Wild Strawberry: An emerging model for ecological and evolutionary genomics

Wild Strawberry: An emerging model for ecological and evolutionary genomics

Aaron ListonDept. of Botany & Plant Pathology

Oregon State University

Why Strawberry?

Campos-de Quiroz 2002. Plant genomics: an

overview. Biological Research 35:385-399.

1. Small Genome Size

Fragaria vesca 236-244 Mbp

Arabidopsis thaliana 153-166 Mbp

1. Small Genome Size2. Easily Grown and Cloned

Why Strawberry?

Fragaria moupinensis in Sichuan, China

1. Small Genome Size2. Easily Grown and Cloned3. Short Generation Time & Stature

Why Strawberry?

1. Small Genome Size2. Easily Grown and Cloned3. Short Generation Time & Stature4. Efficient Creation of Transgenic Plants

Pantazis et al. 2013. Development of an efficient transformation method by Agrobacterium

tumefaciens and high throughput spray assay to identify transgenic plants for woodland

strawberry (Fragaria vesca) using NPTII selection. Plant Cell Reports 32: 329-337.

Why Strawberry?

1. Small Genome Size2. Easily Grown and Cloned3. Short Generation Time & Stature4. Efficient Creation of Transgenic Plants5. Unique Fruit Development

Why Strawberry?

1. Small Genome Size2. Easily Grown and Cloned3. Short Generation Time & Stature4. Efficient Creation of Transgenic Plants5. Unique Fruit Development6. Related to an Edible, High-Value Crop

Why Strawberry?

Cultivated Strawberry is an

Octoploid

Why Strawberry is a Bad Idea

Drawing by Pierre Dénys de Montfort (1801)

Bo & Davis. 2011. Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae). BMC Plant Biology 11: 157.

Fluorescence in situ hybridization (FISH) with 5S (green) and 25S (red) rDNA probes.

Fragaria vesca 2n=14

Fragaria virginiana 2n=56

Cultivated Strawberry is an

Octoploid

30% of 100 bp reads did

not distinguish between the

two progenitor genomes of

cultivated cotton.

“Using an arbitrary length of 1000

bp, we found 47,399 unique loci

where sequence reads of the AT-

genome and DT-genome were

indistinguishable when compared to

each other and to the reference

genome. Assuming sequence read

lengths <500bp, these regions would

likely co-assemble during a de novo

whole genome shotgun assembly

with current read lengths.”

Page et al. 2013.

G3. Early Online.

454, SOLiD and Illumina genome & transcriptome

39X coverage, 25-365 bp reads

202 million base pair (Mbp) assembly

3263 scaffolds 34,809 predicted genes

Fragaria vesca ssp. vesca “Hawaii 4”

“Any sequenced genome is simply a parts list. It is a comprehensive accounting of the

components that make up the genetic basis of the organism and the elements that control

their expression and activity”.

“Any sequenced genome is simply a parts list. It is a comprehensive accounting of the

components that make up the genetic basis of the organism and the elements that control

their expression and activity”.

“Any sequenced genome is simply a parts list. It is a comprehensive accounting of the

components that make up the genetic basis of the organism and the elements that control

their expression and activity”.

“The strawberry plant may be thought of as a factory that takes

water, sunlight, carbon dioxide, and a pinch of minerals to

assemble a desirable product. If you want to understand the

product and how to make it better, cheaper, or faster, you need to

understand the mechanics of the factory at a nuts-and-bolts level.

This level of understanding comes quite quickly if you have the

blueprints. Blueprints show you how parts are assembled and

interact. Unfortunately, blueprint-level resolution of the strawberry

is still decades in the future”.

“Any sequenced genome is simply a parts list. It is a comprehensive accounting of the

components that make up the genetic basis of the organism and the elements that control

their expression and activity”.

“The strawberry plant may be thought of as a factory that takes

water, sunlight, carbon dioxide, and a pinch of minerals to

assemble a desirable product. If you want to understand the

product and how to make it better, cheaper, or faster, you need to

understand the mechanics of the factory at a nuts-and-bolts level.

This level of understanding comes quite quickly if you have the

blueprints. Blueprints show you how parts are assembled and

interact. Unfortunately, blueprint-level resolution of the strawberry

is still decades in the future”.

“Any sequenced genome is simply a parts list. It is a comprehensive accounting of the

components that make up the genetic basis of the organism and the elements that control

their expression and activity”.

