Cold reduces maize (Zea mays L.) production and delays sowings. Cold tolerance in maize is very limited, and breeding maize for cold tolerance is still a major challenge. Our objective was to detect QTL for cold tolerance at germination and seedling stages. We evaluated, under cold and control conditions, 919 Dent and 1009 Flint inbred lines from two nested association mapping designs consisting in 24 double-haploid populations, genotyped with 56,110 SNPs. We found a large diversity of maize cold tolerance within these NAM populations. We detected one QTL for plant weight and four for fluorescence under cold conditions, as well as one for plant weight and two for chlorophyll content under control conditions in the Dent-NAM. There were fewer significant QTL under control conditions than under cold conditions, and half of the QTL were for quantum efficiency of photosystem II. Our results supported the large genetic discrepancy between optimal and low temperatures, as the quantity and the position of the QTL were very variable between control and cold conditions. Furthermore, as we have not found alleles with significant effects on these NAM designs, further studies are needed with other experimental designs to find favorable alleles with important effects for improving cold tolerance in maize.

Temperate grasslands provide both habitats and flower resources for pollinators in agricultural landscapes. Plant-pollinator networks change according to local and landscape variables, which are important to identify to help conserve pollinators in grasslands. We analysed plant-pollinator networks in 18 grasslands on experimental dairy farms located in three French regions contrasting by their climate, altitude, landscape or management. We combined visual surveys and pollen DNA barcoding. Our objectives were to determine which environmental factors influence pollinator taxa abundance and diversity and differences among the visual plant-pollinator networks in the three farming regions. Flower-visiting insects were trapped in six grasslands per farm during three sessions from mid-April to mid-July along fixed 400 m(2) transects. Insects were identified individually to the lowest taxonomic rank possible. Pollen carried by insects was identified using nuclear ribosomal ITS2 sequences belonging to the NCBI nucleotide database. The size and diversity of plant-pollinator networks were much larger and higher in permanent grasslands at the two farms located in lowlands (Mirecourt) and mountains (Marcenat) than those at the farm with temporary grasslands and a crop landscape (Lusignan), but the degree of specialisation (H2') was relatively similar and low (mean of 0.46). Diptera, especially Empididae and Syrphidae, represented most plant-pollinator interactions in Mirecourt and Marcenat, while Hymenoptera were more abundant at Lusignan. The percentage of semi-natural habitats in 500 m buffers and vegetation height explained 23% of the variance in pollinator abundance. Ranunculus sp. Knautia arvensis, Centaurea jacea and Trifolium repens were key plant species in the networks. DNA metabarcoding of pollen loads identified 114 genera in addition to those identified by visual observations (+34-42 per site), reflecting insects' floral pathways and differences in the immediate landscape among farms. This study highlighted the importance of Diptera in plant-pollinator networks and the need to conserve permanent grassland diversity to conserve pollinators.

Next-Generation Sequencing (NGS) technologies, by reducing the cost and increasing the throughput of sequencing, have opened doors to generate genomic data in a range of previously poorly studied species. In this study, we propose a method for the rapid development of a large-scale molecular resources for orphan species. We studied as an example the true lavender (Lavandula angustifolia Mill.), a perennial sub-shrub plant native from the Mediterranean region and whose essential oil have numerous applications in cosmetics, pharmaceuticals, and alternative medicines. The heterozygous clone "Maillette" was used as a reference for DNA and RNA sequencing. We first built a reference Unigene, compound of coding sequences, thanks to de novo RNA-seq assembly. Then, we reconstructed the complete genes sequences (with introns and exons) using an Unigene-guided DNA-seq assembly approach. This aimed to maximize the possibilities of finding polymorphism between genetically close individuals despite the lack of a reference genome. Finally, we used these resources for SNP mining within a collection of 16 commercial lavender clones and tested the SNP within the scope of a genetic distance analysis. We obtained a cleaned reference of 8, 030 functionally in silico annotated genes. We found 359K polymorphic sites and observed a high SNP frequency (mean of 1 SNP per 90 bp) and a high level of heterozygosity (more than 60% of heterozygous SNP per genotype). On overall, we found similar genetic distances between pairs of clones, which is probably related to the out-crossing nature of the species and the restricted area of cultivation. The proposed method is transferable to other orphan species, requires little bioinformatics resources and can be realized within a year. This is also the first reported large-scale SNP development on Lavandula angustifolia. All the genomics resources developed herein are publicly available and provide a rich pool of molecular resources to explore and exploit lavender genetic diversity in breeding programs.

