Genomics of maize resistance to kernel contamination with fumonisins using a multiparental advanced generation InterCross maize population (MAGIC).

Maize kernel is exposed to several fungal species, most notably Fusarium verticillioides, which can contaminate maize kernels with fumonisins. In an effort to increase genetic gains and avoid the laborious tasks of conventional breeding, the use of marker-assisted selection or genomic selection programs was proposed. To this end, in the present study a Genome Wide Association Study (GWAS) was performed on 339 RILs of a Multiparental Advanced Generation InterCross (MAGIC) population that had previously been used to locate Quantitative Trait Locus (QTL) for resistance to Fusarium Ear Rot (FER). Six QTLs for fumonisin content were detected in the bins 3.08, 4.07, 4.10, 7.03-7.04, 9.04-9.05 and 10.04-10.5. Five of the six QTLs collocate in regions where QTLs for FER were also found. However, the genetic variation for fumonisin content in kernel is conditioned by many other QTLs of small effect that could show QTL x environment interaction effects. Although a genomic selection approach to directly reduce fumonisin content in the kernel could be suitable, improving resistance to fumonisin content by genomic selection for FER would be more advisable.

Mapping of resistance to corn borers in a MAGIC population of maize.

BACKGROUND: Corn borers constitute an important pest of maize around the world; in particular Sesamia nonagrioides Lefèbvre, named Mediterranean corn borer (MCB), causes important losses in Southern Europe. Methods of selection can be combined with transgenic approaches to increase the efficiency and durability of the resistance to corn borers. Previous studies of the genetic factors involved in resistance to MCB have been carried out using bi-parental populations that have low resolution or using association inbred panels that have a low power to detect rare alleles. We developed a Multi-parent Advanced Generation InterCrosses (MAGIC) population to map with high resolution the genetic determinants of resistance to MCB. RESULTS: We detected multiple single nucleotide polymorphisms (SNPs) of low effect associated with resistance to stalk tunneling by MCB. We dissected a wide region related to stalk tunneling in multiple studies into three smaller regions (at ~ 150, ~ 155, and ~ 165 Mb in chromosome 6) that closely overlap with regions associated with cell wall composition. We also detected regions associated with kernel resistance and agronomic traits, although the co-localization of significant regions between traits was very low. This indicates that it is possible the concurrent improvement of resistance and agronomic traits. CONCLUSIONS: We developed a mapping population which allowed a finer dissection of the genetics of maize resistance to corn borers and a solid nomination of candidate genes based on functional information. The population, given its large variability, was also adequate to map multiple traits and study the relationship between them.

Fine analysis of a genomic region involved in resistance to Mediterranean corn borer.

BACKGROUND: Sesamia nonagrioides Lefebvere (Mediterranean corn borer, MCB) is the main pest of maize in the Mediterranean area. QTL for MCB stalk tunneling and grain yield under high MCB infestation had been located at bin 8.03-8.05 (4-21 cM and 10-30 cM respectively) in a previous analysis of the EP42 x EP39 RILs mapping population. The objective of the present work was to study with higher resolution those QTL, and validating and estimating with higher precision their locations and effects. To achieve this objective, we developed a set of 38 heterogeneous inbred families (HIFs) which were near-homozygous in the genome, except in the region under study. The HIFs were evaluated in multiple environments under artificial infestation with MCB and genotyped with SNPs. RESULTS: The QTL for grain yield under high infestation was confirmed with higher precision and improved reliability at 112.6-116.9 Mb. On the contrary, the location of the QTL for stalk tunneling was not validated probably due to the fixation of some genomic regions during the development of the HIFs. Our study confirmed that the co-localization of the QTL for stalk tunneling and grain yield in the previous study was due to linked genes, not to pleiotropic effects. So, the QTL for grain yield can be used for improving grain yield without undesirable effect on stalk tunneling. CONCLUSIONS: The HIF analysis is useful for validating QTL and for conducting deeper studies in traits related to corn borer resistance.

Defensive changes in maize leaves induced by feeding of Mediterranean corn borer larvae.

