18 INTRODUCTION 19 MATERIALS AND METHODS 25 RESULTS 31 DISCUSSION 35 REFERENCES 48 PAPER 2. MOLECULAR-MARKER-FACILITATED 52 INVESTIGATION OF HOST-PLANT RESPONSE TO EXSEROHILUM TURCICUM IN MAIZE {ZEA MAYS L.): COMPONENTS OF RESISTANCE. SUMMARY 54 MATERIALS AND METHODS 59 RESULTS 66 DISCUSSION 71 REFERENCES 91 GENERAL CONCLUSIONS 95 LITERATURE CITED 98 ACKNOWLEDGEMENTS 101 APPENDIX 103 1 GENERAL INTRODUCTION An Explanation of Dissertation Format This dissertation includes two manuscripts. These

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... e preceded by a general introduction. The first manuscript reports the creation of an RFLP map and its use to obtain estimates of chromosomal locations of loci determining hostplant response to Exserohilum turcicum in maize; in particular to make comparisons with the locations, where known, of loci defined by alleles with qualitative effects. The second manuscript reports on the genetic effects of quantitative trait loci (QTL) and discusses the resolution of host-plant response into separate components. The manuscripts are followed by a general summary. References cited in the general introduction and general summary are listed in the literature cited section. The appendix includes additional materials pertinent to the main text. Molecular-Marker-Facilitated Investigations of Host-Plant Response to Plant Pathogens Restriction fragment length polymorphisms (RFLPs) represent base pair differences in homologous segments of DNA from genetically distinct individuals. Typically these differences are observed once the DNA has been cleaved by restriction enzymes and hybridized to a radiolabeled DNA probe using the Southern hybridization technique (Watkins, 1988; Helentjaris, 1987). Restriction enzymes only cleave DNA at 5 66:69-70, 1992). Bubeck et al. (1992) used RFLPs to study host-plant response to gray leaf spot in three populations; QTL identified were not consistent over environments or populations. RFLPs are not the only method available to map disease resistance genes: aneuploids, substitution lines or translocation stocks may also be used, depending on the plant species. However, RFLPs can provide more precise estimates of chromosomal regions important in determining host-plant response, and in addition, provide information on the genetic effects of the QTL. Recessive gene action, which will elude translocation mapping, may be identified with RFLP based mapping. In addition RFLP mapping enables much more complete coverage of the genome than translocation mapping. There are many components of resistance, for example, lesion size, lesion number, infection efficiency, incubation period, latent period, lesion expansion rate, and sporulation capacity, that can be considered when assessing host-plant response to Exserohilum turcicum (Brewster et al., 1992). The relationships between the different components and their relative importance in disease assessments will vary, however, with crop and pathogen. Precise measurements on a large number of components is generally not feasible for replicated field evaluations. Some mapping studies involve a combination of methodologies targeting specific loci, but there are few where field testing of segregating populations is involved. 6 Greenhouse and seedling tests may be used; however, these may not be an adequate prediction of adult plant response, particularly in the case of the pathogen of interest in this study (Brewster et al., 1992). The choice of a disease assessment method is of primary concern. The use of a qualitative score, for example R (resistant) or S (susceptible), to rate populations segregating for quantitatively inherited resistance, appears imprudent. Visual estimates, for example, of disease severity, are an alternative but these may be subject to over or under estimation, even when experienced scorers are used (Sherwood et al., 1983). Scaler rating systems (eg. 1-9) may also be inappropriate. Is a linear scale more appropriate than a logarithmic scale? Inadvertently, the precision may be diminished and researchers may be imposing assumptions about the inheritance of the resistance they are trying to map. A non-subjective rating system based on either precise measurements or remote sensing, for example percent reflectance, is generally superior. These assessment methods, however, have an inherent cost either in time or capital outlay which accounts for, in part, reliance on quicker and less objective methods. 7 Statistical Analysis Experimental design The population used in this study was created by crossing two inbred maize lines, B52 (female) and Mol7 (male). The F, hybrid was self-pollinated and 169 random Fg plants were retained. These were self-pollinated to generate the 150 unselected F^.