Comparative genomics of Mycoplasma feriruminatoris, a fast-growing pathogen of wild Caprinae.

Mycoplasma feriruminatoris is a fast-growing Mycoplasma species isolated from wild Caprinae and first described in 2013. M. feriruminatoris isolates have been associated with arthritis, kerato conjunctivitis, pneumonia and septicemia, but were also recovered from apparently healthy animals. To better understand what defines this species, we performed a genomic survey on 14 strains collected from free-ranging or zoo-housed animals between 1987 and 2017, mostly in Europe. The average chromosome size of the M. feriruminatoris strains was 1,040±0,024 kbp, with 24 % G+C and 852±31 CDS. The core genome and pan-genome of the M. feriruminatoris species contained 628 and 1312 protein families, respectively. The M. feriruminatoris strains displayed a relatively closed pan-genome, with many features and putative virulence factors shared with species from the M. mycoides cluster, including the MIB-MIP Ig cleavage system, a repertoire of DUF285 surface proteins and a complete biosynthetic pathway for galactan. M. feriruminatoris genomes were found to be mostly syntenic, although repertoires of mobile genetic elements, including Mycoplasma Integrative and Conjugative Elements, insertion sequences, and a single plasmid varied. Phylogenetic- and gene content analyses confirmed that M. feriruminatoris was closer to the M. mycoides cluster than to the ruminant species M. yeatsii and M. putrefaciens. Ancestral genome reconstruction showed that the emergence of the M. feriruminatoris species was associated with the gain of 17 gene families, some of which encode defence enzymes and surface proteins, and the loss of 25 others, some of which are involved in sugar transport and metabolism. This comparative study suggests that the M. mycoides cluster could be extended to include M. feriruminatoris. We also find evidence that the specific organization and structure of the DnaA boxes around the oriC of M. feriruminatoris may contribute to drive the remarkable fast growth of this minimal bacterium.

Fruit fly phylogeny imprints bacterial gut microbiota.

One promising avenue for reconciling the goals of crop production and ecosystem preservation consists in the manipulation of beneficial biotic interactions, such as between insects and microbes. Insect gut microbiota can affect host fitness by contributing to development, host immunity, nutrition, or behavior. However, the determinants of gut microbiota composition and structure, including host phylogeny and host ecology, remain poorly known. Here, we used a well-studied community of eight sympatric fruit fly species to test the contributions of fly phylogeny, fly specialization, and fly sampling environment on the composition and structure of bacterial gut microbiota. Comprising both specialists and generalists, these species belong to five genera from to two tribes of the Tephritidae family. For each fly species, one field and one laboratory samples were studied. Bacterial inventories to the genus level were produced using 16S metabarcoding with the Oxford Nanopore Technology. Sample bacterial compositions were analyzed with recent network-based clustering techniques. Whereas gut microbiota were dominated by the Enterobacteriaceae family in all samples, microbial profiles varied across samples, mainly in relation to fly identity and sampling environment. Alpha diversity varied across samples and was higher in the Dacinae tribe than in the Ceratitinae tribe. Network analyses allowed grouping samples according to their microbial profiles. The resulting groups were very congruent with fly phylogeny, with a significant modulation of sampling environment, and with a very low impact of fly specialization. Such a strong imprint of host phylogeny in sympatric fly species, some of which share much of their host plants, suggests important control of fruit flies on their gut microbiota through vertical transmission and/or intense filtering of environmental bacteria.

Evidence of local adaptation despite strong drift in a Neotropical patchily distributed bromeliad.

