Bradshaw Lab  
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Our research on the genetic basis of adaptation and speciation in natural populations takes advantage of the powerful tools available in the Mimulus (monkeyflower) experimental system:

  • Tremendous variation within the ~150 species of Mimulus -- flower color, pattern, size, shape, symmetry, pollinators, mating system, habitat preference, growth habit, ... (check out the montage on the home page!)
  • A large collection of EMS-induced mutants with stunning floral phenotypes
  • Short generation time (12 weeks seed to seed), high fecundity (1000-2000 seeds per pollination), easy greenhouse propagation, seed storage at room temperature for decades, inbred lines for many species, ~500Mbp genome size
  • Complete short-read genome sequences for M. lewisii and M. cardinalis, and a fully assembled genome for their congener M. guttatus
  • In planta transformation of M. lewisii and M. cardinalis, making it possible to test the function of individual genes (and even individual mutations) via loss-of-function (RNAi) and gain-of-function transgenes
  • More than 80 years of fieldwork on the ecology of M. lewisii and M. cardinalis -- sister taxa reproductively isolated in sympatry by pollinator preference (Ramsey et al. 2003) -- making it feasible to validate lab and greenhouse experiments in the native range and natural habitat of the two Mimulus species (e.g., Schemske & Bradshaw 1999; Bradshaw & Schemske 2003)

We have three main projects in progress:

  • Identifying the molecular genetic basis of traits involved in reproductive isolation between M. lewisii and M. cardinalis (e.g., flower color, scent, placement of reproductive organs), using a combination of QTL mapping, NIL construction, transgenesis, and field experiments
  • Identifying the molecular genetic and developmental bases of variation in floral form using EMS-induced mutants of M. lewisii
  • Designing and synthesizing (via mutation, introgression, and/or transgenesis) new Mimulus species with novel trait combinations that produce reproductive isolation from their progenitors

See below for more details!

ROSE INTENSITY1, a MYB repressor, is a major gene controlling the difference in anthocyanin (pink) pigment between M. lewisii and M. cardinalis flowers

We know from field experiments that anthocyanin concentration is important to both bumblebees and hummingbirds -- bumblebees prefer paler flowers, and hummingbirds prefer darker flowers (Schemske & Bradshaw 1999).  In a backcross to M. cardinalis, we mapped the major QTL controlling ~40% of the phenotypic variance in anthocyanin concentration to a locus we named ROSE INTENSITY1 (ROI1).  The dominant ROI1 allele in M. lewisii suppresses anthocyanin deposition in the flower petals.

We created a NIL with the recessive M. cardinalis roi1 allele in the M. lewisii background (see photo above), and used this as the founder of a backcross population to fine-map ROI1.  By direct PCR on seedlings, we screened 2714 backcross plants at two markers (MgSTS158 and MgSTS55) flanking ROI1.  Recombination breakpoints narrowed the interval containing ROI1 to just 5 genes, of which the most promising candidate was a single-domain MYB distantly related to other known repressors (e.g., CAPRICE in Arabidopsis).  The QTL and fine-mapping work was supported by NSF FIBR grant 0328636 (John Willis, PI).   See http://www.mimulusevolution.org/ for more on the maps, markers, and other genetics/genomics resources developed for Mimulus with NSF FIBR funding.

We constructed an RNAi transgene to knock down/out the candidate MYB in M. lewisii.  The transgenics had the predicted darker-pink flower phenotype, identical to the NIL homozygous for the recessive roi1 allele. 

To confirm the identity of ROI1, the wild-type M. lewisii allele was transformed into the roi1 NIL background.  The resulting transgenics had the lighter-pink phenotype characteristic of the dominant ROI1 allele.

Similar mapping/transgenesis experiments are in progress for several genes contributing to color and pattern differences between M. lewisii and M. cardinalis that matter to pollinators in the field -- YELLOW UPPER, YELLOW LOWER, and LIGHT AREAS.  For more on these projects, read the NIH grant that is funding this research.

 

The major difference in floral scent between M. lewisii and M. cardinalis flowers is controlled by two monoterpene synthases

In collaboration with Jeff Riffell, we are identifying the genetic basis of differences in floral scent between M. lewisii and M. cardinalis, and assessing the contribution of scent to reproductive isolation mediated by pollinator preference. 

Volatile components of the floral bouquet were collected by dynamic headspace sorption and analyzed by GC-MS.  M. lewisii flowers produce ~100x as much scent as M. cardinalis, consistent with the observation that bumblebees rely on olfaction much more than hummingbirds do.  The vast majority (92%) of the volatiles in the M. lewisii bouquet are monoterpenes, with limonene as the most prevalent of the 11 monoterpenes detected.

To determine which of the monoterpenes are most attractive to bumblebees, we performed simultaneous GC with electroantennogram recording and multi-unit recordings in the antennal lobe of bumblebee brains.  Limonene, myrcene, and ocimene elicited the strongest neurophysiological response.

