Julie Ferreira De Carvalho

Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima

Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5′-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies.

Reference transcriptomes and detection of duplicated copies in hexaploid and allododecaploid Spartina species (Poaceae)

Abstract In this study, we report the assembly and annotation of five reference transcriptomes for the European hexaploid Spartina species (S. maritima, S. alterniflora and their homoploid hybrids S. x townsendii and S. x neyrautii) and the allododecaploid invasive species S. anglica. These transcriptomes were constructed from various leaf and root cDNA libraries that were sequenced using both Roche-454 and Illumina technologies. Considering the high ploidy levels of the Spartina genomes under study, and considering the absence of diploid reference genome and the need of an appropriate analytical strategy, we developed generic bioinformatics tools to (1) detect different haplotypes of each gene within each species and (2) assign a parental origin to haplotypes detected in the hexaploid hybrids and the neo-allopolyploid. The approach described here allows the detection of putative homeologs from sets of short reads. Synonymous substitution rate (KS) comparisons between haplotypes from the hexaploid species revealed the presence of one KS peak (likely resulting from the tetraploid duplication event). The procedure developed in this study can be applied for future differential gene expression or genomics experiments to study the fate of duplicated genes in the invasive allododecaploid S. anglica.

Transcriptome response of the foundation plant Spartina alterniflora to the Deepwater Horizon oil spill

Despite the severe impacts of the Deepwater Horizon oil spill, the foundation plant species Spartina alterniflora proved resilient to heavy oiling, providing an opportunity to identify mechanisms of response to the anthropogenic stress of crude oil exposure. We assessed plants from oil‐affected and unaffected populations using a custom DNA microarray to identify genomewide transcription patterns and gene expression networks that respond to crude oil exposure. In addition, we used T‐DNA insertion lines of the model grass Brachypodium distachyon to assess the contribution of four novel candidate genes to crude oil response. Responses in S. alterniflora to hydrocarbon exposure across the transcriptome as well as xenobiotic specific response pathways had little overlap with those previously identified in the model plant Arabidopsis thaliana. Among T‐DNA insertion lines of B. distachyon, we found additional support for two candidate genes, one (ATTPS21) involved in volatile production, and the other (SUVH5) involved in epigenetic regulation of gene expression, that may be important in the response to crude oil. The architecture of crude oil response in S. alterniflora is unique from that of the model species A. thaliana, suggesting that xenobiotic response may be highly variable across plant species. In addition, further investigations of regulatory networks may benefit from more information about epigenetic response pathways.