Genetic variability of aquaporin expression in maize: from eQTLs to a MITE insertion regulating PIP2; 5 expression.

Plant aquaporins are involved in numerous physiological processes, such as cellular homeostasis, tissue hydraulics, transpiration, and nutrient supply, and are key players of the response to environmental cues. While varying expression patterns of aquaporin genes have been described across organs, developmental stages and stress conditions, the underlying regulation mechanisms remain elusive. Hence, this work aimed to shed light on the expression variability of four plasma membrane intrinsic protein (PIP) genes in maize (Zea mays) leaves, and its genetic causes, through eQTL (expression quantitative trait locus) mapping across a 252-hybrid diversity panel. Significant genetic variability in PIP transcript abundance was observed to different extents depending on the isoforms. The genome-wide association study mapped numerous eQTLs, both local and distant, thus emphasizing the existing natural diversity of PIP gene expression across the studied panel and the potential to reveal regulatory actors and mechanisms. One eQTL associated with PIP2; 5 expression variation was characterized. Genomic sequence comparison and in vivo reporter assay attributed, at least partly, the local eQTL to a transposon-containing polymorphism in the PIP2; 5 promoter. This work paves the way to the molecular understanding of PIP gene regulation and its possible integration into larger networks regulating physiological and stress-adaptation processes.

Genotyping of DNA pools identifies untapped landraces and genomic regions to develop next-generation varieties.

Landraces, that is, traditional varieties, have a large diversity that is underexploited in modern breeding. A novel DNA pooling strategy was implemented to identify promising landraces and genomic regions to enlarge the genetic diversity of modern varieties. As proof of concept, DNA pools from 156 American and European maize landraces representing 2340 individuals were genotyped with an SNP array to assess their genome-wide diversity. They were compared to elite cultivars produced across the 20th century, represented by 327 inbred lines. Detection of selective footprints between landraces of different geographic origin identified genes involved in environmental adaptation (flowering times, growth) and tolerance to abiotic and biotic stress (drought, cold, salinity). Promising landraces were identified by developing two novel indicators that estimate their contribution to the genome of inbred lines: (i) a modified Roger's distance standardized by gene diversity and (ii) the assignation of lines to landraces using supervised analysis. It showed that most landraces do not have closely related lines and that only 10 landraces, including famous landraces as Reid's Yellow Dent, Lancaster Surecrop and Lacaune, cumulated half of the total contribution to inbred lines. Comparison of ancestral lines directly derived from landraces with lines from more advanced breeding cycles showed a decrease in the number of landraces with a large contribution. New inbred lines derived from landraces with limited contributions enriched more the haplotype diversity of reference inbred lines than those with a high contribution. Our approach opens an avenue for the identification of promising landraces for pre-breeding.

Maize adaptation across temperate climates was obtained via expression of two florigen genes.

Expansion of the maize growing area was central for food security in temperate regions. In addition to the suppression of the short-day requirement for floral induction, it required breeding for a large range of flowering time that compensates the effect of South-North gradients of temperatures. Here we show the role of a novel florigen gene, ZCN12, in the latter adaptation in cooperation with ZCN8. Strong eQTLs of ZCN8 and ZCN12, measured in 327 maize lines, accounted for most of the genetic variance of flowering time in platform and field experiments. ZCN12 had a strong effect on flowering time of transgenic Arabidopsis thaliana plants; a path analysis showed that it directly affected maize flowering time together with ZCN8. The allelic composition at ZCN QTLs showed clear signs of selection by breeders. This suggests that florigens played a central role in ensuring a large range of flowering time, necessary for adaptation to temperate areas.

Independent introductions and admixtures have contributed to adaptation of European maize and its American counterparts.

