Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi).

BACKGROUND: The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection. METHODOLOGY: In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper. RESULTS: Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils. CONCLUSION: Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.

Divergent island hybrids mixed waves of ancient gene flow.

In this issue of Molecular Ecology, Salter et al. (2023) discovered that the Cuban Northern Bobwhite subspecies, Colinus virginianus cubanensis (Gould, 1850), is an ancient hybrid population formed due to historical hybridization potentially brought by waves of historical human migration. This study revealed a complex mixture of gene flow from distinct spatiotemporal origins underlying a seemingly semi-independent evolutionary trajectory. Hybridization can be more common and complex than we thought.

Molecular phylogenetic and in silico analysis of glyceraldeyde-3-phosphate dehydrogenase (GAPDH) gene from northern bobwhite quail (Colinus virginianus).

Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR products. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame (ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon-intron pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes. Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this preliminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.

A Highly Contiguous Reference Genome for Northern Bobwhite (Colinus virginianus).

Northern bobwhites (Colinus virginianus) are small quails in the New World Quail family (Odontophoridae) and are one of the most phenotypically diverse avian species. Despite extensive research on bobwhite ecology, genomic studies investigating the evolution of phenotypic diversity in this species are lacking. Here, we present a new, highly contiguous assembly for bobwhites using tissue samples from a vouchered, wild, female bird collected in Louisiana. By performing a de novo assembly and scaffolding the assembly with Dovetail Chicago and HiC libraries and the HiRise pipeline, we produced an 866.8 Mb assembly including 1,512 scaffolds with a scaffold N50 of 66.8 Mb, a scaffold L90 of 17, and a BUSCO completeness score of 90.8%. This new assembly represents approximately 96% of the non-repetitive and 84% of the entire bobwhite genome size, greatly improves scaffold lengths and contiguity compared to an existing draft bobwhite genome, and provides an important tool for future studies of evolutionary and functional genomics in bobwhites.

Annotated Draft Genome Assemblies for the Northern Bobwhite (Colinus virginianus) and the Scaled Quail (Callipepla squamata) Reveal Disparate Estimates of Modern Genome Diversity and Historic Effective Population Size.

Northern bobwhite (Colinus virginianus; hereafter bobwhite) and scaled quail (Callipepla squamata) populations have suffered precipitous declines across most of their US ranges. Illumina-based first- (v1.0) and second- (v2.0) generation draft genome assemblies for the scaled quail and the bobwhite produced N50 scaffold sizes of 1.035 and 2.042 Mb, thereby producing a 45-fold improvement in contiguity over the existing bobwhite assembly, and ≥90% of the assembled genomes were captured within 1313 and 8990 scaffolds, respectively. The scaled quail assembly (v1.0 = 1.045 Gb) was ∼20% smaller than the bobwhite (v2.0 = 1.254 Gb), which was supported by kmer-based estimates of genome size. Nevertheless, estimates of GC content (41.72%; 42.66%), genome-wide repetitive content (10.40%; 10.43%), and MAKER-predicted protein coding genes (17,131; 17,165) were similar for the scaled quail (v1.0) and bobwhite (v2.0) assemblies, respectively. BUSCO analyses utilizing 3023 single-copy orthologs revealed a high level of assembly completeness for the scaled quail (v1.0; 84.8%) and the bobwhite (v2.0; 82.5%), as verified by comparison with well-established avian genomes. We also detected 273 putative segmental duplications in the scaled quail genome (v1.0), and 711 in the bobwhite genome (v2.0), including some that were shared among both species. Autosomal variant prediction revealed ∼2.48 and 4.17 heterozygous variants per kilobase within the scaled quail (v1.0) and bobwhite (v2.0) genomes, respectively, and estimates of historic effective population size were uniformly higher for the bobwhite across all time points in a coalescent model. However, large-scale declines were predicted for both species beginning ∼15-20 KYA.

Two recently sequenced vertebrate genomes are contaminated with apicomplexan species of the Sarcocystidae family.

This paper highlights a general problem, namely that host genome sequences can easily be contaminated with parasite sequences, thus careful isolation of genetic material and careful bioinformatics analysis are needed in all cases. Two recently published genomes are shown here to be contaminated with sequences of apicomplexan parasites which belong to the Sarcocystidae family. Sequences of the characteristic apicomplexan organelle, the apicoplast, were used as queries in BLASTN searches against nucleotide sequences of various animal groups looking for possible contamination. Draft genomes of a bird, Colinus virginianus (Halley et al., 2014), and a bat, Myotis davidii (Zhang et al., 2013) were found to contain at least six and 17 contigs, respectively, originating from the apicoplast of an apicomplexan species, and other genes specific to this phylum can also be found in the published genomes. Obviously, the sources of the genetic material, the muscle and the kidney of the animals, respectively, contained the parasitic cysts. Phylogenetic analyses using 18S rRNA and internal transcribed spacer 1 genes show that the parasite contaminating C. virginianus is a species of Sarcocystis related to ones known to cycle between avian and mammalian hosts. In the case of M. davidii it belongs to the Nephroisospora genus, the only member of which, Nephroisospora eptesici, has been recently identified from the kidney of big brown bats (Eptesicus fuscus).

