Developing Bracken Genomic Resources

Developing genomic resources in the bracken fern, Pteridium aquilinum Joshua Der Dept. Biology, Utah State University

Systems Biology • Shift from reductionist to holistic approach

to understanding complex biological systems

• From single genes and proteins to genomes, transcriptomes and proteomes

• genome structure • gene content, regulation, expression and subsequent processes

• molecular/cellular interaction networks

Genomics • Complete human genome sequence ushered in a new era of genetic investigation (2003)

• Explosion in complete genome sequences • Genome-wide expression studies (microarrays) • Genomics has primarily focused on microbes, model organisms , and important crop plants

Genomics for non-model organisms • de novo genome sequencing is prohibitive, especially for large genomes

• Expressed Sequence Tags (ESTs) • genome-wide transcript tags • Floral Genome Project: bridging genomic information between model organisms

• Next-generation sequencing technologies (Illumina Solexa, ABI SOLID, Roche 454)

Fern evolution • Evolutionarily intermediate between

non-vascular plants (bryophytes) and seed plants (gymnosperms and flowering plants)

• Evolved and maintained independent, photosynthetic, gametophyte and sporophyte generations

Alternation of generations Sporophyte (2n) meiosis

zygote (2n)

syngamy

egg (n)

Generalized plant life cycle sperm (n)

Gametophyte (n)

haploid spores (n)

Alternation of generations meiosis

zygote (2n)

syngamy

egg (n)

Fern life cycle sperm (n)

haploid spores (n)

Fern genetics • Recessive alleles are not masked in haploid gametophytes • Gametophytes can be vegetatively propagated • Controlled crosses and double haploid lines (selfed individuals are homozygous at all loci) • Gametic-phase segregation and recombination can be directly observed from gametophytes • Genome function can be examined in haploid and diploid phases independently

Challenges in fern genetics • Large genome sizes (ave. 5000 MB) • Large chromosome numbers (ave. n=57) • History of polyploidy and hybridization • No fern genomes have been sequenced or proposed for sequencing

• Linkage map and ESTs for Ceratopteris

Bracken fern, Pteridium aquilinum • • • • • •

Worldwide distribution Economically important Highly adaptable and phenotypically plastic Well established culture techniques Model for fern gametophyte development and pheromonal sex determination Ancient polyploid with diploid gene expression (isozymes)

Research questions • What genes and ontology groups are expressed in the reproductive haploid phase in ferns? • Are any of these genes homologous with • •

reproductive genes in bryophytes or flowering plants? Is there a signature of past polyploidy in the transcriptome? Are genes expressed in the haploid phase under purifying selection?

Next-gen sequencing: Roche 454 • Massively parallel, high-throughput platform • 400-600 million bp per run, 400+ bp long • Applications include genome sequencing,

metagenomics, genotype determination, and transcriptome sequencing

• Basically a shotgun sequencing approach that utilizes bioluminescence for sequence determination (pyrosequencing)

Experimental design: Transcriptome sequencing • Total RNA extraction • cDNA synthesis enriched for full length mRNA • cDNA library normalization (enriched for rare transcripts) • Roche 454 library preparation: fragmentation, adapter ligation, emPCR • 1/2 plate Roche 454 Titanium

Experimental design: Bioinformatics • de novo transcriptome assembly • Scaffold assembly against a reference library • Unigene identification, annotation, functional classification • Functional description of gametophyte transcriptome • Identify homologous genes involved in reproduction in moss and seed plants • Analysis of duplicate genes and past polyploidy

Future work •

•

Microsatellites: mined from transcriptome Population genetics: gene flow, demographics, population structure Test for selective sweeps to ID candidate genes for adaptation, reproductive isolation, and speciation Linkage maps Sporophyte expression patterns Custom oligonucleotide arrays Quantitative PCR Transcriptome sequencing...

• • • • • •

Acknowledgments Paul Wolf - support, design/objectives feedback, spores Aaron Duffy - gametophyte culture techniques Mike Pfrender - lab space and equipment for RNA extraction Center for Integrated Biosystems, Utah State University - CIBR students program support VP for Research, Utah State University - matching funds for library preparation and sequencing Center for Genomics and Bioinformatics, Indiana University - library preparation and sequencing My Family: Kristal and Cora - moral support and keeping me grounded

Questions?