Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

Application of infrared thermal technology to assess the level of heat stress and milk yield reduction of cows in tropical smallholder dairy farms.

Panting score (PS) is a common research tool used to assess the physiological state of cows exposed to heat stress, but it is subjective. Infrared temperature (IRT), measured by either infrared thermometers or cameras, may be a more objective and reliable alternative. Very few studies thus far have evaluated the associations between PS, IRT, and milk production. We investigated the applicability of IRT compared with PS as a means of assessing heat stress and milk yield reduction in dairy cows in tropical smallholder dairy farms (SDF). In autumn 2017, SDF located across 4 typical dairy regions of Vietnam were each visited once to collect farm (n = 32) and individual cow data (n = 344). For each SDF, heat load index (HLI) inside the cowsheds, an indicator of environmental heat load calculated from ambient temperature, humidity, and wind speed, was measured. For each cow, PS (0 indicates a cow breathing normally, not panting; 4.5 indicates an extremely heat-stressed cow with excessive panting, tongue fully extended, and excessive drooling), IRT of the cow's body, single-day energy-corrected milk yield (ECM), body weight, and body condition score were measured. Cow genotype, age, lactation number, and days in milk were recorded. The IRT of the cows' inner vulval lip (IVuT) were measured with an infrared thermometer; and the IRT of the cows' vulval surface (OVuT), inner tail base surface (ITBT), ocular area, muzzle, armpit area, paralumbar fossa area, fore udder, rear udder, fore hoof, and hind hoof were also measured with an infrared camera. Multivariate mixed-effects models were used to assess the associations between HLI with PS and IRT, and associations between PS and IRT with ECM while accounting for the effects of other cow variables. All IRT correlated positively with PS (Pearson correlation, r = 0.23-0.50). Each unit increase in HLI was associated with increases of 0.07 units in PS and 0.09 to 0.23°C in IRT. Each degree (°C) increase in IVuT, OVuT, and ITBT was associated with decreases of 0.75, 0.87, and 0.70 kg/cow per day in ECM, respectively, whereas PS and other IRT were not significantly associated with ECM. Thus, all IRT showed potential to assess the heat stress level of cows; and IVuT, OVuT, and ITBT, but not PS and other IRT, showed potential to predict ECM reduction in cows during heat stress. First cross (F1) Holstein Brown Swiss and F1 Holstein Jersey showed lower PS and yielded higher ECM than the third backcross (B3) Holstein Zebu (7/8 Holstein + 1/8 Zebu) and pure Holstein. Thus, F1 Holstein Brown Swiss and F1 Holstein Jersey could be more suitable for tropical SDF than B3 Holstein Zebu and pure Holstein.

Multivariate analysis identifying the main factors associated with cow productivity and welfare in tropical smallholder dairy farms in Vietnam.

This study aimed to rank potential drivers of cow productivity and welfare in tropical smallholder dairy farms (SDFs) in Vietnam. Forty-one variables were collected from 32 SDFs located in four geographically diverse dairy regions, with eight SDFs per region. Twelve variables, including milk yield (MILK), percentages of milk fat (mFA), protein (mPR), dry matter (mDM), energy-corrected milk yield (ECM), heart girth (HG), body weight (BW), ECM per 100 kg BW (ECMbw), body condition score (BCS), panting score (PS), inseminations per conception (tAI), and milk electrical resistance (mRE) of cows, were fitted as outcome variables in the models. Twenty-one other variables describing farm altitude, housing condition, and diet for the cows, cow genotypes, and cow physiological stage were fitted as explanatory variables. Increased farm altitude was associated with increases in ECM and mRE and with decreases in PS and tAI (P < 0.05). Increases in roof heights and percentage of shed side open were associated with increases in ECM, mFA, and mDM (P < 0.05). Increased dry matter intake and dietary densities of dry matter and fat were associated with increased MILK, ECM, and ECMbw and decreased tAI (P < 0.05). Increased dietary lignin density was associated with increased PS. Increased genetic proportion of Brown Swiss in the herd was associated with increased MILK, ECM, and ECMbw (P < 0.05). Thus, to improve cow productivity and welfare in Vietnamese SDFs, the following interventions were identified for testing in future cause-effect experiments: increasing floor area per cow, roof heights, shed sides open, dry matter intake, dietary fat density, and the genetic proportion of Brown Swiss and decreasing dietary lignin density.

