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Poster 158

Background Despite the crucial role that heterosis has played in crop improvement, its genetic and molecular bases are still elusive. Several types of structured populations were used to discover the genetic architecture underlying complex phenotypes, and several QTL related to heterosis were detected. However, such analyses generally lacked the statistical power required for the detailed characterization of individual QTL. Currently, QTL introgression into near-isogenic materials is considered the most effective strategy to this end, despite such materials inevitably contain a variable, unknown and undesired proportion of non-isogenic genome. An introgression program based on residual heterozygous lines allowed us to develop five pairs of maize (Zea mays L.) near-isogenic lines (NILs) suitable for the fine characterization of three major heterotic QTL previously detected. Here we describe the results of the detailed genomic characterization of these NILs that we undertook to establish their genotypic structure, to verify the presence of the expected genotypes within target QTL regions, and to determine the extent and location of residual non-isogenic genomic regions. Results The SNP genotyping approach allowed us to determine the parent-of-origin allele for 14,937 polymorphic SNPs and to describe in detail the genotypic structure of all NILs. The correct introgression was confirmed for all target QTL in the respective NIL and several non-isogenic regions were detected genome-wide. Possible linkage drag effects associated to the specific introgressed regions were observed. The extent and position of other non-isogenic regions varied among NIL pairs, probably deriving from random segregating sections still present at the separation of lineages within pairs. Conclusions The results of this work strongly suggest that the actual isogenicity and the genotypic architecture of near-isogenic materials should be monitored both during the introgression procedure and on the final materials as a paramount requisite for a successful mendelization of target QTL. The information here gathered on the genotypic structure of NILs will be integrated in future experimental programs aimed at the fine mapping and isolation of major heterotic QTL, a crucial step towards the understanding of the molecular bases of heterosis in maize.

Recent advances in high-throughput sequencing technologies have triggered a shift toward single-nucleotide polymorphism (SNP) markers. A systematic bias can be introduced if SNPs are ascertained in a small panel of genotypes and then used for characterizing a larger population (ascertainment bias). With the objective of evaluating a potential ascertainment bias of the Illumina MaizeSNP50 array with respect to elite European maize dent and flint inbred lines, we compared the genetic diversity among these materials based on 731 amplified fragment length polymorphisms (AFLPs), 186 simple sequence repeats (SSRs), 41,434 SNPs of the MaizeSNP50 array (SNP-A), and two subsets of it, i.e., 30,068 Panzea (SNP-P) and 11,366 Syngenta markers (SNP-S). We evaluated the bias effects on major allele frequency, allele number, gene diversity, modified Roger's distance (MRD), and on molecular variance (AMOVA). We revealed ascertainment bias in SNP-A, compared to AFLPs and SSRs. It affected especially European flint lines analyzed with markers (SNP-S) specifically developed to maximize differences among North American dent germplasm. The bias affected all genetic parameters, but did not substantially alter the relative distances between inbred lines within groups. For these reasons, we conclude that the SNP markers of the MaizeSNP50 array can be employed for breeding purposes in the investigated material. However, attention should be paid in case of comparisons between genotypes belonging to different heterotic groups. In this case, it is advisable to prefer a marker subset with potentially low ascertainment bias, like in our case the SNP-P marker set.

Genomics approaches and platforms offer novel opportunities to identify and clone the loci that control root architecture in maize. Mutants for root features have been described and in some cases the relevant genes have been cloned. A number of studies have also described quantitative trait loci (QTLs) that provide access to valuable genetic diversity for the morpho-physiological features that characterize root functionality. Nonetheless, although a number of major QTLs have been identified, none of these QTLs has been cloned so far, mainly due to the difficulty to accurately phenotype the target traits in a sufficiently large number of plants. It is foreseeable that QTL cloning will be facilitated by the adoption of high-throughput phenomics platforms as well as by the information made available through the sequencing of the maize genome and the profiling of the transcriptome, proteome, and metabolome, all of which will contribute to the identification of plausible candidate genes. Allele mining in germplasm and mutant collections through forward- and reverse-genetics approaches, coupled with marker-assisted backcrossing and/or genetic engineering, will further facilitate the introgression of novel genetic variation in elite materials. New QTL-based modeling approaches will improve our capacity to understand genotype × environment interaction at varying water and nutrient regimes, thus contributing to define the most promising “molecular” ideotypes. This notwithstanding, a sizeable impact of marker-assisted selection and other genomics approaches in tailoring the ideal root architecture in maize will only be possible through a deeper knowledge of root functions under a broad range of growing conditions and an integration with conventional breeding methodologies.

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.

The exploitation of heterosis is one of the most outstanding advancements in plant breeding, although its genetic basis is not well understood yet. This research was conducted on the materials arising from the maize single cross B73 × H99 to study heterosis by procedures of classical genetic and quantitative trait loci (QTL) analyses. 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. For seedling weight (SW), number of kernels per plant (NK), and grain yield (GY), heterosis was >100% and the average degree of dominance was >1. Epistasis was significant for SW and NK but not for GY. Several QTL were identified and in most cases they were in the additive–dominance range for traits with low heterosis and mostly in the dominance–overdominance range for plant height (PH), SW, NK, and GY. Only a few QTL with digenic epistasis were identified. The importance of dominance effects was confirmed by highly significant correlations between heterozygosity level and phenotypic performance, especially for GY. Some chromosome regions presented overlaps of overdominant QTL for SW, PH, NK, and GY, suggesting pleiotropic effects on overall plant vigor.