BACKGROUND: Plants can respond to insect attack via defense mechanisms that reduce insect performance. In this study, we examined the effects of several treatments applied to two maize genotypes (one resistant, one susceptible) on the subsequent growth and survival of Sesamia nonagrioides Lef. (Mediterranean corn borer, MCB) larvae. The treatments were infestation with MCB larvae, application of MCB regurgitant upon wounding, wounding alone, or exposure to methyl jasmonate, and they were applied at the V6-V8 stage of maize development. We also monitored changes in the concentrations of compounds known to be involved in constitutive resistance, such as cell wall-bound hydroxycinnamates and benzoxazinoids. RESULTS: In both maize genotypes, the leaves of plants pre-infested with MCB larvae were less suitable for larval development than those from untreated plants. Application of MCB regurgitant upon wounding, and wounding itself, resulted in leaf tissues becoming less suitable for larval growth than those of pre-infested plants, suggesting that there could be herbivore-associated effector molecules that suppress some wounding responses. A single application of MCB regurgitant did not seem to mimic feeding by MCB larvae, although the results suggested that regurgitant deposited during feeding may have enhanced ferulates and diferulates synthesis in infested vs. control plants. Jasmonic acid may play a role in mediating the maize response to MCB attack, but it did not trigger hydroxycinnamate accumulation in the leaves to a level comparable to that induced by larval leaf feeding. The EP39 maize genotype showed an increase in leaf cell wall strength by increasing hemicellulose cross-linking in response to MCB attack, while induced defenses in the EP42 plants appeared to reflect a broader array of resistance mechanisms. CONCLUSIONS: The results indicated that leaf feeding by MCB larvae can increase leaf antibiosis against MCB in two maize genotypes with contrasting levels of resistance against this borer. Also, the larval regurgitant played a positive role in eliciting a defense response. We determined the effects of the plant response on larval growth, and detected defense compounds related to borer resistance.

Identification of QTL for resistance to Mediterranean corn borer in a maize tropical line to improve temperate germplasm.

BACKGROUND: A QTL mapping study for maize resistance to the Mediterranean corn borer (MCB) was performed with a RIL population derived from the cross B73 × CML103. To develop commercial inbreds of maize resistant to the MCB for use in Europe, it would be useful to transfer resistance from tropical germplasm like the subtropical inbred CML103 to temperate lines. The inbred B73 was chosen as representative of the Stiff Stock heterotic group, a major heterotic group used in hybrid grown in both North American and Europe. The objectives were to study the architecture of genetic factors for resistance to MCB and to check the feasibility of using marker-assisted selection (MAS) for transferring those genetic factors. RESULTS: Eight quantitative trait loci (QTL) were declared significant for resistance traits and eight QTL were located for agronomic traits. Alleles from CML103 at QTL significant for tunnel length could reduce tunnel length made for MCB in inbred B73 in more than 8 cm; favorable alleles for yield were also found in CML103 and no genetic correlation coefficient between tunnel length and yield was detected. CONCLUSIONS: MAS for transferring resistance genes to corn borer attack from CML103 to B73 could be successful based on cross validation results and a negative effect on yield would not be expected.

Genome-wide association study reveals a set of genes associated with resistance to the Mediterranean corn borer (Sesamia nonagrioides L.) in a maize diversity panel.

BACKGROUND: Corn borers are the primary maize pest; their feeding on the pith results in stem damage and yield losses. In this study, we performed a genome-wide association study (GWAS) to identify SNPs associated with resistance to Mediterranean corn borer in a maize diversity panel using a set of more than 240,000 SNPs. RESULTS: Twenty five SNPs were significantly associated with three resistance traits: 10 were significantly associated with tunnel length, 4 with stem damage, and 11 with kernel resistance. Allelic variation at each significant SNP was associated with from 6 to 9% of the phenotypic variance. A set of genes containing or physically close to these SNPs are proposed as candidate genes for borer resistance, supported by their involvement in plant defense-related mechanisms in previously published evidence. The linkage disequilibrium decayed (r(2) < 0.10) rapidly within short distance, suggesting high resolution of GWAS associations. CONCLUSIONS: Most of the candidate genes found in this study are part of signaling pathways, others act as regulator of expression under biotic stress condition, and a few genes are encoding enzymes with antibiotic effect against insects such as the cystatin1 gene and the defensin proteins. These findings contribute to the understanding the complex relationship between plant-insect interactions.

