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a Genetics Area Program, Univ. of Missouri, Columbia, MO 65211
b Richard B. Russell Research Center, Athens, GA 31793
c USDA-ARS, Plant Genetics Research Unit and Dep. of Agronomy, Univ. of Missouri, Columbia, MO 65211
* Corresponding author (mcmullenm{at}missouri.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: CEW, corn earworm • CGA, chlorogenic acid • CHS, chalcone synthase • CIM, composite interval mapping • cM, centimorgans • LOD, log-odds ratio • MLM, multiple locus model • QTL, quantitative trait locus • SSR, simple sequence repeat.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Quantitative trait locus analysis is a valuable tool for genome exploration and the investigation of multigenic traits. The C-glycosyl flavone, maysin, found in maize silks, is a product of the flavone pathway and has antinutritive effects on CEW larvae (Waiss et al., 1979; Elliger et al., 1980). Chlorogenic acid, a product of the phenylpropanoid pathway, has also been implicated in CEW antibiosis (Isman and Duffy, 1983). The flavonoid and phenylpropanoid pathways share biochemical intermediates, and the concentrations of maysin and CGA in maize silks are quantitative traits (Byrne et al., 1996a,b, 1998; Lee et al., 1998; McMullen et al., 1998).
Quantitative trait locus studies using populations constructed to explore variation between inbred lines have identified regions of the maize genome important for flavone biosynthesis (Byrne et al., 1996a, b, 1998; McMullen et al., 1998; Lee et al., 1998; Bushman et al., 2002). It has been possible to assign candidate gene identities to many of these regions on the basis of the known locations of genes involved in the well-studied flavonoid biosynthetic pathway. The p1 locus on the short arm of chromosome 1 has consistently been identified as a major QTL for flavone biosynthesis. P1, a Myb-like transcription factor, regulates genes involved in flavonoid biosynthesis; inducing expression of phlobaphene pigmentation in the pericarp and cob, 3-deoxyanthocyanins, and the C-glycosyl flavones (Grotewold et al., 1998). P1 can act without other factors to induce transcription, unlike the related C1 and PL1 Myb-like transcription factors, which require R1 and/or B1 Myc-like transcription factors to induce expression (Chandler et al., 1989; Ludwig et al., 1989; Perrot and Cone, 1989). The repeated detection of large quantitative variance caused by p1 has demonstrated the importance of regulatory loci in quantitative traits.
Additional QTL for maysin were detected on the long arm of chromosomes 2 and 4 (Byrne et al., 1998). These regions include the locations of the whp1 and c2 loci, a pair of duplicate genes encoding CHS in maize. Chalcone synthase catalyzes the first committed step in flavonoid biosynthesis and represents a branch-point between the flavonoid and phenylpropanoid pathways. The regulation of c2 and whp1 gene expression may be a focal point for control of these pathways, and the role of individual CHS loci in flavone synthesis is not known. Considered to be independently regulated and tissue specific, c2 and whp1 may also play different roles in both flavonoid and phenylpropanoid biosynthesis. Nonfunctional alleles, determined by their effects on the anthocyanin pathway, are available at both of these loci, and provide ideal sources of variation.
We performed QTL analysis to examine silk maysin and CGA concentrations in three related populations segregating for nonfunctional alleles of the duplicate CHS loci, c2 and whp1. Our objective was to identify the genetic effects of these duplicate loci on flavone and phenylpropanoid biosynthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Simple Sequence Repeat Analysis
Simple sequence repeat (SSR) markers were used to gather genotypic information for populations. Procedures for DNA extraction and PCR were as in http://www.agron.missouri.edu/ssr.html (verified April 2, 2002). Super Fine Resolution (Amresco, Solon, OH) agarose gels at concentrations ranging from 3.5 to 5% (w/v) were used to resolve amplification products. Gels were visually scored twice independently to assure accurate genetic information.
Statistical Analysis
Linkage maps were generated by MAPMAKER/EXP version 3.0 (Lander et al., 1987) for UNIX. A minimum likelihood of odds (LOD) score of 3.0 and maximum distance of 50 centimorgans (cM) was used as the map generation threshold. Interval mapping with MAPMAKER/QTL (Lander et al., 1987) and composite interval mapping (CIM) with QTL CARTOGRAPHER (http://statgen.ncsu.edu) were used for the identification of significant QTLs. Genome wide thresholds (P = 0.05) for statistical significance for each trait and population were identified by permutation analysis with QTL CARTOGRAPHER (Churchill and Doerge, 1994).
