Emmanuel Buschiazzo

Microsatellite Tandem Repeats Are Abundant in Human Promoters and Are Associated with Regulatory Elements

Tandem repeats are genomic elements that are prone to changes in repeat number and are thus often polymorphic. These sequences are found at a high density at the start of human genes, in the gene’s promoter. Increasing empirical evidence suggests that length variation in these tandem repeats can affect gene regulation. One class of tandem repeats, known as microsatellites, rapidly alter in repeat number. Some of the genetic variation induced by microsatellites is known to result in phenotypic variation. Recently, our group developed a novel method for measuring the evolutionary conservation of microsatellites, and with it we discovered that human microsatellites near transcription start sites are often highly conserved. In this study, we examined the properties of microsatellites found in promoters. We found a high density of microsatellites at the start of genes. We showed that microsatellites are statistically associated with promoters using a wavelet analysis, which allowed us to test for associations on multiple scales and to control for other promoter related elements. Because promoter microsatellites tend to be G/C rich, we hypothesized that G/C rich regulatory elements may drive the association between microsatellites and promoters. Our results indicate that CpG islands, G-quadruplexes (G4) and untranslated regulatory regions have highly significant associations with microsatellites, but controlling for these elements in the analysis does not remove the association between microsatellites and promoters. Due to their intrinsic lability and their overlap with predicted functional elements, these results suggest that many promoter microsatellites have the potential to affect human phenotypes by generating mutations in regulatory elements, which may ultimately result in disease. We discuss the potential functions of human promoter microsatellites in this context.

Slow but not low: genomic comparisons reveal slower evolutionary rate and higher dN/dS in conifers compared to angiosperms

BackgroundComparative genomics can inform us about the processes of mutation and selection across diverse taxa. Among seed plants, gymnosperms have been lacking in genomic comparisons. Recent EST and full-length cDNA collections for two conifers, Sitka spruce (Picea sitchensis) and loblolly pine (Pinus taeda), together with full genome sequences for two angiosperms, Arabidopsis thaliana and poplar (Populus trichocarpa), offer an opportunity to infer the evolutionary processes underlying thousands of orthologous protein-coding genes in gymnosperms compared with an angiosperm orthologue set.ResultsBased upon pairwise comparisons of 3,723 spruce and pine orthologues, we found an average synonymous genetic distance (dS) of 0.191, and an average dN/dS ratio of 0.314. Using a fossil-established divergence time of 140 million years between spruce and pine, we extrapolated a nucleotide substitution rate of 0.68 × 10-9 synonymous substitutions per site per year. When compared to angiosperms, this indicates a dramatically slower rate of nucleotide substitution rates in conifers: on average 15-fold. Coincidentally, we found a three-fold higher dN/dS for the spruce-pine lineage compared to the poplar-Arabidopsis lineage. This joint occurrence of a slower evolutionary rate in conifers with higher dN/dS, and possibly positive selection, showcases the uniqueness of conifer genome evolution.ConclusionsOur results are in line with documented reduced nucleotide diversity, conservative genome evolution and low rates of diversification in conifers on the one hand and numerous examples of local adaptation in conifers on the other hand. We propose that reduced levels of nucleotide mutation in large and long-lived conifer trees, coupled with large effective population size, were the main factors leading to slow substitution rates but retention of beneficial mutations.

Conservation of Human Microsatellites across 450 Million Years of Evolution

The sequencing and comparison of vertebrate genomes have enabled the identification of widely conserved genomic elements. Chief among these are genes and cis-regulatory regions, which are often under selective constraints that promote their retention in related organisms. The conservation of elements that either lack function or whose functions are yet to be ascribed has been relatively little investigated. In particular, microsatellites, a class of highly polymorphic repetitive sequences considered by most to be neutrally evolving junk DNA that is too labile to be maintained in distant species, have not been comprehensively studied in a comparative genomic framework. Here, we used the UCSC alignment of the human genome against those of 11 mammalian and five nonmammalian vertebrates to identify and examine the extent of conservation of human microsatellites in vertebrate genomes. Out of 696,016 microsatellites found in human sequences, 85.39% were conserved in at least one other species, whereas 28.65% and 5.98% were found in at least one and three nonprimate species, respectively. An exponential decline of microsatellite conservation with increasing evolutionary time, a comparable distribution of conserved versus nonconserved microsatellites in the human genome, and a positive correlation between microsatellite conservation and overall sequence conservation, all suggest that most microsatellites are only maintained in genomes by chance, although exceptionally conserved human microsatellites were also found in distant mammals and other vertebrates. Our findings provide the first comprehensive survey of microsatellite conservation across deep evolutionary timescales, in this case 450 Myr of vertebrate evolution, and provide new tools for the identification of functional conserved microsatellites, the development of cross-species microsatellite markers and the study of microsatellite evolution above the species level.

