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.

Unraveling the history of the genus Gallus through whole genome sequencing.

The genus Gallus is distributed across a large part of Southeast Asia and has received special interest because the domestic chicken, Gallus gallus domesticus, has spread all over the world and is a major protein source for humans. There are four species: the red junglefowl (G. gallus), the green junglefowl (G. varius), the Lafayette's junglefowl (G. lafayettii) and the grey junglefowl (G. sonneratii). The aim of this study is to reconstruct the history of these species by a whole genome sequencing approach and resolve inconsistencies between well supported topologies inferred using different data and methods. Using deep sequencing, we identified over 35 million SNPs and reconstructed the phylogeny of the Gallus genus using both distance (BioNJ) and maximum likelihood (ML) methods. We observed discrepancies according to reconstruction methods and genomic components. The two most supported topologies were previously reported and were discriminated by using phylogenetic and gene flow analyses, based on ABBA statistics. Terminology fix requested by the deputy editor led to support a scenario with G. gallus as the earliest branching lineage of the Gallus genus, instead of G. varius. We discuss the probable causes for the discrepancy. A likely one is that G. sonneratii samples from parks or private collections are all recent hybrids, with roughly 10% of their autosomal genome originating from G. gallus. The removal of those regions is needed to provide reliable data, which was not done in previous studies. We took care of this and additionally included two wild G. sonneratii samples from India, showing no trace of introgression. This reinforces the importance of carefully selecting and validating samples and genomic components in phylogenomics.

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.

Defensive behaviour of Apis mellifera against Vespa velutina in France: testing whether European honeybees can develop an effective collective defence against a new predator.

We investigated the prey-predator interactions between the European honeybee, Apis mellifera, and the invasive yellow-legged hornet, Vespa velutina, which first invaded France in 2004 and thereafter spread to neighbouring European countries (Spain, Portugal and Italy). Our goal was to determine how successfully honeybees are able to defend their colonies against their new predator in Europe. Experiments were conducted in the southwest of France-the point of entry of the hornet in Europe-under natural and semi-controlled field conditions. We investigated a total of eight apiaries and 95 colonies subjected to either low or high levels of predation. We analyzed hornet predatory behaviour and collective response of colonies under attack. The results showed that A. mellifera in France exhibit an inefficient and unorganized defence against V. velutina, unlike in other regions of Europe and other areas around the globe where honeybees have co-evolved with their natural Vespa predators.

Geographic variation of melanisation patterns in a hornet species: genetic differences, climatic pressures or aposematic constraints?

Coloration of stinging insects is often based on contrasted patterns of light and black pigmentations as a warning signal to predators. However, in many social wasp species, geographic variation drastically modifies this signal through melanic polymorphism potentially driven by different selective pressures. To date, surprisingly little is known about the geographic variation of coloration of social wasps in relation to aposematism and melanism and to genetic and developmental constraints. The main objectives of this study are to improve the description of the colour variation within a social wasp species and to determine which factors are driving this variation. Therefore, we explored the evolutionary history of a polymorphic hornet, Vespa velutina Lepeletier, 1836, using mitochondrial and microsatellite markers, and we analysed its melanic variation using a colour space based on a description of body parts coloration. We found two main lineages within the species and confirmed the previous synonymy of V. auraria Smith, 1852, under V. velutina, differing only by the coloration. We also found that the melanic variation of most body parts was positively correlated, with some segments forming potential colour modules. Finally, we showed that the variation of coloration between populations was not related to their molecular, geographic or climatic differences. Our observations suggest that the coloration patterns of hornets and their geographic variations are determined by genes with an influence of developmental constraints. Our results also highlight that Vespa velutina populations have experienced several convergent evolutions of the coloration, more likely influenced by constraints on aposematism and Müllerian mimicry than by abiotic pressures on melanism.

Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives.

Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective.

Deciduous trees and the application of universal DNA barcodes: a case study on the circumpolar Fraxinus.

The utility of DNA barcoding for identifying representative specimens of the circumpolar tree genus Fraxinus (56 species) was investigated. We examined the genetic variability of several loci suggested in chloroplast DNA barcode protocols such as matK, rpoB, rpoC1 and trnH-psbA in a large worldwide sample of Fraxinus species. The chloroplast intergenic spacer rpl32-trnL was further assessed in search for a potentially variable and useful locus. The results of the study suggest that the proposed cpDNA loci, alone or in combination, cannot fully discriminate among species because of the generally low rates of substitution in the chloroplast genome of Fraxinus. The intergenic spacer trnH-psbA was the best performing locus, but genetic distance-based discrimination was moderately successful and only resulted in the separation of the samples at the subgenus level. Use of the BLAST approach was better than the neighbor-joining tree reconstruction method with pairwise Kimura's two-parameter rates of substitution, but allowed for the correct identification of only less than half of the species sampled. Such rates are substantially lower than the success rate required for a standardised barcoding approach. Consequently, the current cpDNA barcodes are inadequate to fully discriminate Fraxinus species. Given that a low rate of substitution is common among the plastid genomes of trees, the use of the plant cpDNA "universal" barcode may not be suitable for the safe identification of tree species below a generic or sectional level. Supplementary barcoding loci of the nuclear genome and alternative solutions are proposed and discussed.