Microbial Community Profiling Protocol with Full-length 16S rRNA Sequences and Emu.

16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species-level resolution. This pipeline provides a low-cost solution for characterizing microbiome composition by exploiting real-time, long-read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full-length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu.

A new method for molecular sex identification in the emu (Dromaius novaehollandiae).

1. Sex chromosomes of emus are largely homomorphic. Therefore, the standard methodology for molecular sexing based on screening intron length variations in sex-linked genes is not applicable. However, emu sexing requires costly and time-consuming PCR-RFLP or multiplex PCR methods.2. This experiment used a directed PCR amplification and capillary electrophoresis sexing protocol. Two distinct peaks were observed in females (ZW), while only one peak was observed in males (ZZ).3. This sexing technique proved to be rapid, non-invasive, and highly sensitive and may be useful for verifying the sex ratio and breeding management of emus.

Genetic relationships among emu populations in Japanese farms based on mitochondrial and microsatellite DNA polymorphisms.

Emus (Dromaius novaehollandiae) are expected to become a novel poultry species for producing eggs, meat, and oil. In our previous studies, Japanese emu populations were predicted to have reduced genetic diversity through inbreeding. For a sustainable emu industry in Japan, it is necessary to understand the current genetic structure and relationships in dispersed farms. In this study, we investigated the genetic structure and relationships of six Japanese emu farms based on mitochondrial DNA and microsatellite polymorphisms. We analyzed the DNA sequences of the mitochondrial D-loop region in 157 individuals and detected four haplotypes with four nucleotide substitution sites (Hap-a, Hap-b, Hap-c, and Hap-d). Analysis of molecular variance revealed that 43.6% of total variance was "among population," and the FST value was 0.436 with significant genetic differentiation (P < 0.001). In microsatellite analysis, the expected (HE ) and observed (HO ) heterozygosities were 0.53-0.64 and 0.44-0.59, respectively. Phylogenetic trees and STRUCTURE analysis revealed that the six Japanese farmed emu populations could be divided into four genetically differentiated groups. Therefore, we identified genetic resources that may be useful in extending the genetic diversity of Japanese farms and are predicted to contribute to the conservation and reconstruction of populations.

Seasonal and sex-dependent gene expression in emu (Dromaius novaehollandiae) fat tissues.

Emu (Dromaius novaehollandiae) farming has been gaining wide interest for fat production. Oil rendered from this large flightless bird's fat is valued for its anti-inflammatory and antioxidant properties for uses in therapeutics and cosmetics. We analyzed the seasonal and sex-dependent differentially expressed (DE) genes involved in fat metabolism in emus. Samples were taken from back and abdominal fat tissues of a single set of four male and four female emus in April, June, and November for RNA-sequencing. We found 100 DE genes (47 seasonally in males; 34 seasonally in females; 19 between sexes). Seasonally DE genes with significant difference between the sexes in gene ontology terms suggested integrin beta chain-2 (ITGB2) influences fat changes, in concordance with earlier studies. Six seasonally DE genes functioned in more than two enriched pathways (two female: angiopoietin-like 4 (ANGPTL4) and lipoprotein lipase (LPL); four male: lumican (LUM), osteoglycin (OGN), aldolase B (ALDOB), and solute carrier family 37 member 2 (SLC37A2)). Two sexually DE genes, follicle stimulating hormone receptor (FSHR) and perilipin 2 (PLIN2), had functional investigations supporting their influence on fat gain and loss. The results suggested these nine genes influence fat metabolism and deposition in emus.

Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data.

16S ribosomal RNA-based analysis is the established standard for elucidating the composition of microbial communities. While short-read 16S rRNA analyses are largely confined to genus-level resolution at best, given that only a portion of the gene is sequenced, full-length 16S rRNA gene amplicon sequences have the potential to provide species-level accuracy. However, existing taxonomic identification algorithms are not optimized for the increased read length and error rate often observed in long-read data. Here we present Emu, an approach that uses an expectation-maximization algorithm to generate taxonomic abundance profiles from full-length 16S rRNA reads. Results produced from simulated datasets and mock communities show that Emu is capable of accurate microbial community profiling while obtaining fewer false positives and false negatives than alternative methods. Additionally, we illustrate a real-world application of Emu by comparing clinical sample composition estimates generated by an established whole-genome shotgun sequencing workflow with those returned by full-length 16S rRNA gene sequences processed with Emu.

