Roberta Roberto

Comparative and demographic analysis of orang-utan genomes

‘Orang-utan’ is derived from a Malay term meaning ‘man of the forest’ and aptly describes the southeast Asian great apes native to Sumatra and Borneo. The orang-utan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orang-utan draft genome assembly and short read sequence data from five Sumatran and five Bornean orang-utan genomes. Our analyses reveal that, compared to other primates, the orang-utan genome has many unique features. Structural evolution of the orang-utan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe a primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orang-utan genome structure. Orang-utans have extremely low energy usage for a eutherian mammal, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400,000 years ago, is more recent than most previous studies and underscores the complexity of the orang-utan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (Ne) expanded exponentially relative to the ancestral Ne after the split, while Bornean Ne declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.

Primate chromosome evolution: Ancestral karyotypes, marker order and neocentromeres

In 1992 the Japanese macaque was the first species for which the homology of the entire karyotype was established by cross-species chromosome painting. Today, there are chromosome painting data on more than 50 species of primates. Although chromosome painting is a rapid and economical method for tracking translocations, it has limited utility for revealing intrachromosomal rearrangements. Fortunately, the use of BAC-FISH in the last few years has allowed remarkable progress in determining marker order along primate chromosomes and there are now marker order data on an array of primate species for a good number of chromosomes. These data reveal inversions, but also show that centromeres of many orthologous chromosomes are embedded in different genomic contexts. Even if the mechanisms of neocentromere formation and progression are just beginning to be understood, it is clear that these phenomena had a significant impact on shaping the primate genome and are fundamental to our understanding of genome evolution. In this report we complete and integrate the dataset of BAC-FISH marker order for human syntenies 1, 2, 4, 5, 8, 12, 17, 18, 19, 21, 22 and the X. These results allowed us to develop hypotheses about the content, marker order and centromere position in ancestral karyotypes at five major branching points on the primate evolutionary tree: ancestral primate, ancestral anthropoid, ancestral platyrrhine, ancestral catarrhine and ancestral hominoid. Current models suggest that between-species structural rearrangements are often intimately related to speciation. Comparative primate cytogenetics has become an important tool for elucidating the phylogeny and the taxonomy of primates. It has become increasingly apparent that molecular cytogenetic data in the future can be fruitfully combined with whole-genome assemblies to advance our understanding of primate genome evolution as well as the mechanisms and processes that have led to the origin of the human genome.

Tracking the complex flow of chromosome rearrangements from the Hominoidea Ancestor to extant Hylobates and Nomascus Gibbons by high-resolution synteny mapping

In this study we characterized the extension, reciprocal arrangement, and orientation of syntenic chromosomal segments in the lar gibbon (Hylobates lar, HLA) by hybridization of a panel of approximately 1000 human BAC clones. Each lar gibbon rearrangement was defined by a splitting BAC clone or by two overlapping clones flanking the breakpoint. A reconstruction of the synteny arrangement of the last common ancestor of all living lesser apes was made by combining these data with previous results in Nomascus leucogenys, Hoolock hoolock, and Symphalangus syndactylus. The definition of the ancestral synteny organization facilitated tracking the cascade of chromosomal changes from the Hominoidea ancestor to the present day karyotype of Hylobates and Nomascus. Each chromosomal rearrangement could be placed within an approximate phylogenetic and temporal framework. We identified 12 lar-specific rearrangements and five previously undescribed rearrangements that occurred in the Hylobatidae ancestor. The majority of the chromosomal differences between lar gibbons and humans are due to rearrangements that occurred in the Hylobatidae ancestor (38 events), consistent with the hypothesis that the genus Hylobates is the most recently evolved lesser ape genus. The rates of rearrangements in gibbons are 10 to 20 times higher than the mammalian default rate. Segmental duplication may be a driving force in gibbon chromosome evolution, because a consistent number of rearrangements involves pericentromeric regions (10 events) and centromere inactivation (seven events). Both phenomena can be reasonably supposed to have strongly contributed to the euchromatic dispersal of segmental duplications typical of pericentromeric regions. This hypothesis can be more fully tested when the sequence of this gibbon species becomes available. The detailed synteny map provided here will, in turn, substantially facilitate sequence assembly efforts.

CYP3 phylogenomics: evidence for positive selection of CYP3A4 and CYP3A7.

