A White Campion (Silene latifolia) floral expressed sequence tag (EST) library: annotation, EST-SSR characterization, transferability, and utility for comparative mapping

<p>Abstract</p> <p>Background</p> <p>Expressed sequence tag (EST) databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. One class of markers derived from EST sequences are simple sequence repeat (SSR) markers, also known as EST-SSRs. These are useful in plant genetic and evolutionary studies because they are located in transcribed genes and a putative function can often be inferred from homology searches. Another important feature of EST-SSR markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. In the present study we constructed a normalized EST library from floral tissue of <it>Silene latifolia </it>with the aim to identify expressed genes and to develop polymorphic molecular markers.</p> <p>Results</p> <p>We obtained a total of 3662 high quality sequences from a normalized <it>Silene </it>cDNA library. These represent 3105 unigenes, with 73% of unigenes matching genes in other species. We found 255 sequences containing one or more SSR motifs. More than 60% of these SSRs were trinucleotides. A total of 30 microsatellite loci were identified from 106 ESTs having sufficient flanking sequences for primer design. The inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. Tests for crossamplification of the EST-SSR loci in other <it>Silene </it>species established their applicability to related species.</p> <p>Conclusion</p> <p>The newly characterized genes and gene-derived markers from our <it>Silene </it>EST library represent a valuable genetic resource for future studies on <it>Silene latifolia </it>and related species. The polymorphism and transferability of EST-SSR markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus <it>Silene</it>.</p

Structure and evolution of Apetala3, a sex-linked gene in Silene latifolia

<p>Abstract</p> <p>Background</p> <p>The evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene <it>AP3 </it>was characterized in the dioecious plant species <it>Silene latifolia</it>. It was suggested that this gene had been transferred from an autosome to the Y chromosome.</p> <p>Results</p> <p>In the present study we provide evidence for the existence of an X linked copy of the <it>AP3 </it>gene. We further show that the Y copy is probably located in a chromosomal region where recombination restriction occurred during the first steps of sex chromosome evolution. A comparison of X and Y copies did not reveal any clear signs of degenerative processes in exon regions. Instead, both X and Y copies show evidence for relaxed selection compared to the autosomal orthologues in <it>S. vulgaris </it>and <it>S. conica</it>. We further found that promoter sequences differ significantly. Comparison of the genic region of <it>AP3 </it>between the X and Y alleles and the corresponding autosomal copies in the gynodioecious species <it>S. vulgaris </it>revealed a massive accumulation of retrotransposons within one intron of the Y copy of <it>AP3</it>. Analysis of the genomic distribution of these repetitive elements does not indicate that these elements played an important role in the size increase characteristic of the Y chromosome. However, <it>in silico </it>expression analysis shows biased expression of individual domains of the identified retroelements in male plants.</p> <p>Conclusions</p> <p>We characterized the structure and evolution of <it>AP3</it>, a sex linked gene with copies on the X and Y chromosomes in the dioecious plant <it>S. latifolia</it>. These copies showed complementary expression patterns and relaxed evolution at protein level compared to autosomal orthologues, which suggests subfunctionalization. One intron of the Y-linked allele was invaded by retrotransposons that display sex-specific expression patterns that are similar to the expression pattern of the corresponding allele, which suggests that these transposable elements may have influenced evolution of expression patterns of the Y copy. These data could help researchers decipher the role of transposable elements in degenerative processes during sex chromosome evolution.</p

Progress and Prospects in Gender Visibility at SMBE Annual Meetings

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Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations

Validation of SNP Allele Frequencies Determined by Pooled Next-Generation Sequencing in Natural Populations of a Non-Model Plant Species

Sequencing of pooled samples (Pool-Seq) using next-generation sequencing technologies has become increasingly popular, because it represents a rapid and cost-effective method to determine allele frequencies for single nucleotide polymorphisms (SNPs) in population pools. Validation of allele frequencies determined by Pool-Seq has been attempted using an individual genotyping approach, but these studies tend to use samples from existing model organism databases or DNA stores, and do not validate a realistic setup for sampling natural populations. Here we used pyrosequencing to validate allele frequencies determined by Pool-Seq in three natural populations of Arabidopsis halleri (Brassicaceae). The allele frequency estimates of the pooled population samples (consisting of 20 individual plant DNA samples) were determined after mapping Illumina reads to (i) the publicly available, high-quality reference genome of a closely related species (Arabidopsis thaliana) and (ii) our own de novo draft genome assembly of A. halleri. We then pyrosequenced nine selected SNPs using the same individuals from each population, resulting in a total of 540 samples. Our results show a highly significant and accurate relationship between pooled and individually determined allele frequencies, irrespective of the reference genome used. Allele frequencies differed on average by less than 4%. There was no tendency that either the Pool-Seq or the individual-based approach resulted in higher or lower estimates of allele frequencies. Moreover, the rather high coverage in the mapping to the two reference genomes, ranging from 55 to 284x, had no significant effect on the accuracy of the Pool-Seq. A resampling analysis showed that only very low coverage values (below 10-20x) would substantially reduce the precision of the method. We therefore conclude that a pooled re-sequencing approach is well suited for analyses of genetic variation in natural populations