Amino acid sequence of Japanese quail (Coturnix japonica) and northern bobwhite (Colinus virginianus) myoglobin.

Myoglobin has an important physiological role in vertebrates, and as the primary sarcoplasmic pigment in meat, influences quality perception and consumer acceptability. In this study, the amino acid sequences of Japanese quail and northern bobwhite myoglobin were deduced by cDNA cloning of the coding sequence from mRNA. Japanese quail myoglobin was isolated from quail cardiac muscles, purified using ammonium sulphate precipitation and gel-filtration, and subjected to multiple enzymatic digestions. Mass spectrometry corroborated the deduced protein amino acid sequence at the protein level. Sequence analysis revealed both species' myoglobin structures consist of 153 amino acids, differing at only three positions. When compared with chicken myoglobin, Japanese quail showed 98% sequence identity, and northern bobwhite 97% sequence identity. The myoglobin in both quail species contained eight histidine residues instead of the nine present in chicken and turkey.

A draft de novo genome assembly for the northern bobwhite (Colinus virginianus) reveals evidence for a rapid decline in effective population size beginning in the Late Pleistocene.

Wild populations of northern bobwhites (Colinus virginianus; hereafter bobwhite) have declined across nearly all of their U.S. range, and despite their importance as an experimental wildlife model for ecotoxicology studies, no bobwhite draft genome assembly currently exists. Herein, we present a bobwhite draft de novo genome assembly with annotation, comparative analyses including genome-wide analyses of divergence with the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genomes, and coalescent modeling to reconstruct the demographic history of the bobwhite for comparison to other birds currently in decline (i.e., scarlet macaw; Ara macao). More than 90% of the assembled bobwhite genome was captured within <40,000 final scaffolds (N50 = 45.4 Kb) despite evidence for approximately 3.22 heterozygous polymorphisms per Kb, and three annotation analyses produced evidence for >14,000 unique genes and proteins. Bobwhite analyses of divergence with the chicken and zebra finch genomes revealed many extremely conserved gene sequences, and evidence for lineage-specific divergence of noncoding regions. Coalescent models for reconstructing the demographic history of the bobwhite and the scarlet macaw provided evidence for population bottlenecks which were temporally coincident with human colonization of the New World, the late Pleistocene collapse of the megafauna, and the last glacial maximum. Demographic trends predicted for the bobwhite and the scarlet macaw also were concordant with how opposing natural selection strategies (i.e., skewness in the r-/K-selection continuum) would be expected to shape genome diversity and the effective population sizes in these species, which is directly relevant to future conservation efforts.

Quail Genomics: a knowledgebase for Northern bobwhite.

BACKGROUND: The Quail Genomics knowledgebase (http://www.quailgenomics.info) has been initiated to share and develop functional genomic data for Northern bobwhite (Colinus virginianus). This web-based platform has been designed to allow researchers to perform analysis and curate genomic information for this non-model species that has little supporting information in GenBank. DESCRIPTION: A multi-tissue, normalized cDNA library generated for Northern bobwhite was sequenced using 454 Life Sciences next generation sequencing. The Quail Genomics knowledgebase represents the 478,142 raw ESTs generated from the sequencing effort in addition to assembled nucleotide and protein sequences including 21,980 unigenes annotated with meta-data. A normalized MySQL relational database was established to provide comprehensive search parameters where meta-data can be retrieved using functional and structural information annotation such as gene name, pathways and protein domain. Additionally, blast hit cutoff levels and microarray expression data are available for batch searches. A Gene Ontology (GO) browser from Amigo is locally hosted providing 8,825 unigenes that are putative orthologs to chicken genes. In an effort to address over abundance of Northern bobwhite unigenes (71,384) caused by non-overlapping contigs and singletons, we have built a pipeline that generates scaffolds/supercontigs by aligning partial sequence fragments against the indexed protein database of chicken to build longer sequences that can be visualized in a web browser. CONCLUSION: Our effort provides a central repository for storage and a platform for functional interrogation of the Northern bobwhite sequences providing comprehensive GO annotations, meta-data and a scaffold building pipeline. The Quail Genomics knowledgebase will be integrated with Japanese quail (Coturnix coturnix) data in future builds and incorporate a broader platform for these avian species.

From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite.

The limited availability of genomic tools and data for nonmodel species impedes computational and systems biology approaches in nonmodel organisms. Here we describe the development, functional annotation, and utilization of genomic tools for the avian wildlife species Northern bobwhite (Colinus virginianus) to determine the molecular impacts of exposure to 2,6-dinitrotoluene (2,6-DNT), a field contaminant of military concern. Massively parallel pyrosequencing of a normalized multitissue library of Northern bobwhite cDNAs yielded 71,384 unique transcripts that were annotated with gene ontology (GO), pathway information, and protein domain analysis. Comparative genome analyses with model organisms revealed functional homologies in 8,825 unique Northern bobwhite genes that are orthologous to 48% of Gallus gallus protein-coding genes. Pathway analysis and GO enrichment of genes differentially expressed in livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT revealed several impacts validated by RT-qPCR including: prostaglandin pathway-mediated inflammation, increased expression of a heme synthesis pathway in response to anemia, and a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively. This research effort provides the first comprehensive annotated gene library for Northern bobwhite. Transcript expression analysis provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT exposure case study. Furthermore, the systemic impact of dinitrotoluenes on liver function appears conserved across species as PPAR signaling is similarly affected in fathead minnow liver tissue after exposure to 2,4-DNT.