Objective CYP3A metabolizes 50% of currently prescribed drugs and is frequently involved in clinically relevant drug interactions. The understanding of roles and regulations of the individual CYP3A genes in pharmacology and physiology is incomplete. Methods Using genomic sequences from 16 species we investigated the evolution of CYP3 genomic loci over a period of 450 million years. Results CYP3A genes in amniota evolved from two ancestral CYP3A genes. Upon the emergence of eutherian mammals, one of them was lost, whereas, the other acquired a novel genomic environment owing to translocation. In primates, CYP3A underwent rapid evolutionary changes involving multiple gene duplications, deletions, pseudogenizations, and gene conversions. The expansion of CYP3A in catarrhines (Old World monkeys, great apes, and humans) differed substantially from New World primates (e.g. common marmoset) and strepsirrhines (e.g. galago). We detected two recent episodes of particularly strong positive selection acting on primate CYP3A protein-coding sequence: (i) on CYP3A7 early in hominoid evolution, which was accompanied by a restriction of its hepatic expression to fetal period and (ii) on human CYP3A4 following the split of the chimpanzee and human lineages. In agreement with these findings, three out of four positively selected amino acids investigated in previous biochemical studies of CYP3A affect the activity and regioselectivity. Conclusions CYP3A7 and CYP3A4 may have acquired catalytic functions especially important for the evolution of hominoids and humans, respectively.

Refinement of macaque synteny arrangement with respect to the official rheMac2 macaque sequence assembly

We have compared the synteny block organization of the official macaque genome sequence assembly (Jan. 2006; rheMac2) with an independent assembly that used a molecular cytogenetic approach. The mapping of four synteny segments, ranging in size from 4 Mb to 24 Mb, was found to be inconsistent between the two datasets. We specifically investigated these discrepancies by appropriate co-hybridization FISH experiments with validated reference probes located outside the area under study. We found that in the macaque rheMac2 release three synteny segments were wrongly mapped and one segment was incorrectly oriented.

DDX11L: a novel transcript family emerging from human subtelomeric regions.

BackgroundThe subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members.ResultsDuring a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin.ConclusionOur data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.

Thrombocytopenia-absent-radius syndrome in a child showing a larger 1q21.1 deletion than the one in his healthy mother, and a significant downregulation of the commonly deleted genes

Thrombocytopenia-absent-radius (TAR) syndrome is a rare condition characterized by thrombocytopenia and bilateral absence of the radii with presence of both thumbs. The phenotype has a variable expression. A 200 kb minimal deletion at 1q21.1 is present in all patients. However, the microdeletion, ranging up to 1100 kb in length, is not sufficient to cause the disease. Indeed it is present in 75-80% of unaffected parents. It is assumed that the phenotype develops only in the presence of one or more additional, as-yet-unknown, deletion modifiers (mTARs). We report here on a child affected by TAR syndrome associated with Langerhans cell histiocytosis. Unexpectedly, he showed a 2.029 kb deletion at 1q21.1, almost twice that of the unaffected mother (957 kb). Interestingly, the mother-to-son increased size of the deleted region was already observed in two cases of constitutional diseases, although both resulting as chromosomal terminal deletions. Noteworthy, qPCR experiments, never before performed for patients with TAR syndrome, disclosed that the proband had a statistically significant downregulation of the majority of the genes mapping inside the part of the deletion shared with the mother. The mother, on the contrary, did not show the same downregulation. In summary, the present report adds new insights on the pathogenesis of TAR syndrome, that may represent fruitful directions for future research.

Does Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) host Serratia symbiotica Moran (Bacteria: Enterobacteriaceae)?

The euonymus scale Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) is a pest of spindle that exhibits a strong preference for Euonymus, although it has been detected on at least 18 genera in 13 plant families (Buxus, Camellia, Celastrus, Daphne, Eugenia, Euonymus, Hibiscus, Ilex, Jasminum, Ligustrum, Lonicera, Olea, Pachistima, Pachysandra, Perychmenum, Prunus and Syringa) (Salisbury et al., 2013). Heavy infestation by this pest may lead to the death of the host plant and consequential loss of income from the cultivation of ornamental plants (Kaygin et al., 2008). U. euonymi is an armored scale insect originally from mild Eastern Asia and probably introduced into Europe in the 20th century (Pellizzari & Germain, 2010). Its lifecycle, depending on climate conditions, comprises two-three generations a year and the control measures to limit its diffusion mainly rely on the use of insecticides or the growing of resistant cultivars. The insects can engage mutualistic interactions or symbioses with a variety of bacteria that can profoundly affect the host’s biology. Apart from obligate symbionts (maternally transmitted), a growing number of facultative or secondary symbionts (that can be horizontally transmitted) have been identified (Sandstrom et al., 2001, Moran et al., 2008). Despite not being essential for the host’s life cycle, this last type of symbiont can strongly influence their fitness (Oliver et al., 2003, Jaenike & Brekke, 2011). Additionally, the mutualistic association between insects and bacteria may play a role in the evolution of the latter as described for some groups of Entereobacteriaceae (Moran et al., 2005). A number of genomic and phylogenetic studies on mutualistic associations between Enterobacteriaceae and aphids, psyllids, scale insects, whiteflies, weevils and other insects have been reported (Lefevre et al., 2004, Thao & Baumann, 2004). Here we report the identification of Serratia symbiotica (strain UESS2016) in U. euonymi adult females collected from Sofia (Bulgaria) in 2013.

