SAINT PAUL, MINNESOTA
University of Minnesota and USDA-ARS
Oat-maize chromosome manipulation for the physical mapping of maize sequences --Kynast, RG, Okagaki, RJ, Odland, WE, Stec, A, Russell, CD, Zaia, H, Livingston, SM, Rines, HW, Phillips, RL We have developed a complete set of oat-maize chromosome additions to map maize sequences and study expression of maize genes in the genetic background of oats. A total of 37 monosomic and disomic addition lines that involve five oat (chromosome recipient) and three maize (chromosome donor) lines were generated, as described in detail by Riera-Lizarazu et al. (TAG 93:123-135, 1996), and recovered and identified as described in detail by Kynast et al. (Plant Physiol, in press, 2001). Because each recovered addition line represents its own distinct retention event, we developed a nomenclature for identification of the addition lines. OMAxy.z is an abbreviation for Oat-Maize Addition extended by three alpha numericals. The x position indicates the maize chromosome constitution, which will be d for disomic or m for monosomic. The y position is the number of the maize chromosome that is added, namely 1 to 10. The z position is a number that identifies a particular version of the maize chromosome that traces back to the original recovery event. Table 1 summarizes the identity and status of addition lines that are currently available to the scientific community. Fertile lines include a total of 30 single disomic additions (2n = 6x+2 = 44) for maize chromosomes 1, 2, 3, 4, 6, 7, 8, and 9, one single monosomic addition (2n = 6x+1 = 43) for maize chromosome 8, and two double disomic additions (2n = 6x+2+2 = 46) for maize chromosomes 1+9 and 4+6. Four original haploid additions are maintained as tiller-clones because in those recovered haploid monosomic additions (n = 3x+1 = 22), the added maize chromosome did not transmit to the F2 offspring. The clones include one addition plant with maize chromosome 1, two addition plants each with maize chromosome 5, and one addition plant with maize chromosome 10.

We evaluated the practicability of OMAs for physical mapping with 50 molecular markers that had been previously placed on linkage maps by Southern hybridization. Primer pairs were designed for the 50 markers and used for PCR analysis against the genomic DNAs of a complete set of OMAs. Forty-eight markers mapped to their expected chromosome. The two remaining markers were cloned and sequenced. They appeared to identify duplicates of previously genetically mapped loci. Four duplicate loci were identified that had not been previously mapped.

For evaluating the mapping of new maize sequences, primers have been designed and tested for EST sequences from ZmDB (http:www.zmdb.iastate.edu/) and for STS sequences derived from unmethylated regions of the maize genome described by Rabinowicz et al. (Nature Genet 23:305-308, 1999). To date, 300 EST and 50 STS markers have been mapped to chromosome. These markers include 72 on chromosome 1, 47 on chromosome 2, 45 on chromosome 3, 42 on chromosome 4, 56 on chromosome 5, 46 on chromosome 6, 43 on chromosome 7, 52 on chromosome 8, 42 on chromosome 9, and 20 on chromosome 10. Of these markers, 75% mapped to one chromosome, 20% mapped to two chromosomes, and 5% mapped to three to nine chromosomes.

Mapping at high resolution is possible with oat-maize radiation hybrids (RHs) through the use of panels of lines created by radiation-induced breakage. These lines contain chromosome rearrangements and sub-chromosomal segments of maize DNA. The first RH panel was made from an OMA maize chromosome 9 line and has been described by Riera-Lizarazu et al. (Genetics 156:327-339, 2000).

Two types of panels are being assembled for future mapping with RHs for maize chromosome 9. The first is a high-resolution panel with more than 40 lines that can give a mapping resolution of approximately 5 Mbp. The second panel is a low-resolution panel of five lines that have overlapping segments and can allocate sequences to discrete locations on the chromosome. Figure 1 is a graphic representation of how this latter panel is designed. The strategy of the low-resolution panel is to map hundreds or thousands of sequences to chromosomal regions.

Initial RH panels are being evaluated for maize chromosomes 2 and 4. Both of these panels have more than 30 candidate lines that have been identified as containing maize segments and are currently being characterized with markers to evaluate their usefulness. Monosomic OMA seed have been produced, irradiated, and are being grown for characterization of RHs for maize chromosomes 3, 6, and 7.

New technologies are being employed and evaluated to create higher throughput efficiency for identifying sequences in OMA and RH lines. These technologies include using robotics to process samples, Invader Assay (Third Wave Technologies, Inc.), and microarrays. The use of OMA and RH lines in conjunction with these technologies can provide an efficient and reliable means to map thousands of maize sequences to chromosome and discrete segments within a chromosome.

This material is based upon work supported by the National Science Foundation under Grant No. 9872650.


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