[Plasmablastic lymphoma: a case of rectal disease with bone marrow involvement in a HIV positive patient]. / Linfoma plasmablástico: un caso con enfermedad rectal y compromiso de médula ósea en un paciente VIH positivo.

Plasmablastic lymphoma is an aggressive form of lymphoma diffuse large B cell Lymphoma, initially described in HIV positive patients associated with lesions in the oral cavity. It is about 2% of NHL associated with HIV. This entity currently represents a challenge for the diagnosis and treatment, showing a poor long-term prognosis. This report describes a patient with VIH on HAART and CD4 count in 490 cells/ml associated with Plasmablastic lymphoma that involves rectum and bone marrow. The patient received 6 cycles of EPOCH regimen with complete response.

Genetic and Phenotypic Evaluation of European Maize Landraces as a Tool for Conservation and Valorization of Agrobiodiversity.

The ECPGR European Evaluation Network (EVA) for Maize involves genebanks, research institutions, and private breeding companies from nine countries focusing on the valorization of maize genetic resources across Europe. This study describes a diverse collection of 626 local landraces and traditional varieties of maize (Zea mays L.) from nine European genebanks, including criteria for selection of the collection and its genetic and phenotypic diversity. High-throughput pool genotyping grouped the landraces into nine genetic groups with a threshold of 0.6 admixture, while 277 accessions were designated admixed and likely to have resulted from previous breeding activities. The grouping correlated well with the geographic origins of the collection, also reflecting the various pathways of introduction of maize to Europe. Phenotypic evaluations of 588 accessions for flowering time and plant architecture in multilocation trials over three years confirmed the great diversity within the collection, although phenotypic clusters only partially correlated with the genetic grouping. The EVA approach promotes conservation of genetic resources and opens an opportunity to increase genetic variability for developing improved varieties and populations for farmers, with better adaptation to specific environments and greater tolerance to various stresses. As such, the EVA maize collection provides valuable sources of diversity for facing climate change due to the varieties' local adaptation.

Genetic regulation of cold-induced albinism in the maize inbred line A661.

In spite of multiple studies elucidating the regulatory pathways controlling chlorophyll biosynthesis and photosynthetic activity, little is known about the molecular mechanism regulating cold-induced chlorosis in higher plants. Herein the characterization of the maize inbred line A661 which shows a cold-induced albino phenotype is reported. The data show that exposure of seedlings to low temperatures during early leaf biogenesis led to chlorophyll losses in this inbred. A661 shows a high plasticity, recovering resting levels of photosynthesis activity when exposed to optimal temperatures. Biochemical and transcriptome data indicate that at suboptimal temperatures chlorophyll could not be fully accommodated in the photosynthetic antenna in A661, remaining free in the chloroplast. The accumulation of free chlorophyll activates the expression of an early light inducible protein (elip) gene which binds chlorophyll to avoid cross-reactions that could lead to the generation of harmful reactive oxygen species. Higher levels of the elip transcript were observed in plants showing a cold-induced albino phenotype. Forward genetic analysis reveals that a gene located on the short arm of chromosome 2 regulates this protective mechanism.

Is it possible to control fumonisin contamination in maize kernels by using genotypes resistant to the Mediterranean corn borer?

Insect activity has long been associated with Fusarium infection. The objectives of the current study were 1) to estimate the impact of Mediterranean corn borer, Sesamia nonagrioides Lefèbvre, damage on fumonisin contamination in the maize kernel by comparing fumonisin contamination under infestation and protected conditions, and 2) to measure the potential use of genotypes resistant to this borer as controlling factors of fumonisin contamination. Genotypes with increased kernel damage by borers tended to increase fumonisin accumulation under infestation conditions. In particular environments, other factors influenced fumonisin contamination more than damage by borers. When ear damage by borers is significant, maize resistance to ear damage could contribute to the reduction of fumonisin contamination in the kernels. Genotype such as EP42 x EP77 that combines low ear damage by borers and low fumonisin level across environments is a good choice to control fumonisin contamination. The use of an applicable methodology to identify Mediterranean corn borer-resistant genotypes to ear attack under artificial infestations might be a promising approach.

