Transcriptome sequencing, characterization and

polymorphism detection in Big Sagebrush (Artemisia

tridentata) subspecies

Prabin Bajgain, Joshua Udall (BYU, Provo)

Bryce Richardson (USDA-RMRS, Provo)

- Ecologically, one of the most important shrub species in the intermountain United States

- Three main widespread subspecies – ssp. tridentata (basin ecotype), ssp. vaseyana(mountain ecotype), ssp. wyomingensis (wyoming ecotype); two less common subspecies –ssp. spiciformis, ssp. Xericensis

- Numerous mammals, insects and birds are dependent on big sagebrush for food and shelter – some are obligates while some are semi-obligates

- Human encroachment and wildfires followed by cheatgrass invasion are threatening big sagebrush habitat, and those dependent on it

Big Sagebrush (Artemisia tridentata)

Goals

Entrez records

Database nameSubtree

linksDirectlinks

Nucleotide 32 31

Protein 14 14

Popset 3 3

SNP* 20,953 20,953

PubMed Central 34 34

Taxonomy 2 1

- To create a reliable and relatively large sequence database for big sagebrush

- Develop markers on the gene sequences

- Make the data publicly available for population, ecological and evolutionary studies NCBI Taxonomy Browser, Feb 17 2011

* Bajgain et al., ‘Transcriptome characterization and polymorphism detection in subspecies of Artemisia tridentata (big sagebrush)’ (in press)

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=Search&dopt=DocSum&term=txid55611[Organism:exp]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=Search&dopt=DocSum&term=txid55611[Organism:noexp]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein&cmd=Search&dopt=DocSum&term=txid55611[Organism:exp]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein&cmd=Search&dopt=DocSum&term=txid55611[Organism:noexp]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Popset&cmd=Search&dopt=DocSum&term=txid55611[Organism:exp]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Popset&cmd=Search&dopt=DocSum&term=txid55611[Organism:noexp]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PMC&cmd=Search&dopt=DocSum&term=txid55611[Organism:exp]&pmfilter_Fulltext=offhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PMC&cmd=Search&dopt=DocSum&term=txid55611[Organism:noexp]&pmfilter_Fulltext=offhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=taxonomy&cmd=Search&dopt=DocSum&term=txid55611[Subtree]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=taxonomy&cmd=Search&dopt=DocSum&term=55611[uid]

RNA extraction

and cDNA library

prep

454-sequencing(sspp. tridentata & vaseyana)

Sequence assembly

Pfam & BLASTx

search

Gene annotation(using BLASTxresults)

Marker detection

SNP mapping

Secondary

metabolite genes

Illumina sequencing(ssp. wyomingensis)

Hybridization

theory

Workflow

EST Sequence assembly

Assembly Count Average length Total bases

ssp.

tridentata

(basin)

Reads 823,392 403.91 332,578,737

Singletons 191,745 403.62 77,391,754

Contigs 20,357 716 14,587,705

ssp.

vaseyana

(mtn)

Reads 702,001 333.13 233,854,535

Singletons 179,189 331.51 59,402,844

Contigs 20,250 624 12,641,189

ssp.

combined

Reads 1,525,393 371.34 566,433,272

Singletons 275,866 370.18 102,121,262

Contigs 29,541 796 23,521,465

Summary report of individual and combined de novo assembly

Assembly annotation

• BLASTx:• against NR protein database• e-value of 1e-15

• BLAST2GO for annotation• 21,436 (72.6%) sequences had hits

Biological Process

Molecular Function

Cellular Component

EnzymesNo. of hits

(ssp. tridentata)

No. of hits

(ssp. vaseyana)

MEP

pathway

1-deoxy-D-xylulose 5-phosphate synthase (DXS) 51 100

1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP) 83 118

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCT) 22 22

4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK) 63 126

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) 22 22

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) 0 0

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) 20 12

isopentenyl diphosphate/ dimethylallyl diphosphate isomerase (IDI) 36 20

isopentenyl diphosphate/ dimethylallyl diphosphate synthase (IDS) 0 0

MVA

pathway

acetoacetyl-coenzyme A thiolase (AACT) 39 21

3-hydroxy-methylglutaryl coenzyme A synthase (HMGS) 0 0

3-hydroxy-methylglutaryl coenzyme A reductase (HMGR) 0 0

Mevalonate kinase (MK) 0 50

phosphomevalonate kinase (PMK) 0 0

mevalonate disphosphate decarboxylase (MDC) 20 0Coumarin

biosynthesis

pathway

phenylalanine ammonia lyase 29 45cinnamate 4-hydroxylase 28 704-coumarate CoA ligase 322 215

Secondary metabolite genes

SNP detection

• SNP = Single Nucleotide Polymorphism• parameters:

• 8x coverage; 90% nucleotide frequency; 20% minor allele frequency• 20,952 ‘true’ SNPs, average coverage 20x

0

500

1000

1500

2000

2500

8 12 16 20 24 28 32 36 40 44 48 52 56 60

Nu

mb

er

of S

NP

s

SNP coverage depth

Distribution of the number of SNPs by read coverage depth

SNP detection

tridentataSNPS

vaseyanaSNPS

Both SNP types

Montana wyomingensis

138 306 251

Utah wyomingensis

157 424 458

• suggests origin of tetraploid ssp. wyomingensis via mixed ancestry• more similar to ssp. vaseyana

total

695

1,039

SSR detection

• parameters• 2-7 3-5 4-5 5-5 6-5 7-5 8-5 9-4 10-4 (SSR motif length – repeat number) • 100 bp interruption distance

• 1,003 SSRs in basin• 507 SSRs in mtn

Frequency and distribution of SSRs in two big sagebrush subspecies

0

100

200

300

400

500

600

700

800

di tri tetra penta hexa

Nu

mb

er

of r

ep

eat

s

Repeat motif

tridentata

vaseyana

From here?

- Evolution, intermixing and more evolution of big sagebrush subspecies

- Phylogenetic relationship among big sagebrush populations distributed inthe intermountain US

- Sequence capture approach (~350 genes, 55 populations)

- Common garden studies to look at variation among the populations

- Later, link traits with genes in Artemisia tridentata populations

From here?

Acknowledgements

- Funding: USDA-FS, RMRS, National Fire Plan, GBNPSIP

- Rich Cronn

- Jared Price

- Nancy Shaw

- Covey Jones

- Brian Knaus

- Felix Jimenez

- Scott Yourstone