A new emu genome illuminates the evolution of genome configuration and nuclear architecture of avian chromosomes.

Emu and other ratites are more informative than any other birds in reconstructing the evolution of the ancestral avian or vertebrate karyotype because of their much slower rate of genome evolution. Here, we generated a new chromosome-level genome assembly of a female emu, and estimated the tempo of chromosome evolution across major avian phylogenetic branches, by comparing it to chromosome-level genome assemblies of 11 other bird and one turtle species. We found ratites exhibited the lowest numbers of intra- and inter-chromosomal changes among birds since their divergence with turtles. The small-sized and gene-rich emu microchromosomes have frequent inter-chromosomal contacts that are associated with housekeeping genes, which appears to be driven by clustering their centromeres in the nuclear interior, away from the macrochromosomes in the nuclear periphery. Unlike nonratite birds, only less than one-third of the emu W Chromosome regions have lost homologous recombination and diverged between the sexes. The emu W is demarcated into a highly heterochromatic region (WS0) and another recently evolved region (WS1) with only moderate sequence divergence with the Z Chromosome. WS1 has expanded its inactive chromatin compartment, increased chromatin contacts within the region, and decreased contacts with the nearby regions, possibly influenced by the spreading of heterochromatin from WS0. These patterns suggest that alteration of chromatin conformation comprises an important early step of sex chromosome evolution. Overall, our results provide novel insights into the evolution of avian genome structure and sex chromosomes in three-dimensional space.

Development of 49 novel microsatellite markers from Next-generation sequencing data and a robust method for parentage tests in the emu (Dromaius novaehollandiae).

The emu is a useful and new breed of poultry, but their genetic improvement has not advanced yet due to their very recent domestication. Pedigree information is difficult to record because of their complex reproduction system (polyandry). To identify parent-offspring relationships in the emu, parentage test based on polymorphic DNA markers have to be developed. In this study, we isolated more than 25,000 microsatellite (simple sequence repeat, SSR) regions from Next-generation sequencing data via the QDD pipeline and developed 49 SSR markers with polymorphism in the Japanese farmed emu. The dinucleotide motifs, (AC)n, (AT)n and (AG)n, were the most frequently detected and were found on 10,167 (38.55%), 8,114 (30.76%) and 4,796 (18.18%) contigs, respectively. Forty-nine novel SSR markers were characterized in 20 individuals and showed NA ranged from 2 to 12, with an average of 4.2. HE/HO ranged from 0.389/0.071 to 0.702/1.000 with an average of 0.601/0.515. PIC value ranged from 0.059 to 0.886 with an average of 0.528, and 17 of 49 markers showed a higher polymorphism than 0.500. Thirty-four individuals were genotyped using 12 markers, and CERVUS simulations based on genotype showed that parents of all offspring were identified with 0.9995-1.0 probability. Thus, 49 novel SSR markers and a robust method for parentage test for the Japanese emu were developed.

Mitochondrial DNA variations in Japanese farmed emu populations.

The emu (Dromaius novaehollandiae) is a new poultry. In this study, we investigated the haplotype composition of mitochondrial DNA among emu populations farmed in Japan. We sequenced the D-loop region in 109 individuals, and detected four substitution sites and three haplotypes (Hap-a, -b, and -c). Hap-a was the most frequently observed haplotype in the Japanese populations. Although Hap-c was a rare haplotype in not only Japanese but also Australian populations, it was detected with high frequency in the Japanese farmed population. The AMOVA indicated that 9% of total variance was "among population". The FST value was 0.087 and genetic differentiation was significant (P<0.01). These results may contribute to conserving the genetic resources available for the Japanese emu industry.

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations.