Both genetic drift and divergent selection are predicted to be drivers of population differentiation across patchy habitats, but the extent to which these forces act on natural populations to shape traits is strongly affected by species' ecological features. In this study, we infer the genomic structure of Pitcairnia lanuginosa, a widespread herbaceous perennial plant with a patchy distribution. We sampled populations in the Brazilian Cerrado and the Central Andean Yungas and discovered and genotyped SNP markers using double-digest restriction-site associated DNA sequencing. In addition, we analyzed ecophysiological traits obtained from a common garden experiment and compared patterns of phenotypic and genetic divergence (PST-FST comparisons) in a subset of populations from the Cerrado. Our results from molecular analyses pointed to extremely low genetic diversity and a remarkable population differentiation, supporting a major role of genetic drift. Approximately 0.3% of genotyped SNPs were flagged as differentiation outliers by at least two distinct methods, and Bayesian generalized linear mixed models revealed a signature of isolation by environment in addition to isolation by distance for high-differentiation outlier SNPs among the Cerrado populations. PST-FST comparisons suggested divergent selection on two ecophysiological traits linked to drought tolerance. We showed that these traits vary among populations, although without any particular macro-spatial pattern, suggesting local adaptation to differences in micro-habitats. Our study shows that selection might be a relevant force, particularly for traits involved in drought stress, even for populations experiencing strong drift, which improves our knowledge on eco-evolutionary processes acting on non-continuously distributed species.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20-40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

From microsatellites to single nucleotide polymorphisms for the genetic monitoring of a critically endangered sturgeon.

The use of genetic information is crucial in conservation programs for the establishment of breeding plans and for the evaluation of restocking success. Short tandem repeats (STRs) have been the most widely used molecular markers in such programs, but next-generation sequencing approaches have prompted the transition to genome-wide markers such as single nucleotide polymorphisms (SNPs). Until now, most sturgeon species have been monitored using STRs. The low diversity found in the critically endangered European sturgeon (Acipenser sturio), however, makes its future genetic monitoring challenging, and the current resolution needs to be increased. Here, we describe the discovery of a highly informative set of 79 SNPs using double-digest restriction-associated DNA (ddRAD) sequencing and its validation by genotyping using the MassARRAY system. Comparing with STRs, the SNP panel proved to be highly efficient and reproducible, allowing for more accurate parentage and kinship assignments' on 192 juveniles of known pedigree and 40 wild-born adults. We explore the effectiveness of both markers to estimated relatedness and inbreeding, using simulated and empirical datasets. Interestingly, we found significant correlations between STRs and SNPs at individual heterozygosity and inbreeding that give support to a reasonable representation of whole genome diversity for both markers. These results are useful for the conservation program of A. sturio in building a comprehensive studbook, which will optimize conservation strategies. This approach also proves suitable for other case studies in which highly discriminatory genetic markers are needed to assess parentage and kinship.

Development of Target Sequence Capture and Estimation of Genomic Relatedness in a Mixed Oak Stand.

Anticipating the evolutionary responses of long-lived organisms, such as trees, to environmental changes, requires the assessment of genetic variation of adaptive traits in natural populations. To this end, high-density markers are needed to calculate genomic relatedness between individuals allowing to estimate the genetic variance of traits in wild populations. We designed a targeted capture-based, next-generation sequencing assay based on the highly heterozygous pedunculate oak (Quercus robur) reference genome, for the sequencing of 3 Mb of genic and intergenic regions. Using a mixed stand of 293 Q. robur and Q. petraea genotypes we successfully captured over 97% of the target sequences, corresponding to 0.39% of the oak genome, with sufficient depth (97×) for the detection of about 190,000 SNPs evenly spread over the targeted regions. We validated the technique by evaluating its reproducibility, and comparing the genomic relatedness of trees with their known pedigree relationship. We explored the use of the technique on other related species and highlighted the advantages and limitations of this approach. We found that 92.07% of target sequences in Q. suber and 70.36% of sequences in Fagus sylvatica were captured. We used this SNP resource to estimate genetic relatedness in the mixed oak stand. Mean pairwise genetic relatedness was low within each species with a few values exceeding 0.25 (half-sibs) or 0.5 (full-sibs). Finally, we applied the technique to a long-standing issue in population genetics of trees regarding the relationship between inbreeding and components of fitness. We found very weak signals for inbreeding depression for reproductive success and no signal for growth within both species.