Through the local selection of landraces, humans have guided the adaptation of crops to a vast range of climatic and ecological conditions. This is particularly true of maize, which was domesticated in a restricted area of Mexico but now displays one of the broadest cultivated ranges worldwide. Here, we sequenced 67 genomes with an average sequencing depth of 18x to document routes of introduction, admixture and selective history of European maize and its American counterparts. To avoid the confounding effects of recent breeding, we targeted germplasm (lines) directly derived from landraces. Among our lines, we discovered 22,294,769 SNPs and between 0.9% to 4.1% residual heterozygosity. Using a segmentation method, we identified 6,978 segments of unexpectedly high rate of heterozygosity. These segments point to genes potentially involved in inbreeding depression, and to a lesser extent to the presence of structural variants. Genetic structuring and inferences of historical splits revealed 5 genetic groups and two independent European introductions, with modest bottleneck signatures. Our results further revealed admixtures between distinct sources that have contributed to the establishment of 3 groups at intermediate latitudes in North America and Europe. We combined differentiation- and diversity-based statistics to identify both genes and gene networks displaying strong signals of selection. These include genes/gene networks involved in flowering time, drought and cold tolerance, plant defense and starch properties. Overall, our results provide novel insights into the evolutionary history of European maize and highlight a major role of admixture in environmental adaptation, paralleling recent findings in humans.

High throughput genotyping of structural variations in a complex plant genome using an original Affymetrix® axiom® array.

BACKGROUND: Insertions/deletions (InDels) and more specifically presence/absence variations (PAVs) are pervasive in several species and have strong functional and phenotypic effect by removing or drastically modifying genes. Genotyping of such variants on large panels remains poorly addressed, while necessary for approaches such as association mapping or genomic selection. RESULTS: We have developed, as a proof of concept, a new high-throughput and affordable approach to genotype InDels. We first identified 141,000 InDels by aligning reads from the B73 line against the genome of three temperate maize inbred lines (F2, PH207, and C103) and reciprocally. Next, we designed an Affymetrix® Axiom® array to target these InDels, with a combination of probes selected at breakpoint sites (13%) or within the InDel sequence, either at polymorphic (25%) or non-polymorphic sites (63%) sites. The final array design is composed of 662,772 probes and targets 105,927 InDels, including PAVs ranging from 35 bp to 129kbp. After Affymetrix® quality control, we successfully genotyped 86,648 polymorphic InDels (82% of all InDels interrogated by the array) on 445 maize DNA samples with 422,369 probes. Genotyping InDels using this approach produced a highly reliable dataset, with low genotyping error (~ 3%), high call rate (~ 98%), and high reproducibility (> 95%). This reliability can be further increased by combining genotyping of several probes calling the same InDels (< 0.1% error rate and > 99.9% of call rate for 5 probes). This "proof of concept" tool was used to estimate the kinship matrix between 362 maize lines with 57,824 polymorphic InDels. This InDels kinship matrix was highly correlated with kinship estimated using SNPs from Illumina 50 K SNP arrays. CONCLUSIONS: We efficiently genotyped thousands of small to large InDels on a sizeable number of individuals using a new Affymetrix® Axiom® array. This powerful approach opens the way to studying the contribution of InDels to trait variation and heterosis in maize. The approach is easily extendable to other species and should contribute to decipher the biological impact of InDels at a larger scale.

Genotyping-by-sequencing and SNP-arrays are complementary for detecting quantitative trait loci by tagging different haplotypes in association studies.