Selection of Genome-Wide SNPs for Pooled Allelotyping Assays Useful for Population Monitoring.

Parasitic worms are serious pests of humans, livestock, and crops worldwide. Multiple management strategies are employed in order to reduce their impact, and some of these may affect their genome and population allelic frequency distribution. The evolution of chemical resistance, ecological changes, and pest dispersal has allowed an increasing number of pests to become difficult to control with current management methods. Their lifestyle limits the use of ecological and individual-based management of populations. There is a need to develop rapid, affordable, and simple diagnostics to assess the efficacy of management strategies and delay the evolution of resistance to these strategies. This study presents a multilocus, equal-representation, whole-genome pooled single nucleotide polymorphisms (SNPs) selection approach as a monitoring tool for the ovine nematode parasite Haemonchus contortus. The SNP selection method used two reference genomes of different quality, then validated these SNPs against a high-quality recent genome assembly. From over 11 million high-quality SNPs identified, 334 SNPs were selected, of which 262 were species-specific, yielded similar allele frequencies when assessed as multiple individuals or as pools of individuals, and suitable to distinguish mixed nematode isolate pools from single isolate pools. As a proof-of-concept, 21 Australian H. contortus populations with various phenotypes and genotypes were screened. This analysis confirmed the overall low level of genetic differentiation between populations collected from the field, but clearly identifying highly inbred populations, and populations showing genetic signatures associated with chemical resistance. The analysis showed that 66% of the SNPs were necessary for stability in assessing population genetic patterns, and SNP pairs did not show linkage according to allelic frequencies across the 21 populations. This method demonstrates that ongoing monitoring of parasite allelic frequencies and genetic changes can be achieved as a management assessment tool to identify drug-treatment failure, population incursions, and inbreeding signatures due to selection. The SNP selection method could also be applied to other parasite species.

Genomic diversity and breed composition of Vietnamese smallholder dairy cows.

Vietnamese smallholder dairy cows (VDC) are the result of crossbreeding between different zebu (ZEB) and taurine dairy breeds through many undefined generations. Thus, the predominant breed composition of VDC is currently unknown. This study aimed to evaluate the level of genetic diversity and breed composition of VDC. The SNP data of 344 animals from 32 farms located across four dairy regions of Vietnam were collected and merged with genomic reference data, which included three ZEB breeds: Red Sindhi, Sahiwal and Brahman, three taurine breeds: Holstein (HOL), Jersey (JER) and Brown Swiss (BSW), and a composite breed: Chinese Yellow cattle. Diversity and admixture analyses were applied to the merged data set. The VDC were not excessively inbred, as indicated by very low inbreeding coefficients (Wright's FIS ranged from -0.017 to 0.003). The genetic fractions in the test herds suggested that the VDC are primarily composed of HOL (85.0%); however, JER (6.0%), BSW 5.3%) and ZEB (4.5%) had also contributed. Furthermore, major genotype groupings in the test herds were pure HOL (48%), B3:15/16HOL_1/16ZEB (22%) and B2:7/8HOL_1/8ZEB (12%). The genetic makeup of the VDC is mainly components of various dairy breeds but also has a small percentage of ZEB; thus, the VDC could be a good genetic base for selecting high milk-producing cows with some degree of adaptation to tropical conditions.

Assessment of Performance and Some Welfare Indicators of Cows in Vietnamese Smallholder Dairy Farms.

