Mimulus guttatus: a resource for
comparative genomics in the asterids
UNC Chapel Hill: Todd Vision et al.
(Toby Clarke, Amy Bouck, Eric Ganko*, Andrew Morgan, Stefanie Hartmann, Jason Phillips)
Duke University: John Willis et al
University of Washington: Toby Bradshaw et al
University of Montana: Lila Fishman et al
CUGI: Jeff Tomkins* et al
JGI: Dan Rokhshar et al
Stanford*: Jeremy Schmutz, Jane Grimwood
Funded by: NSF FIBR, DOE CSP
Outline
• Background on Mimulus and the asterids
• Status of genomic resources
 ESTs, BACs, physical and genetic maps
 JGI whole-genome shotgun assembly
 Status of annotation and high-density mapping
• Comparative genomics
 Polyploidy and synteny
 Phylogenetic relationship of Solanaceae relative to
Mimulus and coffee
The lamiid (euasterid I) clade
Coffea
Solanaceae
Antirrhinum
Mimulus
106-119 Mya
Striga
Sesamum
Bremer et al (2002) Mol Phyl Evol 24, 274
Bremer et al (2004) Syst Bio 53, 496
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~160 species in the Phrymaceae
 ex Scrophulariaceae
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Multiple species complexes
Diversity of floral morphologies
and mating systems
Habitat and life history diversity
 from sea level to 10,000 ft
 serpentine and mine tailings
 aquatics, woody shrubs
The genus Mimulus
M. guttatus as a lab organism
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Easy to cultivate & clone
Small size
Generation time ~2 mos.
High seed yield
Can be easily selfed or crossed
Small genome (430 Mb)
High recombination (1 cM ~ 250 kb)
Rich in SNP/indel polymorphism
Really nice field sites
Iron Mountain, OR
Chronology of an emerging
model
Various investigators study:
• the genetics of speciation
Ecological genetic studies begun by • inbreeding depression
Permanent mapping populations
Clausen, Keck & Heisey
• mating system evolution
ESTs, gene based markers
• ecological adaptation
BAC-based physical maps
• cytological evolution
Molecular phylogeny of genus
Genome sequence
Biosystematic studies
begun by Robert Vickery
First markers &
linkage maps
1940
1950
1960
1970
1980
1990
2000
~4K records for “Mimulus + genetics” in Google Scholar
Some ongoing studies in M. guttatus
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Dobzhansky-Muller factors
Cytoplasmic male sterility
Meiotic drive
Copper tolerance & reproductive isolation
Floral pigmentation
Inbreeding depression
Water use efficiency
Flowering time & life history
Candidate genes for floral diversification in Mimulus
EMS mutants of M. lewisii
Christina Pince, Bradshaw lab
M. lewisii mutant
M. lewisii wt
M. lewisii mutant
M. lewisii mutant
M. inconspicuus
M. douglasii
M. lewisii mutant
M. bifidus
M. parishii
M. lewisii mutant M. cardinalis
M. lewisii and M. cardinalis
Evolution of prezygotic isolation
M. lewisii
M. cardinalis
Elevation
mid-high
low-mid
Pollinator
bee
hummingbird
Petal color
pink
red
Corolla width
wide
narrow/tubular
Stigma/anther
inserted
exserted
Nectar volume
1-2 ml
40-100 ml
Toby Bradshaw and Doug Schemske
Genome projects in Mimulus
• NSF Frontiers in Biological Research (2003)
 Integrated ecological and genomic analysis of
speciation in Mimulus
 To enable positional cloning of ecologically and
evolutionarily interesting genes, particularly for
speciation and reproductive isolation
 Physical and genetic maps, markers,
transformation protocols
• DOE Joint Genome Institute
 Community Sequencing Program (2005)
 Whole genome shotgun of M. guttatus IM62
 A number of supporting sequencing projects
Physical maps
• BAC Libraries
 M. guttatus: two 11X libraries
 M. lewisii: one 10X library
• HICF contigs
• End-sequences for all BACs
• Overgos (~750)
CUGI & JGI
Expressed sequence tags
