Single‐nucleotide polymorphism discovery in Leptographium longiclavatum,a mountain pine beetle‐associated symbiotic fungus,using whole‐genome resequencing

Single‐nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost‐effective approaches to uncover genome‐wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole‐genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N₅₀ = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17 266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina^® Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species.     

Development and evaluation of a genome‐wide Coffee 8.5K SNP array and its application for high‐density genetic mapping and for investigating the origin of Coffea arabica L.

Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single‐nucleotide polymorphism (SNP) array for coffee trees. A total of 8580 unique and informative SNPs were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNPs (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high‐density genetic map by adding 1307 SNP markers, whereas 945 SNPs were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora‐derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high‐density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.     

Interactive effects of ozone and drought stress on pigments and activities of antioxidative enzymes in Pinus halepensis

The aim of this study was to investigate the interactive effects of ozone (O₃) and drought on pigments and antioxidant enzymes of Aleppo pine (Pinus halepensis). Two‐year‐old seedlings were exposed in open‐top chambers to charcoal‐filtered air or non‐filtered air plus an additional 40 nL L^?1 of ozone. After 20 months of O₃ exposure, a subset of plants was subjected to drought stress by withholding water supply for 11 d. Ozone induced higher guaiacol peroxidase, catalase and KCN‐resistant superoxide dismutase (SOD) activities in young needles, while drought stress increased glutathione reductase and CuZnSOD. One‐year‐old needles showed lower capacity to activate these enzymes in response to stress. Both ozone and drought activated the xanthophyll cycle pool and reduced chlorophyll contents in both current and 1‐year‐old needles. The combined effects of ozone and drought decreased antioxidant enzyme activities and the capacity of recovering after re‐watering. Similarly, interactive effects of O₃ and drought reduced xanthophyll‐mediated photoprotection capacity in 1‐year‐old needles but induced a higher conversion of the cycle in current‐year needles. These results showed that ozone modified the Aleppo pine response to drought stress, suggesting that this pollutant might be reducing the ability of this species to withstand other environmental stresses.     

Development and mapping of SNP assays in allotetraploid cotton

A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin–streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an N₅₀ of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency), a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating F₂ population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were able to identify within which genome (‘A’ or ‘D’) each SNP resided using diploid species sequence data. Genetic maps generated by these newly identified markers are being used to locate quantitative, economically important regions within the cotton genome.     

SNP discovery using Paired‐End RAD‐tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae)

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N₅₀ = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.     

Genomic atolls of differentiation in coral reef fishes (Hypoplectrus spp., Serranidae)

Because the vast majority of species are well diverged, relatively little is known about the genomic architecture of speciation during the early stages of divergence. Species within recent evolutionary radiations are often minimally diverged from a genomic perspective, and therefore provide rare opportunities to address this question. Here, we leverage the hamlet radiation (Hypoplectrus spp., brightly coloured reef fishes from the tropical western Atlantic) to characterize genomic divergence during the early stages of speciation. Transect surveys and spawning observations in Belize, Honduras and Panama confirm that sympatric barred (H. puella), black (H. nigricans) and butter (H. unicolor) hamlets are phenotypically distinct and reproductively isolated, although hybrid spawnings and individuals with intermediate phenotypes are seen on rare occasions. A survey of approximately 100 000 restriction site‐associated SNPs in 126 samples from the three species across the three replicate populations reveals extremely slight genomewide divergence among species (F_ST = 0.0038), indicating that ecomorphological differences and functional reproductive isolation are maintained in sympatry in a backdrop of extraordinary genomic similarity. Nonetheless, a very small proportion of SNPs (0.05% on average) are identified as F_ST outliers among sympatric species. Remarkably, a single SNP is identified as an outlier in repeated populations for the same species pair. A minicontig assembled de novo around this SNP falls into the genomic region containing the HoxCa10 and HoxCa11 genes in 10 teleost species, suggesting an important role for Hox gene evolution in this radiation. This finding, if confirmed, would provide a better understanding of the links between micro‐ and macroevolutionary processes.     

A survey of genome‐wide single nucleotide polymorphisms through genome resequencing in the Périgord black truffle (Tuber melanosporum Vittad.)