Smallholder dairy farms (SDFs) are distributed widely across lowland and highland regions in Vietnam, but data on the productivity and welfare status of these cows remains limited. This cross-sectional study was conducted to describe and compare the productivity and welfare status of SDF cows across contrasting regions. It was conducted in autumn 2017 on 32 SDFs randomly selected from four typical but contrasting dairy regions (eight SDFs per region); a south lowland, a south highland, a north lowland, and a north highland region. Each farm was visited over a 24-h period (an afternoon followed by a morning milking and adjacent husbandry activities) to collect data of individual lactating cows (n = 345) and dry cows (n = 123), which included: milk yield and concentrations, body weight (BW), body condition score (BCS, 5-point scale, 5 = very fat), inseminations per conception, and level of heat stress experienced (panting score, 4.5-point scale, 0 = no stress). The high level of heat stress (96% of lactating cows were moderate to highly heat-stressed in the afternoon), low energy corrected milk yield (15.7 kg/cow/d), low percentage of lactating cows (37.3% herd), low BW (498 and 521 kg in lactating and dry cows, respectively), and low BCS of lactating cows (2.8) were the most important productivity and welfare concerns determined and these were most serious in the south lowland. By contrast, cows in the north lowland, a relatively hot but new dairying region, performed similarly to those in the south highland; a region historically considered to be one of the most suitable for dairy cows in Vietnam due to its cool environment. This indicates the potential to mitigate heat stress through new husbandry strategies. Cows in the north highland had the highest BW (535 and 569 kg in lactating and dry cows, respectively) and the highest energy corrected milk yield (19.2 kg/cow/d). Cows in all regions were heat-stressed during the daytime, although less so in the highlands compared to the lowlands. Opportunities for research into improving the productivity and welfare of Vietnamese SDF cows are discussed.

Comparison of Genetic Merit for Weight and Meat Traits between the Polled and Horned Cattle in Multiple Beef Breeds.

Breeding for polled animals is deemed the most practical solution to eradicate horns naturally and circumvent management costs and risks on health and welfare. However, there has been a historical reluctance by some farmers to select polled animals due to perceived lower productivity of their calves. This study has compared estimated breeding values (EBVs) between horned and polled animals (N = 2,466,785) for 12 production and carcass traits to assess historical (before 2000) and recent (2000-2018) genetic implications of poll breeding. Older generations of the polled animals in most breeds had significantly lower (Bonferroni-corrected p = 0.05) genetic merits for live (birth to maturity) and carcass weights, milk, meat quality, and fat content traits. Substantial gains of genetic potential were achieved during 2000 to 2018 in each breed, such that polled animals have significantly improved for the majority of traits studied. Generally, polled cohorts showed advantageous EBVs for live and carcass weights irrespective of the lower birth weights in some breeds. While Polled Brahman showed inferior production parameters, the poll genetics' effect size (d) and correlation (r) were very small on recent birth weight (d = -0.30, r = -0.08), 200 days (-0.19, -0.05), 400 days (-0.06, -0.02), 600 days (-0.05, -0.01), mature cow live weight (-0.08, -0.02), and carcass weight (-0.19, -0.05). In conclusion, although there is some evidence that historical selection for polled breeding animals may have reduced productivity, there is strong evidence that more recent selection for polled genotypes in the breeds studied has not resulted in any adverse effects on genetic merit.

Issues of Feeding Strategy for Lactating Cows in Vietnamese Smallholder Dairy Farms.

A limited literature suggests relatively simple feeding regimes and diet formulation strategies for dairy cows in Vietnamese smallholder dairy farms (SDFs). This study aimed to classify and compare feeding regimes and nutrient balance for lactating cows between four typical dairy regions (south lowland, south highland, north lowland, and north highland) in Vietnam and evaluate the possibility of systematic dietary imbalance. Eight SDFs from each of the four regions were visited for two adjacent milking periods per farm. For each visit, frequency and methods of feed and water supply to the lactating cows were recorded, and individual fat corrected milk yield (ECM) of lactating cows were calculated from milk yield and fat concentration. The amount of each diet ingredient offered and refused by each lactating group was weighed and sampled for calculation of dry matter intake per cow (DMI) and analysis of nutrient composition in the component offered. PCDairy, a diet formulation computer model, was used to calculate actual and recommended dietary nutrient concentrations and predict potential milk production. Factor analysis, cluster analysis, and ANOVA were applied to determine grouping effects across as well as between regions. Feeding regimes and diets were grouped into three and nine clusters, respectively. Farmers in the same region tended to apply similar diets and feeding regimes. Across regions, only 47% of all SDFs supplied water ad libitum to the cows. The most used roughages including Napier grass, corn silage, fresh corn with cob, and rice straw were all relatively high in neutral detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin (ADL). The diets in all regions were excessive in crude protein, NDF, ADF, ADL, and most minerals (Ca, P, Mg, K, Na, S, Fe, Zn, Cu, and Mn) but insufficient in net energy and non-fibre carbohydrate. Feed efficiency (1.06 kg FCM/kg DMI) of the diets were sub-optimal. Feeding regimes and dietary nutrient balance of the south lowland SDFs were most problematic. Increasing dietary net energy concentration by increasing the use of starch and fat and decreasing dietary fibre concentration by decreasing the use of Napier grass or rice straw to balance the diets might help improve the milk production and thereby increase feed efficiency.

