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Mitochondrial genome sequences illuminate maternal lineages of conservation concern in a rare carnivore

  


Science-based wildlife management relies on genetic information to infer population connectivity and identify conservation units. The most commonly used genetic marker for characterizing animal biodiversity and identifying maternal lineages is the mitochondrial genome. Mitochondrial genotyping figures prominently in conservation and management plans, with much of the attention focused on the non-coding displacement ("D") loop. We used massively parallel multiplexed sequencing to sequence complete mitochondrial genomes from 40 fishers, a threatened carnivore that possesses low mitogenomic diversity. This allowed us to test a key assumption of conservation genetics, specifically, that the D-loop accurately reflects genealogical relationships and variation of the larger mitochondrial genome.

Knaus et al. BMC Ecology 2011, 11:10

Mitochondrial genomes (at NCBI):
http://www.ncbi.nlm.nih.gov/nuccore/GU121228
http://www.ncbi.nlm.nih.gov/nuccore/GU121229
http://www.ncbi.nlm.nih.gov/nuccore/GU121230
http://www.ncbi.nlm.nih.gov/nuccore/GU121231
http://www.ncbi.nlm.nih.gov/nuccore/GU121232
http://www.ncbi.nlm.nih.gov/nuccore/GU121233
http://www.ncbi.nlm.nih.gov/nuccore/GU121234
http://www.ncbi.nlm.nih.gov/nuccore/GU121235
http://www.ncbi.nlm.nih.gov/nuccore/GU121236
http://www.ncbi.nlm.nih.gov/nuccore/GU121237
http://www.ncbi.nlm.nih.gov/nuccore/HQ705176
http://www.ncbi.nlm.nih.gov/nuccore/HQ705177
http://www.ncbi.nlm.nih.gov/nuccore/HQ705178
http://www.ncbi.nlm.nih.gov/nuccore/HQ705179

Short read (Illumina) libraries (at EBI):
http://www.ebi.ac.uk/ena/data/view/ERP000590

Analyses of mitochondrial genome variation reveals the distinctiveness of western Fisher.
Analyses of mitochondrial genome variation reveals the distinctiveness of western Fisher.

Transcriptome characterization and polymorphism detection between subspecies of big sagebrush (Artemisia tridentata)

  
  
Big sagebrush (Artemisia tridentata) is one of the most widely distributed and ecologically important shrub species in western North America. This species serves as a critical habitat and food resource for many animals and invertebrates. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is a serious threat to big sagebrush ecosystem sustainability. Lack of genomic data has limited our understanding of theevolutionary history and ecological adaptation in this species. Here, we report on the sequencing of expressed sequence tags (ESTs) and detection of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in subspecies of big sagebrush. We have produced a large EST dataset for Artemisia tridentata, which contains a large sample of the big sagebrush leaf transcriptome. SNP mapping among the three subspecies suggest the origin of ssp. wyomingensis via mixed ancestry. A large number of SNP and SSR markers provide the foundation for future research to address questions in big sagebrush evolution, ecological genetics, and conservation using genomic approaches.

Bajgain P, Richardson BA, Price JC, Cronn RC, Udall JA. 2011. Transcriptome characterization and polymorphism detection between subspecies of big sagebrush (Artemisia tridentata). BMC Genomics 12:370.

Udall, J.A., Bajgain, P., Richardson, B.A. 2011. Natural sequence diversity of Artemisia tridentata in the western United States. Botany: Healing the plant, Botanical Society of America. July 9-13, 2011, St. Louis, MO.

Richardson, B.A., Bajgain, P. Udall, J.A., Shaw, N.L. 2011. Assembling the adaptive genetic and genomic pieces in Intermountain shrub species to provide conservation and restoration guidelines. Western Forest Genetics Association. July 25-28, 2011. Portland, OR.

Artemisia tridentata project page at NCBI: http://www.ncbi.nlm.nih.gov/SNP/
Identified SNPs: Includes ss-id starting at ss252842630, and ending at ss252863858.

Artemisia de novo assembly: basin_mtn_contigs.fasta

Raw Roche/454 reads, mtn big sagebrush: http://www.ncbi.nlm.nih.gov/sra/SRX022251
Raw Roche/454 reads, basin big sagebrush: http://www.ncbi.nlm.nih.gov/sra/SRX022255

SRA accession for Illumina reads: SRA020571.3

Transcriptome sequencing has identified over 21,000 genes from Big Sagebrush.
Transcriptome sequencing has identified over 21,000 genes from Big Sagebrush.

