Orthologous Loci Research Articles

Enhancer polymorphisms at the IKZF1 susceptibility locus for acute lymphoblastic leukemia impact B-cell proliferation and differentiation in both Down syndrome and non-Down syndrome genetic backgrounds.

Children with Down syndrome have an approximately 10-fold increased risk of developing acute lymphoblastic leukemia and this risk is influenced by inherited genetic variation. Genome-wide association studies have identified IKZF1 as a strong acute lymphoblastic leukemia susceptibility locus in children both with and without Down syndrome, with association signals reported at rs4132601 in non-Down syndrome and rs58923657 in individuals with Down syndrome (r2 = 0.98 for these two loci). Expression quantitative trait locus analysis in non-Down syndrome lymphoblastoid cell lines has demonstrated an association between the rs4132601 risk allele and decreased IKZF1 mRNA levels. In this study, we provide further mechanistic evidence linking the region encompassing IKZF1-associated polymorphisms to pro-leukemogenic effects in both human lymphoblastoid cell lines and murine hematopoietic stem cells. CRISPR/Cas9-mediated deletion of the region encompassing the rs17133807 major allele (r2 with rs58923657 = 0.97) resulted in significant reduction of IKZF1 mRNA levels in lymphoblastoid cell lines, with a greater effect in Down syndrome versus non-Down syndrome cells. Since rs17133807 is highly conserved in mammals, we also evaluated the orthologous murine locus at rs263378223, in hematopoietic stem cells from the Dp16(1)Yey mouse model of Down syndrome as well as non-Down syndrome control mice. Homozygous deletion of the region encompassing rs263378223 resulted in significantly reduced Ikzf1 mRNA, confirming that this polymorphism maps to a strong murine Ikzf1 enhancer, and resulted in increased B-lymphoid colony growth and decreased B-lineage differentiation. Our results suggest that both the region encompassing rs17133807 and its conserved orthologous mouse locus have functional effects that may mediate increased leukemia susceptibility in both the Down syndrome and non-Down syndrome genetic backgrounds.

Genetic insights into the evolution of genera with the eastern Asia-eastern North America floristic disjunction: a transcriptomics analysis.

Large disjunctions in species distributions provide excellent opportunities to study processes that shape biogeographic patterns. One such disjunction is the eastern Asia-eastern North America (EA-ENA) floristic disjunction. For many genera with this disjunction, species richness is greater in EA than in ENA; this pattern has been attributed, in part, to higher rates of molecular evolution and speciation in EA. Longer branch lengths have been found in some EA clades, relative to their ENA sister clades, suggesting that the EA lineages have evolved at a higher rate, possibly due to environmental heterogeneity, potentially contributing to the species richness anomaly. To evaluate whether rates of molecular evolution are elevated in EA relative to ENA, we used transcriptomes from species in 11 genera displaying this disjunction. Rates of molecular evolution were estimated for up to 385 orthologous nuclear loci per genus. No statistically significant differences were identified in pairwise comparisons between EA and ENA sister species, suggesting equal rates of molecular evolution for both species; the data also suggest similar selection pressures in both regions. For larger genera, evidence likewise argues against more species-rich clades having higher molecular evolutionary rates, regardless of region. Our results suggest that genes across multiple gene ontology categories are evolving at similar rates under purifying selection in species in both regions. Our data support the hypothesis that greater species richness in EA than ENA is due to factors other than an overall increase in rates of molecular evolution in EA.

Pod indehiscence in common bean is associated with the fine regulation of PvMYB26.

In legumes, pod shattering occurs when mature pods dehisce along the sutures, and detachment of the valves promotes seed dispersal. In Phaseolus vulgaris (L)., the major locus qPD5.1-Pv for pod indehiscence was identified recently. We developed a BC4/F4 introgression line population and narrowed the major locus down to a 22.5 kb region. Here, gene expression and a parallel histological analysis of dehiscent and indehiscent pods identified an AtMYB26 orthologue as the best candidate for loss of pod shattering, on a genomic region ~11 kb downstream of the highest associated peak. Based on mapping and expression data, we propose early and fine up-regulation of PvMYB26 in dehiscent pods. Detailed histological analysis establishes that pod indehiscence is associated with the lack of a functional abscission layer in the ventral sheath, and that the key anatomical modifications associated with pod shattering in common bean occur early during pod development. We finally propose that loss of pod shattering in legumes resulted from histological convergent evolution and that it is the result of selection at orthologous loci.

