Quantitative trait loci and metabolic pathways (Zea mays L.yf lavonoidyf lavoneyinsect resistanceyHelicoverpa zea)

The interpretation of quantitative trait locus (QTL) studies is limited by the lack of information on metabolic pathways leading to most economic traits. Inferences about the roles of the underlying genes with a pathway or the nature of their interaction with other loci are generally not possible. An exception is resistance to the corn earworm Helicoverpa zea (Boddie) in maize (Zea mays L.) because of maysin, a C-glycosyl f lavone synthesized in silks via a branch of the well characterized f lavonoid pathway. Our results using f lavone synthesis as a model QTL system indicate: (i) the importance of regulatory loci as QTLs, (ii) the importance of interconnecting biochemical pathways on product levels, (iii) evidence for ‘‘channeling’’ of intermediates, allowing independent synthesis of related compounds, (iv) the utility of QTL analysis in clarifying the role of specific genes in a biochemical pathway, and (v) identification of a previously unknown locus on chromosome 9S affecting f lavone level. A greater understanding of the genetic basis of maysin synthesis and associated corn earworm resistance should lead to improved breeding strategies. More broadly, the insights gained in relating a defined genetic and biochemical pathway affecting a quantitative trait should enhance interpretation of the biological basis of variation for other quantitative traits. The past decade has seen an explosion of information on the structure, organization, and functions of the maize (Zea mays L.) genome, including the development of high density molecular marker maps. One application of new mapping technologies has been the genetic dissection of quantitative traits with much greater precision than was previously possible (1, 2). Still, the quantitative trait loci (QTLs) detected are generally rather poorly defined regions, and the size of a QTL’s phenotypic effect is sometimes confounded with its location relative to the nearest marker or to a nearby QTL. For most traits, genetic and biochemical information on metabolic pathways is extremely limited, and, therefore, it is difficult to interpret QTL results in terms of regulatory and structural genes, duplicate function loci, feedback inhibition, branched pathways, or other phenomena affecting trait expression. Our goal in this research project is to analyze the genetic control of a quantitative trait of economic importance [antibiosis to the corn earworm (CEW)] and to interpret the results in terms of the well characterized flavonoid pathway. The CEW Helicoverpa zea (Boddie) is a major insect pest of maize and other crops (cotton, soybeans, peanuts) in the United States and elsewhere in the Western Hemisphere (3, 4). Corn earworm eggs are laid on the silks, and the larvae access the ear by feeding through the silk channel. Host–plant resistance to CEW by antibiosis is caused by the presence of the C-glycosyl f lavones maysin, apimaysin, and methoxymaysin and related compounds (Fig. 1) in maize silks (5, 6). Although these flavones occur in various proportions in different maize lines and populations, maysin is the predominant compound in most genotypes (M.E.S., unpublished data). Upon ingestion by CEW, the flavones are oxidized to quinones, which bind amino acids, making them unavailable and thus inhibiting larval growth (7). The branches of the flavonoid pathway leading to the synthesis of C-glycosyl f lavones (including maysin and related CEW resistance factors), phlobaphenes (responsible for red cob and pericarp pigments), and 3-deoxyanthocyanins are regulated by the p1 locus. The p1 locus encodes a myb-like transcription factor. That locus also affects the silk-browning trait, whereby silks of some genotypes turn brown after wounding (8, 9). Based on our current understanding, maysin synthesis requires appropriate alleles at p1, c2, andyor whp1 [encoding chalcone synthases (10, 11)], chi1 [encoding chalcone isomerase (12)], pr1, [controlling the 39-hydroxylation of the flavonoid B-ring to convert monohydroxy to dihydroxy compounds (13)], and unidentified additional loci encoding flavone synthase, C-glycosyl transferase, glucose oxidase, rhamnosyl transferase, and an enzyme such as glutathione Stransferase for transport to the vacuole (14, 15) (Fig. 1). Our approach has been to develop F2 or F2:3 populations from crosses of inbred lines chosen to address the contribution of specific genes of the flavonoid pathway on maysin synthesis and antibiotic earworm resistance as measured in the laboratory by larval weight bioassay (16, 17). F2 plants or F2:3 family rows are grown in the field, and silks are collected 2–3 days after silk emergence. The silks are weighed, frozen, and freeze-dried (for recent populations), and concentrations of maysin, apimaysin, and other analogs are determined by reversed-phase HPLC (18). Whorl tissue is collected from F2 or bulked from F2:3 plants, DNA is extracted (19), and genotypes of the individuals in the population are determined by restriction fragment length polymorphism (RFLP) analysis by using primarily the University of Missouri-Columbia core RFLP markers (20, 21) and probes for genes of the flavonoid pathway (22). Analysis of the phenotypic data for silk chemical constituents and bioassay results is carried out primarily with ANOVA (analysis of variance), general linear model, and correlation procedures of SAS statistical analysis software (23). Linkage maps for the populations are constructed with the computer program MAPMAKERyEXP (24), which uses a © 1998 by The National Academy of Sciences 0027-8424y98y951996-5$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: QTL, quantitative trait locus; CEW, corn earworm; RFLP, restriction fragment length polymorphism. ‡To whom reprint requests should be addressed. e-mail: mcmullen@ teosinte.agron.missouri.edu.