In a backcross (to M. cardinalis) mapping population, we extracted volatiles directly from petal tissue and measured the concentrations of limonene, myrcene, and ocimene by GC.  Limonene and myrcene production mapped to a single locus, and ocimene to another (unlinked) locus.  The orthologous regions in the assembled M. guttatus genome contain clusters of predicted terpene synthases.  Candidate genes from the mapped regions in M. lewisii were tested by RT-PCR for expression in petal tissue.  Expressed cDNAs were subcloned into the E. coli protein expression vector pET100, and assayed for terpene synthase activity in vitro.  In this way we identified the two genes that underlie the difference in scent between M. lewisii and M. cardinalis: a bifunctional LIMONENE-MYRCENE SYNTHASE and OCIMENE SYNTHASE.

Transgenic M. lewisii containing RNAi constructs to knock down/out LIMONENE-MYRCENE SYNTHASE and OCIMENE SYNTHASE were constructed and tested for their effects on bumblebee visitation in a greenhouse experiment. The LIMONENE-MYRCENE SYNTHASE knockout had no effect on bumblebee preference relative to the wild-type, but the OCIMENE SYNTHASE knockout reduced bumblebee visitation by 6%. OCIMENE SYNTHASE joins a short but important list of "speciation genes"!

 

GUIDELESS, an R2R3 MYB, is required for the development of nectar guides in the flowers of  M. lewisii

We have a very large collection of EMS-induced mutants in M. lewisii, with an amazing range of variation in floral form.  The first of these mutants to be characterized genetically is the recessive allele guideless, which (when homozygous) prevents the formation of nectar guides -- the yellow stripes in the corolla throat that we have shown are important both for attracting bumblebees and assuring their proper orientation as the bumblebees crawl into the flower (Owen & Bradshaw 2011).

GUIDELESS was mapped using bulked segregant analysis and Illumina sequencing.  The guideless mutant in the LF10 inbred line genetic background was crossed to another M. lewisii inbred line, SL9.  The F1 was selfed to produce an F2 population segregating for guideless, as well as for all of the DNA sequence polymorphisms that distinguish LF10 and SL9.  DNA was extracted from 100 F2 plants homozygous for guideless, and sequenced to 40x depth.  Using the SL9 genome sequence as a reference, the pooled F2 guideless DNA sample was scanned for regions containing a high (>95%) frequency of LF10 SNPs, presumably tightly linked to guideless.  The small region containing guideless was then compared to the LF10 reference genome sequence to find all of the EMS-induced mutations, and those mutations were examined for their presumed effect (i.e., coding/non-coding, synonymous/non-synonymous/nonsense, splice site). 

At the end of this filtering process, only one candidate remained -- an R2R3 MYB with a frameshift mutation in the coding region.  Transgenic M. lewisii LF10 plants carrying an RNAi construct were use to knock out/down the candidate.  The transgenic plants had the expected guideless phenotype, confirming the identity of GUIDELESS.

We are applying the same mapping strategy to identify many of our mutants, three of which are shown below.

 

 

 

 

Speciation by synthesis -- rational design of a new hawkmoth-pollinated Mimulus species derived from  M. cardinalis  

Perhaps the most stringent test of our understanding of speciation is to design and synthesize a new species.  There are many examples in nature of sister species of plants reproductively isolated by pollinator preference.  We propose to engineer a hawkmoth-pollinated Mimulus species using the hummingbird-pollinated M. cardinalis as the ancestor.  The transition from hummingbird pollination to hawkmoth pollination is a common theme in the flora of western North America, but this transition has never occurred in the Erythranthe section of Mimulus, where all species are either bumblebee-pollinated (M. lewisii), hummingbird-pollinated (M. cardinalis, M. verbenaceus, M. eastwoodiae, M. rupestris, M. nelsonii), or selfing (M. parishii).

Nearly all hawkmoth-pollinated flowers are white, radially symmetrical, fragrant, and nectar-rich.  M. cardinalis produces copious quantities of nectar, but is otherwise poorly adapted to attract hawkmoths.  Indeed, in experiments with captive hawkmoths (Manduca sexta), M. cardinalis flowers are almost never visited.

To engineer a white M. cardinalis flower, two pigments must be eliminated.  The dominant YELLOW UPPER (YUP) allele from M. lewisii prevents the deposition of yellow carotenoid pigments in the petal lobes.  We have introgressed YUP into the M. cardinalis background, producing a pink flower.  The recessive pinkless (pnk) allele is found in some yellow-flowered M. cardinalis populations in the Siskiyou mountains of Oregon and on islands off the coast of Baja California.  pnk homozygotes lack anthocyanins in the petal lobes, though they are still able to produce spots in the corolla throat.

Presumably, a YUP/___ pnk/pnk M. cardinalis flower would be white with red spots in the corolla throat, and attractive to hawkmoths.  Unfortunately, YUP and pnk are very tightly linked, so we have yet to produce the desired genotype.

To generate a radialized M. cardinalis flower, we have introgressed the recessive trumpet allele recovered in our mutant screen of M. lewisii.

Engineering the appropriate fragrance into M. cardinalis will be a challenge, since M. cardinalis flowers do not produce significant quantities of the oxygenated aromatic and aliphatic volatiles that are attractive to hawkmoths.  One possibility would be to transfer one or more genes from hawkmoth-pollinated Nicotiana (tobacco) species.

Stay tuned!

 

Last updated on 1 September 2013.  Send questions or comments to toby@uw.edu