Mimicking the natural doping of migrant sandpipers in sedentary quails: effects of dietary n-3 fatty acids on muscle membranes and PPAR expression.

Wild semipalmated sandpipers (Calidris pusilla) eat n-3 fatty acids to prime their muscles for long migrations. Sedentary bobwhite quails (Colinus virginianus) were used as a model to investigate the mechanisms for this natural doping. Our goal was to characterize the stimulating effects of n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA) on oxidative capacity. Mechanisms linked to changes in membrane composition and in gene expression for peroxisome proliferator-activated receptors (PPAR) were investigated. Dietary n-3 fatty acids stimulated the activities of oxidative enzymes by 58-90% (citrate synthase, cytochrome oxidase, carnitine palmitoyl transferase and hydroxyacyl dehydrogenase), and sedentary quails showed the same changes in membrane composition as sandpipers preparing for migration. EPA and DHA have the same doping effect. The substitution of n-6 arachidonic acid by n-3 EPA in membrane phospholipids plays an important role in mediating the metabolic effects of the diet, but results provide no significant support for the involvement of PPARs (as determined by changes in gene expression). The fatty acid composition of mitochondrial membranes and sarcoplasmic reticulum can be monitored by measuring total muscle phospholipids because all phospholipids are equally affected by diet. Only extreme regimes of endurance training can lead to increments in oxidative capacity matching those induced here by diet. As they prepare for long migrations, semipalmated sandpipers improve their physical fitness by eating! Choosing n-3 fatty acid doping over endurance training strikes us as a better strategy to boost aerobic capacity when rapid storage of energy is critical.

Examination of the dietary methionine requirements of breeding northern bobwhite, Colinus virginianus.

Adult Northern bobwhite were used to test the hypothesis that dietary methionine levels recommended by the NRC for breeding quail are excessive for wild bobwhite. We tested the hypothesis by comparing immunocompetence, reproductive performance, and chick viability of Northern bobwhite hens fed diets containing low (0.31%), moderate (0.39%), or high (0.47%) concentrations of methionine. Chick viability was determined by assessing immunocompetence, including evaluating the ability of hens to passively transfer immunity to their chicks. Hens were fed the experimental diets for 6 wk on an ad libitum basis. After 6 wk, methionine treatment had no measurable effect (P > or = 0.20) on hen phytohemagglutinin wing web indices, organ weights, or serum anti-Pasteurella multocida titer indices. Mean egg weight, percentage egg production, total cumulative egg production, yolk weight, yolk volume, and percentage fertile and percentage hatch of fertile eggs did not differ (P > or = 0.12) among diet treatments. Amount of albumen in eggs produced by hens fed the high methionine diet averaged 0.27 g more (P = 0.003) than eggs of hens fed the low methionine diet. Anti-P. multocida titer of yolks from eggs in Week 6 were not different (P = 0.36) between birds fed the high and the low methionine diets. The mortality rate of chicks after challenge with 23 cfu of P. multocida was not different (P > or = 0.05) among diets. Chicks hatched from eggs laid by vaccinated hens during Weeks 2 and 3, however, had lower (P < 0.05) mortality than chicks of unvaccinated hens. It appears a dietary methionine concentration of 0.3% may be sufficient for wild Northern bobwhite to produce viable chicks.

Heterosynapsis and axial equalization of the sex chromosomes of the northern bobwhite quail.

The pairing behavior of the Z and W chromosomes in the female northern bobwhite quail (Colinus virginianus) was analyzed by electron microscopy of silver-stained synaptonemal complexes (SCs). After autosomal pairing was completed, synapsis of the sex chromosomes initiated at the short-arm end of the W chromosome and one end of the Z chromosome. Synapsis then progressed unidirectionally, producing a sex bivalent in which the entire length of the W axis was paired with an equivalent length of the Z axis. Progressive contraction and asymmetrical twisting of the Z axis ultimately resulted in a fully paired configuration with aligned axial ends. Further contraction of the Z axis reduced the extent of asymmetrical twisting such that only the nonaligned centromeric regions distinguished the SC of the ZW bivalent from SCs of similar-sized autosomes in late-pachytene nuclei. Quantitative analyses indicated that the length of the Z axis shortened significantly during the adjustment process, whereas no significant difference occurred in the length of the W axis. The nonalignment of the centromeric regions during transitional stages of ZW synapsis indicates that direct heterosynapsis of nonhomologous segments, followed by axial equalization of the length inequality, is responsible for the length adjustment during synapsis in the sex chromosomes of the bobwhite quail.