Evolutionary History of Alpha Satellite DNA Repeats Dispersed within Human Genome Euchromatin

Major human alpha satellite DNA repeats are preferentially assembled within (peri)centromeric regions but are also dispersed within euchromatin in the form of clustered or short single repeat arrays. To study the evolutionary history of single euchromatic human alpha satellite repeats (ARs), we analyzed their orthologous loci across the primate genomes. The continuous insertion of euchromatic ARs throughout the evolutionary history of primates starting with the ancestors of Simiformes (45–60 Ma) and continuing up to the ancestors of Homo is revealed. Once inserted, the euchromatic ARs were stably transmitted to the descendant species, some exhibiting copy number variation, whereas their sequence divergence followed the species phylogeny. Many euchromatic ARs have sequence characteristics of (peri)centromeric alpha repeats suggesting heterochromatin as a source of dispersed euchromatic ARs. The majority of euchromatic ARs are inserted in the vicinity of other repetitive elements such as L1, Alu, and ERV or are embedded within them. Irrespective of the insertion context, each AR insertion seems to be unique and once inserted, ARs do not seem to be subsequently spread to new genomic locations. In spite of association with (retro)transposable elements, there is no indication that such elements play a role in ARs proliferation. The presence of short duplications at most of ARs insertion sites suggests site-directed recombination between homologous motifs in ARs and in the target genomic sequence, probably mediated by extrachromosomal circular DNA, as a mechanism of spreading within euchromatin.

New Insights Into the Nature of Interspecific Hybrid Sterility in Rice.

Interspecific and intraspecific hybrid sterility is a typical and common phenomenon of postzygotic reproductive barrier in rice. This is an indicator of speciation involved in the formation of new species or subspecies, and it significantly hampers the utilization of favorable genes from distant parents for rice improvement. The Oryza genus includes eight species with the same AA genome and is a model plant for studying the nature of hybrid sterility and its relationship with speciation. Hybrid sterility in rice is mostly controlled by nuclear genes, with more than 50 sterility loci genetically identified to date, of which 10 hybrid sterility loci or pairs were cloned and characterized at the molecular level. Comparing the mapping results for all sterility loci reported indicated that some of these loci from different species should be allelic to each other. Further research revealed that interactions between the multiple alleles at the hybrid sterility locus caused various genetic effect. One hypothesis for this important phenomenon is that the hybrid sterility loci are orthologous loci, which existed in ancient ancestors of rice. When one or more ancestors drifted to different continents, genetic divergence occurred because of adaptation, selection, and isolation among them such that various alleles from orthologous loci emerged over evolutionary time; hence, interspecific hybrid sterility would be mainly controlled by a few orthologous loci with different alleles. This hypothesis was tested and supported by the molecular characterization of hybrid sterility loci from S1, S5, Sa, qHMS7, and S27. From this, we may further deduce that both allelic and non-allelic interactions among different loci are the major genetic basis for the interspecific hybrid sterility between O. sativa and its AA genome relatives, and the same is true for intraspecific hybrid sterility in O. sativa. Therefore, it is necessary to raise the near-isogenic lines with various alleles/haplotypes and pyramided different alleles/haplotypes from sterile loci in the same genetic background aiming to study allelic and non-allelic interaction among different hybrid sterility loci in the AA genome species. Furthermore, the pyramiding lines ought to be used as bridge parents to overcome hybrid sterility for rice breeding purposes.

Double-Locking Mechanism of Self-Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding Region in the Male Specificity Gene.

