Simple sequence repeats in zebra finch (Taeniopygia guttata) expressed sequence tags: a new resource for evolutionary genetic studies of passerines

Background Passerines (perching birds) are widely studied across many biological disciplines including ecology, population biology, neurobiology, behavioural ecology and evolutionary biology. However, understanding the molecular basis of relevant traits is hampered by the paucity of passerine genomics tools. Efforts to address this problem are underway, and the zebra finch (Taeniopygia guttata) will be the first passerine to have its genome sequenced. Here we describe a bioinformatic analysis of zebra finch expressed sequence tag (EST) Genbank entries. Results A total of 48,862 ESTs were downloaded from GenBank and assembled into contigs, representing an estimated 17,404 unique sequences. The unique sequence set contained 638 simple sequence repeats (SSRs) or microsatellites of length ≥20 bp and purity ≥90% and 144 simple sequence repeats of length ≥30 bp. A chromosomal location for the majority of SSRs was predicted by BLASTing against assembly 2.1 of the chicken genome sequence. The relative exonic location (5' untranslated region, coding region or 3' untranslated region) was predicted for 218 of the SSRs, by BLAST search against the ENSEMBL chicken peptide database. Ten loci were examined for polymorphism in two zebra finch populations and two populations of a distantly related passerine, the house sparrow Passer domesticus. Linkage was confirmed for four loci that were predicted to reside on the passerine homologue of chicken chromosome 7. Conclusion We show that SSRs are abundant within zebra finch ESTs, and that their genomic location can be predicted from sequence similarity with the assembled chicken genome sequence. We demonstrate that a useful proportion of zebra finch EST-SSRs are likely to be polymorphic, and that they can be used to build a linkage map. Finally, we show that many zebra finch EST-SSRs are likely to be useful in evolutionary genetic studies of other passerines

A comparison of SNPs and microsatellites as linkage mapping markers: lessons from the zebra finch (Taeniopygia guttata)

Background: Genetic linkage maps are essential tools when searching for quantitative trait loci (QTL). To maximize genome coverage and provide an evenly spaced marker distribution a combination of different types of genetic marker are sometimes used. In this study we created linkage maps of four zebra finch (Taeniopygia guttata) chromosomes (1, 1A, 2 and 9) using two types of marker, Single Nucleotide Polymorphisms (SNPs) and microsatellites. To assess the effectiveness and accuracy of each kind of marker we compared maps built with each marker type separately and with both types of marker combined. Linkage map marker order was validated by making comparisons to the assembled zebra finch genome sequence. Results: We showed that marker order was less reliable and linkage map lengths were inflated for microsatellite maps relative to SNP maps, apparently due to differing error rates between the two types of marker. Guidelines on how to minimise the effects of error are provided. In particular, we show that when combining both types of marker the conventional process of building linkage maps, whereby the most informative markers are added to the map first, has to be modified in order to improve map accuracy. Conclusions: When using multiple types and large numbers of markers to create dense linkage maps, the least error prone loci (SNPs) rather than the most informative should be used to create framework maps before the addition of other potentially more error prone markers (microsatellites). This raises questions about the accuracy of marker order and predicted recombination rates in previous microsatellite linkage maps which were created using the conventional building process, however, provided suitable error detection strategies are followed microsatellite-based maps can continue to be regarded as reasonably reliable

