Global analysis of exon creation versus loss and the role of alternative splicing in 17 vertebrate genomes

Association of alternative splicing (AS) with accelerated rates of exon evolution in some organisms has recently aroused widespread interest in its role in evolution of eukaryotic gene structure. Previous studies were limited to analysis of exon creation or lost events in mouse and/or human only. Our multigenome approach provides a way for (1) distinguishing creation and loss events on the large scale; (2) uncovering details of the evolutionary mechanisms involved; (3) estimating the corresponding rates over a wide range of evolutionary times and organisms; and (4) assessing the impact of AS on those evolutionary rates. We use previously unpublished independent analyses of alternative splicing in five species (human, mouse, dog, cow, and zebrafish) from the ASAP database combined with genomewide multiple alignment of 17 genomes to analyze exon creation and loss of both constitutively and alternatively spliced exons in mammals, fish, and birds. Our analysis provides a comprehensive database of exon creation and loss events over 360 million years of vertebrate evolution, including tens of thousands of alternative and constitutive exons. We find that exon inclusion level is inversely related to the rate of exon creation. In addition, we provide a detailed in-depth analysis of mechanisms of exon creation and loss, which suggests that a large fraction of nonrepetitive created exons are results of ab initio creation from purely intronic sequences. Our data indicate an important role for alternative splicing in creation of new exons and provide a useful novel database resource for future genome evolution research.     

Whole-genome resequencing analyses of five pig breeds,including Korean wild and native,and three European origin breeds

Pigs have been one of the most important sources of meat for humans, and their productivity has been substantially improved by recent strong selection. Here, we present whole-genome resequencing analyses of 55 pigs of five breeds representing Korean native pigs, wild boar and three European origin breeds. 1,673.1 Gb of sequence reads were mapped to the Swine reference assembly, covering ∼99.2% of the reference genome, at an average of ∼11.7-fold coverage. We detected 20,123,573 single-nucleotide polymorphisms (SNPs), of which 25.5% were novel. We extracted 35,458 of non-synonymous SNPs in 9,904 genes, which may contribute to traits of interest. The whole SNP sets were further used to access the population structures of the breeds, using multiple methodologies, including phylogenetic, similarity matrix, and population structure analysis. They showed clear population clusters with respect to each breed. Furthermore, we scanned the whole genomes to identify signatures of selection throughout the genome. The result revealed several promising loci that might underlie economically important traits in pigs, such as the CLDN1 and TWIST1 genes. These discoveries provide useful genomic information for further study of the discrete genetic mechanisms associated with economically important traits in pigs.     

Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process.     

High-Throughput,Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5′ and the 3′ vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.Integration of the retroviral DNA provirus into the host genome is an obligatory step in the retroviral life cycle. Because of this unique property, retroviruses have been adapted as vectors (24, 26) and used successfully to correct genetic diseases, such as X-linked severe combined immunodeficiency (SCID), adenosine deaminase (ADA)-deficient SCID, and X-linked adrenoleukodystrophy, by stable genetic modification of hematopoietic progenitor/stem cells (HPSC) (1, 2, 5, 6, 13, 27, 29). However, the risk of insertional mutagenesis from therapeutic vectors has been demonstrated in several cases in which integration events near or within proto-oncogenes triggered leukemia (8, 12, 14, 16, 34). Therefore, it is important to understand the mechanisms for complex hematopoietic repopulation in humans and to study the behaviors of engineered HPSC clones following transplant.Since retrovirus vectors uniquely “mark” individual HPSC by vector integration sites (VIS), clonal repopulation by HPSC can be analyzed by tracking the VIS. Restriction enzyme-based assays are commonly used for the clonal tracking, where genomic DNA is digested with restriction enzymes to generate VIS DNA fragments of different lengths that can be detected by Southern blotting (9, 17, 18, 22) or nucleotide sequencing via linker-mediated PCR (LM-PCR) (32), inverse PCR (INV-PCR) (33), or linear amplification-mediated PCR (LAM-PCR) (30). These approaches have been widely used in biological and clinical research to study composition of the HPSC pool, stem cell engraftment, regulatory decisions of individual stem cells, and genotoxicity of retroviral vectors (17, 22, 23, 25, 29, 31, 35). While mouse HPSC repopulation is typically mono- or oligoclonal (17), the number of HPSC clones repopulating in humans or nonhuman primates is much larger, manifesting several hundreds to thousands of repopulating clones posttransplant (5, 31, 35). Recent advances in sequencing technology have enabled high-throughput and parallel clonality analysis through large-scale VIS sequencing and enumeration of VIS sequences (5, 15, 35, 36). However, these methods can detect only VIS that are proximal to restriction enzyme sites, and additional experimental limitations may exist (10, 15, 36). As a result, current assays can only roughly estimate clonal frequencies, so the current standard is to perform clone-specific real-time PCR for sensitive and accurate quantification. Recently, novel clonal tracking assays that do not require restriction enzyme usage have been described (10, 11). However, these methods involve experimental steps that are technically challenging, and they require further optimization to achieve reliable, high-throughput quantification.Here, we present a novel VIS detection and quantification system based on 454 pyrosequencing and accompanying guidelines for high-throughput quantification of multiple clonal populations. We used a novel bidirectional PCR method to concurrently analyze both the 5′ (left) and the 3′ (right) vector-host junctions in peripheral blood repopulating cells (PBC) in a rhesus macaque transplanted with autologous HPSC transduced with lentivirus vectors (3). The reproducibility and conditions for reliable quantification were tested by two independent experiments conducted on the same PBC collected at four posttransplant time points. The lengths of VIS PCR amplicons, the amount of genomic DNA for analysis, and the intensity of sequencing are important factors influencing the reliability and the sensitivity of the assay. Of 964 unique vector integrants analyzed, the relative quantities of a 398-member subset were determined, demonstrating heterogeneous and dynamic clonal frequency changes over time. Clonal frequencies were further confirmed by clone-specific real-time PCR. We show that this assay detects the majority of VIS that are present in a given clonal population and accurately measures their relative frequencies.     

Exploring the Genetic Signature of Body Size in Yucatan Miniature Pig

Since being domesticated about 10,000–12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig.     

Dynamic genetic features of chromosomes revealed by comparison of soybean genetic and sequence-based physical maps

Despite the intensive soybean [Glycine max (L.) Merrill] genome studies, the high chromosome number (20) of the soybean plant relative to many other major crops has hindered the development of a high-resolution genomewide genetic map derived from a single population. Here, we report such a map, which was constructed in an F₁₅ population derived from a cross between G. max and G. soja lines using indel polymorphisms detected via a G. soja genome resequencing. By targeting novel indel markers to marker-poor regions, all marker intervals were reduced to under 6 cM on a genome scale. Comparison of the Williams 82 soybean reference genome sequence and our genetic map indicated that marker orders of 26 regions were discrepant with each other. In addition, our comparison showed seven misplaced and two absent markers in the current Williams 82 assembly and six markers placed on the scaffolds that were not incorporated into the pseudomolecules. Then, we showed that, by determining the missing sequences located at the presumed beginning points of the five major discordant segments, these observed discordant regions are mostly errors in the Williams 82 assembly. Distributions of the recombination rates along the chromosomes were similar to those of other organisms. Genotyping of indel markers and genome resequencing of the two parental lines suggested that some marker-poor chromosomal regions may represent introgression regions, which appear to be prevalent in soybean. Given the even and dense distribution of markers, our genetic map can serve as a bridge between genomics research and breeding programs.