BACKGROUND: Single Nucleotide Polymorphism (SNP) array and re-sequencing technologies have different properties (e.g. calling rate, minor allele frequency profile) and drawbacks (e.g. ascertainment bias). This lead us to study their complementarity and the consequences of using them separately or combined in diversity analyses and Genome-Wide Association Studies (GWAS). We performed GWAS on three traits (grain yield, plant height and male flowering time) measured in 22 environments on a panel of 247 F1 hybrids obtained by crossing 247 diverse dent maize inbred lines with a same flint line. The 247 lines were genotyped using three genotyping technologies (Genotyping-By-Sequencing, Illumina Infinium 50 K and Affymetrix Axiom 600 K arrays). RESULTS: The effects of ascertainment bias of the 50 K and 600 K arrays were negligible for deciphering global genetic trends of diversity and for estimating relatedness in this panel. We developed an original approach based on linkage disequilibrium (LD) extent in order to determine whether SNPs significantly associated with a trait and that are physically linked should be considered as a single Quantitative Trait Locus (QTL) or several independent QTLs. Using this approach, we showed that the combination of the three technologies, which have different SNP distributions and densities, allowed us to detect more QTLs (gain in power) and potentially refine the localization of the causal polymorphisms (gain in resolution). CONCLUSIONS: Conceptually different technologies are complementary for detecting QTLs by tagging different haplotypes in association studies. Considering LD, marker density and the combination of different technologies (SNP-arrays and re-sequencing), the genotypic data available were most likely enough to well represent polymorphisms in the centromeric regions, whereas using more markers would be beneficial for telomeric regions.

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.

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies.

BACKGROUND: As for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies. RESULTS: Starting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance). CONCLUSIONS: Our association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment.

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.

Non-random distribution of extensive chromosome rearrangements in Brassica napus depends on genome organization.

Chromosome rearrangements are common, but their dynamics over time, mechanisms of occurrence and the genomic features that shape their distribution and rate are still poorly understood. We used allohaploid Brassica napus (AC, n = 19) as a model to analyze the effect of genomic features on the formation and diversity of meiotically driven chromosome rearrangements. We showed that allohaploid B. napus meiosis leads to extensive new structural diversity. Almost every allohaploid offspring carried a unique combination of multiple rearrangements throughout the genome, and was thus structurally differentiated from both its haploid parent and its sister plants. This large amount of genome reshuffling was remarkably well-tolerated in the heterozygous state, as neither male nor female fertility were strongly reduced, and meiosis behavior was normal in most cases. We also used a quantitative statistical model, which accounted for 75% of the observed variation in rearrangement rates, to show that the distribution of meiotically driven chromosome rearrangements was not random but was shaped by three principal genomic features. In descending order of importance, the rate of marker loss increased strongly with genetic distance from the centromere, the degree of collinearity between chromosomes, and the genome of origin (A < C). Overall, our results demonstrate that B. napus accumulates a large number of genetic changes, but these rearrangements are not randomly distributed in the genome. The structural genetic diversity produced by the allohaploid pathway and its role in the evolution of polyploid species compared to diploid meiosis are discussed.

Genetic diversity of maize landraces from the South-West of France.

From the 17th century until the arrival of hybrids in 1960s, maize landraces were cultivated in the South-West of France (SWF), a traditional region for maize cultivation. A set of landraces were collected in this area between the 1950s and 1980s and were then conserved ex situ in a germplam collection. Previous studies using molecular markers on approx. twenty landraces from this region suggested that they belonged to a Pyrenees-Galicia Flint genetic group and originated from hybridizations between Caribbean and Northern Flint germplasms introduced to Europe. In this study, we assessed the structure and genetic diversity of 194 SWF maize landraces to better elucidate their origin, using a 50K SNP array and a bulk DNA approach. We identified two weakly differentiated genetic groups, one in the Western part and the other in the Eastern part of the studied region. We highlighted the existence of a longitudinal gradient along the SWF area that was probably maintained through the interplay between genetic drifts and restricted gene flows. The contact zone between the two groups observed near the Garonne valley may be the result of these evolutionnary forces. We found in landraces from the East part of the region significant cases of admixture between landraces from the Northern Flint group and landraces from either the Caribbean, Andean or Italian groups. We then assumed that SWF landraces had a multiple origin with a predonderance of Northern Flint germplasm for the two SWF groups, notably for the East part.

Deciphering the Genetic Diversity of Landraces With High-Throughput SNP Genotyping of DNA Bulks: Methodology and Application to the Maize 50k Array.