Correction: Linkage and Association Mapping for Two Major Traits Used in the Maritime Pine Breeding Program: Height Growth and Stem Straightness.

[This corrects the article DOI: 10.1371/journal.pone.0165323.].

Linkage and Association Mapping for Two Major Traits Used in the Maritime Pine Breeding Program: Height Growth and Stem Straightness.

BACKGROUND: Increasing our understanding of the genetic architecture of complex traits, through analyses of genotype-phenotype associations and of the genes/polymorphisms accounting for trait variation, is crucial, to improve the integration of molecular markers into forest tree breeding. In this study, two full-sib families and one breeding population of maritime pine were used to identify quantitative trait loci (QTLs) for height growth and stem straightness, through linkage analysis (LA) and linkage disequilibrium (LD) mapping approaches. RESULTS: The populations used for LA consisted of two unrelated three-generation full-sib families (n = 197 and n = 477). These populations were assessed for height growth or stem straightness and genotyped for 248 and 217 markers, respectively. The population used for LD mapping consisted of 661 founders of the first and second generations of the breeding program. This population was phenotyped for the same traits and genotyped for 2,498 single-nucleotide polymorphism (SNP) markers corresponding to 1,652 gene loci. The gene-based reference genetic map of maritime pine was used to localize and compare the QTLs detected by the two approaches, for both traits. LA identified three QTLs for stem straightness and two QTLs for height growth. The LD study yielded seven significant associations (P ≤ 0.001): four for stem straightness and three for height growth. No colocalisation was found between QTLs identified by LA and SNPs detected by LD mapping for the same trait. CONCLUSIONS: This study provides the first comparison of LA and LD mapping approaches in maritime pine, highlighting the complementary nature of these two approaches for deciphering the genetic architecture of two mandatory traits of the breeding program.

Performance of genomic prediction within and across generations in maritime pine.

BACKGROUND: Genomic selection (GS) is a promising approach for decreasing breeding cycle length in forest trees. Assessment of progeny performance and of the prediction accuracy of GS models over generations is therefore a key issue. RESULTS: A reference population of maritime pine (Pinus pinaster) with an estimated effective inbreeding population size (status number) of 25 was first selected with simulated data. This reference population (n = 818) covered three generations (G0, G1 and G2) and was genotyped with 4436 single-nucleotide polymorphism (SNP) markers. We evaluated the effects on prediction accuracy of both the relatedness between the calibration and validation sets and validation on the basis of progeny performance. Pedigree-based (best linear unbiased prediction, ABLUP) and marker-based (genomic BLUP and Bayesian LASSO) models were used to predict breeding values for three different traits: circumference, height and stem straightness. On average, the ABLUP model outperformed genomic prediction models, with a maximum difference in prediction accuracies of 0.12, depending on the trait and the validation method. A mean difference in prediction accuracy of 0.17 was found between validation methods differing in terms of relatedness. Including the progenitors in the calibration set reduced this difference in prediction accuracy to 0.03. When only genotypes from the G0 and G1 generations were used in the calibration set and genotypes from G2 were used in the validation set (progeny validation), prediction accuracies ranged from 0.70 to 0.85. CONCLUSIONS: This study suggests that the training of prediction models on parental populations can predict the genetic merit of the progeny with high accuracy: an encouraging result for the implementation of GS in the maritime pine breeding program.

Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies.

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

Nucleotide polymorphisms in a pine ortholog of the Arabidopsis degrading enzyme cellulase KORRIGAN are associated with early growth performance in Pinus pinaster.

