Genetic causes and genomic consequences of breakdown of distyly in Linum trigynum.

Distyly is an iconic floral polymorphism governed by a supergene, which promotes efficient pollen transfer and outcrossing through reciprocal differences in the position of sexual organs in flowers, often coupled with heteromorphic self-incompatibility (SI). Distyly has evolved convergently in multiple flowering plant lineages, but has also broken down repeatedly, often resulting in homostylous, self-compatible populations with elevated rates of self-fertilization. Here, we aimed to study the genetic causes and genomic consequences of the shift to homostyly in Linum trigynum, which is closely related to distylous Linum tenue. Building on a high-quality genome assembly, we show that L. trigynum harbors a genomic region homologous to the dominant haplotype of the distyly supergene conferring long stamens and short styles in L. tenue, suggesting that loss of distyly first occurred in a short-styled individual. In contrast to homostylous Primula and Fagopyrum, L. trigynum harbors no fixed loss-of-function mutations in coding sequences of S-linked distyly candidate genes. Instead, floral gene expression analyses and controlled crosses suggest that mutations downregulating the S-linked LtWDR-44 candidate gene for male SI and/or anther height could underlie homostyly and self-compatibility (SC) in L. trigynum. Population genomic analyses of 224 whole-genome sequences further demonstrate that L. trigynum is highly self-fertilizing, exhibits significantly lower genetic diversity genome-wide, and is experiencing relaxed purifying selection and less frequent positive selection on nonsynonymous mutations relative to L. tenue. Our analyses shed light on the loss of distyly in L. trigynum, and advance our understanding of a common evolutionary transition in flowering plants.

Genomic analyses of the Linum distyly supergene reveal convergent evolution at the molecular level.

Supergenes govern multi-trait-balanced polymorphisms in a wide range of systems; however, our understanding of their origins and evolution remains incomplete. The reciprocal placement of stigmas and anthers in pin and thrum floral morphs of distylous species constitutes an iconic example of a balanced polymorphism governed by a supergene, the distyly S-locus. Recent studies have shown that the Primula and Turnera distyly supergenes are both hemizygous in thrums, but it remains unknown whether hemizygosity is pervasive among distyly S-loci. As hemizygosity has major consequences for supergene evolution and loss, clarifying whether this genetic architecture is shared among distylous species is critical. Here, we have characterized the genetic architecture and evolution of the distyly supergene in Linum by generating a chromosome-level genome assembly of Linum tenue, followed by the identification of the S-locus using population genomic data. We show that hemizygosity and thrum-specific expression of S-linked genes, including a pistil-expressed candidate gene for style length, are major features of the Linum S-locus. Structural variation is likely instrumental for recombination suppression, and although the non-recombining dominant haplotype has accumulated transposable elements, S-linked genes are not under relaxed purifying selection. Our findings reveal remarkable convergence in the genetic architecture and evolution of independently derived distyly supergenes, provide a counterexample to classic inversion-based supergenes, and shed new light on the origin and maintenance of an iconic floral polymorphism.

Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing.

BACKGROUND: Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. RESULTS: We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20-25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20-25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. CONCLUSION: We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

The Carniolan Honeybee from Slovenia-A Complete and Annotated Mitochondrial Genome with Comparisons to Closely Related Apis mellifera Subspecies.

The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera-a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies' conservation and preservation as well as its breeding and selection.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

BACKGROUND: The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map. RESULTS: Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor > 98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features. CONCLUSIONS: The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping.

BACKGROUND: It remains a challenge to perform de novo assembly using next-generation sequencing (NGS). Despite the availability of multiple sequencing technologies and tools (e.g., assemblers) it is still difficult to assemble new genomes at chromosome resolution (i.e., one sequence per chromosome). Obtaining high quality draft assemblies is extremely important in the case of yeast genomes to better characterise major events in their evolutionary history. The aim of this work is two-fold: on the one hand we want to show how combining different and somewhat complementary technologies is key to improving assembly quality and correctness, and on the other hand we present a de novo assembly pipeline we believe to be beneficial to core facility bioinformaticians. To demonstrate both the effectiveness of combining technologies and the simplicity of the pipeline, here we present the results obtained using the Dekkera bruxellensis genome. METHODS: In this work we used short-read Illumina data and long-read PacBio data combined with the extreme long-range information from OpGen optical maps in the task of de novo genome assembly and finishing. Moreover, we developed NouGAT, a semi-automated pipeline for read-preprocessing, de novo assembly and assembly evaluation, which was instrumental for this work. RESULTS: We obtained a high quality draft assembly of a yeast genome, resolved on a chromosomal level. Furthermore, this assembly was corrected for mis-assembly errors as demonstrated by resolving a large collapsed repeat and by receiving higher scores by assembly evaluation tools. With the inclusion of PacBio data we were able to fill about 5 % of the optical mapped genome not covered by the Illumina data.

Transcriptome of the alternative ethanol production strain Dekkera bruxellensis CBS 11270 in sugar limited, low oxygen cultivation.