Genebanks harbor original landraces carrying many original favorable alleles for mitigating biotic and abiotic stresses. Their genetic diversity remains, however, poorly characterized due to their large within genetic diversity. We developed a high-throughput, cheap and labor saving DNA bulk approach based on single-nucleotide polymorphism (SNP) Illumina Infinium HD array to genotype landraces. Samples were gathered for each landrace by mixing equal weights from young leaves, from which DNA was extracted. We then estimated allelic frequencies in each DNA bulk based on fluorescent intensity ratio (FIR) between two alleles at each SNP using a two step-approach. We first tested either whether the DNA bulk was monomorphic or polymorphic according to the two FIR distributions of individuals homozygous for allele A or B, respectively. If the DNA bulk was polymorphic, we estimated its allelic frequency by using a predictive equation calibrated on FIR from DNA bulks with known allelic frequencies. Our approach: (i) gives accurate allelic frequency estimations that are highly reproducible across laboratories, (ii) protects against false detection of allele fixation within landraces. We estimated allelic frequencies of 23,412 SNPs in 156 landraces representing American and European maize diversity. Modified Roger's genetic Distance between 156 landraces estimated from 23,412 SNPs and 17 simple sequence repeats using the same DNA bulks were highly correlated, suggesting that the ascertainment bias is low. Our approach is affordable, easy to implement and does not require specific bioinformatics support and laboratory equipment, and therefore should be highly relevant for large-scale characterization of genebanks for a wide range of species.

Homeologous recombination plays a major role in chromosome rearrangements that occur during meiosis of Brassica napus haploids.

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid x euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A-A and/or C-C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.

Testing the link between genome size and growth rate in maize.

Little is known about the factors driving within species Genome Size (GS) variation. GS may be shaped indirectly by natural selection on development and adaptative traits. Because GS variation is particularly pronounced in maize, we have sampled 83 maize inbred lines from three well described genetic groups adapted to contrasted climate conditions: inbreds of tropical origin, Flint inbreds grown in temperate climates, and Dent inbreds distributed in the Corn Belt. As a proxy for growth rate, we measured the Leaf Elongation Rate maximum during nighttime (LERmax) as well as GS in all inbred lines. In addition we combined available and new nucleotide polymorphism data at 29,090 sites to characterize the genetic structure of our panel. We found significant variation for both LERmax and GS among groups defined by our genetic structuring. Tropicals displayed larger GS than Flints while Dents exhibited intermediate values. LERmax followed the opposite trend with greater growth rate in Flints than in Tropicals. In other words, LERmax and GS exhibited a significantly negative correlation (r = - 0.27). However, this correlation was driven by among-group variation rather than within-group variation-it was no longer significant after controlling for structure and kinship among inbreds. Our results indicate that selection on GS may have accompanied ancient maize diffusion from its center of origin, with large DNA content excluded from temperate areas. Whether GS has been targeted by more intense selection during modern breeding within groups remains an open question.

Genetic regulation of meiotic cross-overs between related genomes in Brassica napus haploids and hybrids.

Although the genetic regulation of recombination in allopolyploid species plays a pivotal role in evolution and plant breeding, it has received little recent attention, except in wheat (Triticum aestivum). PrBn is the main locus that determines the number of nonhomologous associations during meiosis of microspore cultured Brassica napus haploids (AC; 19 chromosomes). In this study, we examined the role played by PrBn in recombination. We generated two haploid x euploid populations using two B. napus haploids with differing PrBn (and interacting genes) activity. We analyzed molecular marker transmission in these two populations to compare genetic changes, which have arisen during meiosis. We found that cross-over number in these two genotypes was significantly different but that cross-overs between nonhomologous chromosomes showed roughly the same distribution pattern. We then examined genetic recombination along a pair of A chromosomes during meiosis of B. rapa x B. napus AAC and AACC hybrids that were produced with the same two B. napus genotypes. We observed significant genotypic variation in cross-over rates between the two AAC hybrids but no difference between the two AACC hybrids. Overall, our results show that PrBn changes the rate of recombination between nonhomologous chromosomes during meiosis of B. napus haploids and also affects homologous recombination with an effect that depends on plant karyotype.