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.

Effectiveness of introgression of resistance loci for Gibberella ear rot from two European flint landraces into adapted elite maize (Zea mays L.).

European flint landraces are a major class of maize possessing favorable alleles for improving host resistance to Gibberella ear rot (GER) disease which reduces yield and contaminates the grains with mycotoxins. However, the incorporation of these landraces into breeding programs requires a clear understanding of the effectiveness of their introgression into elite materials. We evaluated 15 pre-selected doubled haploid (DH) lines from two European flint landraces, "Kemater Landmais Gelb" (KE) and "Petkuser Ferdinand Rot" (PE), together with two adapted elite flint lines and seven standard lines for GER severity as the main trait, and several adaptation traits (plant height, days to silking, seed-set, plant vigor) across four environments. From this evaluation, three KE DH lines and one PE DH line, with the lowest GER severity, were selected and used as donor parents that were crossed with the two adapted and GER susceptible flint lines (Flint1 and Flint2) to develop six bi-parental DH populations with 34-145 DH lines each. Each DH population was evaluated across two locations. Correlations between GER severity, which was the target trait, and adaptation traits were weak (-0.02 to 0.19). GER severity of lines from PE landrace was on average 2-fold higher than lines from KE landrace, indicating a clear superiority of the KE landrace lines. Mean GER severity of the DH populations was 39.4-61.0% lower than the adapted elite flint lines. All KE-derived DH populations were on average more resistant (27.0-36.7%) than the PE-derived population (51.0%). Highly resistant lines (1.3-5.2%) were found in all of the populations, suggesting that the DH populations can be successfully integrated into elite breeding programs. The findings demonstrate that selected KE landrace lines used as donors were effective in improving GER resistance of the adapted elite inbreds.

Alleles of a wall-associated kinase gene account for three of the major northern corn leaf blight resistance loci in maize.

Northern corn leaf blight, caused by the fungal pathogen Setosphaeria turcica (anamorph Exserohilum turcicum), is one of the most devastating foliar diseases of maize (Zea mays). Four genes Ht1, Ht2, Ht3 and Htn1 represent the major sources of genetic resistance against the hemibiotrophic fungus S. turcica. Differential maize lines containing these genes also form the basis to classify S. turcica races. Here, we show that Ht2 and Ht3 are identical and allelic to the previously cloned Htn1 gene. Using a map-based cloning approach and Targeting Induced Local Lesions in Genomes (TILLING), we demonstrate that Ht2/Ht3 is an allele of the wall-associated receptor-like kinase gene ZmWAK-RLK1. The ZmWAK-RLK1 variants encoded by Htn1 and Ht2/Ht3 differ by multiple amino acid polymorphisms that particularly affect the putative extracellular domain. A diversity analysis in maize revealed the presence of dozens of ZmWAK-RLK1 alleles. Ht2, Ht3 and Htn1 have been described over decades as independent resistance loci with different race spectra and resistance responses. Our work demonstrates that these three genes are allelic, which has major implications for northern corn leaf blight resistance breeding and nomenclature of S. turcica pathotypes. We hypothesize that genetic background effects have confounded the classical description of these disease resistance genes in the past.

Exploiting genetic diversity in two European maize landraces for improving Gibberella ear rot resistance using genomic tools.

KEY MESSAGE: High genetic variation in two European maize landraces can be harnessed to improve Gibberella ear rot resistance by integrated genomic tools. Fusarium graminearum (Fg) causes Gibberella ear rot (GER) in maize leading to yield reduction and contamination of grains with several mycotoxins. This study aimed to elucidate the molecular basis of GER resistance among 500 doubled haploid lines derived from two European maize landraces, "Kemater Landmais Gelb" (KE) and "Petkuser Ferdinand Rot" (PE). The two landraces were analyzed individually using genome-wide association studies and genomic selection (GS). The lines were genotyped with a 600-k maize array and phenotyped for GER severity, days to silking, plant height, and seed-set in four environments using artificial infection with a highly aggressive Fg isolate. High genotypic variances and broad-sense heritabilities were found for all traits. Genotype-environment interaction was important throughout. The phenotypic (r) and genotypic ([Formula: see text]) correlations between GER severity and three agronomic traits were low (r = - 0.27 to 0.20; [Formula: see text]= - 0.32 to 0.22). For GER severity, eight QTLs were detected in KE jointly explaining 34% of the genetic variance. In PE, no significant QTLs for GER severity were detected. No common QTLs were found between GER severity and the three agronomic traits. The mean prediction accuracies ([Formula: see text]) of weighted GS (wRR-BLUP) were higher than [Formula: see text] of marker-assisted selection (MAS) and unweighted GS (RR-BLUP) for GER severity. Using KE as the training set and PE as the validation set resulted in very low [Formula: see text] that could be improved by using fixed marker effects in the GS model.

