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Heterosis, i.e., the superiority of hybrids over parental lines, is sizable for allogamous species, especially maize (Zea mays L.). Heterosis has been extensively exploited but, despite a century of investigations, its genetic basis is not completely understood, yet. To gain information on its genetic control, we undertook a long term research in maize, aimed at providing a framework of comprehensive quantitative trait locus (QTL) phenotyping, to be integrated with map based cloning. As a first step, we jointly applied classical genetic and QTL analysis in a set of recombinant inbred lines (RILs), derived from the heterotic single cross B73 × H99. RILs were crossed to the three testers B73, H99 and B73 × H99, following a North Carolina III (NCIII) mating design and testcrosses were evaluated, together with the RILs, in three environments. Level of heterosis for several agronomic traits and underlying genetic effects (allelic and non-allelic interactions) were estimated. Several QTL with heterotic effects on agronomic traits were detected and most of them were characterized by dominant or overdominant gene action, whereas non-allelic interaction proved to be of minor importance. We then developed genetic materials suitable for validation and precise estimate of the effects of six heterotic QTL chosen for their appreciable additive and dominance effects. For this purpose, we adopted a residual heterozygous lines (RHL)-based introgression program to produce pairs of near-isogenic lines (NILs) homozygous either for one or the other parental inbred allele (i.e. B73 or H99) at the selected heterotic QTL regions. In addition, during the process of NILs production, we were able to preliminarily validate the phenotypic effects of two major QTL for heterosis, mapped on chromosome 3 (bin 05) and 4 (bin 10). Once NILs were obtained, we then approached a study aimed at verifying and characterizing QTL heterotic effects. The six pairs of NILs were crossed with the two parental inbred lines B73 and H99. The 24 testcrosses are now being evaluated in a multi-year research conducted over several environments at low and high plant densities (4.5 and 9.0 plants m-2, respectively). This investigation is warranted because, in several studies, the level of heterosis has proven to be particularly important in coping with environmental stress. Results obtained in the first year of testing allowed us to confirm additive and dominance effects of heterotic QTL for traits that showed strong heterosis in our previous studies. From non-stress to stress condition, for yield per plant and other agronomic traits the contribution of additive effects declined, while that of dominance increased. These preliminary findings thus confirm the importance of heterosis in coping with stress and its possible role in enhancing crop sustainability. The ultimate objective of QTL mapping is to identify the causal genes that underlie these QTL. Starting from F1 hybrids obtained by crossing contrasting NILs, we produced large F2 populations, each segregating only for one QTL region. We limited our attention to the introgressed QTL mapped on chromosome 4 (bin 10) and 10 (bin 03) because of their sizable effects on yield and other traits. F2 populations were genotyped at markers flanking the segregating QTL, and F3 families are now being produced by selfing informative recombinant F2 individuals. The so obtained F3 families will be evaluated for agronomic traits in order to fine map the heterotic QTL.

Mapping genetics fators affecting heterosis in maize Elisabetta Frascaroli*, Maria Angela Canè*, Pierangelo Landi*, Giorgio Pea†, Michele Morgante‡, Mario Enrico Pè† * Dipartimento di Scienze e Tecnologie Agroambientali, Università di Bologna, Bologna, Italy † Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Milano, Italy ‡ Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, and Istituto di Genomica Applicata Parco Scientifico e Tecnologico di Udine "Luigi Danieli", Udine, Italy Abstract The term heterosis describes the superiority of heterozygous genotypes for one or more characteristics in comparison with the corresponding parental homozygotes. The higher productivity of the heterozygotes is exploited through the development of hybrid varieties in several crop species, and historically it represented one of the most outstanding advancement in plant improvement. Despite the long history of successes, especially in maize (Zea mays L.), the genetic bases of heterosis are not well understood yet. The application of molecular markers for the dissection of the genetic basis of many quantitative traits prompted the development of different approaches for the evaluation of heterosis. Our research was conducted on the genetic material developed from the single cross between maize inbred lines H99 and B73 in order to: (i) study the level of heterosis for traits of agronomic importance; (ii) identify the genomic regions (QTL quantitative trait loci) most involved in heterosis; (iii) investigate the relationships between the level of molecular marker heterozygosity and the phenotypic performance; (iv) estimate the genetic effects involved (i.e., allelic and non-allelic interactions). Materials were the basic generations, the derived 142 recombinant inbred lines (RILs) and the three testcross populations obtained by crossing the 142 RILs to each parent and their F1. RIL population was genotyped with a total of 158 marker loci arranged in a genetic linkage map. All genotypes were field-tested in three locations. The field layout was a randomized complete block design for basic generations, and a modified split-plot design for the four populations. Least square means over locations were used for subsequent analyses. Classical genetic analises were conducted following the North Caroline Model III (NCIII) and Triple Test Cross (TTC) designs. Moreover, Composite Interval Mapping was performed to search for QTL by combining plant’s phenotypic and marker’s genotypic data. QTL analysis was conducted on testcross populations’ means and on derived datasets concerning additive and dominance effects only. Thresholds for declaring putative QTL were defined by permutations. A mixed linear model was then used to map digenic epistatic QTL. Among the examined traits, seedling weight (SW), number of kernels per plant (NK) and grain yield (GY) showed heterosis greater than 100% and dominance degree higher than one. Several QTL were identified prevailingly in the additive-dominance range, for traits with low heterosis, and prevailingly in the dominance-overdominance range for plant height (PH), SW, NK and GY. Only a few QTL with digenic epistasis were identified. Some chromosome regions presented overlaps of overdominant QTL for SW, PH, NK and GY, suggesting pleiotropic effects on the overall plant vigor.