“The strawberry plant may be thought of as a factory that takes

water, sunlight, carbon dioxide, and a pinch of minerals to

assemble a desirable product. If you want to understand the

product and how to make it better, cheaper, or faster, you need to

understand the mechanics of the factory at a nuts-and-bolts level.

This level of understanding comes quite quickly if you have the

blueprints. Blueprints show you how parts are assembled and

interact. Unfortunately, blueprint-level resolution of the strawberry

is still decades in the future”.

Reference guided assembly De novo assembly

Genome Assembly

Genome Coverage

Low Coverage High Coverage

Targeted sequence capture approach

Cronn et al. 2012 Amer J Bot

Solution Phase Hybridization(e.g., Mycroarray MyBait)

• ‘Baits’ synthesized on arrays

• 80-120 bp RNA probes

• Hybridization in solution

• 100 – 500 ng DNA of input library

• Immobilization via biotin-streptavidin bead capture

Targeted sequence capture advantages

• Ability to target SNPs known to be informative in parents

• Efficient data generation-1000s of markers in multiple individuals

• Low variation in locus recovery

• Rapid means for comparative analysis between related species

Cronn et al. 2012 Amer J Bot

vescaiinumae nilgerrensis pentaphyllanipponica viridis

Fragaria Morphological “Diversity”

Leaf and flower Images courtesy of USDA National Clonal Germplasm Repository.

Variation in Sexual Systems

female hermaphrodite

Fragaria vesca subsp. bracteata

Fragaria virginiana

gynodioecious

dioecious

Genetic Linkage Mapping of Male Sterility

female hermaphrodite

Fragaria vesca ssp. bracteata

Genetic Linkage Mapping

www.lifesciencesfoundation.org

Targeted sequence linkage mapping in Fragaria vesca ssp. bracteata

Maternal genome 5.4x

coverage

Paternal genome 3.2x

coverage

Probes targeting 6575 informative variants

48 F1

offspring

~2Mb

sequenced in

each offspring

Mybaits:

3 overlaping

100bp

Sum of all baits

=1% genome

Mean read

depth = 120x

Tennessen et al. 2013

Log 1

0o

f o

dd

s (L

OD

) t

hat

lin

kage

did

no

t o

ccu

r b

y ch

ance

Linkage Groupaxis = recombination events

Linkage between markers on chromosome 4 and male sterility (40 flowering progeny)

Tennessen et al. 2013

Tennessen et al. 2013

Identification of a 2.4 Mbp gap in the assembly

Fine Mapping to a 338 kb locus (95 progeny)

Tennessen et al. 2013

57 Candidate Genes

Tennessen et al. 2013

Chromosomal Rearrangements

or Assembly Artifacts?

Tennessen et al. 2013

SNP detection:

diploid vs octoploid

Polymorphism in the octoploids

Avg. read depth 120x

1:1 1:7

2 adjacent SNPs

that satisfy criteria

Read depth

>32x

Octoploid Linkage Map

Octoploid Linkage Map

Tennessen et al.

Fragaria chiloensis

Fragaria virginiana

Fragaria ananassa1759Philip MillerGardener’s Dictionary

Origin of the Cultivated Strawberry

Introduced to Europe in 1714

Introduced to Europe by 1629

F. virginiana

F. chiloensis F. ananassa var. cuneifolia

Acknowledgements

Rich Cronn – US Forest Service PNW Research Station

Tia-Lynn Ashman – University of Pittsburg

Laboratory & Bioinformatics:

Matt Parks – Griffith Univ, Brisbane Shannon Straub – Oregon State U.Jacob Tennessen – Oregon State U.Brian Knaus – USDA ARSRajanikanth Govindarajulu – U. PittsburghKevin Weitemier – Oregon State U.Chris Edwards – Oregon State U.Zach Foster – Oregon State U.Kimberly Hansen – Northern Ariz U. Laura Mealy – South Dakota State U.

OSU Center for Genome Research & Biocomputing:

Mark DasenkoChris SullivanMathew Peterson

Plants:

Katherine Schuller – U. Pittsburgh

John Syring – Linfield College

USDA NCGR

Nahla Bassil

April Nyberg

Kim Hummer

Funding:

US National Science Foundation

Zach Foster

Kevin Weitemier

Laura Mealy

Kimberly Hansen

Stephen Meyers

Matt Parks

Shannon Straub

Jacob Tennessen

Rich Cronn

Tia-Lynn Ashman

Chris Edwards