Cushla J. Metcalfe

Building the sugarcane genome for biotechnology and identifying evolutionary trends.

BackgroundSugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome.ResultsThree hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences.ConclusionThis release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

BackgroundSugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out.ResultsSixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gyps y/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements.ConclusionsIndividual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNAs that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

Evolution of the Australian Lungfish (Neoceratodus forsteri) Genome: A Major Role for CR1 and L2 LINE Elements

Haploid genomes greater than 25,000 Mb are rare, within the animals only the lungfish and some of the salamanders and crustaceans are known to have genomes this large. There is very little data on the structure of genomes this size. It is known, however, that for animal genomes up to 3,000 Mb, there is in general a good correlation between genome size and the percent of the genome composed of repetitive sequence and that this repetitive component is highly dynamic. In this study, we sampled the Australian lungfish genome using three mini-genomic libraries and found that with very little sequence, the results converged on an estimate of 40% of the genome being composed of recognizable transposable elements (TEs), chiefly from the CR1 and L2 long interspersed nuclear element clades. We further characterized the CR1 and L2 elements in the lungfish genome and show that although most CR1 elements probably represent recent amplifications, the L2 elements are more diverse and are more likely the result of a series of amplifications. We suggest that our sampling method has probably underestimated the recognizable TE content. However, on the basis of the most likely sources of error, we suggest that this very large genome is not largely composed of recently amplified, undetected TEs but may instead include a large component of older degenerate TEs. Based on these estimates, and on Thomsons (Thomson K. 1972. An attempt to reconstruct evolutionary changes in the cellular DNA content of lungfish. J Exp Zool. 180:363-372) inference that in the lineage leading to the extant Australian lungfish, there was massive increase in genome size between 350 and 200 mya, after which the size of the genome changed little, we speculate that the very large Australian lungfish genome may be the result of a massive amplification of TEs followed by a long period with a very low rate of sequence removal and some ongoing TE activity.

Phylogenetic relationships of rock-wallabies, Petrogale (Marsupialia: Macropodidae) and their biogeographic history within Australia

The rock-wallaby genus Petrogale comprises a group of habitat-specialist macropodids endemic to Australia. Their restriction to rocky outcrops, with infrequent interpopulation dispersal, has been suggested as the cause of their recent and rapid diversification. Molecular phylogenetic relationships within and among species of Petrogale were analysed using mitochondrial (cytochrome oxidase c subunit 1, cytochrome b, NADH dehydrogenase subunit 2) and nuclear (omega-globin intron, breast and ovarian cancer susceptibility gene) sequence data with representatives that encompassed the morphological and chromosomal variation within the genus, including for the first time both Petrogale concinna and Petrogale purpureicollis. Four distinct lineages were identified, (1) the brachyotis group, (2) Petrogale persephone, (3) Petrogalexanthopus and (4) the lateralis-penicillata group. Three of these lineages include taxa with the ancestral karyotype (2n=22). Paraphyletic relationships within the brachyotis group indicate the need for a focused phylogeographic study. There was support for P. purpureicollis being reinstated as a full species and P. concinna being placed within Petrogale rather than in the monotypic genus Peradorcas. Bayesian analyses of divergence times suggest that episodes of diversification commenced in the late Miocene-Pliocene and continued throughout the Pleistocene. Ancestral state reconstructions suggest that Petrogale originated in a mesic environment and dispersed into more arid environments, events that correlate with the timing of radiations in other arid zone vertebrate taxa across Australia.

Accommodating the load: The transposable element content of very large genomes.

