Correlates of male fitness in captive zebra finches - a comparison of methods to disentangle genetic and environmental effects

Bolund E, Schielzeth H, Forstmeier W. Correlates of male fitness in captive zebra finches - a comparison of methods to disentangle genetic and environmental effects. BMC Evolutionary Biology. 2011;11(1): 327.Backgound. It is a common observation in evolutionary studies that larger, more ornamented or earlier breeding individuals have higher fitness, but that body size, ornamentation or breeding time does not change despite of sometimes substantial heritability for these traits. A possible explanation for this is that these traits do not causally affect fitness, but rather happen to be indirectly correlated with fitness via unmeasured non-heritable aspects of condition (e.g. undernourished offspring grow small and have low fitness as adults due to poor health). Whether this explanation applies to a specific case can be examined by decomposing the covariance between trait and fitness into its genetic and environmental components using pedigree-based animal models. We here examine different methods of doing this for a captive zebra finch population where male fitness was measured in communal aviaries in relation to three phenotypic traits (tarsus length, beak colour and song rate). Results. Our case study illustrates how methods that regress fitness over breeding values for phenotypic traits yield biased estimates as well as anti-conservative standard errors. Hence, it is necessary to estimate the genetic and environmental covariances between trait and fitness directly from a bivariate model. This method, however, is very demanding in terms of sample sizes. In our study parameter estimates of selection gradients for tarsus were consistent with the hypothesis of environmentally induced bias (ßA = 0.035 ± 0.25 (SE), ßE = 0.57 ± 0.28 (SE)), yet this differences between genetic and environmental selection gradients falls short of statistical significance. Conclusions. To examine the generality of the idea that phenotypic selection gradients for certain traits (like size) are consistently upwardly biased by environmental covariance a meta-analysis across study systems will be needed

Condition dependence and fitness consequences of sexual traits in zebra finches

The focus of this thesis is on sexual selection within a behavioural and evolutionary framework. Sexual selection is concerned with the factors that determine reproductive success. I have used a laboratory population of zebra finches to address fundamental questions that remain unanswered despite decades of research on this model organism in studies of sexual selection in monogamous species. The aim of this thesis was to perform a rigorous investigation of how male zebra finches achieve reproductive success. To do this, I tried to evaluate the signalling value of candidate traits and investigate the mechanisms that lead to reproductive success through both intra- and intersexual selection. The honesty of signals has had a special place in my work. Current theory suggests that secondary sexual signals should be costly to be able to function as honest indicators of male quality in a mate choice context. If ornaments are costly to develop or maintain, this would lead to condition dependent expression of ornaments. I tested the condition dependence of a broad range of traits so that I could compare the condition dependence of sexual and non-sexual traits and between traits in males and females (chapter 1). I found that zebra finches are remarkably resilient to stressful conditions during early development and can develop normally in most traits with very little consequences for fitness or longevity. These results indicate that not all traits that appear to be sexually selected characters will exhibit condition dependence. To study fitness consequences, I used an aviary breeding set-up. This allowed me to study the process of sexual selection in a socially complex environment where natural selection pressures had been relaxed. Since only selection acting on the genetic component of a trait can lead to evolutionary change, I used a quantitative genetic approach to look at selection pressures. Contrary to the prevailing view, I found that beak colour and courtship song rate and song complexity were not subject to female choice and not relevant for reproductive success (chapter 3). Beak colour was also not important in male-male competition (chapter 2). These findings necessitate a re-evaluation of the function of these traits. I suggest that courtship song rate reflects male reproductive strategies, while song structure is important for individual recognition in this highly social species and the beak colour might function as a signal of breeding status. The strongest candidate for a condition-dependent trait with consequences for fitness was body size (measured as tarsus length). Tarsus length was dependent on early condition (chapter 1), played a role in intrasexual competition (chapter 2) and was related to reproductive success in the aviaries (chapter 3). However, the quantitative genetic approach revealed that the selection was acting mainly on the environmental, not the genetic component of tarsus length, explaining why sexual size dimorphism has not evolved. Contrary to the expectations of honest signalling theory, males with a higher undirected song rate (i.e. non-courtship song) were less successful in the aviaries (chapter 4). However, females invested more into reproduction when paired to a high undirected song rate male, indicating that this type of song might function in reproductive stimulation of the partner and maintenance of the pair bond. Furthermore, female reproductive investment can be influenced by male quality. In contrast to previous studies, I found that females invested more when paired to a less attractive male (chapter 5). This investment pattern fits with the breeding system of this species, since life-time monogamy and a short lifespan means that the chances of breeding with a higher quality male in the future is very low. Thus, females should do the ‘best of a bad job’ and compensate by investing more when paired to a less attractive partner. Collectively, this work has provided several unexpected insights that call for a reinterpretation of several of the classical views of this model organism. This illustrates how novel experimental approaches can provide new insights into the process of sexual selection, also in well-studied model systems

Estimation of tumor heterogeneity using CGH array data

<p>Abstract</p> <p>Background</p> <p>Array-based comparative genomic hybridization (CGH) is a commonly-used approach to detect DNA copy number variation in whole genome-wide screens. Several statistical methods have been proposed to define genomic segments with different copy numbers in cancer tumors. However, most tumors are heterogeneous and show variation in DNA copy numbers across tumor cells. The challenge is to reveal the copy number profiles of the subpopulations in a tumor and to estimate the percentage of each subpopulation.</p> <p>Results</p> <p>We describe a relation between experimental data and exact DNA copy number and develop a statistical method to reveal the heterogeneity of tumors containing a mixture of different-stage cells. Furthermore, we validate the method on simulated data and apply the method to 29 pairs of breast primary tumors and their matched lymph node metastases.</p> <p>Conclusion</p> <p>We demonstrate a new method for CGH array analysis that allows a tumor sample to be classified according to its heterogeneity. The method gives an interpretable series of copy number profiles, one for each major subpopulation in a tumor. The profiles facilitate identification of copy number alterations in cancer development.</p

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

AbstractHuman fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated. These SAM-iPSCs provide a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes

Sequencing bias: comparison of different protocols of MicroRNA library construction

<p>Abstract</p> <p>Background</p> <p>MicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored.</p> <p>Results</p> <p>We assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r = 0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar.</p> <p>Conclusions</p> <p>The sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation.</p

Fusion of SpCas9 to E.coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from E. coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells