Using RescueMu Sequences - What Do I Do Next?
The Maize Gene Discovery Project provides several routes for identifying mutations in genes that interest you.
- Check GenBank and ZmDB for sequence matches to your favorite gene.
- How can I decide if a RescueMu "hit" corresponds to a germinal insertion?
- After I have a "hit", how do I confirm which plant(s) have that insertion?
- What else can I do with a library plate?
- Any hope that the RescueMu insertion caused a phenotype?
- How do I order free seed for further research?
- Check GenBank GSS database for sequence matches to your favorite gene or check the ZmDB assembly of ESTs and RescueMu genomic sequences for a match in a type of gene that interests you.
Where do the DNA sequences next to transposed RescueMu elements come from?
We are sequencing maize genomic DNA next to each side of RescueMu elements. We recover RescueMu plasmids from pools of plants planted in rows and columns in tagging grids. The sequenced grids are listed on the progress page. Generally we sequence several hundred plasmids from each of the rows of a grid and from 4 columns; we used the matches between the row and column sequences to calculate the "transposition" frequency of RescueMu and to calibrate the depth of sampling we achieved in the row sequencing. By the end of the project in September 2003 we estimate that ~15,000 independent likely germinal insertions will have been identified.
- How can I decide if a "hit" corresponds to a germinal insertion?
- Row & Column Match
If you find the same RescueMu insertion site on both a row and a column from the same grid you are very likely to have identified a germinal insertion. And, you know the exact plant that has the insertion -- the row and column address within a grid are a specific plant (i.e. Row 17, Cosumn 4).
- Two or more sequence entries for the same type of plasmid
For most grids sequencing was conducted so that there was a 90-95% probability that any germinal insertion would be sampled at least once. Any item seen twice should be germinal insertion, because most somatic insertions are very late in development and result in tiny sectors. Through progeny analysis by DNA blot hybridization and PCR screening we have established that 25/25 examples of plasmids found two or more times are heritable germinal insertions (plasmids found twice or more within a row or in two or more rows or in both a column and a row). Two grids were sampled at a much more shallow depth of about 50% (Grids M and S).
- Only one sequence entry in your favorite gene
For all grids finding just a single example of a plasmid that interests you is worth investigating further, because sampling depth was always incomplete. Many of the single plasmid recoveries are also germinal insertions (transmissible to the next generation). Once you have the library plate of a particular grid, you can screen many times for specific genes -- remember that most genomic insertions are 3 - 8 kb. Even a "somatic insertion" plasmid may be valuable as a template for further sequencing of your gene.
- After I have a match, how do I confirm which plant(s) have the insertion?
http://www.zmdb.iastate.edu/zmdb/library-plate/screening.html Consult this page for protocols on PCR screening of grid library plates.
To determine the exact row and column location and dence exact plant that contains a heritable insertion, you must perform a PCR screen on the library plate prepared from the grid (A thpough CC) from which the sequence was obtained. You can also screen library plates with PCR primers to identify plasmids that were not sequenced successfully; provided you obtain a product of identical size in both a row and a column of the library plate, you have identified a plant carrying that insertion. Note that we will prepare library platies from ~10 grids that have not been sequenced -- these plates should provide identification of an additional ~15,000 germinal insertions.
Approximately 20-40% (depending on the grid) of the heritable insertions were rewovered in two or more rows and are likely to represent small sectors in the ear or tassel of the grid founder plant(s). These events will field a positive PCR product in several rows and or several columns.
- What else can I do with a library plate?
Gene Representation in Library Plates
Each library plate well has hundreds of different plasmids in it, and RescueMu insertion sites are highly enriched for genes and gene-like sequences. A typical 96 well plate representing a grid will have ~15,000 different "genes" represented in it. A collection of 6-8 plates, even those from grids not sequenced, should have most of the genes subject to Mu insertion mutagenesis represented. You can pool aliquots of gris wells to make a library of the insertion plasmids for plating and plasmid recovery through hybridization.
- Any hope that the RescueMu insertion caused a phenotype?
http://www.zmdb.iastate.edu/zmdb/phenotypeDB/index.htm
The Maize Gene Discovery Project has constructed PhenotypeDB, which reports our notes on thousands of families generated by self-pollination of grid plants. These data are reported with the Row & Column Address of the grid plant so that you have direct access to the seed because of your interest in a phenotype.
Because the majority of mutations are caused by standard Mu elements in the grid populations and we selected for a high forward transposition rate of RescueMu, our populations have exceptionally high visible mutation frequencies. For segregating seed defects the frequency is 5 - 10% per grid, for seedling traits it is 10 - 28%, and for adult traits including floral charactecristics and fertility it is 10 - 20 %. We estimate that 5% of the visible mutations will be caused by RescueMu.
By project termination in September 2003 we anticipate finishing screening of ~40,000 ears for seed traits, 15,000 seedling families, and 10,000 adult plant families. Data on mutant characteristics, including photos of unique phenotypes, are provided in PhenotypeDB, a searchable database for maize geneticists. Given the high frequency of visible mutations at all life stages, screening thus far has identified lines with thousands of likely tagged alleles in genes that give a visible phenotype. Individual groups with the Maize Gene Discovery Project anticipate continuing both the seedling and adult screens for several more years to enrich the collection of documented visible traits. Once you have found families segregating for traits of interest, you can order the corresponding seed from the Maize Coop.
- How do I order seed?
http://w3.aces.uiuc.edu/maize-coop/
Project seed stocks are distributed through the Maize Coop and are available without "strings" for your research. You must have a valid APHIS permit covering shipment of transgenic seed containing RescueMu from Illinois to your home state for the Coop to ship seed to you.
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