Maize kernel metabolome involved in resistance to fusarium ear rot and fumonisin contamination.

Fusarium verticillioides poses a threat to worldwide maize production due to its ability to infect maize kernel and synthesize fumonisins that can be accumulated above safety levels for humans and animals. Maize breeding has been proposed as key tool to decrease kernel contamination with fumonisins, but metabolic studies complementary to genomic approaches are necessary to disclose the complexity of maize resistance. An untargeted metabolomic study was proposed using inbreds genetically related but with contrasting levels of resistance in order to uncover pathways implicated in resistance to Fusarium ear rot (FER) and fumonisin contamination in the maize kernel and to look for possible biomarkers. Metabolite determinations were performed in kernels collected at 3 and 10 days after inoculation with F. verticillioides (dat). Discriminant metabolites between resistant and susceptible RILs were rather found at 10 than 3 dat, although metabolite differences at later stages of colonization could be driven by subtle variations at earlier stages of infection. Within this context, differences for membrane lipid homeostasis, methionine metabolism, and indolacetic acid conjugation seemed highly relevant to distinguish between resistant and susceptible inbreds, confirming the polygenic nature of resistance to FER and fumonisin contamination in the maize kernels. Nevertheless, some specific metabolites such as the polyamine spermidine and/or the alkaloid isoquinoline seemed to be promising indirect selection traits to improve resistance to FER and reduce fumonisin accumulation. Therefore, in vitro and in vivo experiments will be necessary to validate the inhibitory effects of these compounds on fumonisins biosynthesis.

Inbred Selection for Increased Resistance to Kernel Contamination with Fumonisins.

In temperate world-wide regions, maize kernels are often infected with the fumonisin-producing fungus Fusarium verticillioides which poses food and feed threats to animals and humans. As maize breeding has been revealed as one of the main tools with which to reduce kernel contamination with fumonisins, a pedigree selection program for increased resistance to Fusarium ear rot (FER), a trait highly correlated with kernel fumonisin content, was initiated in 2014 with the aim of obtaining inbred lines (named EPFUM) with resistance to kernel contamination with fumonisins and adapted to our environmental conditions. The new released EPFUM inbreds, their parental inbreds, hybrids involving crosses of one or two EPFUM inbreds, as well as commercial hybrids were evaluated in the current study. The objectives were (i) to assess if inbreds released by that breeding program were significantly more resistant than their parental inbreds and (ii) to examine if hybrids derived from EPFUM inbreds could be competitive based on grain yield and resistance to FER and fumonisin contamination. Second-cycle inbreds obtained through this pedigree selection program did not significantly improve the levels of resistance to fumonisin contamination of their parental inbreds; however, most EPFUM hybrids showed significantly better resistance to FER and fumonisin contamination than commercial hybrids did. Although European flint materials seem to be the most promising reservoirs of alleles with favorable additive and/or dominance effects for resistance to kernel contamination with fumonisins, marketable new Reid × Lancaster hybrids have been detected as they combine high resistance and yields comparable to those exhibited by commercial hybrids. Moreover, the white kernel hybrid EPFUM-4 × EP116 exploits the genetic variability within the European flint germplasm and can be an alternative to dent hybrid cultivation because white flint grain can lead to higher market prices.

Genomic versus phenotypic selection to improve corn borer resistance and grain yield in maize.

Introduction: The study of yield and resistance/tolerance to pest are related traits fundamental for maize breeding programs. Genomic selection (GS), which uses all marker information to calculate genomic breeding values, is presented as an emerging alternative to phenotypic and marker-assisted selections for improving complex traits controlled by many genes with small effects. Therefore, although phenotypic selection (PS) has been effective for increasing resistance and yield under high infestation with maize stem borers, higher genetic gains are expected to be obtained through GS based on the complex architecture of both traits. Our objective was to test whether GS is more effective than PS for improving resistance and/or tolerance to maize stem borers and grain yield. Methods: For this, we compared different selection programs based on phenotype and genotypic value for a single trait, resistance or yield, and for both traits together. Results and discussion: We obtained that GS achieved the highest genetic gain for yield, meanwhile phenotypic selection for yield was the program that achieved the highest reduction of tunnel length, but was ineffective for increasing yield. However, phenotypic or genomic selection for increased resistance may be more effective in improving both traits together; although the gains per cycle would be small for both traits.