Douglas-fir Transcriptome Observatory

  
  
Douglas-fir is finely attuned to its local environment, making it critical to match seed sources and planting locations. Despite the ecological and economic importance of local adaptation, genes responsible for adaptation are unknown. Western Transcriptome funding helped us assemble a "transcriptome reference" that identifies expressed genes in diverse tissues. This reference includes over 38,000 genes, roughly twice as many genes as are in humans! The USDA-NIFA Plant Genome program funded the "Douglas-fir Transcriptome Observatory", which will track gene expression in well-adapted and poorly adapted trees as related to seasonal weather changes over a complete growing season.

Presentation at Plant and Animal Genomes XIX (2011): psme_transcriptome_v2.pdf.

Douglas-fir needle transcriptome, bzipped fasta (11MB): psme_strandspec_trinity.fasta.bz2

Transcriptome sequencing has identified over 38,000 genes from Douglas-fir.
Transcriptome sequencing has identified over 38,000 genes from Douglas-fir.

Gene expression in the tanoak-Phytophthora ramorum interaction.

  
  
Disease processes are dynamic, and involve a suite of changes in gene expression in both the host and the pathogen. As such, they lend themselves well to transcriptomic analysis. Moreover, a single tissue contains both host and pathogen RNA, allowing for the study of the transcriptional responses of both species to each other. Here we focus on a generalist invasive pathogen (Phytophthora ramorum) and its most susceptible California Floristic Province native host (tanoak, Notholithocarpus densiflorus). The advent of new sequencing technologies has made the study of non-model systems possible at a scale never before possible. We argue that this non-model system is ideal for studying the interactions between host and pathogen using massively parallel mRNA sequencing, in part because the P. ramorum genome has been fully sequenced.

Poster presented at conference: Hayden_Transcription_v2.pdf
Poster presented at the Society for the Study of Evolution conference 2011: Wright_SSE_Poster.pdf

Preliminary work funded by the U.S. Forest Service has contributed to the funding of the continuation of this work by the Gordon and Betty Moore Foundation.

Interactions between Tan Oak and Sudden Oak-Death are revealed by mRNA sequencing. .
Interactions between Tan Oak and Sudden Oak-Death are revealed by mRNA sequencing.

Aspen transcriptome

  
  
As part of the collaborative Western Forest Transcriptome Survey, we have sequenced the transcriptome of Populus tremuloides (quaking aspen), a close relative of the fully- sequenced model organism, Populus trichocarpa (black cottonwood). P. tremuloides has the largest latitudinal distribution of any North American tree, and is an excellent study system for adaptation and climate change.

This study can be summarized by two major objectives:
1) to provide the first blueprint of the expressed portion of the aspen genome
2) to characterize variation present within and among natural populations of aspen, across a latitudinal gradient.

To address these goals, we have collected and propagated aspen roots from locations in Arizona and Montana. Total RNA was extracted from mature, mid-season leaves of clones grown in a common environment. cDNA from a total of six individuals (three each from Arizona and Montana) were bar-coded and sequenced using an Illumina Genome Analyser. Bar-coded sequences were pooled to address our first objective and separated for the second. Preliminary results indicate that the transcriptome of P. tremuloides has greater than 90% sequence similarity to the annotated coding regions of P. trichocarpa genome. We will present results from our search for variation in genes and gene networks across a latitudinal gradient, summarizing evolutionary rate differences in genes from P. trichocarpa vs. P. tremuloides and expression differences within and between P. tremuloides populations. The addition of our transcriptome data to the burgeoning knowledge of Populus genetics further extends the utility of this long-lived angiosperm model organism.

Latitudinal Gene Expression Variation in A Widely Distributed Tree Species. Presentation at Plant and Animal Genomes XIX (2011): HR_PAG_2011_18Jan11.pdf

Aspen has one of the largest latitudinal distribution of any N. American tree.
Aspen has one of the largest latitudinal distribution of any N. American tree.

Sugar pine transcriptome

  
  
Sugar pine is an ecologically and economically important tree species in the West. It ranges from Mexico to Oregon. We sampled and sequenced the transcriptome of 3 individuals, one from Mexico, one from Oregon and one from California, all growing within 3 meters of each other in a common garden. With this data we hope to capture the range of gene expression variation across the species in this shared environment.

Poster presented at conference: JWW_GordonConference2011.pdf
Sugar pine transcriptome, bzipped fasta (11MB): pila_Trinity_name.fa.bz2

Sugar pine is an ecologically and economically important tree species in which ranges from Mexico to Oregon.
Sugar pine is an ecologically and economically important tree species in which ranges from Mexico to Oregon.