Nestling diet, secondary sexual traits and fitness in the zebra finch

We examined the effect of nestling diet quality on a suite of physiological, morphological and life-history traits in adult male zebra finches,Taeniopygia guttata. Compared with birds reared on a supplemented diet, nestlings reared on a seed-only diet showed a reduced rate of growth and reduced cell-mediated immune function as measured by an in vivo response to aT lymphocyte-dependent mitogen. There were no differences between birds reared on the two diets in any of the following adult traits: body size, primary sexual traits (testes mass, numbers of stored sperm, sperm function, velocity and morphology), secondary sexual traits (beak colour and song rate), serological traits or immunological traits. The only differences we detected were a lower body mass and a greater proportion of individuals with plumage abnormalities among those reared on a seed-only diet (this latter effect was transient). The fact that male zebra finches reared on a seed-only diet were, as adults, virtually indistinguishable from those reared on a supple- mented diet, despite having reduced growth and immune function as nestlings, demonstrates that they subsequently compensated through the di¡erential allocation of resources. Our results indicate that differ- ential allocation is costly in terms of fitness since birds reared on a seed-only diet experienced a significantly greater mortality rate than those reared on a supplemented diet. This in turn suggests the existence of a trade-of between the development of traits important for reproduction, such as primary and secondary sexual traits and longevity

Long sperm fertilize more eggs in a bird

Sperm competition, in which the ejaculates of multiple males compete to fertilize a female's ova, results in strong selection on sperm traits. Although sperm size and swimming velocity are known to independently affect fertilization success in certain species, exploring the relationship between sperm length, swimming velocity and fertilization success still remains a challenge. Here, we use the zebra finch (Taeniopygia guttata), where sperm size influences sperm swimming velocity, to determine the effect of sperm total length on fertilization success. Sperm competition experiments, in which pairs of males whose sperm differed only in length and swimming speed, revealed that males producing long sperm were more successful in terms of (i) the number of sperm reaching the ova and (ii) fertilizing those ova. Our results reveal that although sperm length is the main factor determining the outcome of sperm competition, complex interactions between male and female reproductive traits may also be important. The mechanisms underlying these interactions are poorly understood, but we suggest that differences in sperm storage and utilization by females may contribute to the outcome of sperm competition

Sperm mobility: mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus

When females are sexually promiscuous, sexual selection continues after insemination through sperm competition and cryptic female choice, and male traits conveying an advantage in competitive fertilization are selected for. Although individual male and ejaculate traits are known to influence paternity in a competitive scenario, multiple mechanisms co-occur and interact to determine paternity. The way in which different traits interact with each other and the mechanisms through which their heritability is maintained despite selection remain unresolved. In the promiscuous fowl, paternity is determined by the number of sperm inseminated into a female, which is mediated by male social dominance, and by the quality of the sperm inseminated, measured as sperm mobility. Here we show that: (i) the number of sperm inseminated determines how many sperm reach the female sperm-storage sites, and that sperm mobility mediates the fertilizing efficiency of inseminated sperm, mainly by determining the rate at which sperm are released from the female storage sites, (ii) like social status, sperm mobility is heritable, and (iii) subdominant males are significantly more likely to have higher sperm mobility than dominant males. This study indicates that although the functions of social status and sperm mobility are highly interdependent, the lack of phenotypic integration of these traits may maintain the variability of male fitness and heritability of fertilizing efficiency

Egg shape in the Common Guillemot Uria aalge and Brunnich’s Guillemot U. lomvia: not a rolling matter?

The adaptive significance of avian egg shape is poorly understood, and has been studied only in those species producing pyriform (pear-shaped, or pointed) eggs: waders and guillemots (murres) Uria spp., albeit to a limited extent. In the latter, it is widely believed that the pyriform shape has evolved to minimise their likelihood of rolling off a cliff ledge: the idea being that the more pointed the egg, the narrower the arc in which it rolls, and the less likely it is it will fall from a cliff ledge. Previous research also claimed that the rolling trajectory—the diameter of the arc they describe—of Common Guillemot U. aalge eggs is influenced not only by its shape but also by its mass, with heavier (i.e. larger) eggs describing a wider arc than lighter eggs. The finding that both shape and mass determined the rolling trajectory of Common Guillemot eggs (the shape–mass hypothesis) was used to explain the apparent anomaly that Bru¨nnich’s Guillemot U. lomvia produce eggs that are less pointed, yet breed on narrower ledges than Common Guillemots. They are able to do this, it was suggested, because Bru¨nnich’s Guillemot eggs are smaller and lighter in mass than those of Common Guillemots. However, since some populations of Bru¨nnich’s Guillemots produce eggs that are as large or larger than those of some Common Guillemot populations, the shape–mass hypothesis predicts that that (1) larger (i.e. heavier) eggs of both guillemot species will be more pyriform (pointed) in shape, and (2) that eggs of the two species of same mass should be similarly pointed. We tested these predictions and found: (1) only a weak, positive association between egg volume and pointedness in both guillemot species (\3% of the variation in egg shape explained by egg volume), and (2) no evidence that eggs of the two species of similar mass were more similar in shape: regardless of their mass, Brunnich’s Guillemot eggs were less pointed than Common Guillemot eggs. Overall, our results call into question the long-held belief that protection from rolling is the main selective factor driving guillemot egg shape