Inducible maize defense mechanisms against the corn borer Sesamia nonagrioides: a transcriptome and biochemical approach.

In spite of multiple studies elucidating individual defense mechanisms against stalk borer feeding, little information is available about the plant response to these members of Lepidoptera. Four maize inbred lines were cultivated in a greenhouse and challenged with larvae of the corn borer Sesamia nonagrioides. Transcriptome and biochemical analyses were performed to elucidate the maize response mechanisms to this insect. General plant defense mechanisms were activated, including the jasmonic acid biosynthetic pathway, proteinase inhibitors, and four defense-related transcription factors. Interestingly, gene ontology analysis shows that maize plants undergo cell-wall reorganization after being attacked. These results were confirmed through biochemical analyses showing that the concentration of some cell-wall-related compounds significantly changed after plant infestation in a genotype-dependent way. In conclusion, maize plants respond to the attack of the corn borer S. nonagrioides through cell-wall fortification, activating genes involved in cell-wall organization, which finally is reflected in a higher concentration of some cell-wall components, especially in resistant genotypes.

Genomics and Pathways Involved in Maize Resistance to Fusarium Ear Rot and Kernel Contamination With Fumonisins.

Fusarium verticillioides is a causal agent of maize ear rot and produces fumonisins, which are mycotoxins that are toxic to animals and humans. In this study, quantitative trait loci (QTLs) and bulk-segregant RNA-seq approaches were used to uncover genomic regions and pathways involved in resistance to Fusarium ear rot (FER) and to fumonisin accumulation in maize kernels. Genomic regions at bins 4.07-4.1, 6-6.01, 6.04-6.05, and 8.05-8.08 were related to FER resistance and/or reduced fumonisin levels in kernels. A comparison of transcriptomes between resistant and susceptible inbred bulks 10 days after inoculation with F. verticillioides revealed 364 differentially expressed genes (DEGs). In the resistant inbred bulks, genes involved in sink metabolic processes such as fatty acid and starch biosynthesis were downregulated, as well as those involved in phytosulfokine signaling and many other genes involved in cell division; while genes involved in secondary metabolism and compounds/processes related to resistance were upregulated, especially those related to cell wall biosynthesis/rearrangement and flavonoid biosynthesis. These trends are indicative of a growth-defense trade-off. Among the DEGs, Zm00001d053603, Zm00001d035562, Zm00001d037810, Zm00001d037921, and Zm00001d010840 were polymorphic between resistant and susceptible bulks, were located in the confidence intervals of detected QTLs, and showed large differences in transcript levels between the resistant and susceptible bulks. Thus, they were identified as candidate genes involved in resistance to FER and/or reduced fumonisin accumulation.

Role of hydroxycinnamic acids in the infection of maize silks by Fusarium graminearum Schwabe.

In the current study, the hydroxycinnamic acids in silks of diverse maize inbred lines differing in Fusarium resistance were determined at several times after inoculation with Fusarium graminearum or sterile water as control. The main objective was to determine the possible relationship between the hydroxycinnamic acid changes in silks and ear rot resistance. Several changes in the cell-wall-bound hydroxycinnamic acid concentrations were observed after inoculation with F. graminearum, although these changes were not directly correlated with genotypic resistance to this fungus. Ester-bound ferulic acid decreased, probably due to degradation of hemicellulose by hydrolytic enzymes produced by Fusarium spp., while p-coumaric acid and diferulates showed slight increases that, in conjunction, did not result in delayed F. graminearum progression through the silks. It is important to note that the decrease of ferulic acid in the F. graminearum treatment was faster in susceptible than in resistant genotypes, suggesting a differential hemicellulose degradation in silk tissues. Therefore, the ability of the maize genotypes to slow down that process through hemicellulose structural features or xylanase inhibitors needs to be addressed in future studies.

Is the basal area of maize internodes involved in borer resistance?

BACKGROUND: To elucidate the role of the length of the internode basal ring (LIBR) in resistance to the Mediterranean corn borer (MCB), we carried out a divergent selection program to modify the LIBR using two maize synthetic varieties (EPS20 and EPS21), each with a different genetic background. We investigated the biochemical mechanisms underlying the relationship between the LIBR and borer resistance. Selection to lengthen or shorten the LIBR was achieved for each synthetic variety. The resulting plants were analyzed to determine their LIBR response, growth, yield, and borer resistance. RESULTS: In the synthetic variety EPS20 (Reid germplasm), reduction of the LIBR improved resistance against the MCB. The LIBR selection was also effective in the synthetic variety EPS21 (non-Reid germplasm), although there was no relationship detected between the LIBR and MCB resistance. The LIBR did not show correlations with agronomic traits such as plant height and yield. Compared with upper sections, the internode basal ring area contained lower concentrations of cell wall components such as acid detergent fiber (ADF), acid detergent lignin (ADL), and diferulates. In addition, some residual 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3-(4H)-one (DIMBOA), a natural antibiotic compound, was detected in the basal area at 30 days after silking. CONCLUSION: We analyzed maize selections to determine whether the basal area of maize internodes is involved in borer resistance. The structural reinforcement of the cell walls was the most significant trait in the relationship between the LIBR and borer resistance. Lower contents of ADF and ADL in the rind of the basal section facilitated the entry of larvae in this area in both synthetic varieties, while lower concentrations of diferulates in the pith basal section of EPS20 facilitated larval feeding inside the stem. The higher concentrations of DIMBOA may have contributed to the lack of correlation between the LIBR and borer resistance in EPS21. This novel trait could be useful in maize breeding programs to improve borer resistance.

Unraveling the role of maize (Zea mays L.) cell-wall phenylpropanoids in stem-borer resistance.

The cell wall putatively plays a role in host-plant resistance to phytopathogens. Here, we investigated which cell wall-bound phenolic compounds have determining roles in maize (Zea mays) resistance to attack by the Mediterranean corn borer Sesamia nonagrioides (Lefèbvre). Diverse sets of maize genotypes having contrasting hydroxycinnamate contents and borer resistance levels were evaluated. The interdependent relationships among some cell wall-bound phenolic compounds, such as ferulic acid and its dimers, or p-coumaric acid and syringyl lignin subunits, were analyzed. Both p-coumaric acid and syringyl momoners showed significant negative correlations with damage, as assessed by tunnel lengths, caused by S. nonagrioides larvae. Thus, the use of cell wall-bound p-coumaric acid in pest-resistant crop breeding programs is advisable.

QTL mapping for Mediterranean corn borer resistance in European flint germplasm using recombinant inbred lines.

BACKGROUND: Ostrinia nubilalis (ECB) and Sesamia nonagrioides (MCB) are two maize stem borers which cause important losses in temperate maize production, but QTL analyses for corn borer resistance were mostly restricted to ECB resistance and maize materials genetically related (mapping populations derived from B73). Therefore, the objective of this work was to identify and characterize QTLs for MCB resistance and agronomic traits in a RILs population derived from European flint inbreds. RESULTS: Three QTLs were detected for stalk tunnel length at bins 1.02, 3.05 and 8.05 which explained 7.5% of the RILs genotypic variance. The QTL at bin 3.05 was co-located to a QTL related to plant height and grain humidity and the QTL at bin 8.05 was located near a QTL related to yield. CONCLUSIONS: Our results, when compared with results from other authors, suggest the presence of genes involved in cell wall biosynthesis or fortification with effects on resistance to different corn borer species and digestibility for dairy cattle. Particularly, we proposed five candidate genes related to cell wall characteristics which could explain the QTL for stalk tunnelling in the region 3.05. However, the small proportion of genotypic variance explained by the QTLs suggest that there are also many other genes of small effect regulating MCB resistance and we conclude that MAS seems not promising for this trait. Two QTLs detected for stalk tunnelling overlap with QTLs for agronomic traits, indicating the presence of pleitropism or linkage between genes affecting resistance and agronomic traits.

Genetic variation at bx1 controls DIMBOA content in maize.

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.