Genomic prediction within and across maize landrace derived populations using haplotypes.

Genomic prediction (GP) using haplotypes is considered advantageous compared to GP solely reliant on single nucleotide polymorphisms (SNPs), owing to haplotypes' enhanced ability to capture ancestral information and their higher linkage disequilibrium with quantitative trait loci (QTL). Many empirical studies supported the advantages of haplotype-based GP over SNP-based approaches. Nevertheless, the performance of haplotype-based GP can vary significantly depending on multiple factors, including the traits being studied, the genetic structure of the population under investigation, and the particular method employed for haplotype construction. In this study, we compared haplotype and SNP based prediction accuracies in four populations derived from European maize landraces. Populations comprised either doubled haploid lines (DH) derived directly from landraces, or gamete capture lines (GC) derived from crosses of the landraces with an inbred line. For two different landraces, both types of populations were generated, genotyped with 600k SNPs and phenotyped as lines per se for five traits. Our study explores three prediction scenarios: (i) within each of the four populations, (ii) across DH and GC populations from the same landrace, and (iii) across landraces using either DH or GC populations. Three haplotype construction methods were evaluated: 1. fixed-window blocks (FixedHB), 2. LD-based blocks (HaploView), and 3. IBD-based blocks (HaploBlocker). In within population predictions, FixedHB and HaploView methods performed as well as or slightly better than SNPs for all traits. HaploBlocker improved accuracy for certain traits but exhibited inferior performance for others. In prediction across populations, the parameter setting from HaploBlocker which controls the construction of shared haplotypes between populations played a crucial role for obtaining optimal results. When predicting across landraces, accuracies were low for both, SNP and haplotype approaches, but for specific traits substantial improvement was observed with HaploBlocker. This study provides recommendations for optimal haplotype construction and identifies relevant parameters for constructing haplotypes in the context of genomic prediction.

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.

Natural alleles of the abscisic acid catabolism gene ZmAbh4 modulate water use efficiency and carbon isotope discrimination in maize.

Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.

Physiological adaptive traits are a potential allele reservoir for maize genetic progress under challenging conditions.

Combined phenomic and genomic approaches are required to evaluate the margin of progress of breeding strategies. Here, we analyze 65 years of genetic progress in maize yield, which was similar (101 kg ha-1 year-1) across most frequent environmental scenarios in the European growing area. Yield gains were linked to physiologically simple traits (plant phenology and architecture) which indirectly affected reproductive development and light interception in all studied environments, marked by significant genomic signatures of selection. Conversely, studied physiological processes involved in stress adaptation remained phenotypically unchanged (e.g. stomatal conductance and growth sensitivity to drought) and showed no signatures of selection. By selecting for yield, breeders indirectly selected traits with stable effects on yield, but not physiological traits whose effects on yield can be positive or negative depending on environmental conditions. Because yield stability under climate change is desirable, novel breeding strategies may be needed for exploiting alleles governing physiological adaptive traits.

Genetic diversity of European maize landraces: Dataset on the molecular and phenotypic variation of derived doubled-haploid populations.

Genetic variation is the basis of selection, evolution and breeding. Maize landraces represent a rich source of allelic diversity, but their efficient utilization in breeding and research has been hampered by their heterogeneous and heterozygous nature and insufficient information about most accessions. While molecular inventories of germplasm repositories are growing steadily, linking these data to meaningful phenotypes for quantitative traits is challenging. Here, we present comprehensive molecular and phenotypic data for ∼1,000 doubled-haploid (DH) lines derived from three pre-selected European maize landraces. Due to their full homozygosity, the DH lines can be multiplied ad libitum and represent a powerful biological resource available to the community. The DH lines allow high-precision phenotyping in repeated experiments and reveal the full additive genetic variance of the population. The DH lines were evaluated for nine agronomically important, quantitative traits in multi-environment field trials comprising seven locations and two years. The DH populations revealed high genetic variance and high heritability for the analysed traits. The DH lines were genotyped with 600k SNP markers. After stringent quality filtering 500k markers remained for further analyses. This is the largest resource of landrace derived DH material in maize, unprecedented in its structure and dimension. The presented data are ideal for linking molecular variation to meaningful phenotypes. They can be used for genome-wide association studies, genomic prediction, and population genetic analyses as well as for developing and testing statistical methods. All plant material is available to the community for conducting additional experiments, extending the panel of traits and environments, and for testing the landrace-derived lines in combination with other genetic material.