Statistical analysis was performed by Statistical Analysis System (SAS for Windows v. 7.0, SAS Institute Inc., Cary, NC). When QTL detection software identified significant regions of the genome flanked by genetic markers, analysis was performed to examine the genomic region between the markers. This was accomplished with only those individuals in which no known recombination had occurred, as determined by inheritance of marker genotypes from the same parent flanking the region of interest. In this manner, the QTL, rather than association with an individual genetic marker could be identified.
Multiple-locus models (MLM) were created with PROC GLM in SAS. Markers to be tested were selected from QTL CARTOGRAPHER by stepwise regression and CIM results, single-factor ANOVA, and EPISTACY (Holland, 1998). Stepwise regression thresholds P(F-in) and P(F-out) were 0.01 for selection of markers with main effects. Analysis of all possible two-way interactions between marker loci was conducted by EPISTACY (Holland, 1998). A significance threshold of P = 0.001 was established to account for the large number of comparisons. Individual markers and significant interactions were included in the multiple-effects model if the Type III sums of squares P-value was less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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QTL Detection
Maysin
In Populations 1 and 2, a majority of the phenotypic variance in flavone biosynthesis was explained by a QTL detected by CIM on chromosome 1 in bin 1.03 (Table 2
, Fig. 1)
. The nearest marker loci to this QTL were phi001 in Population 1 and bnlg2238 in Population 2. These populations segregated for the p1-www and p1-wwb alleles. Inheritance of the functional p1-wwb region led to marked elevation in silk maysin and CGA concentration. We believe the identity of this major QTL to be the transcription factor p1. The p1 locus was not significant for maysin accumulation in Population 3, which segregated for p1-wwb and p1-wrb alleles.
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Composite interval mapping detected a QTL in Population 1 on the short arm of chromosome 9 for maysin accumulation. A QTL in the same region was detected in Population 2 for maysin and CGA. This region was not mapped in Population 3. The chromosome 9 QTL explained only a small percentage of the phenotypic variance in each population.
Chlorogenic Acid
A QTL on chromosome 1 corresponds to the p1 locus and, as for maysin, accounted for the greatest portion of phenotypic variance for CGA accumulation in Populations 1 and 2 (Table 2). This region explained 17.0% of the phenotypic variance in CGA levels in Population 1 and 42.7% in Population 2. In Population 3, segregation at p1 was also significant for CGA accumulation, but only explained, 2.9% of the phenotypic variance.
In Populations 2 and 3, the chromosome 4 QTL was also significant for CGA levels (Table 2, Fig. 1). Nonfunctional alleles at the c2 locus inherited from the W23 parental line demonstrated an additive contribution toward higher amounts of CGA. This QTL explained 17.8% of the phenotypic variance for CGA in Population 2 and 8.1% in Population 3.
A common major QTL for CGA accumulation was detected in all three populations in the centromeric region of chromosome 2. In Population 1, the nearest SSR marker to this QTL was bnlg1036 (2.05) and the interval of bnlg108 (2.05) and umc1065 (2.05) contained this QTL in Populations 2 and 3. This region explained a large portion of the phenotypic variance in CGA accumulation (25%) in Populations 2 and 3 (Table 2). This QTL is consistent with the locus designated qtl2 in the companion paper (Bushman et al., 2002). The other chromosome 2 QTL thought to be whp1 influenced CGA accumulation in a dosage-sensitive manner in Population 1. Gene action of the whp1 QTL behaved similarly to the c2 region on chromosome 4.
Regions of chromosome 3 that affected CGA accumulation were detected in all three populations. A single QTL was detected in Population 1 and two QTL were detected in both Populations 2 and 3. Evidence suggests that three QTL may exist for CGA throughout chromosome 3, but it was not possible to resolve the three QTL in any one population (this paper, Bushman et al. 2002, and McMullen, unpublished data).
Multiple Locus Models
Multiple locus models were generated for maysin and CGA using marker data from Populations 1 and 2. For Population 1, the MLM for maysin had a total R2 of 65.5% and included major QTL and several interactions (Table 3)
. Main effect loci for maysin were p1, c2, whp1, and the chromosome 9 QTL associated with marker bnlg1209 (bin 9.04). The interactions p1 x c2, bnlg2277 (bin 2.03) x whp1, and bnlg1724 (bin 9.00) x whp1 were significant and retained in the Population 1 MLM for maysin. A similar MLM for maysin was generated for Population 2 with an R2 of 46.3%. Main effect loci included p1, c2, and the chromosome 9 QTL (bnlg1209, bin 9.04) with the interactions p1 x c2, p1 x bnlg1209 (bin 9.04), and c2 x bnlg125 (bin 2.03) retained in the model.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In Population 3, a red-cobbed (p1-wrb) line was crossed with a white-cobbed (p1-wwb) line to reduce effects of the p1 locus in silks. Both of these alleles were considered to be functional in silks, as flavone and CGA levels were high compared with p1-www alleles. Although the combination of p1-wrb x p1-wwb alleles was not significant for silk maysin levels, the p1 region was significant for CGA accumulation. Increased dosage of the p1-wwb allele increased CGA accumulation.