Intercontinental trials reveal stable QTL for Northern corn leaf blight resistance in Europe and in Brazil.

KEY MESSAGE: NCLB is the most devastating leaf disease in European maize, and the introduction of Brazilian resistance donors can efficiently increase the resistance levels of European maize germplasm. Northern corn leaf blight (NCLB) is one of the most devastating leaf pathogens in maize (Zea mays L.). Maize cultivars need to be equipped with broad and stable NCLB resistance to cope with production intensification and climate change. Brazilian germplasm is a great source to increase low NCLB resistance levels in European materials, but little is known about their effect in European environments. To investigate the usefulness of Brazilian germplasm as NCLB resistance donors, we conducted multi-parent QTL mapping, evaluated the potential of marker-assisted selection as well as genome-wide selection of 742 F1-derived DH lines. The line per se performance was evaluated in one location in Brazil and six location-by-year combinations (= environments) in Europe, while testcrosses were assessed in two locations in Brazil and further 10 environments in Europe. Jointly, we identified 17 QTL for NCLB resistance explaining 3.57-30.98% of the genotypic variance each. Two of these QTL were detected in both Brazilian and European environments indicating the stability of these QTL in contrasting ecosystems. We observed moderate to high genomic prediction accuracies between 0.58 and 0.83 depending on population and continent. Collectively, our study illustrates the potential use of tropical resistance sources to increase NCLB resistance level in applied European maize breeding programs.

Fungal resistance mediated by maize wall-associated kinase ZmWAK-RLK1 correlates with reduced benzoxazinoid content.

Wall-associated kinases (WAKs) have recently been identified as major components of fungal and bacterial disease resistance in several cereal crop species. However, the molecular mechanisms of WAK-mediated resistance remain largely unknown. Here, we investigated the function of the maize gene ZmWAK-RLK1 (Htn1) that confers quantitative resistance to northern corn leaf blight (NCLB) caused by the hemibiotrophic fungal pathogen Exserohilum turcicum. ZmWAK-RLK1 was found to localize to the plasma membrane and its presence resulted in a modification of the infection process by reducing pathogen penetration into host tissues. A large-scale transcriptome analysis of near-isogenic lines (NILs) differing for ZmWAK-RLK1 revealed that several differentially expressed genes are involved in the biosynthesis of the secondary metabolites benzoxazinoids (BXs). The contents of several BXs including DIM2 BOA-Glc were significantly lower when ZmWAK-RLK1 is present. DIM2 BOA-Glc concentration was significantly elevated in ZmWAK-RLK1 mutants with compromised NCLB resistance. Maize mutants that were affected in overall BXs biosynthesis or content of DIM2 BOA-Glc showed increased NCLB resistance. We conclude that Htn1-mediated NCLB resistance is associated with a reduction of BX secondary metabolites. These findings suggest a link between WAK-mediated quantitative disease resistance and changes in biochemical fluxes starting with indole-3-glycerol phosphate.

Low validation rate of quantitative trait loci for Gibberella ear rot resistance in European maize.