^ lines used in the field experiments. A sets within replications design was used with 27 entries nested within each of 6 sets. Two replications (12 sets) were grown at Ames, Iowa and two replications at Urbana, Illinois in the summer of 1991. Check inbreds (both parents) were included in each set. The sets within replications design was analyzed using the model presented by Schutz and Cockerham (1962): ^ijk = ^^ + S. + r, + g.. + s,; + (gr).j^ ; where the effects S are for sets, r for replications, g for entries in sets, s for environmental differences of sets in replications, and gr for replication x entry in set interactions. The subscript i varies from one to s, j varies from one to g and k varies from one to r. ABSTRACT RFLPs at 103 loci were used to identify the location of quantitative sources of resistance to Exserohilum turcicum in 150 Fj.j lines of a B52/Mol7 maize population. Host-plant response was measured in terms of average number of lesions per leaf, average percent leaf tissue diseased (severity) and average size of lesions. The location of QTL were compared with loci having known qualitative effects, namely Htl, Ht2 and bxl. Chromosomal regions containing the Htl and Ht2 loci showed a small contribution in determining lesion size, even though alleles'with dominant, qualitative effects at these loci have never been reported in either inbred parent. Similar effects were not observed for number of lesions or disease severity. Likewise some contribution was observed for chromosomal regions encompassing the bxl locus in determining lesion size but not number of lesions or disease severity. Overall the contribution of loci in the vicinity of Htl, Ht2 and bxl was small relative to variation attributable to loci with quantitative effects identified in this study. RFLP mapping concurred with reciprocal translocation mapping on the importance of chromosomes 3, 5 and 7, despite the fact that these studies utilized diverse sources of resistant germplasm. 4^ w Apuya, N.R., B.L. Frazier, P. Keim, E.J. Roth, and K.G. Lark. 1988. Restriction fragment length polymorphisms as genetic markers in soybean. Glycine max(L.) merrill. Theor. Appl. Genet. 75:889-901. Bentolila, S., C. Guitton, N. Bouvet, A. Sailland, S. Nykaza, and G. Freyssinet. 1991. Identification of an RFLP marker tightly linked to the Htl gene in maize-Theor. Appl. Genet. 82:393-398. Brewster, V.A., M.L. Carson, and Z. Wicks III. 1992. Mapping components of partial resistance to northern leaf blight of maize using reciprocal translocations. Phytopathology 82:225-229. Bubeck, D.M., M.M. Goodman, W.D. Beavis, and D. Grant. 1992. Quantitative trait loci controlling resistance to gray leaf spot in maize. In press. Edwards, M.D., C.W. Stuber, and J.F. Wendel. 1987. Molecular-marker-facilitated investigations of quantitative-trait loci in maize. I. Numbers, genomic distribution and types of gene action. Genetics 116: 113-125. Hallauer, A.R., and J.B. Miranda. 1988. Quantitative genetics in maize breeding. 2nd ed. Iowa State University Press, Ames, lA. Helentjaris, T. 1987. A genetic linkage map for maize based on RFLPs. Trends In Genetics 3 (8):217-221. Hilu, H.M., and A.L. Hooker. 1964. Host-pathogen relationship of Helminthosporium turcicum in resistant and susceptible corn seedlings. Phytopathology 54:570-575. Hooker, A.L., and S.K. Kim. 1973. Monogenic and multigenic resistance to Helminthosporium turcicum in corn. Plant Dis. Reptr. 57:586-589. Knapp, S.J., W.W. Stroup, and W.M. Ross. 1985. Exact confidence intervals for heritability on a progeny mean basis. Crop Soi. 25:192-194. Lander, E.S-, and D. Botstein. 1989. Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121:185-199. 99 Landry, B.S., R.V. Kesseli, B. Farrara, and R.W. Michelmore, 1987. A genetic map of lettuce (Lactuca sativa L.) with restriction fragment length polymorphism, isozyme, disease resistance and morphological markers. Genetics 116:331-337. Lincoln, S.E. and E.S. Lander. 1990. Mapping genes controlling quantitative traits using MAPMAKER/QTL. Whitehead Institute for Biomedical Research, Cambridge, MA. McMullen, M.D., and R. Louie. 1989. The linkage of molecular markers to a gene controlling the symptom response in maize to maize dwarf mosaic virus. Molecular Plant-Microbe Interactions 2 (6):309-314. Melchinger, A.E. 1990. Use of molecular markers in breeding for oligogenic disease resistances. Plant Breeding 104: 1-19. Mode, C.J., and H.F. Robinson, 1959. Pleiotropism and the genetic variance and covariance. Biometrics 15:518-537. . 1988. Resolution of quantitative traits into mendelian factors by using a complete linkage map of restriction fragment length polymorphisms. Nature 335:721-726. Schutz, W.M., and C.C. Cockerham. 1962. The effect of field blocking on gain from selection. North Carolina State College, Institute of Statistics, Mimeograph Series No. 328. Shapiro, S.S., and M.B. Wilk. 1965. An analysis of variance for normality (complete samples). Biometrika 52:591-611.