Three hundred and eighteen symptomatic potato samples were collected from the main potato growing provinces of Tunisia, in main and late season crops (respectively spring and autumn 2013). Double antibody sandwich ELISA (DAS-ELISA) was used to assess the presence of the five most common potato viruses [ Potato virus A (PVA), Potato virus X (PVX), Potato virus Y (PVY), Potato leafroll virus (PLRV) and Potato virus S (PVS)]. According to ELISA results, the highest overall percentage of infected samples was observed in the late season (84.5%), in comparison to the main season (30.7%). PVY was dominant in all infected plots with detection rates ranging from 20% in the North region (main season) to 100% in Cap Bon (late season). PLRV was the second most detected virus, with 4.3% distributed in the North West and Sahel during the main season, but was not detected in the late season. PVX was found with even lower detection rates of 0.6% and 1.3% in main and late seasons, respectively. No infection by PVA or PVS was detected in these two periods. Positive PVX detection results were confirmed by Reverse Transcription PCR using total RNA extracted from infected leaves. The near-complete nucleotide sequences of three PVX isolates were determined using an Illumina MiSeq sequencer. Phylogenetic analyses on full genome sequences or on coat protein (CP) gene sequences show that Tunisian PVX isolates belong to low diversity clade I.

Because of their abundance and their amenability to high-throughput genotyping techniques, Single Nucleotide Polymorphisms (SNPs) are powerful tools for efficient genetics and genomics studies, including characterization of genetic resources, genome-wide association studies and genomic selection. In wheat, most of the previous SNP discovery initiatives targeted the coding fraction, leaving almost 98% of the wheat genome largely unexploited. Here we report on the use of whole-genome resequencing data from eight wheat lines to mine for SNPs in the genic, the repetitive and non-repetitive intergenic fractions of the wheat genome. Eventually, we identified 3.3 million SNPs, 49% being located on the B-genome, 41% on the A-genome and 10% on the D-genome. We also describe the development of the TaBW280K high-throughput genotyping array containing 280,226 SNPs. Performance of this chip was examined by genotyping a set of 96 wheat accessions representing the worldwide diversity. Sixty-nine percent of the SNPs can be efficiently scored, half of them showing a diploid-like clustering. The TaBW280K was proven to be a very efficient tool for diversity analyses, as well as for breeding as it can discriminate between closely related elite varieties. Finally, the TaBW280K array was used to genotype a population derived from a cross between Chinese Spring and Renan, leading to the construction a dense genetic map comprising 83,721 markers. The results described here will provide the wheat community with powerful tools for both basic and applied research.

Faced with the decline of pollinators, it is relevant to strengthen our understanding of the whole plant-pollinator web in semi-natural grasslands that serve as refuges for pollinator populations. The aim of this study was to explore the diversity of flower-foraging insects involved in pollen transfer in mountain semi-natural grasslands. Insects actively collecting pollen and/or nectar were caught in spring in six mountain semi-natural grasslands displaying a floristic richness gradient. Individual determinations of insects were made at the finest possible taxonomic scale and pollen loads were removed from the insect body. Using next-generation DNA sequencing, pollens were identified through the ribosomal DNA cistron using the ITS2 database and the ITS plant rDNA cistron sequences from Genbank. A total of 236 flower-foraging insects were collected. Diptera represented 82% of the total catches distantly followed by Hymenoptera (15%) and Apoidea (bees) (11%). Visual observations revealed that Diptera foraged on 16 of the 21 flower species visited by insects. DNA metabarcoding showed that 82% (191) of all of the collected insects were carrying pollen and 44% (104) were carrying two genera of plants or more. Our results demonstrate that Diptera are potential key-pollinators in mountain semi-natural grasslands that cannot be overlooked by the scientific community. However difficulties of taxonomic determination due to severe shortage of experts for Diptera have to be urgently overcome. Further studies on the link between pollen transfer and actual pollination in a global change context are also required. Moreover, our results support the idea that DNA metabarcoding provides accurate information about the plants-insects networks but it also pointed out sensitive issues, especially the necessity to build reliable national barcode databases.

