Genome wide SNP discovery, analysis and evaluation in mallard (Anas platyrhynchos)

<p>Abstract</p> <p>Background</p> <p>Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl.</p> <p>Results</p> <p>More than one billion base pairs of sequence information were generated resulting in a 16× coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72.</p> <p>Conclusion</p> <p>We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, ~101,000 SNPs were detected within our wild mallard sequences and ~49,000 were detected between wild and domesticated duck data. In the ~101,000 SNPs we found a subset of ~20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (~150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e.g. disentangling migratory connectivity) and industrial breeding programs.</p

Reference genes for quantitative gene expression studies in multiple avian species.

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species

Development and Validation of a Self-Report Measure of Mentalizing: The Reflective Functioning Questionnaire

Reflective functioning or mentalizing is the capacity to interpret both the self and others in terms of internal mental states such as feelings, wishes, goals, desires, and attitudes. This paper is part of a series of papers outlining the development and psychometric features of a new self-report measure, the Reflective Functioning Questionnaire (RFQ), designed to provide an easy to administer self-report measure of mentalizing. We describe the development and initial validation of the RFQ in three studies. Study 1 focuses on the development of the RFQ, its factor structure and construct validity in a sample of patients with Borderline Personality Disorder (BPD) and Eating Disorder (ED) (n=108) and normal controls (n=295). Study 2 aims to replicate these findings in a fresh sample of 129 patients with personality disorder and 281 normal controls. Study 3 addresses the relationship between the RFQ, parental reflective functioning and infant attachment status as assessed with the Strange Situation Procedure (SSP) in a sample of 136 community mothers and their infants. In both Study 1 and 2, confirmatory factor analyses yielded two factors assessing Certainty (RFQ_C) and Uncertainty (RFQ_U) about the mental states of self and others. These two factors were relatively distinct, invariant across clinical and non-clinical samples, had satisfactory internal consistency and test–retest stability, and were largely unrelated to demographic features. The scales discriminated between patients and controls, and were significantly and in theoretically predicted ways correlated with measures of empathy, mindfulness and perspective-taking, and with both self-reported and clinician-reported measures of borderline personality features and other indices of maladaptive personality functioning. Furthermore, the RFQ scales were associated with levels of parental reflective functioning, which in turn predicted infant attachment status in the SSP. Overall, this study lends preliminary support for the RFQ as a screening measure of reflective functioning. Further research is needed, however, to investigate in more detail the psychometric qualities of the RFQ