• >500K from M. guttatus IM62
 ~ half Sanger, half 454
 Mostly from floral buds and maturing fruits
 Also from roots, seedlings, leaves
• 30K from M. guttatus DUN
• 30K from M. nasutus SF
• 20K from M. lewisii
Other FIBR products
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~1000 genetic (EPIC) markers
Multiple RIL and IL populations
Baseline polymorphism and LD data
Agrobacterium-based transformation
Germplasm collection
Community database
http://openwetware.org/wiki/Mimulus_Community
JGI whole-genome shotgun
• M. guttatus IM62
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inbred line from Iron Mountain, OR
~7X coverage
Mostly 3 kb & 8 kb paired-end Sanger reads
>200K ~30K fosmid end-sequences
>100K BAC end-sequences
• M. guttatus DUN
 inbred line from a coastal population
 ~20X coverage of 454 paired-ends
Assembly statistics
number
tot lgth
N50
L50
scaffolds
2,216
321.7 Mb
81
1.1 Mb
contigs
17,831
300.7 Mb
1,770
45.5 Kb
Scaffolds span ~75% of estimated 430 Mb genome
Gaps comprise ~6.5% of the scaffolds
Number of scaffolds > 50 KB = 512 (which includes 95.7% of
assembly)
Jeremy Schmutz
Genetic mapping of scaffolds
• To order and orient the >2K scaffolds on the
genetic map
• Procedure
 Genome reduction to ~100K markers using
restriction-associated DNA (RAD) - Miller et al
(2007) Genome Res 7, 214
 Nextgen sequencing of ~100 IM62xDUN RIL lines
at ~0.4X coverage each (in pools of 12)
 Infer genotypes in 20-100kb sliding windows and
analyze linkage map
• Model predicts ~50% of scaffolds and >90%
of bp in assembly will be mapped
Toby Clarke
Structural annotation
• Currently ~25,000 predictions (earlier
assembly)
• Planned JGI pipeline
 Mask for repeats
 Homology to Phytozome proteins, Genbank
asterid proteins
 M. guttatus and M. nasutus ESTs, translated M.
lewisii ESTs
 Genomescan, Fgenesh integrated with Pasa
 Filter out TEs & low-confidence genes
 Annotation planned to be available in February
Theresa Mitros, JGI
Repeat composition
• RepeatModeler/RepeatMasker masks ~60%
of the assembly
• Known LTR retrotransposons: 15%
 Most active yet seen is a gypsy with 35 EST hits
• Known centromere linked repeats: 8%
 Cnt728: highly abundant, heterochromatic,
tandem repeat interspersed with LTRs
FISH of pachytene
chromosomes with probes for
DNA (blue), Cnt728 (green)
and a gypsy retrotransposon
(pink).
Eric Ganko and Arpiar Saunders
BAC annotations
• 22 fully-sequenced BACs (2.5 Mb)
• 236 genes -> 40-45K genes in genome
Eric Ganko
Mimulus-rice synteny
Andrew Morgan
Microsynteny with Solanaceae
(ovate region, Wang et al 2008 Genetics)
May be broken into 3 regions in Mimulus
Within each, overall order and orientation well conserved
2 Mimulus genes with no syntenolog in Solanaceae
9 Solanaceae genes with no syntenolog in Mimulus
Eric Ganko
VISTA conservation plot (20/50nt windows)
Mimulus-specific genes (blue)
Mimulus genes with Solanceae syntenologs (orange)
Eric Ganko
Unresolved relationships
among lamiid genomes
Solanum (Solanales)
Mimulus (Lamiales)
Coffee (Gentianales)
All 3 possible relationships for these taxa have been
published
Mimulus is an outgroup to the
Solanaceae-coffee clade
Solanum (Solanales)
100
Coffee (Gentianales)
Mimulus (Lamiales)
Maximum likelihood analysis of concatenated alignment
for 13 gene families totalling 5,691 amino acids
Amy Bouck and Stefanie Hartman
Mimulus as a resource for tomato
• Data should be available by mid-2009
• Assist with gene and repeat structural
annotation
• Early look at asterid-specific biology
 Shared functional annotation
• Evolutionary analysis
 Potentially improve coverage of syntenic blocks
 Could serve as outgoup to reconstruct Solanaceae
ancestor and polarize evolutionary changes between
Solanaceae and coffee