A high-quality reference for the sunflower genome (Helianthus annuus L.) and analysis of gene networks involved in flowering time and oil metabolism provide a basis for nutritional exploitation and analyses of adaptation to climate change. Nicolas Langlade and colleagues report the genome sequence of the domesticated sunflower, Helianthus annuus L., a global oil crop that can maintain stable yields across a wide range of environmental conditions. Their comparative analyses provide insights into the evolutionary history of Asterids. They also analysed transcriptomic data from vegetative and floral organs, re-sequenced 80 domesticated lines and performed genome-wide association studies identifying 35 loci associated with flowering time. These resources will be useful in breeding programs as well as ecological and evolutionary studies. The domesticated sunflower, Helianthus annuus L., is a global oil crop that has promise for climate change adaptation, because it can maintain stable yields across a wide variety of environmental conditions, including drought1. Even greater resilience is achievable through the mining of resistance alleles from compatible wild sunflower relatives2,3, including numerous extremophile species4. Here we report a high-quality reference for the sunflower genome (3.6 gigabases), together with extensive transcriptomic data from vegetative and floral organs. The genome mostly consists of highly similar, related sequences5 and required single-molecule real-time sequencing technologies for successful assembly. Genome analyses enabled the reconstruction of the evolutionary history of the Asterids, further establishing the existence of a whole-genome triplication at the base of the Asterids II clade6 and a sunflower-specific whole-genome duplication around 29 million years ago7. An integrative approach combining quantitative genetics, expression and diversity data permitted development of comprehensive gene networks for two major breeding traits, flowering time and oil metabolism, and revealed new candidate genes in these networks. We found that the genomic architecture of flowering time has been shaped by the most recent whole-genome duplication, which suggests that ancient paralogues can remain in the same regulatory networks for dozens of millions of years. This genome represents a cornerstone for future research programs aiming to exploit genetic diversity to improve biotic and abiotic stress resistance and oil production, while also considering agricultural constraints and human nutritional needs8,9.

The continuing increase in size and quality of the “short reads” raw data is a significant help for the quality of the assembly obtained through various bioinformatics tools. However, building a reference genome sequence for most plant species remains a significant challenge due to the large number of repeated sequences which are problematic for a whole-genome quality de novo assembly. Furthermore, for most SNP identification approaches in plant genetics and breeding, only the “Gene-space” regions including the promoter, exon and intron sequences are considered.

Semi natural grasslands are considered as a vital habitat for wild pollinators, which in return contribute to preserve the floristic diversity of this environment. To study the interactions between pollinators and plants, flower-foraging insects were caught from beginning of May to end of June along walking transects in 6 mountain permanent grasslands in Cantal (France). We developed and test in parallel a method based on DNA barcoding analysis, allowing a quick identification of the insect and its pollen load at the same time. We collected thus 394 flower visitor insects, most of them belonging to Diptera (72%) of which comprised 32% Empididae and 20% Syrphidae, and 20 % belonging to Hymenoptera of which 80% were wild native and domestic bees. Three families of flowers (Asteraceae, Apiaceae and Ranunculaceae) comprised two thirds of the total flowering species from which the insects were collected. DNA barcoding of these insects showed that 87% of the collected insects were carrying pollen and 45% were carrying two genders of plants or more. Results suggest the important role of the Diptera as wild pollinators at this period in such mountain environment. Moreover, our results have demonstrated that the DNA barcoding is a powerful tool to study flower-foraging insects and their pollens loads which will be very soon operational.

AbstractSunflower (Helianthus annuus L.) is used as “seed snacks”, so called “pipas”, in the Mediterranean countries which may offer an interesting opportunity for agricultural diversification of this crop. The morpho-phenological variability already demonstrated in the Tunisian local populations can be a basis for the creation of new varieties well adapted to climate and soil conditions in that region. The molecular characterization of 59 accessions and reference lines generated 194 alleles from 30 SSR loci (3–10 alleles per locus) and 54 haplotypes, built from 117 SNP detected by NGS sequencing of 7 genes (4–16 haplotypes per gene). These data highlight some uniqueness of the Tunisian material compared to 7 control lines but a low genetic dispersion between accessions. However, a core collection of 8 populations, capturing 88 % of the Tunisian genetic diversity, could be proposed for a future sunflower breeding program.