Theoretical and experimental assessment of genome-based prediction in landraces of allogamous crops.

Discovery and enrichment of favorable alleles in landraces are key to making them accessible for crop improvement. Here, we present two fundamentally different concepts for genome-based selection in landrace-derived maize populations, one based on doubled-haploid (DH) lines derived directly from individual landrace plants and the other based on crossing landrace plants to a capture line. For both types of populations, we show theoretically how allele frequencies of the ancestral landrace and the capture line translate into expectations for molecular and genetic variances. We show that the DH approach has clear advantages over gamete capture with generally higher prediction accuracies and no risk of masking valuable variation of the landrace. Prediction accuracies as high as 0.58 for dry matter yield in the DH population indicate high potential of genome-based selection. Based on a comparison among traits, we show that the genetic makeup of the capture line has great influence on the success of genome-based selection and that confounding effects between the alleles of the landrace and the capture line are best controlled for traits for which the capture line does not outperform the ancestral population per se or in testcrosses. Our results will guide the optimization of genome-enabled prebreeding schemes.

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.

Discovery of beneficial haplotypes for complex traits in maize landraces.

Genetic variation is of crucial importance for crop improvement. Landraces are valuable sources of diversity, but for quantitative traits efficient strategies for their targeted utilization are lacking. Here, we map haplotype-trait associations at high resolution in ~1000 doubled-haploid lines derived from three maize landraces to make their native diversity for early development traits accessible for elite germplasm improvement. A comparative genomic analysis of the discovered haplotypes in the landrace-derived lines and a panel of 65 breeding lines, both genotyped with 600k SNPs, points to untapped beneficial variation for target traits in the landraces. The superior phenotypic performance of lines carrying favorable landrace haplotypes as compared to breeding lines with alternative haplotypes confirms these findings. Stability of haplotype effects across populations and environments as well as their limited effects on undesired traits indicate that our strategy has high potential for harnessing beneficial haplotype variation for quantitative traits from genetic resources.

European maize landraces made accessible for plant breeding and genome-based studies.

KEY MESSAGE: Doubled-haploid libraries from landraces capture native genetic diversity for a multitude of quantitative traits and make it accessible for breeding and genome-based studies. Maize landraces comprise large allelic diversity. We created doubled-haploid (DH) libraries from three European flint maize landraces and characterized them with respect to their molecular diversity, population structure, trait means, variances, and trait correlations. In total, 899 DH lines were evaluated using high-quality genotypic and multi-environment phenotypic data from up to 11 environments. The DH lines covered 95% of the molecular variation present in 35 landraces of an earlier study and represent the original three landrace populations in an unbiased manner. A comprehensive analysis of the target trait plant development at early growth stages as well as other important agronomic traits revealed large genetic variation for line per se and testcross performance. The majority of the 378 DH lines evaluated as testcrosses outperformed the commercial hybrids for early development. For total biomass yield, we observed a yield gap of 15% between mean testcross yield of the commercial hybrids and mean testcross yield of the DH lines. The DH lines also exhibited genetic variation for undesirable traits like root lodging and tillering, but correlations with target traits early development and yield were low or nonsignificant. The presented diversity atlas is a valuable, publicly available resource for genome-based studies to identify novel trait variation and evaluate the prospects of genomic prediction in landrace-derived material.

Carbon isotope composition, water use efficiency, and drought sensitivity are controlled by a common genomic segment in maize.

KEY MESSAGE: A genomic segment on maize chromosome 7 influences carbon isotope composition, water use efficiency, and leaf growth sensitivity to drought, possibly by affecting stomatal properties. Climate change is expected to decrease water availability in many agricultural production areas around the globe. Therefore, plants with improved ability to grow under water deficit are urgently needed. We combined genetic, phenomic, and physiological approaches to understand the relationship between growth, stomatal conductance, water use efficiency, and carbon isotope composition in maize (Zea mays L.). Using near-isogenic lines derived from a maize introgression library, we analysed the effects of a genomic region previously identified as affecting carbon isotope composition. We show stability of trait expression over several years of field trials and demonstrate in the phenotyping platform Phenodyn that the same genomic region also influences the sensitivity of leaf growth to evaporative demand and soil water potential. Our results suggest that the studied genomic region affecting carbon isotope discrimination also harbours quantitative trait loci playing a role in maize drought sensitivity possibly via stomatal behaviour and development. We propose that the observed phenotypes collectively originate from altered stomatal conductance, presumably via abscisic acid.