Dekkera bruxellensis can outcompete Saccharomyces cerevisiae in environments with low sugar concentrations. It is usually regarded as a spoilage yeast but has lately been identified as an alternative ethanol production organism. In this study, global gene expression in the industrial isolate D. bruxellensis CBS 11270 under oxygen and glucose limitation was investigated by whole transcriptome sequencing using the AB SOLiD technology. Among other observations, we noted expression of respiratory complex I NADH-ubiquinone reductase although D. bruxellensis is a Crabtree positive yeast. The observed higher expression of NADH-generating enzymes compared to NAD(+)-generating enzymes might be the reason for the previously observed NADH imbalance and resulting Custer effect in D. bruxellensis. Low expression of genes involved in glycerol production is probably the molecular basis for high efficiency of D. bruxellensis metabolism under nutrient limitation. No D. bruxellensis homologs to the genes involved in the final reactions of glycerol biosynthesis were detected. A high number of expressed sugar transporter genes is consistent with the hypothesis that the competitiveness of D. bruxellensis is due to a higher affinity for the limiting substrate.

Xerochrysium gen. nov. and Bettsia, genera encompassing xerophilic species of Chrysosporium.

On the basis of a study of ITS sequences, Vidal et al. (Rev. Iber. Micol. 17: 22, 2000) recommended that the genus Chrysosporium be restricted to species belonging to Onygenales. Using nrLSU genes, we studied the majority of clades examined by Vidal et al. and showed that currently accepted species in Chrysosporium phylogenetically belong in six clades in three orders. Surprisingly, the xerophilic species of Chrysosporium, long thought to be a single grouping away from the majority of Chrysosporium species, occupy two clades, one in Leotiales, the other in Eurotiales. Species accepted in Leotiales are related to the sexual genus Bettsia. One is the type species B. alvei, and related asexual strains classified as C. farinicola, the second is C. fastidium transferred to Bettsia as B. fastidia. Species in the Eurotiales are transferred to Xerochrysium gen. nov., where the accepted species are X. xerophilum and X. dermatitidis, the correct name for C. inops on transfer to Xerochrysium. All accepted species are extreme xerophiles, found in dried and concentrated foods.

Phylogeny and intraspecific variation of the extreme xerophile, Xeromyces bisporus.

The filamentous ascomycete Xeromyces bisporus is an extreme xerophile able to grow down to a water activity of 0.62. We have inferred the phylogenetic position of Xeromyces in relation to other xerophilic and xerotolerant fungi in the order Eurotiales. Using nrDNA and betatubulin sequences, we show that it is more closely related to the xerophilic foodborne species of the genus Chrysosporium, than to the genus Monascus. The taxonomy of X. bisporus and Monascus is discussed. Based on physiological, morphological, and phylogenetic distinctiveness, we suggest that Xeromyces should be retained as a separate genus.

The extreme xerophilic mould Xeromyces bisporus--growth and competition at various water activities.

Little is known about the mould, Xeromyces bisporus, unique in its strong xerophilicity and ability to grow at water activity (a(w)) 0.62, lower than for any other known organism. The linear growth rates of one fast and one slow-growing strain of X. bisporus were assessed at 20, 25, 30 and 37 °C on solid agar media containing a mixture of glucose and fructose to reduce a(w) to 0.94, 0.88, 0.84, 0.80, 0.76 and 0.66. Growth rates of xerophilic species closely related to X. bisporus, viz. Chrysosporium inops, C. xerophilum and Monascus eremophilus, were also assessed. Optimal conditions for growth of both X. bisporus strains were approx. 0.84 a(w) and 30°C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. X. bisporus FRR 2347 even grew well at 0.66 a(w) (0.48 mm/day). C. inops and C. xerophilum were more tolerant of high a(w) than X. bisporus, and could be differentiated from each other based on: the faster growth of C. xerophilum; its preference for temperatures ≥ 30 °C and a(w) ≥ 0.94 (c.f.≤ 25 °C and ~0.88 a(w) for C. inops); and its ability to grow at 0.66 a(w), which is the lowest a(w) reported to date for this species. M. eremophilus grew slowly (max. 0.4mm/day) even in its optimal conditions of ~0.88 a(w) and 25 °C. To investigate the competitive characteristics of X. bisporus at low a(w), both X. bisporus strains were grown in dual-culture with xerotolerant species Aspergillus flavus and Penicillium roqueforti, and xerophilic species A. penicillioides, C. inops, C. xerophilum and Eurotium chevalieri, on glucose-fructose agar plates at 0.94, 0.84, 0.80 and 0.76 a(w) and at 25 °C. Growth rates and types of interactions were assessed. Excretion of inhibitory substances acting over a long-range was not observed by any species; inhibitors acting over a short-range that temporarily slowed competitors' growth or produced a protective zone around the colony were occasionally observed for A. penicillioides, C. inops and C. xerophilum. Instead, rapid growth relative to the competitor was the most common means of dominance. The xerotolerant species, A. flavus and P. roqueforti were dominant over X. bisporus at 0.94 a(w). E. chevalieri was often dominant due to its rapid growth over the entire a(w) range. At a(w)<0.80, X. bisporus was competitive because it grew faster than the other species examined. This supports the concept that its ideal environmental niche is sugary foods with low a(w).