Poster Abstract C.18

Oral Communication Abstract – 4.03

Poster Abstract – H.06

Poster Abstract - D.06

This thesis was aimed to investigate the genetic response to abiotic and biotic stresses in durum wheat (Triticum turgidum L. var. durum), a cultivated tetraploid subspecies used for the production of pasta, couscous and various types of bread. Two research areas were focused: i) the high-throughput phenotyping (HTP) to detect novel drought tolerance quantitative trait loci (QTL) clusters and ii) the Kompetitive Allele Specific Polymerase chain reaction (KASP) marker development for the genetic dissection of Furarium head blight (FHB) resistance. Concerning the first area, I investigated drought adaptive traits on durum wheat elite accessions at Maricopa Agricultural Center (University of Arizona, US) which provided the experimental field and the high-throughput phenotyping platforms. The genome-wide association study (GWAS) detected thirty-one QTL clusters for two or more drought adaptive traits unrelated to the major loci responsible for phenology and plant height. Twelve of them overlapped with the major QTL for grain yield and related traits previously reported in studies carried out across a broad range of soil moisture availability and field drought conditions in wheat. Concerning the second area, I investigated two plant materials: i) 130 durum wheat accessions artificially inoculated with Fusarium culmorum and F. graminearum species and evaluated for incidence (INC), severity (SEV), FHB index, Fusarium-damaged kernels (FDK) and deoxynivalenol (DON) content; ii) 165 F6 recombinant inbred lines (RILs) from the cross between the cultivars Simeto and Levante evaluated for SEV using FG as inoculum. The genetic dissection led to sixteen QTL clusters, in part unrelated to the phenology and unknown in bread wheat, from which specific loci significantly influenced DON content. The haplotype analysis allowed me to validate KASP Single Nucleotide Polymorphisms (SNPs) suitable for marker-assisted selection (MAS) programs and to select cultivars for resistance/tolerance to DON content and other FHB traits.

Given the importance of early cold tolerance in maize (Zea mays L.), we previously conducted a divergent selection for low (L) and high (H) cold tolerance at germination. The source population was the F2 of B73 × IABO78 single cross. Selection was first conducted as recurrent selection (four cycles) and then in inbreeding (three generations). Ten lines were finally selected for L and ten for H; these lines were then evaluated to investigate: (i) their field cold tolerance in the early autotrophic phase and the relationship with the cold tolerance expressed at emergence and in the heterotrophic phase; (ii) the inheritance of these cold tolerance traits; (iii) their agronomic performance as lines per se and as crosses; (iv) their SNP-based genetic characterization. For all cold tolerance traits, the H group of lines was superior to the L group; selection responses were often symmetric, suggesting the prevalence of additive effects. The relationship among cold tolerance traits was appreciable, suggesting a common genetic basis. Differences among testcrosses were largely due to general combining ability effects and the relationship between line per se and testcross performance was high, confirming the prevalence of additive effects. Correlated responses were also obtained for agronomic traits, the H lines being superior to L lines both per se and as crosses. For SNP-marker loci, the B73 allelic frequency was greater in the H group (61.7 %) than in L group (39.7 %). Across field trials, an H line excelled for all traits, thus proving to deserve further investigations.

