SAINT PAUL, MINNESOTA
University of Minnesota and USDA-ARS
A novel maize ditelosome 10 addition to oat cv. Sun II for use in radiation hybrid mapping --Kynast, RG, Okagaki, RJ, Granath, SR, Rines, HW, Phillips, RL A complete set of disomic oat-maize chromosome additions, each with an individual maize chromosome pair added to a complete genome of hexaploid oat, is desirable for chromosomally dissecting and mapping the maize genome and studying maize genes and markers in the genetic background of oat (Kynast et al., Funct. Integrative Genomics 2:60-69, 2002). To date, a total of 46 disomic addition lines have been produced by pollinating more than 80,000 florets of eight different oat lines with four different maize lines. Fertile F1 (oat maize) hybrids and their F2 and subsequent generations were recovered, and the added maize chromosomes cytologically and molecularly identified, as described elsewhere (Kynast et al., Plant Physiol. 125:1228-1235, 2001). The series of fertile disomic addition lines (2n = 6x+2 = 44) include ones for maize chromosomes 1, 2, 3, 4, 5, 6, 7, 8, and 9, with seed and/or DNA available upon request. The maize chromosome 10 has been recovered as a monosomic addition to haploid GAF-Park oat; however, to date it has failed to produce F2 offspring. The chromosome 10 addition is being maintained vegetatively by tiller-cloning for DNA/RNA production. In an attempt to complete the set of disomic oat-maize addition lines for use in the production of radiation hybrids, we extended our crossing program to additional genotypes and culture conditions. From about 8,000 crosses between oat cv. Sun II and maize cv. Seneca 60, we generated 51 new proembryos. Of these, 11 proembryos developed into plantlets of haploid oat with one or more maize chromosome(s) added; 36 proembryos developed into haploid oat plants without maize chromosomes. Four proembryos did not develop enough tissue for an analysis.

Plant F1-0289-1 retained the maize chromosome 10, in addition to all oat chromosomes, based on a positive test for the maize-specific LTR-type retrotransposon Grande 1 and maize chromosome 10 SSR markers. The plant was allowed to self-pollinate, and four maize-positive panicles were found to set seeds (Table 1). Genomic DNA samples of ten F2 offspring from each maize-positive panicle of the F1-0289-1 plant were assayed for presence versus absence of the maize-specific LTR-type retrotransposon Grande 1 (Figure 1). PCR using Grande 1-specific primers detected maize chromatin in nine offspring plants of F1-0289-1/a (designated F2-3776/a-1 to F2-3776/a-9; F2-3776/a-10 was negative), nine offspring plants of F1-0289-1/b (designated F2-3776/b-1 to F2-3776/b-10; F2-3776/b-4 was negative), no offspring plant of F1-0289-1/c (all F2-3776/c-1 to F2-3776/c-10 were negative), and ten offspring plants of F1-0289-1/d (designated F2-3776/d-1 to F2-3776/d-10). We verified the maize chromosome identities by assaying the corresponding DNA samples of the maize-positive F2 offspring plants with two sets of SSRs genetically mapped to maize chromosome 10 from the MaizeDB (http://www.agron.missouri.edu/) (Figure 2). We selected the markers: p-phi041, p-phi117, and p-umc1293 from bin 10.00, and p-umc1249, p-umc1196, p-umc1176 and p-umc1084 from bin 10.07. Since most of the maize-positive plants showed consistent presence of the three markers from the bin 10.00, but absence of the four markers from the bin 10.07, we conclude that the maize chromosome present in each of these offspring is likely a telocentric derivative of the chromosome 10 retained in the original plant F1-0289-1. GISH analysis on root-tip cells using labeled genomic DNA from maize revealed the disomic status and the telocentric nature of the added chromosomes in most of the plants analyzed by PCR (Figure 3). The line was given the designation OMAdt10.20S in accordance with the proposed nomenclature for oat-maize chromosome addition lines (Kynast et al. 2001: Maize Genetics Coop. Newsl. 75, 54-55).

Since there are more seeds to be analyzed for the possible telosomic addition of the long arm of chromosome 10, we will test all seeds with larger numbers of chromosome arm-specific SSRs. At present we are propagating the identified disomic telocentric F2 offspring plants to test for chromosome stability and transmission to F3 offspring. Once more seed are produced, the new addition lines will be available upon request to the scientific community.

This material is based upon work supported by the National Science Foundation under Grant No. 0110134.

Table 1. Seed set of the first fertile oat-maize chromosome 10 addition F1 (Sun II Seneca 60) 0289-1.
 
     
Number of Seeds
 
F1 Plant/Panicle
Grande 1-SSR
Florets
Harvested
Analyzed
F2 Offspring IDs
F1-0289-1/a
+
222
51
10
F2-3776/a
F1-0289-1/b
+
264
59
10
F2-3776/b
F1-0289-1/c
+
156
48
10
F2-3776/c
F1-0289-1/d
+
154
31
10
F2-3776/d

Figure 1. Selected examples of oat offspring possessing maize-chromatin shown by PCR with Grande 1 primers. The presence of the 500 bp band (+) indicates presence of maize chromatin in the DNA extract of the corresponding F2 plant; M=Molecular Weight Marker, A=Sun II, B=Seneca 60, C=No-DNA Control.

Figure 2. Telocentric maize chromosome identification with the short arm-specific SSR, p-phi041 (bin10.00). The presence of the 200 bp band is diagnostic for the short arm of maize chromosome 10 as shown in DNA extracts of the corresponding F2 plants; M=Molecular Weight Marker, A=Sun II, B=Seneca 60, C=No-DNA Control.

Figure 3. GISH of fluorophore-labeled genomic maize DNA to root meristem cells of plant F2-3776/b-6. Arrows point to the two telocentric maize chromosomes (green), which are formed from the short arm of chromosome 10 (smallest maize chromosome). Oat chromosomes (red) are counterstained with propidium iodide.
 
 


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