KEY MESSAGE: Six quantitative trait loci (QTL) for Gibberella ear rot resistance in maize were tested in two different genetic backgrounds; three QTL displayed an effect in few near isogenic line pairs. Few quantitative trait loci (QTL) mapping studies for Gibberella ear rot (GER) have been conducted, but no QTL have been verified so far. QTL validation is prudent before their implementation into marker-assisted selection (MAS) programs. Our objectives were to (1) validate six QTL for GER resistance, (2) evaluate the QTL across two genetic backgrounds, (3) investigate the genetic background outside the targeted introgressions. Pairs of near isogenic lines (NILs) segregating for a single QTL (Qger1, Qger2, Qger10, Qger13, Qger16, or Qger21) were developed by recurrent backcross until generation BC3S2. Donor parents (DP) carrying QTL were backcrossed to a susceptible (UH009) and a moderately resistant (UH007) recurrent parent. MAS was performed using five SNP markers covering a region of 40 cM around each QTL. All NILs were genotyped with the MaizeSNP50 assay and phenotyped for GER severity and deoxynivalenol and zearalenone content. Traits were significantly (P < 0.001) intercorrelated. Out of 34 NIL pairs with the UH009 genetic background, three pairs showed significant differences in at least one trait for three QTL (Qger1, Qger2, Qger13). Out of 25 NIL pairs with the UH007 genetic background, five pairs showed significant differences in at least one trait for two QTL (Qger2, Qger21). However, Qger16, Qger10 and Qger13 were most likely false positives. The genetic background possibly affected NIL pairs comparisons due to linkage drag and/or epistasis with residual loci from the DP in non-target regions. In conclusion, validation rates were disappointingly low, which further indicates that GER resistance is controlled by many low-effect QTL.

The maize disease resistance gene Htn1 against northern corn leaf blight encodes a wall-associated receptor-like kinase.

Northern corn leaf blight (NCLB) caused by the hemibiotrophic fungus Exserohilum turcicum is an important foliar disease of maize that is mainly controlled by growing resistant maize cultivars. The Htn1 locus confers quantitative and partial NCLB resistance by delaying the onset of lesion formation. Htn1 represents an important source of genetic resistance that was originally introduced from a Mexican landrace into modern maize breeding lines in the 1970s. Using a high-resolution map-based cloning approach, we delimited Htn1 to a 131.7-kb physical interval on chromosome 8 that contained three candidate genes encoding two wall-associated receptor-like kinases (ZmWAK-RLK1 and ZmWAK-RLK2) and one wall-associated receptor-like protein (ZmWAK-RLP1). TILLING (targeting induced local lesions in genomes) mutants in ZmWAK-RLK1 were more susceptible to NCLB than wild-type plants, both in greenhouse experiments and in the field. ZmWAK-RLK1 contains a nonarginine-aspartate (non-RD) kinase domain, typically found in plant innate immune receptors. Sequence comparison showed that the extracellular domain of ZmWAK-RLK1 is highly diverse between different maize genotypes. Furthermore, an alternative splice variant resulting in a truncated protein was present at higher frequency in the susceptible parents of the mapping populations compared with in the resistant parents. Hence, the quantitative Htn1 disease resistance in maize is encoded by an unusual innate immune receptor with an extracellular wall-associated kinase domain. These results further highlight the importance of this protein family in resistance to adapted pathogens.

Improving resistance to the European corn borer: a comprehensive study in elite maize using QTL mapping and genome-wide prediction.

KEY MESSAGE: The efficiency of marker-assisted selection for native resistance to European corn borer stalk damage can be increased when progressing from a QTL-based towards a genome-wide approach. Marker-assisted selection (MAS) has been shown to be effective in improving resistance to the European corn borer (ECB) in maize. In this study, we investigated the performance of whole-genome-based selection, relative to selection based on individual quantitative trait loci (QTL), for resistance to ECB stalk damage in European elite maize. Three connected biparental populations, comprising 590 doubled haploid (DH) lines, were genotyped with high-density single nucleotide polymorphism markers and phenotyped under artificial and natural infestation in 2011. A subset of 195 DH lines was evaluated in the following year as lines per se and as testcrosses. Resistance was evaluated based on stalk damage ratings, the number of feeding tunnels in the stalk and tunnel length. We performed individual- and joint-population QTL analyses and compared the cross-validated predictive abilities of the QTL models with genomic best linear unbiased prediction (GBLUP). For all traits, the GBLUP model consistently outperformed the QTL model despite the detection of QTL with sizeable effects. For stalk damage rating, GBLUP's predictive ability exceeded at times 0.70. Model training based on DH line per se performance was efficient in predicting stalk breakage in testcrosses. We conclude that the efficiency of MAS for ECB stalk damage resistance can be increased considerably when progressing from a QTL-based towards a genome-wide approach. With the availability of native ECB resistance in elite European maize germplasm, our results open up avenues for the implementation of an integrated genome-based selection approach for the simultaneous improvement of yield, maturity and ECB resistance.