Genome-Wide Analysis of Yield in Europe: Allelic Effects Vary with Drought and Heat Scenarios.

Assessing the genetic variability of plant performance under heat and drought scenarios can contribute to reduce the negative effects of climate change. We propose here an approach that consisted of (1) clustering time courses of environmental variables simulated by a crop model in current (35 years × 55 sites) and future conditions into six scenarios of temperature and water deficit as experienced by maize (Zea mays L.) plants; (2) performing 29 field experiments in contrasting conditions across Europe with 244 maize hybrids; (3) assigning individual experiments to scenarios based on environmental conditions as measured in each field experiment; frequencies of temperature scenarios in our experiments corresponded to future heat scenarios (+5°C); (4) analyzing the genetic variation of plant performance for each environmental scenario. Forty-eight quantitative trait loci (QTLs) of yield were identified by association genetics using a multi-environment multi-locus model. Eight and twelve QTLs were associated to tolerances to heat and drought stresses because they were specific to hot and dry scenarios, respectively, with low or even negative allelic effects in favorable scenarios. Twenty-four QTLs improved yield in favorable conditions but showed nonsignificant effects under stress; they were therefore associated with higher sensitivity. Our approach showed a pattern of QTL effects expressed as functions of environmental variables and scenarios, allowing us to suggest hypotheses for mechanisms and candidate genes underlying each QTL. It can be used for assessing the performance of genotypes and the contribution of genomic regions under current and future stress situations and to accelerate breeding for drought-prone environments.

Two major quantitative trait loci controlling the number of seminal roots in maize co-map with the root developmental genes rtcs and rum1.

The genetic dissection of root architecture and functions allows for a more effective and informed design of novel root ideotypes and paves the way to evaluate their effects on crop resilience to a number of abiotic stresses. In maize, limited attention has been devoted to the genetic analysis of root architecture diversity at the early stage. The difference in embryonic (including seminal and primary) root architecture between the maize reference line B73 (which mostly develops three seminal roots) and the landrace Gaspé Flint (with virtually no seminal roots) was genetically dissected using a collection of introgression lines grown in paper rolls and pots. Quantitative trait locus (QTL) analysis identified three QTLs controlling seminal root number (SRN) on chromosome bins 1.02, 3.07, and 8.04-8.05, which collectively explained 66% of the phenotypic variation. In all three cases, Gaspé Flint contributed the allele for lower SRN. Primary root dry weight was negatively correlated with SRN (r= -0.52), and QTLs for primary root size co-mapped with SRN QTLs, suggesting a pleiotropic effect of SRN QTLs on the primary root, most probably caused by competition for seed resources. Interestingly, two out of three SRN QTLs co-mapped with the only two known maize genes (rtcs and rum1) affecting the number of seminal roots. The strong additive effect of the three QTLs and the development of near isogenic lines for each QTL in the elite B73 background provide unique opportunities to characterize functionally the genes involved in root development and to evaluate how root architecture affects seedling establishment, early development, and eventually yield in maize.

First steps to understand heat tolerance of temperate maize at adult stage: identification of QTL across multiple environments with connected segregating populations.

KEY MESSAGE: Dents were more heat tolerant than Flints. QTL for heat tolerance with respect to grain yield at field conditions were identified considering multiple populations and environments. High temperatures have the potential to cause severe damages to maize production. This study aims to elucidate the genetic mechanisms of heat tolerance under field conditions in maize and the genome regions contributing to natural variation. In our study, heat tolerance was assessed on a multi-environment level under non-controlled field conditions for a set of connected intra- and interpool Dent and Flint populations. Our findings indicate that Dent are more heat tolerant during adult stage than Flint genotypes. We identified 11 quantitative trait loci (QTL) including 2 loci for heat tolerance with respect to grain yield. Furthermore, we identified six heat-tolerance and 112 heat-responsive candidate genes colocating with the previously mentioned QTL. To investigate their contribution to the response to heat stress and heat tolerance, differential expression and sequence variation of the identified candidate genes should be subjected to further research.

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.

Selection strategies for the development of maize introgression populations.

