Srikar Chamala

Characterization of duplicate gene evolution in the recent natural allopolyploid Tragopogon miscellus by next‐generation sequencing and Sequenom iPLEX MassARRAY genotyping

Tragopogon miscellus (Asteraceae) is an evolutionary model for the study of natural allopolyploidy, but until now has been under‐resourced as a genetic model. Using 454 and Illumina expressed sequence tag sequencing of the parental diploid species of T. miscellus, we identified 7782 single nucleotide polymorphisms that differ between the two progenitor genomes present in this allotetraploid. Validation of a sample of 98 of these SNPs in genomic DNA using Sequenom MassARRAY iPlex genotyping confirmed 92 SNP markers at the genomic level that were diagnostic for the two parental genomes. In a transcriptome profile of 2989 SNPs in a single T. miscellus leaf, using Illumina sequencing, 69% of SNPs showed approximately equal expression of both homeologs (duplicate homologous genes derived from different parents), 22% showed apparent differential expression and 8.5% showed apparent silencing of one homeolog in T. miscellus. The majority of cases of homeolog silencing involved the T. dubius SNP homeolog (164/254; 65%) rather than the T. pratensis homeolog (90/254). Sequenom analysis of genomic DNA showed that in a sample of 27 of the homeologs showing apparent silencing, 23 (85%) were because of genomic homeolog loss. These methods could be applied to any organism, allowing efficient and cost‐effective generation of genetic markers.

Rapid, repeated, and clustered loss of duplicate genes in allopolyploid plant populations of independent origin.

The predictability of evolution is debatable, with recent evidence suggesting that outcomes may be constrained by gene interaction networks [1]. Whole-genome duplication (WGD; polyploidization-ubiquitous in plant evolution [2]) provides the opportunity to evaluate the predictability of genome reduction, a pervasive feature of evolution [3, 4]. Repeated patterns of genome reduction appear to have occurred via duplicated gene (homeolog) loss in divergent species following ancient WGD [5-9], with evidence for preferential retention of duplicates in certain gene classes [8-10]. The speed at which these patterns arise is unknown. We examined presence/absence of 70 homeologous loci in 59 Tragopogon miscellus plants from five natural populations of independent origin; this allotetraploid arose ~80 years ago via hybridization between diploid parents and WGD [11]. Genes were repeatedly retained or lost in clusters, and the gene ontology categories of the missing genes correspond to those lost after ancient WGD in the same family (Asteraceae; sunflower family) [6] and with gene dosage sensitivity [8]. These results provide evidence that the outcomes of WGD are predictable, even in 40 generations, perhaps due to the connectivity of gene products [8, 10, 12]. The high frequency of single-allele losses detected and low frequency of changes fixed within populations provide evidence for ongoing evolution.

Assembly and Validation of the Genome of the Nonmodel Basal Angiosperm Amborella

Shaping Plant Evolution Amborella trichopoda is understood to be the most basal extant flowering plant and its genome is anticipated to provide insights into the evolution of plant life on Earth (see the Perspective by Adams). To validate and assemble the sequence, Chamala et al. (p. 1516) combined fluorescent in situ hybridization (FISH), genomic mapping, and next-generation sequencing. The Amborella Genome Project (p. 10.1126/science.1241089) was able to infer that a whole-genome duplication event preceded the evolution of this ancestral angiosperm, and Rice et al. (p. 1468) found that numerous genes in the mitochondrion were acquired by horizontal gene transfer from other plants, including almost four entire mitochondrial genomes from mosses and algae. Fluorescence in situ hybridization allows for next-generation sequencing of a large, difficult genome. [Also see Perspective by Adams; Research Articles by Amborella Genome Project and Rice et al.] Genome sequencing with next-generation sequence (NGS) technologies can now be applied to organisms pivotal to addressing fundamental biological questions, but with genomes previously considered intractable or too expensive to undertake. However, for species with large and complex genomes, extensive genetic and physical map resources have, until now, been required to direct the sequencing effort and sequence assembly. As these resources are unavailable for most species, assembling high-quality genome sequences from NGS data remains challenging. We describe a strategy that uses NGS, fluorescence in situ hybridization, and whole-genome mapping to assemble a high-quality genome sequence for Amborella trichopoda, a nonmodel species crucial to understanding flowering plant evolution. These methods are applicable to many other organisms with limited genomic resources.