Ejaculate allocation by male sand martins, Riparia riparia

Males of many species allocate sperm to ejaculates strategically in response to variation in the risk and intensity of sperm competition. The notable exception is passerine birds, in which evidence for strategic allocation is absent. Here we report the results of a study testing for strategic ejaculate allocation in a passerine bird, the sand martin (Riparia riparia). Natural ejaculates were collected from males copulating with a model female. Ejaculates transferred in the presence of a rival male contained significantly more sperm than ejaculates transferred in the absence of a rival male. There was no evidence that this difference was due to the confounding effects of the year of ejaculate collection, the identity of the model female, the colony, the stage of season or the period of the day in which ejaculates were collected. A more detailed examination of the ejaculate patterns of individual males, achieved by the DNA profiling of ejaculates, provided additional evidence for strategic allocation of sperm

Sperm morphology, adenosine triphosphate (ATP) concentration and swimming velocity: unexpected relationships in a passerine bird.

The relationship between sperm energetics and sperm function is poorly known, but is central to our understanding of the evolution of sperm traits. The aim of this study was to examine how sperm morphology and ATP content affect sperm swimming velocity in the zebra finch Taeniopygia guttata We exploited the high inter-male variation in this species and created extra experimental power by increasing the number of individuals with very long or short sperm through artificial selection. We found a pronounced quadratic relationship between total sperm length and swimming velocity, with velocity increasing with length up to a point, but declining in the very longest sperm. We also found an unexpected negative association between midpiece length and ATP content: sperm with a short midpiece generally contained the highest concentration of ATP. Low intracellular ATP is therefore unlikely to explain reduced swimming velocity among the very longest sperm (which tend to have a shorter midpiece)

Sperm gatekeeping : 3D imaging reveals a constricted entrance to zebra finch sperm storage tubules

Females across many internally fertilizing taxa store sperm, often in specialized storage organs in their reproductive tracts. In birds, several hundred sperm storage tubules exist in the utero-vaginal junction of the oviduct, and there is growing evidence that sperm storage in these tubules is selective. The mechanisms underlying female sperm storage in birds remain unknown because of our limited ability to make three-dimensional, live observations inside the large, muscular avian oviduct. Here, we describe a new application of fluorescence selective plane illumination microscopy to optically section oviduct tissue from zebra finch Taeniopygia guttata females label free by harnessing tissue autofluorescence. Our data provide the first description of the three-dimensional structure of sperm storage organs in any vertebrate to the best of our knowledge and reveal the presence of gate-like constricted openings that may play a role in sperm selection

Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies

Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised libraries had a larger number of contigs but used fewer reads compared to unnormalised libraries. Considerably more genes were also detected using the contigs produced from normalised cDNA, and microsatellite discovery was up to 73% more efficient in these. There was a positive correlation between the detected expression level of genes in normalised and unnormalised cDNA, and there was no difference in the number of genes identified as being differentially expressed between blood and spleen for the normalised and unnormalised libraries. We conclude that normalised cDNA libraries are preferable for many applications of RNA-Seq and that these can also be used in quantitative gene expression studies