The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan are unknown and the number of microsatellite markers for genetic analysis of the emu is insufficient. In this study, we isolated 16 microsatellites from the emu genome and developed ten new microsatellite markers. These microsatellite markers were used to characterize three farm emu populations in Japan. The number of alleles ranged from 3 to 13 and the expected (HE) and observed heterozygosity (HO) of these microsatellite loci was 0.187-0.802 and 0.179-0.647, respectively. The polymorphic information content ranged from 0.176 to 0.786. Positive inbreeding coefficient (FIS) values were detected in all tested populations, and they ranged from 0.027 to 0.540. These results suggest that farm populations of the emu in Japan resulted from inbreeding. The fixation index (FST) values ranged from 0.026 to 0.061, and phylogenetic trees and population structure analysis confirmed no definitive genetic differentiation among the three populations. Therefore, these populations are at a relatively low level of genetic differentiation at present. The microsatellite markers developed in our study can be utilized for genetic analysis and preservation of genetic resources in the emu.

Attenuated Fgf Signaling Underlies the Forelimb Heterochrony in the Emu Dromaius novaehollandiae.

Powered flight was fundamental to the establishment and radiation of birds. However, flight has been lost multiple times throughout avian evolution. Convergent losses of flight within the ratites (flightless paleognaths, including the emu and ostrich) often coincide with reduced wings. Although there is a wealth of anatomical knowledge for several ratites, the genetic mechanisms causing these changes remain debated. Here, we use a multidisciplinary approach employing embryological, genetic, and genomic techniques to interrogate the mechanisms underlying forelimb heterochrony in emu embryos. We show that the initiation of limb formation, an epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) and myoblast migration into the LPM, occur at equivalent stages in the emu and chick. However, the emu forelimb fails to subsequently proliferate. The unique emu forelimb expression of Nkx2.5, previously associated with diminished wing development, initiates after this stage (concomitant with myoblast migration into the LPM) and is therefore unlikely to cause this developmental delay. In contrast, RNA sequencing of limb tissue reveals significantly lower Fgf10 expression in the emu forelimb. Artificially increasing Fgf10 expression in the emu LPM induces ectodermal Fgf8 expression and a limb bud. Analyzing open chromatin reveals differentially active regulatory elements near Fgf10 and Sall-1 in the emu wing, and the Sall-1 enhancer activity is dependent on a likely Fgf-mediated Ets transcription factor-binding site. Taken together, our results suggest that regulatory changes result in lower expression of Fgf10 and a concomitant failure to express genes required for limb proliferation in the early emu wing bud.

Genetic diversity and drivers of dwarfism in extinct island emu populations.

Australia's iconic emu (Dromaius novaehollandiae novaehollandiae) is the only living representative of its genus, but fossil evidence and reports from early European explorers suggest that three island forms (at least two of which were dwarfs) became extinct during the nineteenth century. While one of these-the King Island emu-has been found to be conspecific with Australian mainland emus, little is known about how the other two forms-Kangaroo Island and Tasmanian emus-relate to the others, or even the size of Tasmanian emus. We present a comprehensive genetic and morphological analysis of Dromaius diversity, including data from one of the few definitively genuine Tasmanian emu specimens known. Our genetic analyses suggest that all the island populations represent sub-populations of mainland Dnovaehollandiae Further, the size of island emus and those on the mainland appears to scale linearly with island size but not time since isolation, suggesting that island size-and presumably concomitant limitations on resource availability-may be a more important driver of dwarfism in island emus, though its precise contribution to emu dwarfism remains to be confirmed.

A simplified protocol for molecular sexing in the emu (Dromaius novaehollandiae).

Male and female emus are nearly identical both as chicks and as adults. Although morphological differences of the internal genital tract can be used to distinguish the sexes, a high degree of diagnostic skill is required for accurate sexing. DNA-based sexing methods are highly accurate and can be used to diagnose sex without requiring a high degree of technical skill. However, conventional PCR-RFLP is time consuming and costly, requiring the digestion of PCR products. In this study, we simplified the protocol for sexing the emu by using multiplex PCR without restriction enzyme treatment. Multiplex PCR based on a W-specific primer, with the commonly designed primer set on both Z and W chromosomes, amplified both 197-bp and 272-bp bands in the female, and only the 272-bp band in the male. Sexing results obtained in this way were completely concordant with results obtained using the conventional PCR-RFLP method. Thus, we succeeded in simplifying the protocol for sexing the emu, and suggest that our protocol improves production efficiency by facilitating rapid pairing and selection of individuals to establish high-quality pedigrees.