MarkerMiner 1.0: A New Application for Phylogenetic Marker Development Using Angiosperm Transcriptomes

Premise of the study: Targeted sequencing using next-generation sequencing (NGS) platforms offers enormous potential for plant systematics by enabling economical acquisition of multilocus data sets that can resolve difficult phylogenetic problems. However, because discovery of single-copy nuclear (SCN) loci from NGS data requires both bioinformatics skills and access to high-performance computing resources, the application of NGS data has been limited. Methods and Results: We developed MarkerMiner 1.0, a fully automated, open-access bioinformatic workflow and application for discovery of SCN loci in angiosperms. Our new tool identified as many as 1993 SCN loci from transcriptomic data sampled as part of four independent test cases representing marker development projects at different phylogenetic scales. Conclusions: MarkerMiner is an easy-to-use and effective tool for discovery of putative SCN loci. It can be run locally or via the Web, and its tabular and alignment outputs facilitate efficient downstream assessments of phylogenetic utility, locus selection, intron-exon boundary prediction, and primer or probe development.

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

BackgroundRecent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.ResultsAnalysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.ConclusionsWhen placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.

Genome-Wide Identification of Evolutionarily Conserved Alternative Splicing Events in Flowering Plants

Alternative splicing (AS) plays important roles in many plant functions, but its conservation across the plant kingdom is not known. We describe a methodology to identify AS events and identify conserved AS events across large phylogenetic distances using RNA-Seq datasets. We applied this methodology to transcriptome data from nine angiosperms including Amborella, the single sister species to all other extant flowering plants. AS events within 40–70% of the expressed multi-exonic genes per species were found, 27,120 of which are conserved among two or more of the taxa studied. While many events are species specific, many others are shared across long evolutionary distances suggesting they have functional significance. Conservation of AS event data provides an estimate of the number of ancestral AS events present at each node of the tree representing the nine species studied. Furthermore, the presence or absence of AS isoforms between species with different whole genome duplication (WGD) histories provides the opportunity to examine the impact of WDG on AS potential. Examining AS in gene families identifies those with high rates of AS, and conservation can distinguish ancient events vs. recent or species specific adaptations. The MADS-box and SR protein families are found to represent families with low and high occurrences of AS, respectively, yet their AS events were likely present in the MRCA of angiosperms.

Making next-generation sequencing work for you: approaches and practical considerations for marker development and phylogenetics

Recent reviews are setting the stage for the use of next-generation sequencing technologies in phylogenetic applications. However, the processes for developing new markers for phylogenetic analyses remain difficult to navigate for many researchers in plant systematics. We review several experimental approaches and practical considerations for developing new phylogenetic markers with next-generation sequencing technologies. We also outline a flexible framework for data acquisition that is readily adaptable to the needs of individual researchers and carefully consider cost-related issues that may be of concern to many laboratories in evolutionary biology. The next-generation and targeted sequencing approaches presented here offer considerable savings of time and money over the traditional PCR and Sanger sequencing approaches currently used in plant systematic research, particularly in cases involving large numbers of taxa and phylogenetic markers. Even with a limited research budget, next-generation sequence data can accommodate exploration of biological questions in ways that were not previously possible.

The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity

Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome.

The Early Stages of Polyploidy: Rapid and Repeated Evolution in Tragopogon

Elucidating the causes and consequences of polyploidy (whole-genome duplication; WGD) is arguably central to understanding the evolution of most eukaryotic lineages. However, much of what we know about these processes is derived from the study of crops and synthetic polyploids. Tragopogon provides the unique opportunity to investigate the genetic and genomic changes that occur across an evolutionary series from F1 hybrids, synthetic allopolyploids, independently formed natural populations of T. mirus and T. miscellus that are 60–80 years post-formation, to older Eurasian polyploids that are dated by molecular clocks at several million years old, and finally to a putative ancient polyploidization thought to have occurred prior to or early in the history of the Asteraceae (40–43 mya). Tragopogon joins other well-studied natural polyploid systems (e.g., Glycine, Nicotiana, Gossypium, Spartina, Senecio), but presents a range of research possibilities that is not available in any other system. We have shown in T. mirus and T. miscellus that upon allopolyploidization, massive gene loss occurs in patterns that are repeated across populations of independent origin and with a bias against genes derived from T. dubius, the diploid parent shared by both new allotetraploids. We have also shown significant changes in gene expression (transcriptomic shock) in the early generations of allopolyploidy in these species. Massive and repeated patterns of chromosomal variation (intergenomic translocations and aneuploidy) have been revealed by fluorescence in situ hybridization. Aneuploidy results in substitutions between homeologous chromosomes, through reciprocal monosomy-trisomy (1:3 copies) or nullisomy-tetrasomy (0:4 copies). We propose that substantial chromosomal instability results in karyotype restructuring, a likely common process following WGD and a driver of allopolyploid speciation, which has largely unexplored implications for gene losses, gains, and expression patterns. But gene loss and expression changes as well as karyotypic changes are ongoing in T. mirus and T. miscellus, in that no population is fixed for any of these events; thus, we have literally caught evolution in the act.

Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations

BackgroundDNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification.ResultsFrom pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps.ConclusionsThe apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes.