Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae.

BACKGROUND: The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. RESULTS: We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary. CONCLUSIONS: Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Single-cell RNA sequencing identifies senescence as therapeutic target in rhabdomyolysis-induced acute kidney injury.

BACKGROUND: The role of macrophages in the development of rhabdomyolysis-induced acute kidney injury (RM-AKI) has been established, but an in-depth understanding of the changes in the immune landscape could help to improve targeted strategies. Whereas senescence is usually associated with chronic kidney processes, we also wished to explore whether senescence could also occur in AKI and whether senolytics could act on immune cells. METHODS: Single-cell RNA sequencing was used in the murine glycerol-induced RM-AKI model to dissect the transcriptomic characteristics of CD45+ live cells sorted from kidneys 2 days after injury. Public datasets from murine AKI models were reanalysed to explore cellular senescence signature in tubular epithelial cells (TECs). A combination of senolytics (dasatinib and quercetin, DQ) was administered to mice exposed or not to RM-AKI. RESULTS: Unsupervised clustering of nearly 17 000 single-cell transcriptomes identified seven known immune cell clusters. Sub-clustering of the mononuclear phagocyte cells revealed nine distinct cell sub-populations differently modified with RM. One macrophage cluster was particularly interesting since it behaved as a critical node in a trajectory connecting one major histocompatibility complex class IIhigh (MHCIIhigh) cluster only present in Control to two MHCIIlow clusters only present in RM-AKI. This critical cluster expressed a senescence gene signature, that was very different from that of the TECs. Senolytic DQ treatment blocked the switch from a F4/80highCD11blow to F4/80lowCD11bhigh phenotype, which correlated with prolonged nephroprotection in RM-AKI. CONCLUSIONS: Single-cell RNA sequencing unmasked novel transitional macrophage subpopulation associated with RM-AKI characterized by the activation of cellular senescence processes. This work provides a proof-of-concept that senolytics nephroprotective effects may rely, at least in part, on subtle immune modulation.

Multi-genome comparisons reveal gain-and-loss evolution of the anti-Mullerian hormone receptor type 2 gene, an old master sex determining gene, in Percidae.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplications (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Genome structures resolve the early diversification of teleost fishes.

Accurate species phylogenies are a prerequisite for all evolutionary research. Teleosts are the largest and most diversified group of extant vertebrates, but relationships among their three oldest extant lineages remain unresolved. On the basis of seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (arapaima, elephantnose fish) in a monophyletic sister group to all other teleosts, i.e., the Clupeocephala lineage (zebrafish, medaka). This finding resolves more than 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies.

Generation of a chromosome-level genome assembly for Pacific halibut (Hippoglossus stenolepis) and characterization of its sex-determining genomic region.

The Pacific halibut (Hippoglossus stenolepis) is a key species in the North Pacific Ocean and Bering Sea ecosystems, where it also supports important fisheries. However, the lack of genomic resources limits our understanding of evolutionary, environmental and anthropogenic forces affecting key life history characteristics of Pacific halibut and prevents the application of genomic tools in fisheries management and conservation efforts. In the present study, we report on the first generation of a high-quality chromosome-level assembly of the Pacific halibut genome, with an estimated size of 602 Mb, 24 chromosome-length scaffolds that contain 99.8% of the assembly and a N50 scaffold length of 27.3 Mb. In the first application of this important resource, we conducted genome-wide analyses of sex-specific genetic variation by pool sequencing and characterized a potential sex-determining region in chromosome 9 with a high density of female-specific SNPs. Within this region, we identified the bmpr1ba gene as a potential candidate for master sex-determining (MSD) gene. bmpr1ba is a member of the TGF-ß family that in teleosts has provided the largest number of MSD genes, including a paralogue of this gene in Atlantic herring. The genome assembly constitutes an essential resource for future studies on Pacific halibut population structure and dynamics, evolutionary history and responses to environmental and anthropogenic influences. Furthermore, the genomic location of the sex-determining region in Pacific halibut has been identified and a putative candidate MSD gene has been proposed, providing further support for the rapid evolution of sex-determining mechanisms in teleost fish.

An ancient truncated duplication of the anti-Müllerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family.

The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12 Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type Ⅱ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100 million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor ß pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.

A duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas).

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFß signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.

A supernumerary "B-sex" chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus.

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species3,4 that have often been considered as selfish genetic elements that behave as genome parasites.5,6 The observation that in some species Bs can be either restricted or predominant in one sex7-14 raised the interesting hypothesis that Bs could play a role in sex determination.15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

Genome structure and content of the rice root-knot nematode (Meloidogyne graminicola).

Discovered in the 1960s, Meloidogyne graminicola is a root-knot nematode species considered as a major threat to rice production. Yet, its origin, genomic structure, and intraspecific diversity are poorly understood. So far, such studies have been limited by the unavailability of a sufficiently complete and well-assembled genome. In this study, using a combination of Oxford Nanopore Technologies and Illumina sequencing data, we generated a highly contiguous reference genome (283 scaffolds with an N50 length of 294 kb, totaling 41.5 Mb). The completeness scores of our assembly are among the highest currently published for Meloidogyne genomes. We predicted 10,284 protein-coding genes spanning 75.5% of the genome. Among them, 67 are identified as possibly originating from horizontal gene transfers (mostly from bacteria), which supposedly contribute to nematode infection, nutrient processing, and plant defense manipulation. Besides, we detected 575 canonical transposable elements (TEs) belonging to seven orders and spanning 2.61% of the genome. These TEs might promote genomic plasticity putatively related to the evolution of M. graminicola parasitism. This high-quality genome assembly constitutes a major improvement regarding previously available versions and represents a valuable molecular resource for future phylogenomic studies of Meloidogyne species. In particular, this will foster comparative genomic studies to trace back the evolutionary history of M. graminicola and its closest relatives.

Sex chromosome and sex locus characterization in goldfish, Carassius auratus (Linnaeus, 1758).

BACKGROUND: Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. Here we used sequencing approaches to better characterize sex determination and sex-chromosomes in an experimental strain of goldfish. RESULTS: Our results confirmed that sex determination in goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18 °C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (~ 11.7 Mb) on goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the coding DNA sequence of the goldfish amh gene. CONCLUSIONS: These results show that our goldfish strain has a relatively large sex locus on LG22, which is likely the Y chromosome of this experimental population. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers, which will be important tools for future research on sex determination in our experimental goldfish population. However, additional work would be needed to explore whether this sex locus is conserved in other populations of goldfish.

Genome Sequence of the Euryhaline Javafish Medaka, Oryzias javanicus: A Small Aquarium Fish Model for Studies on Adaptation to Salinity.

The genus Oryzias consists of 35 medaka-fish species each exhibiting various ecological, morphological and physiological peculiarities and adaptations. Beyond of being a comprehensive phylogenetic group for studying intra-genus evolution of several traits like sex determination, behavior, morphology or adaptation through comparative genomic approaches, all medaka species share many advantages of experimental model organisms including small size and short generation time, transparent embryos and genome editing tools for reverse and forward genetic studies. The Java medaka, Oryzias javanicus, is one of the two species of medaka perfectly adapted for living in brackish/sea-waters. Being an important component of the mangrove ecosystem, O. javanicus is also used as a valuable marine test-fish for ecotoxicology studies. Here, we sequenced and assembled the whole genome of O. javanicus, and anticipate this resource will be catalytic for a wide range of comparative genomic, phylogenetic and functional studies. Complementary sequencing approaches including long-read technology and data integration with a genetic map allowed the final assembly of 908 Mbp of the O. javanicus genome. Further analyses estimate that the O. javanicus genome contains 33% of repeat sequences and has a heterozygosity of 0.96%. The achieved draft assembly contains 525 scaffolds with a total length of 809.7 Mbp, a N50 of 6,3 Mbp and a L50 of 37 scaffolds. We identified 21454 predicted transcripts for a total transcriptome size of 57, 146, 583 bps. We provide here a high-quality chromosome scale draft genome assembly of the euryhaline Javafish medaka (321 scaffolds anchored on 24 chromosomes (representing 97.7% of the total bases)), and give emphasis on the evolutionary adaptation to salinity.

Characterization of a Y-specific duplication/insertion of the anti-Mullerian hormone type II receptor gene based on a chromosome-scale genome assembly of yellow perch, Perca flavescens.

Yellow perch, Perca flavescens, is an ecologically and economically important species native to a large portion of the northern United States and southern Canada and is also a promising candidate species for aquaculture. However, no yellow perch reference genome has been available to facilitate improvements in both fisheries and aquaculture management practices. By combining Oxford Nanopore Technologies long-reads, 10X Genomics Illumina short linked reads and a chromosome contact map produced with Hi-C, we generated a high-continuity chromosome-scale yellow perch genome assembly of 877.4 Mb. It contains, in agreement with the known diploid chromosome yellow perch count, 24 chromosome-size scaffolds covering 98.8% of the complete assembly (N50 = 37.4 Mb, L50 = 11). We also provide a first characterization of the yellow perch sex determination locus that contains a male-specific duplicate of the anti-Mullerian hormone type II receptor gene (amhr2by) inserted at the proximal end of the Y chromosome (chromosome 9). Using this sex-specific information, we developed a simple PCR genotyping assay which accurately differentiates XY genetic males (amhr2by+ ) from XX genetic females (amhr2by- ). Our high-quality genome assembly is an important genomic resource for future studies on yellow perch ecology, toxicology, fisheries and aquaculture research. In addition, characterization of the amhr2by gene as a candidate sex-determining gene in yellow perch provides a new example of the recurrent implication of the transforming growth factor beta pathway in fish sex determination, and highlights gene duplication as an important genomic mechanism for the emergence of new master sex determination genes.

Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.