COLLEGE STATION, TEXAS
Texas A&M University

Utilization of molecular probes to facilitate development of Quality Protein Maize
 --Lin, KR; Bockholt, AJ; Smith, JD
The nutritional quality of maize can be dramatically improved by selecting for mutations in opaque-2 (o2), a recessive and endosperm specific gene that regulates expression of -zein. However, o2 kernels are relatively soft, which results in problems with milling, disease and insect resistance. Quality protein maize (QPM) is a name for o2 maize genetically modified for hard endosperm by selection (of modifier genes) in order to overcome the agronomic constraints. The QPM modifiers behave as a multigenic trait. Their introgression into elite inbreds is complicated because, in addition to maintaining the homozygous o2 gene, multiple modifiers must be selected. Although conventional breeding procedures have been used successfully to convert commercial lines to QPM forms, the procedure is highly inefficient and is not directly oriented toward improvement of grain quality. If genetic markers could be used to identify unknown modifiers that contributed to QPM phenotype, both the efficiency and the potential for grain quality improvement would be significantly increased. Therefore, the objective of this study was to find markers which could facilitate the identification of high quality maize before selection.

Triads of four inbreds, Mo17, B73, T224 and T232, were analyzed. Each triad consisted of 3 forms of the inbred, i.e., the normal inbred, an o2 form that was backcrossed at least 4 times to the normal inbred, and a QPM form that was backcrossed at least twice to the o2 inbred with selection for hard endosperm and agronomic characteristics. These three lines of a triad are genetically similar, but not identical, and the genetic similarity of the original line is greater with the o2/o2 line than the QPM line. However, genetic similarity is much greater within than among triads. The QPM inbred from each triad may contain chromosomal segments introgressed from the original open-pollinated QPM varieties developed from CIMMYT. To the extent that introgressed regions exist in the same chromosomal locations in all four triads, they probably represent chromosomal regions that were retained by selection for seed characteristics during backcrossing. Thus, introgressed DNA that is present in QPM lines has the potential to carry modifier QPM genes.

To detect these heterogeneous introgressed segments, we hybridized a collection of maize RFLP probes (obtained from Dr. David Hoisington, formerly of the University of Missouri, Columbia) to DNA from seedlings of the four triads. DNA isolation, digestion, electrophoresis, blot hybridization, probe labeling and autoradiography were described previously (Gardiner et al., Genetics 134: 917-930, 1993). About 100 UMC clones were selected for coverage of the maize genome at approximately 20 cM intervals based on the maize linkage map (Gardiner et al., 1993).

Fifty-four UMC probes were screened in the four triads with EcoRI and HindIII digests (Fig. 1). Of these probes, 9 detected same size bands in all three forms of all four triads. Polymorphisms (12 out of 54) occurred between the o2 forms and the normal inbreds in one or more triads with either enzyme digest. These probes may reflect the different origins of O2 and o2 alleles. Ten probes (out of 54) were hybridized to different size bands in the normal, o2 and QPM forms in one or more triads with either enzyme digest. About half (26 out of 54) of the UMC clones detected identical bands in the normal and o2 lines, but bands were different in one or more QPM lines in one or two enzyme digests. Practically, a combination of molecular probes that would allow for selection of modifiers prior to selection for agronomic characteristics would be a powerful tool for QPM inbred conversion.


Figure 1. Southern blot pattern using probe umc64. Genomic DNA of four triads was digested with EcoRI (lanes 1-12) and HindIII (lanes 13-24). Size was separated on 1% agarose gel, blotted to a nylon membrane (HybondTM-NC+) and hybridized to a radiolabelled probe. N: Normal maize. o2: opaque-2 maize. Q: Quality protein maize.

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