Armillaria transcriptome

  
  
Armillaria (Fr.) Staude is a widely distributed genus of approximately 40 species (Volk and Burdsall 1995) that display diverse ecological behaviors ranging from saprobe to virulent pathogen. A. solidipes (formerly A. ostoyae; Burdsall and Volk 2008), one of the causal agents of Armillaria root disease, is a virulent primary pathogen with a broad host range in north temperate latitudes (Kile et al. 1991). The fungus attacks sapwood and travels under bark as mycelial fans and between trees as rhizomorphs. The pathogen causes a white rot of infected wood and is responsible for reduced forest yields as a result of direct tree mortality and non-lethal cryptic infections (Cruickshank et al. 2011).

The purpose of this research is to present the first assembly and characterization of a transcriptome from a root disease pathogen. Specifically, our objective is to identify a large number of genes expressed by an active mycelial fan of A. solidipes, focusing on genes that may be associated with pathogenicity (i.e., those that result in a loss or reduction in disease symptoms when disrupted). Detection of putative genes that show homology to annotated genes involved in infection, cuticle and cell wall degradation, response to host environment, production of fungal toxins and signaling will ultimately help inform forest management decisions.

De novo assembly and transcriptome characterization of an Armillaria solidipes mycelial fan. Poster presented at Western International Forest Disease Work Conference.

Armillaria is a widely distributed genus displaying behaviors ranging from saprobe to virulent pathogen.
Armillaria is a widely distributed genus displaying behaviors ranging from saprobe to virulent pathogen.
(Image from wikipedia.org)

Genetic resources in conifers

  
  
Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356 958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5 M (USD).

Jennings, T. N., Knaus, B. J., Mullins, T. D., Haig, S. M. and Cronn, R. C. (2011), Multiplexed microsatellite recovery using massively parallel sequencing. Molecular Ecology Resources. doi: 10.1111/j.1755-0998.2011.03033.x

Software resources: http://brianknaus.com/

Short read (Illumina) libraries (at EBI): http://www.ebi.ac.uk/ena/data/view/ERP000647

Microsatellites were developed for Pfrimer's conure, Gull-billed tern, Micronesian Kingfisher, Yellow rail, Red-cockaded woodpecker, Alaska yellow cedar and Port Orford cedar.
Microsatellites were developed for Pfrimer's conure, Gull-billed tern, Micronesian Kingfisher, Yellow rail, Red-cockaded woodpecker, Alaska yellow cedar and Port Orford cedar.

Port Orford Cedar microsatellites:

  

Microsatellite primers were developed for the Pacific Northwest conifer, Chamaecyparis lawsoniana (Cupressaceae), to enhance efficiencies in disease-resistance breeding and germplasm screening for gene conservation of this rare species. Using multiplexed massively parallel Illumina sequencing, we identified over 300,000 microsatellite-containing sequences from 2 million paired-end microreads. After stringent filtering and primer evaluation, we selected 11 primer pairs and used these to screen variation in four populations of C. lawsoniana. Loci show between three and 10 repeats per locus, with an average of eight. Screening of these markers in the North American relative Callitropsis nootkatensis demonstrated limited marker transferability, but these markers could have utility in Asian species of Chamaecyparis. These microsatellite primers show high polymorphism and should provide a high level of individual discrimination for paternity analysis in defined pedigrees, and routine screening of wild variation in Chamaecyparis lawsoniana.   
Jennings TN, Knaus BJ, Kolpak S, Cronn R (2011) Microsatellite primers for the Pacific Northwest endemic conifer Chamaecyparis lawsoniana (Cupressaceae). American Journal of Botany. doi:10.3732/ajb.1100317

Primer sequences:
Jennings TN, Knaus BJ, Kolpak S, Cronn R (2011) Data from: Microsatellite primers for the Pacific Northwest endemic conifer Chamaecyparis lawsoniana (Cupressaceae). Dryad Digital Repository. doi:10.5061/dryad.bq002

Port Orford cedar is an ecologically and economically important species that has a distribution limited to riparian zones, coastal marine terraces, and serpentine soils of southwestern Oregon and northwestern California and is susceptible to the introduced and highly virulent root pathogen Phytophthora lateralis.
Port Orford cedar is an ecologically and economically important species that has a distribution limited to riparian zones, coastal marine terraces, and serpentine soils of southwestern Oregon and northwestern California and is susceptible to the introduced and highly virulent root pathogen Phytophthora lateralis.

Microsatellites developed for 30 conifers:

  
  
In order to provide new molecular resources for a host of conifers, we've recently used the Jennings et al. (2011) method to develop microsatellites for many conifer taxa. Sequences with designed primers are posted at:

http://openwetware.org/wiki/Conifermicrosat.

High quality genetic markers will benefit whitebark pine conservation and animals dependent on this threatened species.
High quality genetic markers will benefit whitebark pine conservation and animals dependent on this threatened species.