Covariation between line and testcross performance for reduced mycotoxin concentrations in European maize after silk channel inoculation of two Fusarium species.

Fusarium spp. in maize can contaminate grain with mycotoxins harmful to humans and animals. Breeding and growing resistant varieties is one alternative to reduce contamination by mycotoxins. Little is known about the population parameters relevant to resistance breeding. The objectives of this study were to draw conclusions on breeding of reduced mycotoxin concentrations of deoxynivalenol, zearalenone and fumonisins, and resistance to ear rot after silk channel inoculation with F. graminearum or F. verticillioides, respectively. For that, variation and covariation of line and testcross performance and correlations between both species and between mycotoxin concentrations and ear rot resistance were calculated. Means of ear rot after infection with F. graminearum were higher than with F. verticillioides. Moderate phenotypic correlations (r = 0.46-0.65) between resistances to both Fusarium spp. implicate the need of separate testing. Analyses of variance revealed significant (P < 0.01) differences among lines in line and testcross performance for 30-60 entries per maturity group. Multi-environmental trials for accurate selection are necessary due to significant (P < 0.1) genotype × environment interactions. High genotypic correlations between ear rots and mycotoxins (r ≥ 0.90), and similar heritabilities of both traits, revealed the effectiveness of indirect selection for mycotoxin concentrations based on ear rot rating after inoculation. Moderate genotypic correlations between line and testcross performance were found (r = 0.64-0.83). The use of one moderately to highly susceptible tester is sufficient since genotypic correlations between testcrosses of different testers were high (r = 0.80-0.94). Indirect selection for testcross performance based on line performance is less effective than selection based on mycotoxin concentrations. Consequently, selection for resistance to ear rot and mycotoxin accumulation should be started among testcrosses tested first for general combining ability based on ear rot data in parallel with a negative selection for line per se performance.

Population parameters for resistance to Fusarium graminearum and Fusarium verticillioides ear rot among large sets of early, mid-late and late maturing European maize (Zea mays L.) inbred lines.

Infection of maize ears with Fusarium graminearum (FG) and Fusarium verticillioides (FV) reduces yield and quality by mycotoxin contamination. Breeding and growing varieties resistant to both Fusarium spp. is the best alternative to minimize problems. The objectives of our study were to draw conclusions on breeding for ear rot resistance by estimating variance components, heritabilities and correlations between resistances to FV and FG severity and to investigate different inoculation methods. In 2007 and 2008, three maturity groups (early, mid-late, late) each comprising about 150 inbred lines were tested in Germany, France, Italy, and Hungary according to their maturity group. They were silk channel inoculated by FG (early) and FV (all groups). In the late maturity group, additionally kernel inoculation was applied in a separate trial. The percentage of mycelium coverage on the ear was rated at harvest (0-100%). Significant (P < 0.01) genotypic variances of ear rot severity were found in all groups. Inoculation was superior to natural infection because of higher disease severities and heritabilities. In early maturing flints and dents, FG caused significantly (P < 0.01) higher ear rot severity than FV (61.7 and 55.1% FG vs. 18.2 and 11.1% FV ear rot severity, respectively). FV inoculation in Southern Europe (mid-late, late) resulted in similar means between 10.3 and 14.0%. Selection is complicated by significant (P < 0.01) genotype x environment interactions. Correlation between FG and FV severity was moderate in flints and dents (r = 0.59 and 0.49, respectively) but lines resistant to both fungi exist. We conclude that chances for selecting improved European elite maize material within the existing germplasms is promising by multi-environmental inoculation trials.