Biochemical and functional characterization of transiently expressed in neural precursor (TENP) protein in emu egg white.

A protein transiently expressed in the neural precursors of developing tissues (TENP) was found to be present in emu (Dromaius novaehollandiae) egg white as one of the major proteins. Nucleotide analysis of its encoding cDNA revealed a sequence of 452 amino acids including a 19 amino acid peptide signal. Phylogenetic analysis determined that emu TENP was clustered within the bactericidal/permeability-increasing protein (BPI) superfamily together with other avian TENPs. RT-PCR analysis revealed that the emu TENP gene was highly expressed in the magnum of the oviduct, indicating that TENP is a major egg white component. Emu TENP was purified by anion exchange chromatography and ammonium sulfate fractionation. Unlike BPI, emu TENP exhibited antibacterial activity against Gram-positive bacteria, including Micrococcus luteus and Bacillus subtilis, but not against Gram-negative bacteria such as Escherichia coli and Salmonella Typhimurium. The results suggest that emu TENP is a potent novel antibacterial protein with a spectrum distinct from that of BPI.

Sex-biased gene expression at homomorphic sex chromosomes in emus and its implication for sex chromosome evolution.

Sex chromosomes originate from autosomes. The accumulation of sexually antagonistic mutations on protosex chromosomes selects for a loss of recombination and sets in motion the evolutionary processes generating heteromorphic sex chromosomes. Recombination suppression and differentiation are generally viewed as the default path of sex chromosome evolution, and the occurrence of old, homomorphic sex chromosomes, such as those of ratite birds, has remained a mystery. Here, we analyze the genome and transcriptome of emu (Dromaius novaehollandiae) and confirm that most genes on the sex chromosome are shared between the Z and W. Surprisingly, however, levels of gene expression are generally sex-biased for all sex-linked genes relative to autosomes, including those in the pseudoautosomal region, and the male-bias increases after gonad formation. This expression bias suggests that the emu sex chromosomes have become masculinized, even in the absence of ZW differentiation. Thus, birds may have taken different evolutionary solutions to minimize the deleterious effects imposed by sexually antagonistic mutations: some lineages eliminate recombination along the protosex chromosomes to physically restrict sexually antagonistic alleles to one sex, whereas ratites evolved sex-biased expression to confine the product of a sexually antagonistic allele to the sex it benefits. This difference in conflict resolution may explain the preservation of recombining, homomorphic sex chromosomes in other lineages and illustrates the importance of sexually antagonistic mutations driving the evolution of sex chromosomes.

AKT signalling is required for ribosomal RNA synthesis and progression of Eµ-Myc B-cell lymphoma in vivo.

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eµ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eµ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.

Expressed sequence tag analysis of the emu (Dromaius novaehollandiae) pituitary by 454 GS Junior pyrosequencing.

Emus (Dromaius novaehollandiae) are farmed for their oil for pharmaceutical and cosmetic uses. This emu pituitary expressed sequence tag study was undertaken to identify novel transcripts in the emu pituitary to propel their identification and functional studies. By mapping reads derived from the Roche 454 GS Junior pyrosequencer to 8 reference species (human, mouse, chicken, zebra finch, fruit fly, turkey, round worm, and Carolina anole lizard) from the UniGene database, a total of 81,788 reads (53,312 mapped reads) were obtained and assembled with Reference Sequence (RefSeq). We annotated 6,676 potential emu genes by referencing 7 species (excluding lizard) and identified 1,232 potential genes common among 3 species (human, mouse, and chicken) with complete available reference genomes. Gene Ontology analysis revealed 376 Gene Ontology terms showing, with the highest counts, their involvements in biological processes, metabolism, and cellular components. These potential genes were detected to associate with 20 pathways including mitogen-activated protein kinase, insulin, neurotrophin signaling pathways, and carbohydrate digestion and absorption pathway. We also revealed a panel of tissue-specific genes including regulator of G-protein signaling protein (RGS), glucagon-like peptide receptor (GLPR), and growth hormone-inducible transmembrane protein (GHITM). Additionally, fatty acid binding protein (FABP), fatty acid desaturase (FAS), and stearoyl-coenzyme A desaturase (SCD), key enzyme genes in fat metabolism, were found to be also expressed in emu pituitary. This expressed sequence tag study represents the first step in functional characterization of emu pituitary gene expression and SNP identification for the improvement of fat production in the emu.