Self-compatibility in Arabidopsis thaliana represents the relatively recent disruption of ancestral obligate cross pollination, recognized as one of the prevalent evolutionary pathways in flowering plants, as noted by Darwin. Our previous study found that inversion of the male specificity gene (SP11/SCR) disrupted self-incompatibility, which was restored by overexpressing the SCR with the reversed inversion. However, SCR in A. thaliana has other mutations aside from the pivotal inversion, in both promoter and coding regions, with probable effects on transcriptional regulation. To examine the functional consequences of these mutations, we conducted reciprocal introductions of native promoters and downstream sequences from orthologous loci of self-compatible A. thaliana and self-incompatible A. halleri. Use of this inter-species pair enabled us to expand the scope of the analysis to transcriptional regulation and deletion in the intron, in addition to inversion in the native genomic background. Initial analysis revealed that A. thaliana has a significantly lower basal expression level of SCR transcripts in the critical reproductive stage compared to that of A. halleri, suggesting that the promoter was attenuated in inducing transcription in A. thaliana. However, in reciprocal transgenic experiments, this A. thaliana promoter was able to restore partial function if coupled with the functional A. halleri coding sequence, despite extensive alterations due to the self-compatible mode of reproduction in A. thaliana. This represents a synergistic effect of the promoter and the inversion resulting in fixation of self-compatibility, primarily enforced by disruption of SCR. Our findings elucidate the functional and evolutionary context of the historical transition in A. thaliana thus contributing to the understanding of the molecular events leading to development of self-compatibility.

A new classification ofCarex(Cyperaceae) subgenera supported by a HybSeq backbone phylogenetic tree

AbstractThe field of systematics is experiencing a new molecular revolution driven by the increased availability of high-throughput sequencing technologies. As these techniques become more affordable, the increased genomic resources have increasingly far-reaching implications for our understanding of the Tree of Life. With c. 2000 species, Carex (Cyperaceae) is one of the five largest genera of angiosperms and one of the two largest among monocots, but the phylogenetic relationships between the main lineages are still poorly understood. We designed a Cyperaceae-specific HybSeq bait kit using transcriptomic data of Carex siderosticta and Cyperus papyrus. We identified 554 low-copy nuclear orthologous loci, targeting a total length of c. 1 Mbp. Our Cyperaceae-specific kit shared loci with a recently published angiosperm-specific Anchored Hybrid Enrichment kit, which enabled us to include and compile data from different sources. We used our Cyperaceae kit to sequence 88 Carex spp., including samples of all the five major clades in the genus. For the first time, we present a phylogenetic tree of Carex based on hundreds of loci (308 nuclear exon matrices, 543 nuclear intron matrices and 66 plastid exon matrices), demonstrating that there are six strongly supported main lineages in Carex: the Siderostictae, Schoenoxiphium, Unispicate, Uncinia, Vignea and Core Carex clades. Based on our results, we suggest a revised subgeneric treatment and provide lists of the species belonging to each of the subgenera. Our results will inform future biogeographic, taxonomic, molecular dating and evolutionary studies in Carex and provide the step towards a revised classification that seems likely to stand the test of time.

The multicomparative 2-n-way genome suite.

To effectively analyze the increasing amounts of available genomic data, improved comparative analytical tools that are accessible to and applicable by a broad scientific community are essential. We built the “2-n-way” software suite to provide a fundamental and innovative processing framework for revealing and comparing inserted elements among various genomes. The suite comprises two user-friendly web-based modules. The 2-way module generates pairwise whole-genome alignments of target and query species. The resulting genome coordinates of blocks (matching sequences) and gaps (missing sequences) from multiple 2-ways are then transferred to the n-way module and sorted into projects, in which user-defined coordinates from reference species are projected to the block/gap coordinates of orthologous loci in query species to provide comparative information about presence (blocks) or absence (gaps) patterns of targeted elements over many entire genomes and phylogroups. Thus, the 2-n-way software suite is ideal for performing multidirectional, non-ascertainment-biased screenings to extract all possible presence/absence data of user-relevant elements in orthologous sequences. To highlight its applicability and versatility, we used 2-n-way to expose approximately 100 lost introns in vertebrates, analyzed thousands of potential phylogenetically informative bat and whale retrotransposons, and novel human exons as well as thousands of human polymorphic retrotransposons.

Genomic Origin and Diversification of the Glucosinolate MAM Locus

Glucosinolates are a diverse group of plant metabolites that characterize the order Brassicales. The MAM locus is one of the most significant QTLs for glucosinolate diversity. However, most of what we understand about evolution at the locus is focused on only a few species and not within a phylogenetic context. In this study, we utilize a micro-synteny network and phylogenetic inference to investigate the origin and diversification of the MAM/IPMS gene family. We uncover unique MAM-like genes found at the orthologous locus in the Cleomaceae that shed light on the transition from IPMS to MAM. In the Brassicaceae, we identify six distinct MAM clades across Lineages I, II, and III. We characterize the evolutionary impact and consequences of local duplications, transpositions, whole genome duplications, and gene fusion events, generating several new hypothesizes on the function and diversity of the MAM locus.

SNP markers for low molecular glutenin subunits (LMW-GSs) at the Glu-A3 and Glu-B3 loci in bread wheat.

The content and composition of seed storage proteins is largely responsible for wheat end-use quality. They mainly consist of polymeric glutenins and monomeric gliadins. According to their electrophoretic mobility, gliadins and glutenins are subdivided into several fractions. Glutenins are classified as high molecular weight or low molecular weight glutenin subunits (HMW-GSs and LMW-GSs, respectively). LMW-GSs are encoded by multigene families located at the orthologous Glu-3 loci. We designed a set of 16 single-nucleotide polymorphism (SNP) markers that are able to detect SDS-PAGE alleles at the Glu-A3 and Glu-B3 loci. The SNP markers captured the diversity of alleles in 88 international reference lines and 27 Mexican cultivars, when compared to SDS-PAGE and STS markers, however, showed a slightly larger percent of multiple alleles, mainly for Glu-B3. SNP markers were then used to determine the Glu-1 and Glu-3 allele composition in 54 CIMMYT historical lines and demonstrated to be useful tool for breeding programs to improve wheat end product properties.

Insertion of Telomeric Repeats in the Human and Horse Genomes: An Evolutionary Perspective.

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.

Evolutionary and biochemical analyses reveal conservation of the Brassicaceae telomerase ribonucleoprotein complex.

The telomerase ribonucleoprotein complex (RNP) is essential for genome stability and performs this role through the addition of repetitive DNA to the ends of chromosomes. The telomerase enzyme is composed of a reverse transcriptase (TERT), which utilizes a template domain in an RNA subunit (TER) to reiteratively add telomeric DNA at the ends of chromosomes. Multiple TERs have been identified in the model plant Arabidopsis thaliana. Here we combine a phylogenetic and biochemical approach to understand how the telomerase RNP has evolved in Brassicaceae, the family that includes A. thaliana. Because of the complex phylogenetic pattern of template domain loss and alteration at the previously characterized A. thaliana TER loci, TER1 and TER2, across the plant family Brassicaceae, we bred double mutants from plants with a template deletion at AtTER1 and T-DNA insertion at AtTER2. These double mutants exhibited no telomere length deficiency, a definitive indication that neither of these loci encode a functional telomerase RNA. Moreover, we determined that the telomerase components TERT, Dyskerin, and the KU heterodimer are under strong purifying selection, consistent with the idea that the TER with which they interact is also conserved. To test this hypothesis further, we analyzed the substrate specificity of telomerase from species across Brassicaceae and determined that telomerase from close relatives bind and extend substrates in a similar manner, supporting the idea that TERs in different species are highly similar to one another and are likely encoded from an orthologous locus. Lastly, TERT proteins from across Brassicaceae were able to complement loss of function tert mutants in vivo, indicating TERTs from other species have the ability to recognize the native TER of A. thaliana. Finally, we immunoprecipitated the telomerase complex and identified associated RNAs via RNA-seq. Using our evolutionary data we constrained our analyses to conserved RNAs within Brassicaceae that contained a template domain. These analyses revealed a highly expressed locus whose disruption by a T-DNA resulted in a telomeric phenotype similar to the loss of other telomerase core proteins, indicating that the RNA has an important function in telomere maintenance.

Transcriptome Ortholog Alignment Sequence Tools (TOAST) for phylogenomic dataset assembly

BackgroundAdvances in next-generation sequencing technologies have reduced the cost of whole transcriptome analyses, allowing characterization of non-model species at unprecedented levels. The rapid pace of transcriptomic sequencing has driven the public accumulation of a wealth of data for phylogenomic analyses, however lack of tools aimed towards phylogeneticists to efficiently identify orthologous sequences currently hinders effective harnessing of this resource.ResultsWe introduce TOAST, an open source R software package that can utilize the ortholog searches based on the software Benchmarking Universal Single-Copy Orthologs (BUSCO) to assemble multiple sequence alignments of orthologous loci from transcriptomes for any group of organisms. By streamlining search, query, and alignment, TOAST automates the generation of locus and concatenated alignments, and also presents a series of outputs from which users can not only explore missing data patterns across their alignments, but also reassemble alignments based on user-defined acceptable missing data levels for a given research question.ConclusionsTOAST provides a comprehensive set of tools for assembly of sequence alignments of orthologs for comparative transcriptomic and phylogenomic studies. This software empowers easy assembly of public and novel sequences for any target database of candidate orthologs, and fills a critically needed niche for tools that enable quantification and testing of the impact of missing data. As open-source software, TOAST is fully customizable for integration into existing or novel custom informatic pipelines for phylogenomic inference.Software, a detailed manual, and example data files are available through github carolinafishes.github.io

Transcriptome-based target-enrichment baits for stony corals (Cnidaria: Anthozoa: Scleractinia).

Despite the ecological and economic significance of stony corals (Scleractinia), a robust understanding of their phylogeny remains elusive due to patchy taxonomic and genetic sampling, as well as the limited availability of informative markers. To increase the number of genetic loci available for phylogenomic analyses in Scleractinia, we designed 15,919 DNA enrichment baits targeting 605 orthogroups (mean 565 ± SD 366 bp) over 1,139 exon regions. A further 236 and 62 barcoding baits were designed for COI and histone H3 genes respectively for quality and contamination checks. Hybrid capture using these baits was performed on 18 coral species spanning the presently understood scleractinian phylogeny, with two corallimorpharians as outgroup. On average, 74% of all loci targeted were successfully captured for each species. Barcoding baits were matched unambiguously to their respective samples and revealed low levels of cross‐contamination in accordance with expectation. We put the data through a series of stringent filtering steps to ensure only scleractinian and phylogenetically informative loci were retained, and the final probe set comprised 13,479 baits, targeting 452 loci (mean 531 ± SD 307 bp) across 865 exon regions. Maximum likelihood, Bayesian and species tree analyses recovered maximally supported, topologically congruent trees consistent with previous phylogenomic reconstructions. The phylogenomic method presented here allows for consistent capture of orthologous loci among divergent coral taxa, facilitating the pooling of data from different studies and increasing the phylogenetic sampling of scleractinians in the future.

Reconstruction of the Evolutionary Histories of UGT Gene Superfamily in Legumes Clarifies the Functional Divergence of Duplicates in Specialized Metabolism.

Plant uridine 5′-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in Medicago truncatula, Glycine max, Phaseolus vulgaris, Lotus japonicus, and Trifolium pratense genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (UGT73P2: galactosyltransferase and UGT73P10: arabinosyltransferase) and third (UGT91H4: rhamnosyltransferase and UGT91H9: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina–millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-O glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (UGT73F2: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.

Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales

BackgroundThe order Enterobacterales encompasses a broad range of metabolically and ecologically versatile bacterial taxa, most of which are motile by means of peritrichous flagella. Flagellar biosynthesis has been linked to a primary flagella locus, flag-1, encompassing ~ 50 genes. A discrete locus, flag-2, encoding a distinct flagellar system, has been observed in a limited number of enterobacterial taxa, but its function remains largely uncharacterized.ResultsComparative genomic analyses showed that orthologous flag-2 loci are present in 592/4028 taxa belonging to 5/8 and 31/76 families and genera, respectively, in the order Enterobacterales. Furthermore, the presence of only the outermost flag-2 genes in many taxa suggests that this locus was far more prevalent and has subsequently been lost through gene deletion events. The flag-2 loci range in size from ~ 3.4 to 81.1 kilobases and code for between five and 102 distinct proteins. The discrepancy in size and protein number can be attributed to the presence of cargo gene islands within the loci. Evolutionary analyses revealed a complex evolutionary history for the flag-2 loci, representing ancestral elements in some taxa, while showing evidence of recent horizontal acquisition in other enterobacteria.ConclusionsThe flag-2 flagellar system is a fairly common, but highly variable feature among members of the Enterobacterales. Given the energetic burden of flagellar biosynthesis and functioning, the prevalence of a second flagellar system suggests it plays important biological roles in the enterobacteria and we postulate on its potential role as locomotory organ or as secretion system.

Target Nuclear and Off-Target Plastid Hybrid Enrichment Data Inform a Range of Evolutionary Depths in the Orchid Genus Epidendrum.