The genetic dispositions of p1-wrb and p1-wwb could be indicative of multiple factors. Data indicate a second locus, pericarp color2 (p2), exists proximal, yet tightly linked to p1, with high homology to the p1 locus (Zhang et al., 2000). Functional p1 alleles have been demonstrated to affect pigmentation in pericarp and cob, and browning in silk tissue. However, the p2 allele has distinct tissue specificity, conferring only the silk browning phenotype. Silk browning in maize is highly correlated to flavone accumulation in silks (Byrne et al., 1996a; Guo et al., 1999).
Chalcone Synthase Loci
The c2 region on chromosome 4, and less consistently, the whp1 region on chromosome 2, have been identified in previous studies (Byrne et al., 1998; Lee et al., 1998) as candidates of QTL variation for maysin accumulation in maize silks. The populations in this study were constructed to examine specifically the effects of variability at the CHS loci on C-glycosyl flavone and phenylpropanoid biosynthesis. Nonfunctional c2 and whp1 alleles were used as sources of variability at the two CHS loci in maize.
Examination of the bin 2.09 (whp1) and 4.08 (c2) QTLs separately and their interaction (Fig. 2) provided evidence that they corresponded to whp1 and c2. The favorable allele for maysin accumulation in each case was the functional allele. As dosage of the functional CHS loci c2 and whp1 increased, greater amounts of maysin accumulated. The c2 and whp1 regions demonstrated an additive, nonepistatic effect on maysin biosynthesis (Fig. 2). Functional whp1 alleles partially compensated for the absence of functional c2 alleles. In the presence of functional c2, whp1 increased maysin accumulation beyond that observed with c2 alone. The effect of c2 on maysin production required the presence of functional p1 alleles, while p1 had no effect on maysin production through whp1. This result suggests that while c2 is under direct control of p1, whp1 is regulated by a separate genetic mechanism in silks.
The phenylpropanoid and C-glycosyl flavone pathways share biochemical intermediates. As the initial enzymatic reaction in flavonoid biosynthesis, CHS represents the committal step in the pathway (Harborne, 1998). Nonfunctional CHS would theoretically reduce flux towards the production of C-glycosyl flavones, increasing intermediates available for the phenylpropanoid pathway. Parental lines used in Population 1 do not synthesize large amounts of CGA. A slight increase in effect of c2 for CGA was observed in Population 1, but it was well below significance thresholds. Levels of CGA may not have been large enough to produce statistically significant differences between c2 classes. In Population 2, CGA levels follow a marked relationship to CHS genotype. Chlorogenic acid levels in silks increased by 12.9% in homozygous c2 whp1 vs. C2 Whp1 classes.
Genetic effects resulting from shunting of intermediates between pathways was also observed with the dihydroflavanone reductase structural gene anthocyaninless1 (a1) (McMullen et al., 2001). The a1 gene's function is at a branch-point between flavone and 3-deoxyanthocyanin biosynthesis, and individuals homozygous for nonfunctional a1 alleles have markedly increased maysin production when functional p1 allele(s) is/are present. Genetic variability of both a1 and p1 result in an epistatic interaction as we have observed for p1 and c2. Our results confirm that genetic variation altering flux between pathways can be the genetic basis of both major QTL and epistatic effects.
We demonstrated that the p1 locus affected the flavone pathway, exerting a global effect. Complicating QTL detection was the influence on the apparent activity of loci not under P1 regulation through indirect epistatic effects. Detection of QTL by current methodology and statistical thresholds relies heavily on class means and the corresponding relative variance for determination of significance and inclusion in MLM. In the case of minor QTL, segregation of QTL with large effects will increase the variance within the genotypic classes of the minor QTL as demonstrated in Fig. 2. Even though p1 and whp1 are not involved in a detected significant epistatic interaction, the variance within the different whp1 classes increases in a manner dependent on p1 genotype. Our inability to detect whp1 as a QTL in Population 2 may be a result of a limitation in power to detect QTL when highly influential loci are segregating. It was possible to detect the whp1 locus as a QTL in Population 3, in which segregation for functional and nonfunctional p1 alleles did not occur. Populations 2 and 3 differed only in the segregation of alleles at the p1 locus. The requirement of a functional p1 allele may be important to up-regulate other steps within the flavonoid biosynthetic pathway. The resulting increase in biochemical intermediates, accompanied by functional whp1 allele(s) acting independently of p1 regulation circumvents the absence of a functional c2 chalcone synthase.