Nidularia pulvinata (Planchon, 1864) (Hemiptera Kermesidae) gall-inducing attitude

Recurrent Nidularia pulvinata outbreaks off Quercus ilex L. (Fagaceae) led us to scrutinize large amount of infested and damaged trunks, branches and twigs. A long series of detailed observations suggested studying the attitude of the Kermesidae to induce phloem/xylem disorganization by saliva injection, possibly. Evidence show that woody plants tissues near or under the scale insects swell considerably giving the organs a prominent and rising appearance. Infested bark and other plant surfaces are prone to produce crevices and other possible shelters for future crawlers that will find a nice living site nearby. In vivo transverse section, accurate observations corroborated with polarized light microscopy and Scanning Electron Microscopy suggests that the scale stylets strongly disturb the explored plant tissues. Plant tissues reply to the injury with considerable overgrowing and necrosis. By observations and evidenced we discuss the opportunity to consider Nidularia pulvinata a gall-making species, also comparing its attitude with that of other Kermesidae and Asterolecaniidae. Nidularia pulvinata infestation starts from crawlers that set into natural bark crevices. Scales feeding elicits the plant reaction that results in progressive widening of infested crevices. That, in consequence, offers more room for the subsequent broods of the Kermesidae. To evaluate the action of Nidularia feeding on host plant wood, we fell down two small Quercus ilex infested to death. We chose and mark several points before to cut the infested trunks and branches transversally. The action of Nidularia pulvinata feeding on host plant wood is clear in the left and central figure in comparison to right picture of a not infested trunk. Each log was pictured from the transversal section and the corresponding side. Red dashed lines connect corresponding points of the wood. The red arrow points to wood overgrowths for Nidularia infestation, while a blue arrow target a lateral branch bud. Red lines encircle wood overgrowths due to hyperplasia/hypertrophy stimulated by the Kermesidae. On twigs the Nidularia pulvinata feeding results in wood swelling, depression/overgrowth and necrosis.

Nidularia pulvinata (Planchon) (Hemiptera: Kermesidae) urban outbreaks associated with entomopathogenic fungi

Pest outbreaks often give to insect pathogens the opportunity to infect their host species eventually leading them to death. Recent Nidularia outbreaks off urban Quercus ilex L. showed some cases of entomopathogenic fungi virulence, apparently sustained by species of Fusarium and other fungi. Infection is apparent on the Nidularia population because fungi provoke the scale color shifting to orange or dull-green in medium in large patches. After the isolation in an axenic culture of the Kermesid-associate Mycota, ITS genomic regions amplified by PCR using the universal ITS5/ITS4 primers were sequenced by external service (Macrogen, Seoul, South Korea) for molecular identification. Blast analysis (http://blast. ncbi. nlm. nih. gov/Blast. cgi) of the ITS sequence showed a high homology with Quambalaria cyanescens (de Hoog & G. A. de Vries) Z. W. de Beer, Begerow & R. Bauer 2006 (Fungi: Basidiomycota), (identity: 97-96%; e-value 0. 0; coverage 82-75%), Fusarium acuminatum Ellis & Everh. 1895 (Fungi: Ascomycota) and Fusarium avenaceum (Fr. ) Sacc. 1886 (Fungi: Ascomycota) (identity: 99%; e-value 0. 0; coverage 82-80%), and Penicillium sumatraense Svilv. 1936 (Fungi: Ascomycota) (identity: 100%; e-value . 0; coverage 95-91%). We investigate the biological and ecological role of the above-recorded fungi by correlating the age of infected scale and their age. Mass culturing of the most promising pathogen will lead to semi-field trials to demonstrate the isolate entomopathogenic ability. Finally, we discuss the case of homonymy between the fungal Taxon named Nidularia pulvinata (Schwein.) (Gasteromycetes) and the scale Nidularia pulvinata (Planchon) that can lead to some confusion.