Measuring Microsatellite Conservation in Mammalian Evolution with a Phylogenetic Birth–Death Model

Microsatellites make up ∼3% of the human genome, and there is increasing evidence that some microsatellites can have important functions and can be conserved by selection. To investigate this conservation, we performed a genome-wide analysis of human microsatellites and measured their conservation using a binary character birth--death model on a mammalian phylogeny. Using a maximum likelihood method to estimate birth and death rates for different types of microsatellites, we show that the rates at which microsatellites are gained and lost in mammals depend on their sequence composition, length, and position in the genome. Additionally, we use a mixture model to account for unequal death rates among microsatellites across the human genome. We use this model to assign a probability-based conservation score to each microsatellite. We found that microsatellites near the transcription start sites of genes are often highly conserved, and that distance from a microsatellite to the nearest transcription start site is a good predictor of the microsatellite conservation score. An analysis of gene ontology terms for genes that contain microsatellites near their transcription start site reveals that regulatory genes involved in growth and development are highly enriched with conserved microsatellites.

Development of microsatellite markers for the short-beaked echidna using three different approaches

Abstract. We used three different methods, size-selected genomic library, cross-species amplification of a mammal-wide set of conserved microsatellites and genomic sequencing, to develop a panel of 43 microsatellite loci for the short-beaked echidna (Tachyglossus aculeatus). These loci were screened against 13 individuals from three different regions (Tasmania, Kangaroo Island, Perth region), spanning the breadth of the range of the short-beaked echidna. Nine of the 43 tested loci amplified reliably, generated clear peaks on the electropherogram and were polymorphic, with the number of alleles per locus ranging from two to eight (mean = 3.78) in the individuals tested. Polymorphic information content ranged from 0.16 to 0.78, and expected heterozygosity ranged from 0.19 to 0.84. One of the nine microsatellites showed a heterozygote deficit, suggesting a high probability of null alleles. The genomic sequencing approach using data derived from the Roche FLX platform is likely to provide the most promising method to develop echidna microsatellites. The microsatellite markers developed here will be useful tools to study population genetic structure, gene flow, kinship and parentage in Tachyglossus sp. and potentially also in endangered Zaglossus species.

Evolutionary and phylogenetic significance of platypus microsatellites conserved in mammalian and other vertebrate genomes

Building on the recent publication of the first monotreme genome, that of the platypus, and the discovery that many platypus microsatellites are found in the genomes of three mammals (opossum, human, mouse) and two non-mammalian vertebrates (chicken, lizard), we investigated further the evolutionary conservation of microsatellites identified in the monotreme lineage and tested whether the conservation of microsatellites we observe in vertebrates has phylogenetic signal. Most conserved platypus microsatellites (75%) were found in one species, with the platypus sharing many more microsatellites with mammals than with reptiles (83% versus 30%). Within mammals, unexpectedly, many more platypus microsatellites had orthologues in the opossum genome than in that of either human or mouse, which was at odds with the very well supported view that monotremes diverged from a lineage containing both eutherians and marsupials (Theria hypothesis). We investigated the phylogenetic significance of microsatellite conservation through Bayesian and maximum parsimony tree reconstruction using presence/absence data of microsatellite loci conserved in a total of 18 species, including the platypus. Although models of evolution implemented in current phylogenetic reconstruction algorithms are not tailor-made for microsatellite data, we were able to construct vertebrate phylogenies that correspond well to the accepted mammalian phylogeny, with two of our three reconstructions supporting the Theria hypothesis. Our analysis provides ground for new theoretical development in phylogeny-based analyses of conserved microsatellite data.

Design and Implementation of Degenerate Microsatellite Primers for the Mammalian Clade

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ∼1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120–160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced.