Significant differences were observed between the adjusted means of distinct inbred lines, indicating the existance of additive genetic variability for fumonisin contamination resistance in the Embrapa’s tropical maize panel. The heritability estimated in this study was moderate and similar to the one reported by Samayoa et al. (2019) using 270 maize inbred lines (h2 = 0.46). Other studies have reported correlations between Fusarium ear rot resistance and fumonisin contamination ranging from 0.87 to 0.92 (Bolduan et al. 2009, Robertson-Hoyt et al. 2006), suggesting that indirect selection could be an alternative to reduce the fumonisin contamination in maize grains. However, Eller et al. (2008) showed that the selection for Fusarium ear rot resistance is not always successful to reduce fumonisin contamination, requiring more QTL studies to better understand its genetic basis.
It was not possible to conduct out the study of Genotype x Environment (GxE). Giomi et al. (2016)
looked at Genotype x Year (G xY) for maize inbreds Fusarium ear rot in two years trials and found G x Y to be small. The GxY variance components were minor compared to those of principal effects (results not shown) indicating that the ranking of genotypes for disease, severity tended to be stable across years and fungal species. Because of that, the three-field trials conducted were considered similar in the Giomi et al. (Giomi et al. 2016) study. According to Samayoa et al. (2015) in study genome-wide association analysis for fumonisin content in maize kernels the phenotypic mean across environments would finely correspond to genotype performance because genotype x environment significant effects have been rather attributed to the heterogeneity of genotypic variances than to the lack of correlation of genotype performance in different environments. Hence, in a condition such as that, as our phenotypic trait was assessed with reasonable precisions based on our heritability estimates, we do not expect dramatic impacts of additional trials, particularly for QTL.
Linkage disequilibrium (LD) measures are usually specific to the target germplasm panel, varying considerably between distinct genomic regions (Romay et al. 2013) Thus, different values of linkage disequilibrium (LD) decay were reported by Romay et al. (2013) ( r2 = 0.1, 1 kb), Zila et al. (2013) ( r2 = 0.2, 10 kb), and Zila et al. (2014) (r2 = 0.2, 1 kb) in maize. In the present study, the average LD over chromosomes dropped below r2 = 0.1 within approximately 10 kb (Fig. A.1). Lu et al. (2010) have also reported an average LD extension of 10 kb, using haplotype-based analysis of 2,052 SNP markers and 305 inbred lines and obtained 28,791 high-quality SNP markers among the 45,868 SNPs. Similar results of the LD were observed by Wang at al. (2017). Using 45,868 SNPs with an average LD decay of r2 = 0.1, the GWAS of head smut resistance in a panel of 144 inbred lines allowed identified eighteen candidate genes. In relation to other studies, we achieved greater coverage of the genome with 385,654 high-quality SNPs, on average, 38,706 SNPs were obtained per chromosome, ranging from 59,939 for chromosome 1 to 27,910 SNPs for chromosome 10. This measure of LD decay was valuable to identify candidate genes associated to the QTL detected for the fumonisin contamination resistance in the Embrapa’s tropical maize panel.
Nine high-resolution QTLs were significantly associated with fumonisin contamination resistance. Genes located within the genomic interval of 10 kb were considered in LD with the detected QTLs. These candidate genes were classified according to the MaizeGDB genome browser. The QTLs in bins 2.05, 2.08, 3.06, 5.01 and 10.03 presented seven genes (Fig. 3, Table 2), were also associated to fumonisin contamination in other studies (Zila et al. 2014, Zila et al. 2013, Maschietto et al. 2017, Coan et al. 2018, Samayoa et al. 2015) (Online Resource 5). Unveiled four genes in the regiões 2.05, 4,05, and 5.01 (Table 2) i.e. have not been previously described as related to fumonisins contamination resistance (Fig. 3). Some of these candidate genes colocalized with QTLs shown in Table 2, like GRMZM2G013200 (Bin 4.01), GRMZM2G051270 (Bin 5.05), and GRMZM2G083347 (Bin 10.03), were simultaneously detected in the Gene Ontology analysis with annotated functions to resistance to pathogens (Online Resource 5).
There are different immune strategies for defense against pathogens in plants (Wit 2007). Pathogen-associated molecular patterns (PAMPs), for example, are patterns recognized by receptors (pattern-recognition receptors - PRRs), which induce the immune response (pattern triggered immunity - PTI). PRRs can be categorized as receptor kinases localized on plasma membrane (RKs) or receptor-like proteins (RLPs) (Boutrot and Zipfel, 2017; Zhang et al. 2017), which reinforces the host defenses. The effector-triggered immunity (ETI), mediated by resistance proteins (RPs), is a secondary immune response that when activated allows the plant to stop the pathogen development. During the induction of local immune responses, a systemic acquired resistance (SAR) can become activated. The maize, for example, when infected by Fusarium verticillioides, expresses a set of defense genes (Lanubile et al. 2014; Wang et al. 2016). This response seems to play a primary role in the resistance of maize to Fusarium verticillioides, where salicylic acid and jasmonic acid signaling pathways can be involved (Wang et al. 2016). Hence, genes directly involving in the immune response in plants are more suitable as candidate genes for the associations found for fumonisin resistance.
GRMZM2G060216 (176 Mbp, bin 3.06) and GRMZM2G083347 (14 Mbp, bin 10.03) genes were described as involved in response to jasmonic acid (JA) (Online Resource 3). The signaling pathway of JA promotes downstream activation of defense genes responsive at PR (pathogenesis-related) proteins, such as chitinases (Lanubile et al. 2012). Hormonal signalizing via salicylic acid, auxin, abscisic acid, ethylene, and by own jasmonic acid, are orchestrated until they reach the nucleus (Berens et al. 2017; Lanubile et al. 2014; Wang et al. 2016). Besides this, the GRMZM2G060216 gene refers to the transcription factor LG2, which is related to systemic acquired resistance by the salicylic acid-mediated signaling pathway (Chen et al. 2012). Galić et al. (2019) in a study to assess the factors affecting Fusarium ear rot and fumonisin contamination of maize, identified genes that can confer resistance, and found genes that code for NAC transcription factor.
GRMZM2G036708 gene was described as a cysteine synthase (bin 2.05, 107 Mbp). The largest class of resistance proteins involved in ETI response consists of nucleotide-binding-leucine rich repeat (NB-LRR) proteins (Samayoa et al. 2019). Ormancey et al. (2018) demonstrated that the protein acts as a negative regulator of fumonisin B1 induced cell death in Arabidopsis. The cysteine synthase that like other several PRRs, has leucine-rich receptor-like kinases, that were also identified in studies of associated with Fusarium ear rots are one of the greatest challenges for maize consumption chain. So resistance to contamination by fumonisins is a major challenge maize (Lanubile et al. 2014; Wang et al. 2016).
The gene GRMZM2G154156 (107 Mbp, bin 2.05) was described as a protein from the Ubiquitin ligase complex. The regulatory process in ubiquitylation specifically resides in the E3 ligase and the cognate substrate. A number of abiotic stresses are mediated by protein ubiquitylation processes processes (Haak et al. 2017). Members from this family are involved in the regulation of some biological processes, including vegetative growth, plant reproduction, biotic and abiotic stresses tolerance. Furthermore, the ubiquitin ligases ring domain ligase 3 (gene RGLG3) and gene RGLG4 coordinately and positively regulate fumonisin B1 triggered programmed cell death by modulating the Jasmonic acid signaling pathway in a coronatine insensitive 1 (COI1)- and the gene MYC2-dependent manner in Arabidopsis (Zhang et al. 2015).
Coan et al. (2018) also reported SNP significantly associated with Fusarium ear rot in bin 10.03, in SNP physical position 234 Mbp. Wisser et al. (2006) found bin 10.03 to contain a large QTL conditioning resistance to several maize diseases. Therefore is important for resistance since common rust resistance genes rp1 and rp5 were found in this bin (Chen et al. 2016, Coan et al. 2018). Several SNPs associated with the candidate genes presented protein domains that have high similarity to the pathogenesis-related proteins and were reported to improve disease resistance.
The gene GRMZM2G022213 (208 Mbp, bin 2.08 - Table 2) annotated as zinc finger protein MAGPIE, regulates tissue boundaries cell division and asymmetric cell division (Welch et al. 2007). GRMZM2G051270 gene located in the bin 5.05, 7 Mbp, correspond to a sulfate adenylyltransferase cysteine (Table 2). ATP-S could be involved in plant-tolerance to several abiotic stresses via different S-compounds pathogen responses (Álvarez et al. 2012). S-containing compounds is directly or indirectly modulated/regulated by ATP-S and are involved in plant tolerance to both biotic and abiotic stresses (Anjum et al. 2015). There is a high correlation between fumonisin contamination, linoleic acid content and masking action in maize hybrids with higher oleic to linoleic ratio (Dall’Asta et al. 2012). This masking phenomenon consists of the formation of covalent bonds between the tricarballylic groups of fumonisins and sulfhydryl groups of the side chains of amino acids in proteins. The gene GRMZM2G051270 present the sulfate groups and might be related to the increase of fatty acid composition on fumonisin contamination and the occurrence of hidden fumonisins in maize.
The SNPs linked to candidate genes significantly associated with fumonisin resistance could be used as molecular markers to select resistent genotypes and decrease mycotoxin contamination. The unknown genes or not directly involved genes have the potential to be investigated, since they might be involved in resistance, as biochemical and genetic pathways leading to resistance to fumonisin are complex and, for the most part, unknown (Zila et al. 2014). Thus, the QTLs significative associated with fumonisin resistance in this study are promising to be useful for whole-genome selection in tropical maize.
The Lines (410399_19_1, 371056_1, 211_0587_5, 552697_F, 2841, L724, L_228_3x45611_x228_3__2_4_x228_3__1_1, 3821095_5) had a higher frequency of favorable alleles for the resistance to fumonisin concentration (70% RR - alleles that increase the resistance) and on average provided the best resistance (Online Resource 4), also suggesting a predominance of the dominance effects in the resistance to fumonisin contamination in tropical maize.
The complex nature of resistance challenged maize breeders to effectively incorporate novel resistance alleles into adapted breeding pools; as a result, most commercial maize hybrids have lower levels of resistance than desired (Bush et al. 2004). Therefore, inbred lines that presented a higher frequency of favorable alleles and lower fumonisin content could be used in future crosses for the generation of resistant hybrids, supporting advances in plant breeding.