Durum wheat (Triticum turgidum subsp. durum) is a key crop worldwide, and yet, its improvement and adaptation to emerging environmental threats is made difficult by the limited amount of allelic variation included in its elite pool. New allelic diversity may provide novel loci to international crop breeding through quantitative trait loci (QTL) mapping in unexplored material. Here, we report the extensive molecular and phenotypic characterization of hundreds of Ethiopian durum wheat landraces and several Ethiopian improved lines. We test 81 587 markers scoring 30 155 single nucleotide polymorphisms and use them to survey the diversity, structure, and genome-specific variation in the panel. We show the uniqueness of Ethiopian germplasm using a siding collection of Mediterranean durum wheat accessions. We phenotype the Ethiopian panel for ten agronomic traits in two highly diversified Ethiopian environments for two consecutive years and use this information to conduct a genome-wide association study. We identify several loci underpinning agronomic traits of interest, both confirming loci already reported and describing new promising genomic regions. These loci may be efficiently targeted with molecular markers already available to conduct marker-assisted selection in Ethiopian and international wheat. We show that Ethiopian durum wheat represents an important and mostly unexplored source of durum wheat diversity. The panel analysed in this study allows the accumulation of QTL mapping experiments, providing the initial step for a quantitative, methodical exploitation of untapped diversity in producing a better wheat. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Divergently selected genotypes can be used for detecting the genomic regions affecting the selected trait (selection signature). Moreover, the genetic distances (GDs) among divergently selected lines can be correlated with the agronomic performances of the crosses among them. Using as source the maize F2 of B73 × IABO78, we previously conducted four cycles of divergent recurrent selection and three cycles of divergent selection in inbreeding for cold tolerance at germination. We finally obtained 10 lines selected for low (L) and 10 lines selected for high (H) cold tolerance, which exhibited a notable divergence for both the selected and associated traits. Herein, we investigated the 20 lines and the 28 single diallel crosses among eight random lines (four L and four H); the main objectives were to identify the putative regions controlling the selected and associated traits and to study the relationships between crosses performances and GDs among their parental lines. Allele frequencies at 932 recombination blocks based on 19,220 polymorphic SNPs were obtained for the two lines’ groups; the F ST calculated across sliding windows indicated 18 regions highly divergent between groups. The increasing alleles for cold tolerance were contributed by both parents, consistently with the transgressive segregations previously found. Several regions associated to DG also affected various agronomic traits. The cross performances showed some relationships with the genetic distances among parental lines for traits affected by dominance, provided that all crosses were considered, while these relationships vanished when only L × H crosses were examined.

Low-temperature stress is considered as the major abiotic constraint limiting plant’s growth and the potential land cultivation. Crop adaptation to limiting temperature is thus an important breeding objective because it determines yield stability in environment-friendly cultivation practices. Conventional breeding methods had limited success in improving the cold tolerance of important crop plants because of the complexity of stress tolerance traits, low genetic variance, and lack of efficient selection criteria. The knowledge of physiology, of genetics, and of the DNA technology has improved substantially nowadays, and these advancements will allow the breeder to predict the breeding value of best genotypes by using physiology, genetics, and molecular information. The perspective for selecting more effectively cold-tolerant crops will involve efficient genotyping, reliable phenotyping and envirotyping, and adequate statistical models.

Maize originated in tropical areas and improving cold tolerance is an important breeding objective for cultivation in high latitudes. We review the main limitations in understanding and improving cold tolerance in maize and the contribution of genomics in dissecting the genetic basis of the trait and selecting better genotypes. Physiological analyses revealed that non-optimal temperature exerts detrimental effects on a multitude of metabolic functions at different growing stages, each under the control of independent gene sets. Loci controlling cold tolerance at different growing stages have been investigated by means of linkage mapping or genome-wide association, revealing that no major genes are responsible for the trait. This finding was confirmed in transcriptomic studies that always revealed multiple candidates, and a large amount of data is being collected that altogether will make it possible to obtain a more coherent picture of response to cold. To harness the increasing body of information available from the maize genome sequence and gene expression data, new bioinformatics tools will be helpful for integrating the big-data obtained from the large-scale genomics and phenomics experiments. With the enhancement of knowledge, plant science is shifting its focus from “explanatory” to “predictive” and from a plant breeding perspective the focus will be predicting the breeding value of the best genotypes by using molecular information. The future strategies for selection of cold tolerance will involve intensive genotyping, high-precision phenotyping and advanced statistical analyses to predict the optimal genotypes for more time- and cost-efficient breeding strategies.

