RFLP analysis of opaque2 genotypes in segregating populations
--Srinavas R. Kata, Brian H. Taylor, A. J. Bockholt and J. D. Smith

The Texas A&M University Quality Protein Maize (QPM) breeding program has focused on the development of hard, corneous endosperm forms of elite inbreds that are homozygous opaque2. The general approach has been to select inbreds from open pollinated QPM (CIMMYT) and High Lysine (South Africa) populations, which had previously been selected for hard endosperm. These inbreds are used as non-recurrent parents for improvement of seed characteristics of elite lines by backcrossing.

Marker assisted selection procedures are particularly useful in breeding programs involving seed specific, recessive alleles such as o2. Under the assumption that a probe derived from the target gene would minimize recombination, we obtained a full length cDNA of the O2 gene from M. Maddaloni, M (Bergamo, Italy), and developed an RFLP marker assay to identify polymorphisms tightly linked to the o2 locus. Under lab conditions, the presence or absence of specific polymorphisms provided genotypic identification of juvenile plants.

To field test the procedures, Tx5855 was crossed to plants from four hard endosperm o2/o2 populations (POP20, POP21, POP22 and POP23, provided by Dr. Hans Gevers, H) from South Africa and two CIMMYT QPM populations (POB67 and POB68). The F1 plants were selfed and backcrossed to both the parents, and the resultant F2 and BC1 populations were planted on Texas A&M University Research Farm. Individual seedlings were tagged and leaf tissue was collected from juvenile (4-5 weeks after planting) plants. The leaf samples were individually frozen in liquid nitrogen and stored at -80 C. DNA was isolated from the plants using a CTAB extraction procedure, digested with HindIII, size separated on an agarose gel and blotted to polysulfone membrane (Biotrace; Gelman Sciences). The blots were probed and genotypes were identified for individual plants. To compare predicted genotype with the progeny seed phenotype, each plant was selfed and the seed phenotypes were determined using a light box. Genotypic analysis was done for 270 plants in the segregating populations. Then genotypes of individual plants were compared with progeny seed phenotypes. In all the cases where the seed phenotype was clear, the RFLP analysis matched the phenotype prediction. However, the opacity of o2/o2 kernels varied considerably in different genetic backgrounds, and the visual identification of seed phenotypes was not reliable in some cases. Utilization of RFLP analysis in determining the opaque2 genotypes was essential in these lines.

Probe testing of several lines (not used in field test), presumed to be o2/o2 hard endosperm derivatives, revealed that the o2 allele had been lost. This emphasizes the usefulness of the RFLP probe in tracking the o2 allele during conversion of established lines of o2/o2 and vitreous o2 forms. The results of our field tests indicate that RFLP analyses correctly identified opaque2 genotypes in segregating populations with 100% accuracy, and that this assay can be used for genotypic identification in different source materials. 

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