Molecular characterization of goose- and chicken-type lysozymes in emu (Dromaius novaehollandiae): evidence for extremely low lysozyme levels in emu egg white.

Lysozyme (LZ), a bacteriolytic enzyme, is found in the egg white of many avian eggs and plays an important role in host defense; however, LZ activity in emu (Dromaius novaehollandiae) egg white is exceptionally undetectable. We cloned and characterized emu goose-type LZ (LZG) and chicken-type LZ (LZC) genes. RT-PCR analysis revealed very low LZG gene expression levels and absence of LZC gene expression in the emu oviduct. Sequencing of full-length LZG and LZC cDNAs indicated that their amino acid sequences show high similarities to ostrich LZG and LZC, respectively, with conserved catalytic residues for enzymatic activities. Whereas recombinant emu LZG prepared using Pichia pastoris exhibited similar enzyme activity as ostrich LZG, recombinant emu LZC exhibited significantly higher lytic activity than chicken LZC. We concluded that emus have functional genes for both LZG and LZC like many other avians, and the LZG gene is expressed in oviduct probably as in other ratite, however, its expression levels in egg white were low to be detected.

Ancient DNA suggests dwarf and 'giant' emu are conspecific.

BACKGROUND: The King Island Emu (Dromaius ater) of Australia is one of several extinct emu taxa whose taxonomic relationship to the modern Emu (D. novaehollandiae) is unclear. King Island Emu were mainly distinguished by their much smaller size and a reported darker colour compared to modern Emu. METHODOLOGY AND RESULTS: We investigated the evolutionary relationships between the King Island and modern Emu by the recovery of both nuclear and mitochondrial DNA sequences from sub-fossil remains. The complete mitochondrial control (1,094 bp) and cytochrome c oxidase subunit I (COI) region (1,544 bp), as well as a region of the melanocortin 1 receptor gene (57 bp) were sequenced using a multiplex PCR approach. The results show that haplotypes for King Island Emu fall within the diversity of modern Emu. CONCLUSIONS: These data show the close relationship of these emu when compared to other congeneric bird species and indicate that the King Island and modern Emu share a recent common ancestor. King Island emu possibly underwent insular dwarfism as a result of phenotypic plasticity. The close relationship between the King Island and the modern Emu suggests it is most appropriate that the former should be considered a subspecies of the latter. Although both taxa show a close genetic relationship they differ drastically in size. This study also suggests that rates of morphological and neutral molecular evolution are decoupled.

Embryonic development of the emu, Dromaius novaehollandiae.

The chick, Gallus gallus, is the traditional model in avian developmental studies. Data on other bird species are scarce. Here, we present a comparative study of the embryonic development of the chick and the emu Dromaius novaehollandiae, a member of Paleognathae, which also includes the ostrich, rhea, tinamou, kiwi, and cassowary. Emu embryos ranging from Hamburger and Hamilton (HH) equivalent stages 1 to 43 were collected and their gross morphology analyzed. Its early development was studied in detail with time-lapse imaging and molecular techniques. Emu embryos in general take 2-3 times longer incubation time to reach equivalent chicken stages, requiring 1 day for HH2, 2.5 days for HH4, 7 days for limb bud initiation, 23 days for feather germ appearance, and approximately 50-56 days for hatching. Chordin gene expression is similar in emu and chick embryos, and emu Brachyury is not expressed until HH3. Circulation is established at approximately the 27- to 30-somite stage. Forelimb buds are formed and patterned initially, but their growth is severely retarded. The size difference between an emu and a chick embryo only becomes apparent after limb bud formation. Overall, emu and chick embryogenesis proceeds through similar stages, but developmental heterochrony between these two species is widely observed.