Smallholder agriculture involves millions of farmers worldwide. A methodical utilization of their traditional knowledge in modern breeding efforts may help the production of locally adapted varieties better addressing their needs. In this study, a combination of participatory approaches, genomics, and quantitative genetics is used to trace the genetic basis of smallholder farmer preferences of durum wheat traits. Two smallholder communities evaluated 400 Ethiopian wheat varieties, mostly landraces, for traits of local interest in two locations in the Ethiopian highlands. For each wheat variety, farmers provided quantitative evaluations of their preference for flowering time, spike morphology, tillering capacity, and overall quality. Ten agronomic and phenology traits were simultaneously measured on the same varieties, providing the means to compare them with farmer traits. The analysis of farmer traits showed that they were partially influenced by gender and location but were repeatable and heritable, in some cases more than metric traits. The durum wheat varieties were genotyped for more than 80 thousand SNP markers, and the resulting data was used in a genome wide association (GWA) study providing the molecular dissection of smallholder farmers’ choice criteria. We found 124 putative quantitative trait loci (QTL) controlling farmer traits and 30 putative QTL controlling metric traits. Twenty of such QTL were jointly identified by farmer and metric traits. QTL derived from farmer traits were in some cases dependent on gender and location, but were consistent throughout. The results of the GWA study show that smallholder farmers’ traditional knowledge can yield QTL eluding metric measurements of phenotypes. We discuss the potential of including farmer evaluations based on traditional knowledge in crop breeding, arguing for the utilization of this untapped resource to develop better adapted genetic materials for local agriculture.

Bo23 (Reg. No. GP-599, PI 677346) is a maize (Zea mays L.) inbred line developed at the Department of Agricultural Sciences, University of Bologna (Bologna, Italy), using as source the F2 of the cross B73 × IABO78. B73 is a well-known inbred from a US Corn Belt germplasm, and IABO78 is an inbred from Italian germplasm not previously used in breeding. Bo23 was released in 2016 due to its high cold tolerance and agronomic performance when sown early. The breeding procedure leading to Bo23 consisted of four cycles of full-sib recurrent selection and three cycles of selection in inbreeding for germination at low temperature. In the evaluation of selection responses, a line coded as H102 (now Bo23) excelled for cold tolerance in the field and for agronomic traits. Therefore, Bo23 was further evaluated in seven trials conducted in early sowing in the southern Po Valley (Italy). The line again exhibited outstanding performance for cold tolerance and agronomic traits, both per se and as testcross. Bo23 was superior to B73 for most traits, indicating that its performance is also due to alleles of IABO78. This implies that the use of Bo23 in breeding for cold tolerance and agronomic traits can also contribute the beneficial genetic variation from its Italian parent.

Background Maize (Zea mays) is a globally produced crop with broad genetic and phenotypic variation. New tools that improve our understanding of the genetic basis of quantitative traits are needed to guide predictive crop breeding. We have produced the first balanced multi-parental population in maize, a tool that provides high diversity and dense recombination events to allow routine quantitative trait loci (QTL) mapping in maize. Results We produced 1,636 MAGIC maize recombinant inbred lines derived from eight genetically diverse founder lines. The characterization of 529 MAGIC maize lines shows that the population is a balanced, evenly differentiated mosaic of the eight founders, with mapping power and resolution strengthened by high minor allele frequencies and a fast decay of linkage disequilibrium. We show how MAGIC maize may find strong candidate genes by incorporating genome sequencing and transcriptomics data. We discuss three QTL for grain yield and three for flowering time, reporting candidate genes. Power simulations show that subsets of MAGIC maize might achieve high-power and high-definition QTL mapping. Conclusions We demonstrate MAGIC maize’s value in identifying the genetic bases of complex traits of agronomic relevance. The design of MAGIC maize allows the accumulation of sequencing and transcriptomics layers to guide the identification of candidate genes for a number of maize traits at different developmental stages. The characterization of the full MAGIC maize population will lead to higher power and definition in QTL mapping, and lay the basis for improved understanding of maize phenotypes, heterosis included. MAGIC maize is available to researchers. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0716-z) contains supplementary material, which is available to authorized users.

Summary Multiparental cross designs for mapping quantitative trait loci (QTL) provide an efficient alternative to biparental populations because of their broader genetic basis and potentially higher mapping resolution. We describe the development and deployment of a recombinant inbred line (RIL) population in durum wheat (Triticum turgidum ssp. durum) obtained by crossing four elite cultivars. A linkage map spanning 2664 cM and including 7594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs. QTL analysis was carried out by both interval mapping on founder haplotype probabilities and SNP bi-allelic tests for heading date and maturity date, plant height and grain yield from four field experiments. Sixteen QTL were identified across environments and detection methods, including two yield QTL on chromosomes 2BL and 7AS, with the former mapped independently from the photoperiod response gene Ppd-B1, while the latter overlapped with the vernalization locus VRN-A3. Additionally, 21 QTL with environment-specific effects were found. Our results indicated a prevalence of environment-specific QTL with relatively small effect on the control of grain yield. For all traits, functionally different QTL alleles in terms of direction and size of genetic effect were distributed among parents. We showed that QTL results based on founder haplotypes closely matched functional alleles at known heading date loci. Despite the four founders, only 2.1 different functional haplotypes were estimated per QTL, on average. This durum wheat population provides a mapping resource for detailed genetic dissection of agronomic traits in an elite background typical of breeding programmes.