Characteristics of Cowsheds in Vietnamese Smallholder Dairy Farms and Their Associations with Microclimate-A Preliminary Study.

In smallholder dairy farms (SDFs), farmers often build cowsheds using local materials and based on self-accumulated experience without due consideration to reducing the risk of heat stress. This study aimed to characterise the heat stress abatement strategies and microclimate within SDF cowsheds from four typical dairy regions of Vietnam (south lowland, south highland, north lowland and north highland) and identify the housing parameters most associated with the microclimate. The study was conducted on 32 SDFs (eight SDFs per region) in autumn 2017. Twelve housing management variables, illustrating cowshed design and heat stress abatement methods of each SDF, were collected. Six microclimate parameters, collected within the cowshed, were temperature (AT), humidity, air speed (AS), heat load index (HLI), Temperature-humidity index (THI) and accumulated heat load units (AHLU) during a day (06:00 h to 18:00 h). Factor analysis and cluster analysis was applied to group cowsheds of SDFs into clusters where SDFs in the cluster had the same cowshed characteristics. Multivariable linear models were applied to define the parameters most likely to inform future research into heat stress mitigation on SDF. Averaged from 08:00 h to 18:00 h, microclimate inside the cowsheds was considered hot (HLI > 79) in the highland and very hot (HLI > 86) in the lowland regions. Cows in the lowland regions accumulated high heat load (AHLU > 50) by 18:00 h. Cowsheds of SDFs varied widely and grouped into seven cowshed types, but no type was more effective than others in reducing heat stress conditions within cowsheds. Using roof soakers together with fans decreased AT and HLI by 1.3 °C and 3.2 units, respectively, at 14:00 h compared to 11:00 h. Each 100 m increase in altitude was associated with decreases of 0.4 °C in AT, 1.3 units in HLI and 0.8 units in THI (p < 0.001). Each meter increase in the eave height of the cowshed roof was associated with decreases of 0.87 °C in AT, 3.31 units in HLI and 1.42 units in THI, and an increase of 0.14 m/s in AS (p < 0.05). The cowshed parameters that should be prioritised for future research into the amelioration of heat stress in SDF cows include using the roof soakers together with fans, increasing altitude, eave roof height and floor area per cow.

Use of whole-genome sequence data and novel genomic selection strategies to improve selection for age at puberty in tropically-adapted beef heifers.

BACKGROUND: In tropically-adapted beef heifers, application of genomic prediction for age at puberty has been limited due to low prediction accuracies. Our aim was to investigate novel methods of pre-selecting whole-genome sequence (WGS) variants and alternative analysis methodologies; including genomic best linear unbiased prediction (GBLUP) with multiple genomic relationship matrices (MGRM) and Bayesian (BayesR) analyses, to determine if prediction accuracy for age at puberty can be improved. METHODS: Genotypes and phenotypes were obtained from two research herds. In total, 868 Brahman and 960 Tropical Composite heifers were recorded in the first population and 3695 Brahman, Santa Gertrudis and Droughtmaster heifers were recorded in the second population. Genotypes were imputed to 23 million whole-genome sequence variants. Eight strategies were used to pre-select variants from genome-wide association study (GWAS) results using conditional or joint (COJO) analyses. Pre-selected variants were included in three models, GBLUP with a single genomic relationship matrix (SGRM), GBLUP MGRM and BayesR. Five-way cross-validation was used to test the effect of marker panel density (6 K, 50 K and 800 K), analysis model, and inclusion of pre-selected WGS variants on prediction accuracy. RESULTS: In all tested scenarios, prediction accuracies for age at puberty were highest in BayesR analyses. The addition of pre-selected WGS variants had little effect on the accuracy of prediction when BayesR was used. The inclusion of WGS variants that were pre-selected using a meta-analysis with COJO analyses by chromosome, fitted in a MGRM model, had the highest prediction accuracies in the GBLUP analyses, regardless of marker density. When the low-density (6 K) panel was used, the prediction accuracy of GBLUP was equal (0.42) to that with the high-density panel when only six additional sequence variants (identified using meta-analysis COJO by chromosome) were included. CONCLUSIONS: While BayesR consistently outperforms other methods in terms of prediction accuracies, reasonable improvements in accuracy can be achieved when using GBLUP and low-density panels with the inclusion of a relatively small number of highly relevant WGS variants.

