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Project Documentation & Protocols: Maize Gene Discovery Project: Introduction to RescueMu Tagging

http://www.maizegdb.org/documentation/mgdp/library-plate/progress.php - Chart our progress in sequencing grids and producing library plates for you to screen by PCR at this page.

Table of Contents

A. DEFINTIONS

F0 RescueMu founder individual

F0 RescueMu x Mutator source in r-g background with either a1-mum2 or bz2-mu1 reporter alleles => F1 progeny with red-spotted kernels

F1 x r-g tester; r-g x F1; bz2 or a1 testers x F1=> F2 progeny suitable for grids

F2 plants DNA is recovered from pooled leaf discs representing the rows and columns in a field of 2304 individuals; plasmid rescue yields 48 row plus 48 column libraries of RescueMu in E. coli

F2 (X) => F3 progeny screened for phenotypes and deposited into the Maize Coop available to the genetics community.

 

B. Strategy for Selecting F2 individuals for the Summer 1999 Grids

1. Genetic Analysis & Crossing Schemes: Numerous F1 families segregating for transgenic RescueMu were planted in Hawaii in November 1998. Each family started with 23 seeds, and there were 10 . 23 individuals that survived to adulthood. These families segregated1:1 for the presence of the basta resistance marker, which is tightly linked to the RescueMu transgene locus. Laurie Smith and Virginia Walbot painted herbicide into a 3 cm circle on adult leaves, and within 5-7 days sensitive individuals were readily recognized by a large patch or leaf-long streaks of yellow-brown tissue. Scoring was done by Dean Goodman, Damon Lisch and Virginia Walbot. Only the "still green" plants were used in crosses. Each individual was crossed by r-g tester, to r-g tester and to either a1 or bz2 tester depending on which anthocyanin reporter gene was present as a result of the Mutator source used. Lines crossed to a1 had a low copy MuDR background (one or two copies) and few other Mu elements while lines crossed to bz2 have standard MuDR copy number (~5-10 copies) and multiple other Mu elements.

The harvested ears were delivered to Stanford in late March 1999, and then tagged and sorted by family. The crosses by r-g tester were evaluated for RescueMu somatic excision frequency by Gillian Nan and Virginia Walbot, and only those families with most ears with high spotting frequency were selected for further analysis. Three kernels were removed from two or three ears from each of these families, and individual seedling DNA samples were prepared for Southern blot analysis.

2. Goals: Identify families with the highest frequency of RescueMu germinal insertion events. Determine in a large population whether the efficiency of RescueMu germinal insertion is higher through the ear or pollen parent. Screening of F2 individuals for the Hawaii 1999 prototype grid indicated that the frequency was higher through the ear parent, however, this was a small sample and several of the lines tested showed some loss of Mutator activity, some transgene silencing, or both. Epigenetic loss of Mutator and transgene silencing are higher through pollen than through the ear, consequently, confounding the analysis. Prior studies by Don Robertson who scored forward mutation frequency and by Damon Lisch who scored new "insertion bands" on Southern blots indicate that Mutator activity is about twice as high in pollen compared to transmission through the ear in lines that maintain Mutator activity.

C. Procedures

1. Southern blot test on 3 kernels from two or three ears in F2 families in which F1 plant x r-g tester was highly spotted indicating somatic mobility of RescueMu. Determine the frequency of germinal insertions on a per family basis by scoring Southern blots for new fragments. A unique Rhizobium sequence within RescueMu is used for the hybridization probe to simplify analysis.

2. Phenotypic confirmation for families with a reasonable frequency of new insertions, then

  1. Check ear phenotype scoring sheets to determine if this F1 individual crossed to r-g tester also yielded spotted kernels, indicating maintenance of RescueMu mobility through the pollen => more ears worth checking for new insertions.
  2. Check crosses of these families to either a1 or bz2 testers . determines general status of Mutator activity and these ears should also contain new RescueMu insertions

We gratefully acknowledge the excellent assistance of undergraduate Karen Brewer, who performed all of the testcross kernel scoring. Karen is an expert on scoring RescueMu excision sectors, because she has assisted graduate student Manish Raizada for the past three years on the RescueMu project. Because most sectors are tiny (one or just a few cells), a low power dissecting microscope is used; the restoration of R activity confers cell autonomous anthocyanin pigmentation. In contrast, restoration of A1 or BZ2 enzyme activity results in non-cell-autonomous pigmentation, with a dark central cell(s) and a halo of cells; consequently the sectors on a1 and bz2 ears can be scored by eye.

3. Identifying the best families. There could also be some instances of a "good individual" in a family that has some evidence of Mutator loss (low frequency of spotted kernels on bz2 or a1 tester ears) or silencing of the RescueMu transgene (low spotting on the r-g tester crosses). As necessary, such individuals are also checked, but it is better to find families with many individuals that exhibit consistent RescueMu mobility and Mutator activity.

4. Prior to Hawaii 1999-2000 (Grid E) Reconfirmation by Southern blotting. Gillian Nan, who has performed all of the initial screening, made a list of the "likely usable Hawaii families" and then divided the work so that Laura Roy, at UC-San Diego, could help perform the next set of Southern blots.

Prior to constructing grids, we then checked

  • if the frequency of new insertions is different between the female (F1 x r-g) and male direction of cross (tester x F1 plant), and
  • if the frequency of new insertions is similar in additional members of the same F1 family.