Other QTL
qtl2
We detected a highly significant locus in the centromeric region on chromosome 2 that explained a large portion of the phenotypic variability for CGA. This is consistent with the QTL for CGA, qtl2, detected in the companion paper (Bushman et al., 2001). The Mp708 parent contributed the allele at this locus that increased CGA biosynthesis.
Chromosome 9 QTL
The QTL detected on chromosome 9 in Populations 1 and 2 maps to the same location as the recessive enhancer of maysin1 (rem1) locus described previously (Byrne et al., 1996b; Lee et al., 1998) but did not have similar gene action. The rem1 locus was shown to require a functional allele at p1 to exert an effect. No significant interaction with the p1 locus was detected. An additive effect was observed at this locus. This was different from the recessive gene action of rem1 for increased maysin production (Byrne et al., 1996b, 1998). Our current understanding of the alleles identified at the rem1 locus provides evidence against proposing rem1 as a candidate for this QTL. Different alleles that have not yet been identified at the rem1 locus could demonstrate additive gene action.
The Myb-domain transcription factor colored aleurone1 (c1) is also located on the short arm of chromosome 9 within the confidence interval of this QTL in Populations 1 and 2. In other populations in which interactions among loci without a main effect were not analyzed, the c1 region has not been identified as influencing flavone accumulation (McMullen et al., 2001).
Epistasis
Analysis of marker data by means of EPISTACY led to the discovery of several significant interactions between marker loci. Many of the detected interactions were among loci indicated as QTL with main effects and reflected interactions among flavonoid biosynthetic pathway loci. The p1 region was involved in many of the significant interactions. Though other significant interactions were detected between identified QTL, only the interaction between p1 and c2 remained significant in the MLM for maysin in Population 1.
Of particular interest in this study was the identification of significant interactions among loci not identified as QTL or having main effects. EPISTACY identified regions on the short arm of chromosome 9 (bnlg1724, bin 9.00) and the short arm of chromosome 2 (bnlg2277, bin 2.03) as having significant interactions with the whp1 locus. These regions of the genome contain the c1 and colored plant1 (b1) loci involved in regulation of the anthocyanin branch of flavonoid biosynthesis. Neither of these regions was identified as a single-factor QTL, but interactions between these loci and whp1 were included in the MLM.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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From this study, we also demonstrated that QTL with small effects may be masked by QTL explaining a large portion of the phenotypic variance. The method chosen to overcome this obstacle was to eliminate genetic variation at the most phenotypically significant QTL in one of the populations. This poses a dilemma because tailored construction of populations for individual QTL detection is not generally efficient or feasible for applied research. In QTL experiments, the establishment of stringent significance thresholds potentially over-reduces the Type I error rate at the expense of increasing Type II errors. In our genetic system, this has resulted in inconsistent detection of structural loci as QTL. If goals are to maximize short-term gains through identification and utilization of QTL with large effects, then high significance thresholds will suffice in most cases. The results of this study indicate that the effects of duplicate loci, in the context of potential differential regulation, need to be considered together to fully account for the genetic variation influencing a given trait.
In this study, we demonstrated a lack of functional CHS increases CGA accumulation, most likely through redirection of intermediates originally destined for flavone synthesis to the phenylpropaniod pathway. These genetic effects manifest significant epistatic interactions because expression of the variation in structural genes as QTL effects was conditional on the allelic state of regulatory genes controlling the pathways. The complex regulatory networks and variability present in transcription factors may, through epistatic interaction with single factor QTLs, play a major role in explaining the genotype-specific effects often encountered in manipulating QTLs in marker-assisted breeding projects.
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ACKNOWLEDGMENTS |
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Received for publication October 8, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This article has been cited by other articles:
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  B. S. Bushman, M. E. Snook, J. P. Gerke, S. J. Szalma, M. A. Berhow, K. E. Houchins, and M. D. McMullen Two Loci Exert Major Effects on Chlorogenic Acid Synthesis in Maize Silks Crop Sci., September 1, 2002; 42(5): 1669 - 1678. [Abstract] [Full Text] [PDF]
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