Very large genomes, that is, those above 20 Gb, are rare but widely distributed throughout the eukaryotes. They are found within the diatoms, dinoflagellates, metazoans and green plants, but so far have not been found in the excavates. There is a known positive correlation between genome size and the proportion of the genome composed of transposable elements (TEs). Very large genomes may therefore be expected to be almost entirely composed of TEs. Of the large genomes examined, in the angiosperms, gymnosperms and the dinoflagellates only a small portion of the genome was identified as TEs, most of these genomes were unidentified and may be novel or diverse TEs. In the salamanders and lungfish, 25 to 47% of the genome were identifiable retrotransposons, that is, TEs that copy themselves before insertion. However, the predominant class of TEs found in the lungfish was not the same as that found in the salamanders. The little data we have at the moment suggests therefore that the diversity and abundance of TEs is variable between taxa with large genomes, similar to patterns found in taxa with smaller genomes. Based on results from the human genome, we suggest that the ‘missing’ portion of the lungfish and salamander genomes are old, highly divergent, and therefore inactive copies of TEs. The data available indicate that, unlike plants with large genomes, neither the lungfish nor the salamanders show an increased risk of extinction. Based on a slow rate of DNA loss in salamanders it has been suggested that the large salamander genome is the result of run-away genome expansion involving genome size increases via TE proliferation associated with reduced recombination rate. We know of no studies on DNA loss or recombination rates in lungfish genomes, however a similar scenario could describe the process of genome expansion in the lungfish. A series of waves of TE transposition and sequence decay would describe the pattern of TE content seen in both the lungfish and the salamanders. The lungfish and salamanders, therefore, may accommodate their large load of TEs because these TEs have accumulated gradually over a long period of time and have been subject to inactivation and decay.

Phylogeography of Cuban Rivulus: Evidence for allopatric speciation and secondary dispersal across a marine barrier

The genus Rivulus is currently comprised of two species, R. cylindraceus and R. insulaepinorum, which are endemic to Cuba. However, the taxonomic status of the latter species remains dubious because of the poor quality of the original description. In addition, a recent barcoding survey suggests that the two species may be conspecific. The aim of this study was to test the hypothesis that the two species represent a single evolutionary clade. To delimit the species and their evolutionary history, we used a combination of molecular phylogenetic analyses, with both mitochondrial and nuclear sequences, tests of phylogeographic hypotheses, combined with morphological measurements and information on known dispersal barriers and species distribution. None of the data sets support R. insulaepinorum and R. cylindraceus as separate taxa. However, a new species, restricted to the northwestern part of the main island, was identified by phylogenetic analyses, body colour pattern and geographical distribution. The evolutionary distance between the two lineages (cytb, d=15%; CAM-4, d=2.5%) indicates a long period of divergence. Phylogeographic analyses shed light on the dispersal history of R. cylindraceus, which probably originated on the Isla de la Juventud. They also suggest that each lineage had contrasting histories; Rivulus sp. is restricted to a relatively small geographic area whereas R. cylindraceus has dispersed considerably and more than once from its centre of origin, probably facilitated by sea level fluctuations. These results strengthen previous findings, i.e. that the diversity of Cuban freshwater fishes is far from well-known and deserves more in-depth studies, and that vicariance and dispersal events have resulted in a complex biogeographical landscape which has had a significant impact on the freshwater fishes of the Caribbean islands.

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: Evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.

Heterogeneous conservation of Dlx paralog co-expression in jawed vertebrates.

Background The Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model. Results The genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family. Conclusion Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

Genetic differentiation and introgression amongst Thylogale (pademelons) taxa in eastern Australia

Although pademelons (Thylogale) are widespread and common in coastal eastern Australia, they have been largely neglected in population genetic studies. Here we use 10 microsatellite loci and the mitochondrial DNA (mtDNA) cytochrome b (Cytb) gene to examine genetic differentiation amongst populations of the red-necked (Thylogale thetis) and red-legged (T. stigmatica) pademelon in eastern Australia. Evidence of hybridisation was detected between subspecies of T. stigmatica in central Queensland. Specimens sampled between Eungella and Sarina were found to represent a broad (~90km)zoneofintrogression,forbothnuclearandmtDNAmarkers,betweenT.s.stigmatica(WetTropics)andT.s.wilcoxi (south-east Queensland). In addition, individuals sampled from around Proserpine were genetically T. s. stigmatica rather than T. s. wilcoxi, as had previously been assumed. This observation raises some intriguing questions about the dispersal ability of T. stigmatica and the phylogeographic history of moist forest taxa in eastern Australia. Only limited evidence of introgression was detected between sympatric populations of T. thetis and T. s. wilcoxi in south-east Queensland.

Using quantitative PCR with retrotransposon-based insertion polymorphisms as markers in sugarcane

Highlight qPCR-RBIP was used to examine the dosage of particular markers and evolutionary history in Saccharum and the related genera, Erianthus and Miscanthus. It also differentiated between S. spontaneum and S. officinarum.