This requires sampling three kinds of F2 ears:

a. remaining F1 x r-g tester ears of that family

b. r-g tester X F1 individuals of that family

  1. bz2 or a1 tester x F1 individuals of that family

At this stage, we also estimated the number of seed on ears in each type of cross for each family, so that we knew how many seed are available for grid construction. Perform Southern blots on 3 seedling DNA samples on the outcross (b. and c.) and a few more individuals x r-g tester (a.) to certify that a family is appropriate for a grid. These additional data will allow us to decide whether we want to use only F1 x r-g crosses or the outcrosses as well.

5. Set up a grid. Each site needs a grid of 2304 plants and would be advised to plant a 60 x 60 grid of 3600 plants. Each grid is built from 10-15 ears of corn; thus each ear would yield about 4-6 rows in their field. Germination is about 90% in a Mutator outcross population => ~3250 plants. All rows should have at least 48 individuals in them; some minor replanting may be necessary to even up the rows. At each location additional Southern blots are performed while the plants are growing to pick the best 48 rows in terms of gene tagging; up to 12 rows (representing seed from 2-5 ears) can be discarded. The chosen plants are tagged with a wired tagged, labeled by row number and individual number.

6. Low frequency of germinal insertion. Our experience to date indicates that mobilization of RescueMu from the complex transgene loci occurs such that ~10 - 50% of progeny have one germinal RescueMu insertion. These individuals have many new somatic insertions generated during plant growth indicating that Mutator remains active. The low frequency of mobilization from transgenes could reflect transgene silencing (possibly methylation) independent of Mutator status, a low frequency of mobilization of the large RescueMu element (at 4.7 kb it is nearly the length of MuDR, which causes few of the mutations in standard Mutator lines), and/or a difficulty mobilizing RescueMu elements from a transgene arrays with multiple, tandem copies of the transposon.

D. Future Improvements

1. Better MuDR Stocks and Transposed RescueMu. For the future, Vicki Chandler, Carolyn Napoli and Damon Lisch are working on alternative strategies for RescueMu tagging. Southern blot screens of herbicide-sensitive progenies at UC-SD, UC-B and University of Arizona identified individuals with transposed copies of RescueMu that had segregated away from the transgene. These materials were selected to generate populations for a larger screen in Hawaii 1999-2000 for individuals with 2 or more transposed RescueMu. For the future, Vicki Chandler is coordinating development of better tagging lines.

Problem #1. There is mounting evidence that transgene silencing and the tandem inserts present at the RescueMu transgene loci could inhibit transposition. Solution: Pick lines with transposed copies of RescueMu elements and intercross and pick individuals that lack the original transgene locus (now susceptible to herbicide). These lines contain several "mobile" RescueMu elements but no transgenes. We will characterize the new transgene locations to develop a plasmid rescue protocol that biases against recovery of these sites and enriches for recovery of the next generation insertion mutations.

Problem #2. The single copy MuDR line has MuDR in an unfavorable location (work of Robertson & Stinard and Lisch et al.). Lines have been selected in which this MuDR has transposed to new locations; lines with two or three MuDR elements but few other Mu elements have been identified using Southern blot hybridization. Such lines have already been crossed with the RescueMu lines with the original transgene or with transposed copies of RescueMu.

2. New RescueMu Elements. Gillian Nan at Stanford has built new RescueMu plasmids. The shorter 2.1 kb miniMu and 2.7 kb midiMu may transpose more frequently. In addition, the new generation RescueMu element has other features that facilitate plasmid rescue; in particular, inclusion of an 8 bp restriction recognition site (SwaI, blunt cut) next to RescueMu will eliminate the "launching pad" transgenes from plasmid rescue. The new generation RescueMu elements are inserted at the location of a Mu1 element in the bz2::Mu1-mu1 allele. This provides a native maize context for the RescueMu element. The 35S promoter has been eliminated from the construct as well. The new RescueMu elements will be in F1 plants in 2000 so that somatic excision can be scored.

E. Grids F - Q, 2000 - 2001

1. Male Founder plants with trRescueMu In these grids individual male founder plants were identified by Southern blot screening that contained one or more transposed RescueMu (trRescueMu) elements but no transgene array. The once transposed elements move at 50 - 100% frequency, hence progeny of a plant with two trRescueMu typically inherit at least one new insertion site. The original parental trRescueMu element(s) segregates 1:1 in the progeny.

2. trRescueMu can be used to efficiently sequence maize genes Grids with the highest tagging frequencies (G, H, I, M through mid-2002) have been chosen for DNA sequencing after plasmid rescue http://www.maizegdb.org/documentation/mgdp/library-plate/plasmid_rescue.php and preparation of templates containing the flanking genomic DNA http://www.maizegdb.org/documentation/mgdp/library-plate/pcr.php. Sequencing indicates that a high percentage of genomic insertions match maize and other grass ESTs (about 55% of insertions) and to genomic DNA from Arabidopsis and rice. It is clear that trRescueMu preferentially insert into maize genes and gene-like DNA. There is a 16-fold bias against insertions in retrotransposons. We are exploring using trRescueMu templates as substrates for shotgun sequencing of maize genes. This is one of several "gene enrichment" strategies being considered for gene-focused sequencing of maize. It is likely that only ~10% of the 2500 MB maize genome corresponds to genes and their immediate flanking "regulatory" regions. Any method that would allow selective recovery of genes should be considered.

F. Grids R - U, summer 2002 both male and female-derived tagging

To circumvent the problem that pollen-lethal tagged genes will be lost by crossing male founders with trRescueMu to female anthocyanin tester ears, in summer 2001 we identified numerous plants with one or several trRescueMu and used these as female parents. They were sib-crossed or crossed by an anthocyanin tester line to make "low" and "high" copy trRescueMu stocks. In summer 2002 two of the tagging grids will utilize these "female" source populations.


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