Introgression libraries are valuable resources for QTL detection and breeding, but their development is costly and time-consuming. Selection strategies for the development of introgression populations with a limited number of individuals and high-throughput (HT) marker assays are required. The objectives of our simulation study were to design and compare selection strategies for the development of maize introgression populations of 100 lines with population sizes of 360-720 individuals per generation for different DH and S2 crossing schemes. Pre-selection for complete donor chromosomes or donor chromosome halves reduced the number of simultaneous backcross programs. The investigated crossing and selection schemes differed considerably with respect to their suitability to create introgression populations with clearly separated, evenly distributed target donor chromosome segments. DH crossing schemes were superior to S2 crossing schemes, mainly due to complete homozygosity, which greatly reduced the total number of disjunct genome segments in the introgression populations. The S2 crossing schemes were more flexible with respect to selection and provided economic alternatives to DH crossing schemes. For the DH crossing schemes, increasing population sizes gradually over backcross generations was advantageous as it reduced the total number of required HT assays compared to constant population sizes. For the S2 crossing schemes, large population sizes in the final backcross generation facilitated selection for the target segments in the final backcross generation and reduced fixation of large donor chromosome segments. The suggested crossing and selection schemes can help to make the genetic diversity of exotic germplasm available for enhancing the genetic variation of narrow-based breeding populations of crops.

Stable carbon isotope discrimination is under genetic control in the C4 species maize with several genomic regions influencing trait expression.

In plants with C4 photosynthesis, physiological mechanisms underlying variation in stable carbon isotope discrimination (Δ(13)C) are largely unknown, and genetic components influencing Δ(13)C have not been described. We analyzed a maize (Zea mays) introgression library derived from two elite parents to investigate whether Δ(13)C is under genetic control in this C4 species. High-density genotyping with the Illumina MaizeSNP50 Bead Chip was used for a detailed structural characterization of 89 introgression lines. Phenotypic analyses were conducted in the field and in the greenhouse for kernel Δ(13)C as well as plant developmental and photosynthesis-related traits. Highly heritable significant genetic variation for Δ(13)C was detected under field and greenhouse conditions. For several introgression library lines, Δ(13)C values consistently differed from the recurrent parent within and across the two phenotyping platforms. Δ(13)C was significantly associated with 22 out of 164 analyzed genomic regions, indicating a complex genetic architecture of Δ(13)C. The five genomic regions with the largest effects were located on chromosomes 1, 2, 6, 7, and 9 and explained 55% of the phenotypic variation for Δ(13)C. Plant development stage had no effect on Δ(13)C expression, as phenotypic as well as genotypic correlations between Δ(13)C, flowering time, and plant height were not significant. To our knowledge, this is the first study demonstrating Δ(13)C to be under polygenic control in the C4 species maize.

Key impact of Vgt1 on flowering time adaptation in maize: evidence from association mapping and ecogeographical information.

An association study conducted on 375 maize inbred lines indicates a strong relationship between Vgt1 polymorphisms and flowering time, extending former quantitative trait loci (QTL) mapping results. Analysis of allele frequencies in a landrace collection supports a key role of Vgt1 in maize altilatitudinal adaptation.

Quantitative trait loci for early plant vigour of maize grown in chilly environments.

Maize (Zea mays L.) is particularly sensitive to chilling in the early growth stages. The objective of this study was to determine quantitative trait loci (QTL) for early plant vigour of maize grown under cool and moderately warm conditions in Central Europe. A population of 720 doubled haploid (DH) lines was derived from a cross between two dent inbred lines contrasting in early vigour and were genotyped with 188 SSR markers. The DH lines per se and their testcrosses with a flint line were evaluated in field experiments across 11 environments in 2001 and 2002. Plants were harvested after six to eight leaves had been fully developed to assess fresh matter yield as a criterion of early vigour. Seven QTL were detected for line performance and ten QTL for testcross performance, explaining 64 and 49% of the genetic variance. Six out of seven QTL detected in the lines per se were also significant in their testcrosses. Significant QTL x environment interaction was observed, but no relationship existed between the size of the QTL effects and the mean temperature in the individual environment. The correlation between fresh matter yield and days to silking was non-significant, indicating that differences in early plant vigour were not simply caused by maturity differences. For three additional chilling-related traits, leaf chlorosis, leaf purpling, and frost damage seven, six, and five QTL were detected, respectively. Three QTL for leaf chlorosis, two for leaf purpling, and two for frost damage co-localized with QTL for fresh matter yield. Results are considered as a reliable basis for further genetic, molecular, and physiological investigations.