizing real-time PCR data generated in the study of channel catfish physiology. Gene expression and stability were evaluated among 15 channel catfish tissues and within physiologically-relevant tissues in response to experimental manipulation (i.e. LHRH injection, fasting, and acute stress). Expression of the seven candidate reference genes varied across all tissue types tested, indicating that none of the genes could suitably serve as reference genes for cross tissue comparisons. Experimentally altering the physiological state of the fish differentially affected expression of the various reference genes depending on experimental design and tissue type, with 18S unaffected by the experimental treatment in all tissues examined. For example, the selection of a differentially expressed gene, GAPDH, as opposed to 18S, to normalize hepatic growth hormone receptor during fasting resulted in misinterpretation of the data. These results reveal the importance of providing comprehensive details of reference gene validation when publishing real-time PCR results, with this manuscript serving as a basic guideline for reference gene selection in channel catfish research.

Cloning and expression of aromatase in a turtle with temperature-dependent sex determination

It has been hypothesized that estrogen production may play a pivotal role in the sex determination of reptiles with temperature-dependent sex determination (TSD). This hypothesis has been furthered by studies that have shown higher aromatase activity in the developing ovaries in some reptiles. However, other studies have not consistently supported this hypothesis. In the current study we addressed this issue by cloning P450 aromatase cDNA in the turtle, Trachemys scripta, and developing a quantitative competitive RT-PCR for aromatase. This assay was then used to quantify aromatase mRNA levels in adrenal-kidney-gonad complexes (AKG) during TSD. Aromatase mRNA was detected in the AKGs at both male- and female-producing temperatures from the earliest stage of development sampled (stage 15), through hatching (stage 26). However, levels remained relatively constant during the thermosensitive period of TSD. Further, no significant difference was detected between male- and female-producing temperatures during the thermosensitive period. After the thermosensitive period, aromatase mRNA levels increased in females (this coincides with the period during which the ovaries are differentiating). These results are consistent with those of several previous studies of certain reptiles with TSD. The current results suggest that the expression of aromatase may not be a pivotal regulatory step in the sex determination cascade of this turtle.

Expression of Dmrt1 in a turtle with temperature-dependent sex determination

There is a variety of sex determining mechanisms among vertebrates. Many reptiles possess temperature-dependent sex determination (TSD), in which the incubation temperature of the egg determines the sex of the hatchling. The red-eared slider turtle, Trachemys scripta has often been used as a model system for examining the physiology of TSD. In the current study, the expression of Dmrt1 was examined during TSD in this turtle. Dmrt1 is a putative regulator of sex determination/differentiation and has been identified in a variety of vertebrates, including fishes, amphibians, reptiles, birds, and mammals. Specifically, Dmrt1 has been shown to be up-regulated in a male-specific pattern during embryonic development in many vertebrates. In the current study, the expression patterns of Dmrt1 were examined in the developing adrenal-kidney-gonad complexes of T. scripta during embryonic development. Using a quantitative competitive RT-PCR, Dmrt1 was shown to be up-regulated during the thermosensitive period of sex determination in males. In contrast, levels of Dmrt1 remained low in females throughout the thermosensitive period. These data suggest that the up-regulation of Dmrt1 may play a role in male sex determination/sex differentiation during TSD in T. scripta.

Interclutch Variation in Sex Ratios Produced at Pivotal Temperature in the Red-eared Slider, a Turtle With Temperature-dependent Sex Determination

Abstract Many reptiles exhibit temperature-dependent sex determination (TSD) in which the incubation temperature of the egg determines the sex of the embryo. The purpose of the current study was to examine intraspecific variation in temperature sensitivity during TSD. Two aspects of temperature sensitivity were evaluated in the Red-Eared Slider, Trachemys scripta: (1) clutch-specific variation in sex ratios produced at pivotal temperature and (2) variation in clutch sex ratios produced over the nesting season (e.g., early nesting season, midnesting season, late nesting season). Relatively large numbers of clutch sex ratios were examined from two or three time periods (e.g., early nesting season, midnesting season, late nesting season), during each of three nesting seasons. The results indicate that pivotal temperatures vary significantly among clutches, with clutch sex ratios ranging from all male to all female when incubated near the putative pivotal temperature. The results on seasonal variation in clutch sex ratios were more ambiguous. Data from two of the nesting seasons do not reveal significant variation between time periods. During the third nesting season, sex ratios from the first two time periods were not significantly different from one another, but the sex ratios from the very end of the nesting season were significantly different from those of the two earlier time periods. In summary, the results reveal a clutch-specific variation in pivotal temperatures, but the results from the seasonal study of sex ratios did not consistently reveal a shift in sex ratios over each nesting season.

Relationship between Habitat Type, Fire Frequency, and Amblyomma americanum Populations in East-Central Alabama

ABSTRACT: Ticks were collected from 20 sites in the Calhoun, Cherokee, and Cleburne Counties in east-central Alabama areas to determine the relationship between plant physiognomy, environmental variables, and tick populations. Sites investigated included various burning regimes, wildland-urban—interface (WUI), a college campus, and an unmanaged area. Amblyomma americanum (L.) (Acari: Ixodidae) dominated the tick population while Ixodes scapularis Say was not encountered. There were complex differences in tick populations among site conditions. After prescribed burning, the tick population size was small but was larger in subsequent 2- and 5-year post-burn sites. An increase in Odocoileus virginianus foraging in recently burned sites is likely responsible for this phenomenon. WUI areas had the largest tick populations likely due to Odocoileus virginianus activity in an area that provides cover, forage, and a connection to a wildlife refuge. It is possible that the likelihood of humans coming in contact with ticks and tick-borne diseases is greater in WUI areas than in unbroken contiguous forest. A. americanum showed a positive correlation with percent cover of grass and leaf litter mass and a negative relationship with pine sapling density. Variables expected to be strongly correlated with A. americanum populations such as soil moisture, canopy closure, and tree density were found to have weak correlations.

Comparison of the microbial diversity and abundance between the freshwater land-locked lakes of Schirmacher Oasis, and the perennially ice-covered Lake Untersee in East Antarctica

Extreme conditions such as low temperature, dryness, and constant UV-radiation in terrestrial Antarctica are limiting factors to the survival of microbial populations. The objective of this study was to investigate the microbial diversity and enumeration between the open water lakes of Schirmacher Oasis and the permanently ice-covered Lake Untersee. The lakes in Schirmacher Oasis possessed an abundant and diverse group of microorganisms compared to Lake Untersee. Furthermore, the microbial diversity between two lakes in Schirmacher Oasis (Lake L27C and L47) was compared by culture-based molecular approach. It was determined that L27C had a richer microbial diversity representing 4 different phyla and 7 different genera. In contrast L47 consisted of 3 different phyla and 6 different genera. The difference in microbial community could be due to the wide range of pH between L27C (pH 9.1) and L47 (pH 5.7). Most of the microbes isolated from these lakes consisted of adaptive biological pigmentation. Characterization of the microbial community found in the freshwater lakes of East Antarctica is important because it gives a further glimpse into the adaptation and survival strategies found in extreme conditions.