The Périgord black truffle (Tuber melanosporum Vittad.), considered a gastronomic delicacy worldwide, is an ectomycorrhizal filamentous fungus that is ecologically important in Mediterranean French, Italian and Spanish woodlands. In this study, we developed a novel resource of single nucleotide polymorphisms (SNPs) for T. melanosporum using Illumina high‐throughput resequencing. The genome from six T. melanosporum geographical accessions was sequenced to a depth of approximately 20×. These geographical accessions were selected from different populations within the northern and southern regions of the geographical species distribution. Approximately 80% of the reads for each of the six resequenced geographical accessions mapped against the reference T. melanosporum genome assembly, estimating the core genome size of this organism to be approximately 110 Mbp. A total of 442 326 SNPs corresponding to 3540 SNPs/Mbps were identified as being included in all seven genomes. The SNPs occurred more frequently in repeated sequences (85%), although 4501 SNPs were also identified in the coding regions of 2587 genes. Using the ratio of nonsynonymous mutations per nonsynonymous site (pN) to synonymous mutations per synonymous site (pS) and Tajima's D index scanning the whole genome, we were able to identify genomic regions and genes potentially subjected to positive or purifying selection. The SNPs identified represent a valuable resource for future population genetics and genomics studies.     

Single‐nucleotide polymorphism discovery and validation in high‐density SNP array for genetic analysis in European white oaks

An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full‐sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south‐western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x‐axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high‐throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.     

A 34K SNP genotyping array for Populus trichocarpa: Design,application to the study of natural populations and transferability to other Populus species

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost‐effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre‐ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.     

Development of an integrated 200K SNP genotyping array and application for genetic mapping,genome assembly improvement and genome wide association studies in pear (Pyrus)

Pear (Pyrus; 2n = 34), the third most important temperate fruit crop, has great nutritional and economic value. Despite the availability of many genomic resources in pear, it is challenging to genotype novel germplasm resources and breeding progeny in a timely and cost‐effective manner. Genotyping arrays can provide fast, efficient and high‐throughput genetic characterization of diverse germplasm, genetic mapping and breeding populations. We present here 200K AXIOM^® PyrSNP, a large‐scale single nucleotide polymorphism (SNP) genotyping array to facilitate genotyping of Pyrus species. A diverse panel of 113 re‐sequenced pear genotypes was used to discover SNPs to promote increased adoption of the array. A set of 188 diverse accessions and an F₁ population of 98 individuals from ‘Cuiguan’ × ‘Starkrimson’ was genotyped with the array to assess its effectiveness. A large majority of SNPs (166 335 or 83%) are of high quality. The high density and uniform distribution of the array SNPs facilitated prediction of centromeric regions on 17 pear chromosomes, and significantly improved the genome assembly from 75.5% to 81.4% based on genetic mapping. Identification of a gene associated with flowering time and candidate genes linked to size of fruit core via genome wide association studies showed the usefulness of the array in pear genetic research. The newly developed high‐density SNP array presents an important tool for rapid and high‐throughput genotyping in pear for genetic map construction, QTL identification and genomic selection.     

The easy road to genome‐wide medium density SNP screening in a non‐model species: development and application of a 10 K SNP‐chip for the house sparrow (Passer domesticus)

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations.     

Development of an SNP‐based parentage verification panel for lovebirds

The genus Agapornis, or lovebirds, are popular pet parrots worldwide. Currently, breeders are dependent on pedigree records as a selection tool as no molecular parentage verification test is available for any of the nine species. The A. roseicollis reference genome was recently assembled. This was followed by the sequencing of the whole genomes of the parents of the reference genome individual at 30× coverage. The parents’ reads were mapped against the reference genome to identify SNPs. Over 1.6 million SNPs, shared between the parents, were discovered using the Genome Analysis Toolkit pipeline. SNPs were filtered to a panel of 480 SNPs based on Genome Analysis Toolkit parameters. The panel of 480 SNPs was genotyped in a population of 960 lovebirds across seven species. A panel of 262 SNPs was compiled that included SNPs successfully amplified across all species. The 262‐SNP panel was reduced based on the observed heterozygosity (H_O) and minor allele frequency (MAF) values per SNP to include the lowest number of SNPs with the highest exclusion power for parentage verification. Two smaller panels consisting of 195 SNPs with MAF and H_O values >0.1 and 40 SNPs with MAF and H_O values >0.3, were constructed. The panels were verified using 43 families from different species with known relationships to evaluate the exclusion power of each panel. The 195 SNP panel with an average exclusion probability of 99.9% and MAF and H_O values >0.1 was proposed as the routine Agapornis parentage verification panel.