Jeffrey K. Conner, Michigan State University, PI; Shin-Han Shiu, Michigan State University, Co-PI; Yongli Xiao, The Institute for Genomic Research (TIGR), Co-PI
Scientific objectives and approaches: Radish (Raphanus) is an important crop, a major agricultural pest weed on six continents, and an invasive species of natural areas, especially in California. Radish is a model system for studies of ecology and evolution, with major past and ongoing work on population and molecular genetics, plant-insect interactions (both pollination and herbivory), quantitative genetics of floral and life history traits, natural selection through both male and female fitness, potential for adaptation to global change, and the possible role of transgene escape and natural hybridization in the creation of more weedy and invasive genotypes. Thus, we have very broad and deep knowledge of how radish interacts with its abiotic and biotic environment from basic ecology and evolutionary genetics to issues of fundamental applied importance. The wealth of ecological and evolutionary background in this species makes it an excellent candidate to understand adaptation at the molecular genetic level as well as address the applied issues; however, rapid progress in this area is currently hampered by the lack of radish sequence information. In addition, the taxonomic position of radish, as a close relative of Brassica and a more distant member of the same family as Arabidopsis, makes it an ideal candidate for comparative genomics among closely related plant species.
We propose to sequence two cDNA libraries, one from the crop and one from wild radish, from both the 5’ and 3’ ends, to produce abundant EST and full-length cDNA sequence data. We will identify orthologs in Brassica and Arabidopsis, and initiate comparative genomic studies in several key areas including evolution in polyploids, gene retention and loss after duplication, and rates of adaptive evolution at the sequence level in an outcrossing plant. We will mine these data for codominant markers that will enable a number of research groups to understand adaptation of native, weedy, and invasive radish to its environment through fine scale genetic mapping. The cDNA sequence will also facilitate future studies of the mechanisms of phenotypic plasticity, e.g. induction of anti-herbivore defensive chemicals, through measurements of differential gene expression.
Broader impacts:Sequence data of any kind for radish is sorely lacking, so the sequences we will generate will greatly facilitate the work of the radish research community, and likely attract additional ecologists and evolutionary biologists to this species. This project will establish a collaboration between Kellogg Biological Station (KBS), a leading ecological and evolutionary field station that includes an NSF Long-Term Ecological Research (LTER) site as well as an ongoing K-12 educational partnership, with TIGR, a leading sequencing andstructural genomics institutecenter. Such collaborations are unusual, but the marriage of modern genomics with modern field ecology and evolution will greatly advance our understanding of both areas, as well as educational opportunities for students at all levels and their teachers. Because KBS is a key member of GLACEO, the Great Lakes and Central US Ecological Observatory, a member of the Consortium of Regional Ecological Observatories, this collaboration could(will?) impact the NSF National Ecological Observatory Network (NEON) program as well. We will work with high school biology teachers and graduate fellows who are part of the NSF-funded KBS GK-12 project (Conner is a co-PI) to develop a classroom unit on the use of genetic tools in ecology, environmental, and genomic science.
Progress report: NSF DBI-0312656 Large-Scale Analysis of Novel Arabidopsis Genes Predicted by Comparative Genomics; (P.I. C. D. Town; co-P.I. YL. Xiao; 9/03-8/06).
This project was funded to verify the structure of, and produce full-length cDNAs in a Gateway recombination vector for 2,000 Arabidopsis genes that were either annotated as hypothetical or not annotated initially but subsequently predicted by comparative genomics (http://www.tigr.org/tdb/hypos/). The project is currently being completed through a no-cost extension. To date, we have processed over 2,300 genes through the RACE/structure pipeline, validating the structure of ~1,500 annotated genes and providing experimental support for ~500 novel genes. Approximately 2,100 ORFs have been targeted by our highly efficient FL-cDNA pipeline yielding more than 2,000 Gateway entry clones which include hypothetical genes, novel genes and several hundred low-expression genes that have functional annotation but for which there is no evidence for expression in the public ATH1 GeneChip data. Due to the sensitivity of our ORF cloning pipeline and the richness of the cDNA populations employed, we were able to generate Gateway entry clones for many of the “non-expressed” genes. At the start of the project, the ORF clones were produced only in the closed configuration (i.e. with a stop codon) as per our original project description. However, around the mid-point of the project we adopted a degenerate primer strategy developed by Pierre Hilson and colleagues and for the last 800 targets have produced ORF clones in both open and closed configurations (Underwood et al. 2006). Approximately 2/3 of our clones have been deposited at the Arabidopsis Biological Resource Center (ABRC) and the rest are being re-arrayed to complete this process. TIGR produced 152,680 sequence reads for this project and total clear range of sequences is 78,061,332 bp. This community service project has also generated ~5,000 GenBank submissions and one publication with another in preparation.
Overview of the genus Raphanus
The genus Raphanus (radish) includes the cultivated radish, R. sativus, and one of the world’s most economically important weeds, R. raphanistrum (Holm et al. 1997). Raphanus is a model system in plant reproductive ecology and evolution, particularly in the areas of pollination and herbivory (e.g., Agrawal 1998; Agrawal et al. 2002; Agrawal et al. 2004; Bett and Lydiate 2003; Conner 2002; Conner et al. 2003a; Devlin and Ellstrand 1990; Irwin and Strauss 2005; Irwin et al. 2003; Mazer and Schick 1991; Morgan and Conner 2001; Snow et al. 2001; Stanton et al. 1986). The genus originated in the Mediterranean region. The crop radish, R. sativus, may have had multiple origins, probably derived from R. raphanistrum or a recent common ancestor. All radish species or subspecies are highly interfertile, with little segregation distortion or disruption of chromosome pairing in crosses between R. sativus and R. raphanistrum (Bett and Lydiate 2003), and in California most wild radish is the result of hybridization between these two species, so several authors have suggested that all Raphanus are in fact one species (references). In the last 200 years weedy R. raphanistrum has spread to every continent except Antarctica, is an increasingly serious agricultural pest in 17 countries (Holm et al. 1997), and is the worst dicot agricultural weed in southwest Australia (Warwick and Francis; R. Cousens, pers. comm.). The Raphanus genome has nine chromosomes, is fairly small compared to other angiosperms (estimated at 573 Mbp; Johnston et al. 2005; map distance 915 cM; Bett and Lydiate 2003), and is very closely related to the Brassica A and C genomes (Warwick and Black 1991).
The most recent treatment of the large (over 3000 species) and important family Brassicaceae divides most of the family into three large and well-supported clades (Beilstein et al. 2006). One of these clades includes Arabidopsis and Capsella (both currently being sequenced), and Brassica and Raphanus are in one of the other clades. These latter two are sister genera, having shared a common ancestor between 0.9 and 2.2 mya (Yang et al. 1999; Yang et al. 2002) and crosses between these two have been conducted for some time (creating amphidiploid Raphanobrassica; Williams and Hill 1986). The Arabidopsis/Capsella and Raphanus/Brassica clades probably diverged 15 to 20 million years ago, and sequence similarity between Brassica and Arabidopsis ranges from 75%-90% in exons.