BACKGROUND:Breeding for cold tolerance in maize promises to allow increasing growth area and production in temperate zones. The objective of this research was to conduct genome-wide association analyses (GWAS) in temperate maize inbred lines and to find strategies for pyramiding genes for cold tolerance. Two panels of 306 dent and 292 European flint maize inbred lines were evaluated per se and in testcrosses under cold and control conditions in a growth chamber. We recorded indirect measures for cold tolerance as the traits number of days from sowing to emergence, relative leaf chlorophyll content or quantum efficiency of photosystem II. Association mapping for identifying genes associated to cold tolerance in both panels was based on genotyping with 49,585 genome-wide single nucleotide polymorphism (SNP) markers.RESULTS:We found 275 significant associations, most of them in the inbreds evaluated per se, in the flint panel, and under control conditions. A few candidate genes coincided between the current research and previous reports. A total of 47 flint inbreds harbored the favorable alleles for six significant quantitative trait loci (QTL) detected for inbreds per se evaluated under cold conditions, four of them had also the favorable alleles for the main QTL detected from the testcrosses. Only four dent inbreds (EZ47, F924, NK807 and PHJ40) harbored the favorable alleles for three main QTL detected from the evaluation of the dent inbreds per se under cold conditions. There were more QTL in the flint panel and most of the QTL were associated with days to emergence and ΦPSII.CONCLUSIONS:These results open new possibilities to genetically improve cold tolerance either with genome-wide selection or with marker assisted selection.

Le service de pollinisation rendu par l'ecosysteme prairial est tres souvent mis en avant mais il est peu explicite et peu quantifie. L'objectif de cette etude est de quantifier les interactions plante-insecte a l'origine de ce service dans des prairies appartenant a 3 agrosystemes contrastes. Pour cela, nous avons capture les insectes butinant les fleurs sur des transects, a 3 periodes de l'annee, dans 18 prairies presentant un gradient de richesse floristique et appartenant a 3 fermes experimentales INRA situee a Marcenat (Cantal), Mirecourt (Vosges) et Lusignan (Vienne). Nous avons teste en parallele une methode d'analyse basee sur le meta-barcoding, permettant la determination simultanee de l'insecte et des pollens qu'il transporte. Nous avons analyse 979 couples fleur-insecte butineur. Les resultats mettent en evidence le role important des Dipteres dans les prairies permanentes temperees multi-especes en tant que butineurs et pollinisateurs potentiels, notamment en debut de saison de vegetation. Elle montre egalement que les interactions entre familles d'insectes et familles de plantes observees a Marcenat sont plus nombreuses et reparties plus equitablement qu'a Mirecourt et Lusignan. Une forte proportion des insectes captures (82 %) transportait du pollen et l'analyse des barcodes ADN revele que 44 % transportaient en meme temps de 2 a 6 genres botaniques differents. Ces premiers resultats permettent d'identifier les acteurs potentiels du service de pollinisation dans les prairies.

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.

Since mid-2005, Illumina Infinium® genotyping arrays provide data with good accuracy for thousands of SNPs (single nucleotide polymorphism) in thousands individuals for many organisms including plants. These extensive data used in large studies gave new insight in population and molecular genetics. Nevertheless, flexible high density genotyping tools at reasonable costs are still to be developed. Low cost genotyping assays would allow many applications including control of individuals, marker assisted management and genetic approaches in species where molecular developments have a comparatively low value relative to the cost of SNP arrays. The initial cost and/or the minimum sample number requirement are limiting factors for developing a new genotyping tool. An add-on option on already existing product gives a first opportunity to reduce the cost of a beadchip. Designing a multi-species array could be another alternative to increase the number of SNPs and decrease the minimum number of samples for each species, reaching lower genotyping cost assay per species.

The availability of a saturated genetic map of Clementine was identified by the International Citrus Genome Consortium as an essential prerequisite to assist the assembly of the reference whole genome sequence based on a Clementine derived haploid. The primary goals of the present study were to establish a Clementine reference map, and to perform comparative mapping with pummelo and sweet orange. Five parental genetic maps were established with SNPs, SSRs and InDels. A medium density reference map (961 markers for 1084.1 cM) of Clementine was established and used by the ICGC to facilitate the chromosome assembly of the haploid genome sequence. Comparative mapping with pummelo and sweet orange revealed that the linear order of markers was highly conserved. Reasonable inferences of most citrus genomes should be obtained by mapping next-generation sequencing data against the haploid reference genome sequence. Skewed segregations were frequent and higher in the male than female Clementine potentially leading to false interpretation of the genetic determinism of phenotypic traits. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange and identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Introgression of pummelo genome fragments were identified in heterozygosity in each chromosome. Moreover, it appeared that the genome of the haploid Clementine used to establish the citrus reference genome sequence was inherited primarily from the 'Mediterranean' mandarin. The usefulness of this genetic map, anchored in the reference whole genome sequence, is discussed. (Resume d'auteur)