We have carried out a candidate-gene-based association genetic study in Pinus pinaster Aiton and evaluated the predictive performance for genetic merit gain of the most significantly associated genes and single nucleotide polymorphisms (SNPs). We used a second generation 384-SNP array enriched with candidate genes for growth and wood properties to genotype mother trees collected in 20 natural populations covering most of the European distribution of the species. Phenotypic data for total height, polycyclism, root-collar diameter and biomass were obtained from a replicated provenance-progeny trial located in two sites with contrasting environments (Atlantic vs Mediterranean climate). General linear models identified strong associations between growth traits (total height and polycyclism) and four SNPs from the korrigan candidate gene, after multiple testing corrections using false discovery rate. The combined genomic breeding value predictions assessed for the four associated korrigan SNPs by ridge regression-best linear unbiased prediction (RR-BLUP) and cross-validation accounted for up to 8 and 15% of the phenotypic variance for height and polycyclic growth, respectively, and did not improve adding SNPs from other growth-related candidate genes. For root-collar diameter and total biomass, they accounted for 1.6 and 1.1% of the phenotypic variance, respectively, but increased to 15 and 4.1% when other SNPs from lp3.1, lp3.3 and cad were included in RR-BLUP models. These results point towards a desirable integration of candidate-gene studies as a means to pre-select relevant markers, and aid genomic selection in maritime pine breeding programs.

Imprints of natural selection along environmental gradients in phenology-related genes of Quercus petraea.

We explored single nucleotide polymorphism (SNP) variation in candidate genes for bud burst from Quercus petraea populations sampled along gradients of latitude and altitude in Western Europe. SNP diversity was monitored for 106 candidate genes, in 758 individuals from 32 natural populations. We investigated whether SNP variation reflected the clinal pattern of bud burst observed in common garden experiments. We used different methods to detect imprints of natural selection (FST outlier, clinal variation at allelic frequencies, association tests) and compared the results obtained for the two gradients. FST outlier SNPs were found in 15 genes, 5 of which were common to both gradients. The type of selection differed between the two gradients (directional or balancing) for 3 of these 5. Clinal variations were observed for six SNPs, and one cline was conserved across both gradients. Association tests between the phenotypic or breeding values of trees and SNP genotypes identified 14 significant associations, involving 12 genes. The results of outlier detection on the basis of population differentiation or clinal variation were not very consistent with the results of association tests. The discrepancies between these approaches may reflect the different hierarchical levels of selection considered (inter- and intrapopulation selection). Finally, we obtained evidence for convergent selection (similar for gradients) and clinal variation for a few genes, suggesting that comparisons between parallel gradients could be used to screen for major candidate genes responding to natural selection in trees.

High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination.

BACKGROUND: The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. RESULTS: In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. CONCLUSION: This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

Comparative mapping in the Fagaceae and beyond with EST-SSRs.

BACKGROUND: Genetic markers and linkage mapping are basic prerequisites for comparative genetic analyses, QTL detection and map-based cloning. A large number of mapping populations have been developed for oak, but few gene-based markers are available for constructing integrated genetic linkage maps and comparing gene order and QTL location across related species. RESULTS: We developed a set of 573 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12 oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in 5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and Quercus petraea (sessile oak). Consensus maps for the two species were constructed and aligned. They showed a high degree of macrosynteny between these two sympatric European oaks. We assessed the transferability of EST-SSRs to other Fagaceae genera and a subset of these markers was mapped in Castanea sativa, the European chestnut. Reasonably high levels of macrosynteny were observed between oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs, to support further population genetic analyses with gene-based markers. Finally, based on the orthologous relationships between the oak, Arabidopsis, grape, poplar, Medicago, and soybean genomes and the paralogous relationships between the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak chromosomes from the eudicot ancestral karyotype. CONCLUSIONS: This study provides map locations for a large set of EST-SSRs in two oak species of recognized biological importance in natural ecosystems. This first step toward the construction of a gene-based linkage map will facilitate the assignment of future genome scaffolds to pseudo-chromosomes. This study also provides an indication of the potential utility of new gene-based markers for population genetics and comparative mapping within and beyond the Fagaceae.

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. RESULTS: We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 in vitro SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 in silico SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for in silico and in vitro SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. CONCLUSIONS: Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine.

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon x Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0-34.4 and 28.9-31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14-70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.