Universal angiosperm enrichment probe sets designed to enrich hundreds of putatively orthologous nuclear single-copy loci are increasingly being applied to infer phylogenetic relationships of different lineages of angiosperms at a range of evolutionary depths. Studies applying such probe sets have focused on testing the universality and performance of the target nuclear loci, but they have not taken advantage of off-target data from other genome compartments generated alongside the nuclear loci. Here we do so to infer phylogenetic relationships in the orchid genus Epidendrum and closely related genera of subtribe Laeliinae. Our aims are to: 1) test the technical viability of applying the plant anchored hybrid enrichment (AHE) method (Angiosperm v.1 probe kit) to our focal group, 2) mine plastid protein coding genes from off-target reads; and 3) evaluate the performance of the target nuclear and off-target plastid loci in resolving and supporting phylogenetic relationships along a range of taxonomical depths. Phylogenetic relationships were inferred from the nuclear data set through coalescent summary and site-based methods, whereas plastid loci were analyzed in a concatenated partitioned matrix under maximum likelihood. The usefulness of target and flanking non-target nuclear regions and plastid loci was assessed through the estimation of their phylogenetic informativeness. Our study successfully applied the plant AHE probe kit to Epidendrum, supporting the universality of this kit in angiosperms. Moreover, it demonstrated the feasibility of mining plastome loci from off-target reads generated with the Angiosperm v.1 probe kit to obtain additional, uniparentally inherited sequence data at no extra sequencing cost. Our analyses detected some strongly supported incongruences between nuclear and plastid data sets at shallow divergences, an indication of potential lineage sorting, hybridization, or introgression events in the group. Lastly, we found that the per site phylogenetic informativeness of the ycf1 plastid gene surpasses that of all other plastid genes and several nuclear loci, making it an excellent candidate for assessing phylogenetic relationships at medium to low taxonomic levels in orchids.

Episodic positive diversifying selection on key immune system genes in major avian lineages.

The major histocompatibility complex (MHC) of the adaptive immune system and the toll-like receptor (TLR) family of the innate immune system are involved in the detection of foreign invaders, and thus are subject to parasite-driven molecular evolution. Herein, we tested for macroevolutionary signatures of selection in these gene families within and among all three major clades of birds (Paleognathae, Galloanserae, and Neoaves). We characterized evolutionary relationships of representative immune genes (Mhc1 and Tlr2b) and a control gene (ubiquitin, Ubb), using a relatively large and phylogenetically diverse set of species with complete coding sequences (34 orthologous loci for Mhc1, 29 for Tlr2b, and 37 for Ubb). Episodic positive diversifying selection was found in the gene-wide phylogenies of the two immune genes, as well as at specific sites within each gene (8.5% of codon sites in Mhc1 and 2.7% in Tlr2b), but not in the control gene (Ubb). We found 20% of lineages under episodic diversifying selection in Mhc1 versus 9.1% in Tlr2b. For Mhc1, selection was relaxed in the Galloanserae and intensified in the Neoaves relative to the other clades, but no differences were detected among clades in the Tlr2b gene. In summary, we provide evidence of episodic positive diversifying selection in key immune genes and demonstrate differential strengths of selection within Class Aves, with the adaptive gene showing an increased divergence and evolutionary rate over the innate gene, contributing to the growing understanding of vertebrate immune gene evolution.

The phylogenomics of diversification on an island: applying anchored hybrid enrichment to New Zealand Leptospermum scoparium (Myrtaceae)

Abstract Leptospermum scoparium (Myrtaceae) is a morphologically highly variable species found in mainland Australia, Tasmania and New Zealand. For example, in New Zealand up to six morphologically distinct varieties of this species have been described, although only two (var. scoparium and var. incanum) are now formally recognized. In the present study we provide a first examination of genetic diversity in this culturally and commercially important species with the aim of gaining insights into its origins and evolution. We used anchored hybrid enrichment to acquire sequence data from 485 orthologous low-copy nuclear loci for 27 New Zealand and three Australian accessions of L. scoparium and representatives of several other Leptospermum spp. The final concatenated data matrix contained 421 687 nucleotide positions of which 55 102 were potentially informative. Despite the relative large data set, our analyses suggest that a combination of low and incompatible data signal limits the resolution of relationships among New Zealand populations of L. scoparium. Nevertheless, our analyses are consistent with genetic diversity being geographically structured, with three groups of L. scoparium recovered. We discuss the evolutionary and taxonomic implications of our findings.

CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease)

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child’s lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.