We have DNA fingerprinted 56 CIMMYT and 12 other inbred maize lines, using 19 microsatellite (SSR) markers. These were selected from the list of SSR markers defined by Pioneer HiBred International, Inc. (for loci denominated phi...) and by North Carolina State University (nc...) (Senior et al. MNL 70:50-54 70:112-117). We have remapped these markers, and most of the 32 others available at CIMMYT, in tropical maize populations with no discrepancies compared to published data. We fingerprinted a set of 17 CIMMYT lines (CMLs) presently being studied for heterotic relationships in 11 environments by Javier Betrán, 12 CMLs representing 4 tropical heterotic groups, and 37 lines already fingerprinted using RFLP markers (González de León et al. Agron Abstr 1989). Four comparative materials were placed in external lanes, B73, Mo17, a Huayleño (Peruvian highland flour) accession and a new, unique teosinte from Nicaragua.
The 19 SSR markers we used were the more promising ones when examined for clarity and amount of polymorphism on agarose gels using 14 inbred lines. We tested varying proportions of glycerol, MgCl2, primer, target DNA and nucleotides, and determined optimal annealing temperatures. We are examining the potential for multiplexing (joint PCR amplification) of markers, so tried to use uniform conditions.
The agarose was a 1:1 mixture of Metaphor and SeaKem, at 3%. Two CIMMYT- designed internal markers, 66 and 300 base pairs long, were used in all lanes. The PhiX174/HaeIII size marker in lane 15 of the two 30-lane combs served to estimate base pair numbers for amplified products.
Lane bands were scored using enlarged acetate xerograms of the gel photos and a Numonics digitizer, and were recorded using the HyperBlot data entry and analysis system (Hoisington et al. Agron Abstr 1989). HyperBlot then estimated the numbers of nucleotides in each band entry (Southern Anal Bioch 100: 319-323 1979). Bands were clustered into morphs defined by migration distance (see following note) using average cluster linkage. The number of final clusters can be controlled by presetting the minimum cluster distance -- we used 20-35 thousandths-of-an-inch (0.51-0.89 mm) depending on the marker. We found 2-5 morphs per SSR marker. Some morphs seem to contain a small array of "sub-morphs", probably differing by multiples of SSR motif repeats, not distinguishable in agarose gels. Many morphs differ by much more than a few repeats of the motif(s), perhaps involving insertions/deletions in regions between the primed sequences and the SSR itself. Such differences ranged up to 21, 28 and 36 bases. Variation in gel conditions, reading variance and differences between combs must be factors controlling some intra-morph variation, probably resolvable by using polyacrylamide gels.
These data can be used to choose SSR markers for Marker Assisted Selection projects, but so far have not proved useful in relating lines into meaningful patterns.
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