Characterization of the poll allele in Brahman cattle using long-read Oxford Nanopore sequencing.

Brahman cattle (Bos indicus) are well adapted to thrive in tropical environments. Since their introduction to Australia in 1933, Brahman's ability to grow and reproduce on marginal lands has proven their value in the tropical beef industry. The poll phenotype, which describes the absence of horns, has become desirable in the cattle industry for animal welfare and handler safety concerns. The poll locus has been mapped to chromosome one. Four alleles, each a copy number variant, have been reported across this locus in B. indicus and Bos taurus. However, the causative mutation in Brahman cattle has not been fully characterized. Oxford Nanopore Technologies' minION sequencer was used to sequence four homozygous poll (PcPc), four homozygous horned (pp), and three heterozygous (Pcp) Brahmans to characterize the poll allele in Brahman cattle. A total of 98 Gb were sequenced and an average coverage of 3.33X was achieved. Read N50 scores ranged from 9.9 to 19 kb. Examination of the mapped reads across the poll locus revealed insertions approximately 200 bp in length in the poll animals that were absent in the horned animals. These results are consistent with the Celtic poll allele, a 212-bp duplication that replaces 10 bp. This provides direct evidence that the Celtic poll allele is segregating in the Australian Brahman population.

Optimized Genetic Testing for Polledness in Multiple Breeds of Cattle.

Many breeds of modern cattle are naturally horned, and for sound husbandry management reasons the calves frequently undergo procedures to physically remove the horns by disbudding or dehorning. These procedures are however a welfare concern. Selective breeding for polledness - absence of horns - has been effective in some cattle breeds but not in others (Bos indicus genotypes) due in part to the complex genetics of horn phenotype. To address this problem different approaches to genetic testing which provide accurate early-in-life prediction of horn phenotype have been evaluated, initially using microsatellites (MSAT) and more recently single nucleotide polymorphism (SNP). A direct gene test is not effective given the genetic heterogeneity and large-sized sequence variants associated with polledness in different breeds. The current study investigated 39,943 animals of multiple breeds to assess the accuracy of available poll testing assays. While the standard SNP-based test was an improvement on the earlier MSAT haplotyping method, 1999 (9.69%) out of 20,636 animals tested with this SNP-based assay did not predict a genotype, most commonly associated with the Indicus-influenced breeds. The current study has developed an optimized poll gene test that resolved the vast majority of these 1999 unresolved animals, while the predicted genotypes of those previously resolved remained unchanged. Hence the optimized poll test successfully predicted a genotype in 99.96% of samples assessed. We demonstrated that a robust set of 5 SNPs can effectively determine PC and PF alleles and eliminate the ambiguous and undetermined results of poll gene testing previously identified as an issue in cattle.

Multivariate genomic predictions for age at puberty in tropically adapted beef heifers.

Heifers that have an earlier age at puberty often have greater lifetime productivity. Age at puberty is moderately heritable so selection should effectively reduce the number of days to puberty, and improve heifer productivity and profitability as a result. However, recording age at puberty is intensive, requiring repeat ovarian scanning to determine age at first corpus luteum (AGECL). Genomic selection has been proposed as a strategy to select for earlier age at puberty; however, large reference populations of cows with AGECL records and genotypes would be required to generate accurate GEBV for this trait. Reproductive maturity score (RMS) is a proxy trait for age at puberty for implementation in northern Australia beef herds, where large scale recording of AGECL is not feasible. RMS assigns a score of 0 to 5 from a single ovarian scan to describe ovarian maturity at ~600 d. Here we use multivariate genomic prediction to evaluate the value of a large RMS data set to improve accuracy of GEBV for age at puberty (AGECL). There were 882 Brahman and 990 Tropical Composite heifers with AGECL phenotypes, and an independent set of 974 Brahman, 1,798 Santa Gertrudis, and 910 Droughtmaster heifers with RMS phenotypes. All animals had 728,785 real or imputed SNP genotypes. The correlation of AGECL and RMS (h2 = 0.23) was estimated as -0.83 using the genomic information. This result also demonstrates that using genomic information it is possible to estimate genetic correlations between traits collected on different animals in different herds, with minimal or unknown pedigree linkage between them. Inclusion of heifers with RMS in the multi-trait model improved the accuracy of genomic evaluations for AGECL. Accuracy of RMS GEBV generally did not improve by adding heifers with AGECL phenotypes into the reference population. These results suggest that RMS and AGECL may be used together in a multi-trait prediction model to increase the accuracy of prediction for age at puberty in tropically adapted beef cattle.

Optimizing Hybrid de Novo Transcriptome Assembly and Extending Genomic Resources for Giant Freshwater Prawns (Macrobrachium rosenbergii): The Identification of Genes and Markers Associated with Reproduction.

The giant freshwater prawn, Macrobrachium rosenbergii, a sexually dimorphic decapod crustacean is currently the world's most economically important cultured freshwater crustacean species. Despite its economic importance, there is currently a lack of genomic resources available for this species, and this has limited exploration of the molecular mechanisms that control the M. rosenbergii sex-differentiation system more widely in freshwater prawns. Here, we present the first hybrid transcriptome from M. rosenbergii applying RNA-Seq technologies directed at identifying genes that have potential functional roles in reproductive-related traits. A total of 13,733,210 combined raw reads (1720 Mbp) were obtained from Ion-Torrent PGM and 454 FLX. Bioinformatic analyses based on three state-of-the-art assemblers, the CLC Genomic Workbench, Trans-ABySS, and Trinity, that use single and multiple k-mer methods respectively, were used to analyse the data. The influence of multiple k-mers on assembly performance was assessed to gain insight into transcriptome assembly from short reads. After optimisation, de novo assembly resulted in 44,407 contigs with a mean length of 437 bp, and the assembled transcripts were further functionally annotated to detect single nucleotide polymorphisms and simple sequence repeat motifs. Gene expression analysis was also used to compare expression patterns from ovary and testis tissue libraries to identify genes with potential roles in reproduction and sex differentiation. The large transcript set assembled here represents the most comprehensive set of transcriptomic resources ever developed for reproduction traits in M. rosenbergii, and the large number of genetic markers predicted should constitute an invaluable resource for future genetic research studies on M. rosenbergii and can be applied more widely on other freshwater prawn species in the genus Macrobrachium.

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus).

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478bp and N50 length of 506bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

An16-resilin: an advanced multi-stimuli-responsive resilin-mimetic protein polymer.

Engineered protein polymers that display responsiveness to multiple stimuli are emerging as a promising class of soft material with unprecedented functionality. The remarkable advancement in genetic engineering and biosynthesis has created the opportunity for precise control over the amino acid sequence, size, structure and resulting functions of such biomimetic proteins. Herein, we describe the multi-stimuli-responsive characteristics of a resilin-mimetic protein, An16-resilin (An16), derived from the consensus sequence of resilin gene in the mosquito Anopheles gambiae. We demonstrate that An16 is an intrinsically disordered protein that displays unusual dual-phase thermal transition behavior along with responsiveness to pH, ion, light and humidity. Identifying the molecular mechanisms that allow An16 to sense and switch in response to varying environments furthers the ability to design intelligent biomacromolecules.

A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery.

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9ppt which showed best growth performance. Total sequence data generated was 467.8Mbp, consisting of 4,116,424 reads with an average length of 112bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

Evidence for positive selection of taurine genes within a QTL region on chromosome X associated with testicular size in Australian Brahman cattle.

BACKGROUND: Previous genome-wide association studies have identified significant regions of the X chromosome associated with reproductive traits in two Bos indicus-influenced breeds: Brahman cattle and Tropical Composites. Two QTL regions on this chromosome were identified in both breeds as strongly associated with scrotal circumference measurements, a reproductive trait previously shown to be useful for selection of young bulls. Scrotal circumference is genetically correlated with early age at puberty in both male and female offspring. These QTL were located at positions 69-77 and 81-92 Mb respectively, large areas each to which a significant number of potential candidate genes were mapped. RESULTS: To further characterise these regions, a bioinformatic approach was undertaken to identify novel non-synonymous SNP within the QTL regions of interest in Brahman cattle. After SNP discovery, we used conventional molecular assay technologies to perform studies of two candidate genes in both breeds. Non-synonymous SNP mapped to Testis-expressed gene 11 (Tex11) were associated (P < 0.001) with scrotal circumference in both breeds, and associations with percentage of normal sperm cells were also observed (P < 0.05). Evidence for recent selection was found as Tex11 SNP form a haplotype segment of Bos taurus origin that is retained within Brahman and Tropical Composite cattle with greatest reproductive potential. CONCLUSIONS: Association of non-synonymous SNP presented here are a first step to functional genetic studies. Bovine species may serve as a model for studying the role of Tex11 in male fertility, warranting further in-depth molecular characterisation.

A candidate gene association study for growth performance in an improved giant freshwater prawn (Macrobrachium rosenbergii ) culture line.

A candidate gene approach using type I single nucleotide polymorphism (SNP) markers can provide an effective method for detecting genes and gene regions that underlie phenotypic variation in adaptively significant traits. In the absence of available genomic data resources, transcriptomes were recently generated in Macrobrachium rosenbergii to identify candidate genes and markers potentially associated with growth. The characterisation of 47 candidate loci by ABI re-sequencing of four cultured and eight wild samples revealed 342 putative SNPs. Among these, 28 SNPs were selected in 23 growth-related candidate genes to genotype in 200 animals selected for improved growth performance in an experimental GFP culture line in Vietnam. The associations between SNP markers and individual growth performance were then examined. For additive and dominant effects, a total of three exonic SNPs in glycogen phosphorylase (additive), heat shock protein 90 (additive and dominant) and peroxidasin (additive), and a total of six intronic SNPs in ankyrin repeats-like protein (additive and dominant), rolling pebbles (dominant), transforming growth factor-ß induced precursor (dominant), and UTP-glucose-1-phosphate uridylyltransferase 2 (dominant) genes showed significant associations with the estimated breeding values in the experimental animals (P =0.001-0.031). Individually, they explained 2.6-4.8 % of the genetic variance (R²=0.026-0.048). This is the first large set of SNP markers reported for M. rosenbergii and will be useful for confirmation of associations in other samples or culture lines as well as having applications in marker-assisted selection in future breeding programs.

Controlled surface modification of tissue culture polystyrene for selective cell binding using resilin-inspired polypeptides.

Modified tissue culture polystyrene (TCP) surfaces have been fabricated by attachment of recombinant polypeptides based on Drosophila melanogaster resilin and the Anopheles gambiae resilin-like protein. The D. melanogaster polypeptide (Rec-1) was from the first exon of resilin and consisted of 17 very similar repeats of a 15 residue sequence. The A. gambiae polypeptide consisted of 16 repeats of an 11 residue consensus sequence (An16). Polypeptides were attached to the TCP surface through tyrosine-based photo-crosslinking using blue light in combination with (RuII(bpy)3)Cl2 and sodium persulfate. TCP that has been manufactured by mild oxidation has surface phenolic groups that are believed to participate in this crosslinking process. X-ray photoelectron spectroscopy and contact angle analyses were used to demonstrate polypeptide binding. At higher coating concentrations of Rec-1 and An16, the surface was passivated and fibroblasts no longer attached and spread. At coating concentrations of 1 mg ml(-1) for Rec-1 and 0.1 mg ml(-1) for An16, where the surface was fully passivated against fibroblast attachment, addition of a cell attachment peptide, cyclo(Arg-Gly-Asp-D-Tyr-Lys) during coating and photo-crosslinking at >0.1 mg ml(-1), led to the restoration of fibroblast binding that was dependent on the integrin αV chain.