Many signal transduction pathways are involved in hepatocyte proliferation. One is activation of Akt and extracellular signal-regulated kinase (ERK)1/2, which are derived from direct stimulation from growth factors in platelets. The other is signal transducer and activator of transcription-3 (STAT3) activation by interleukin (IL)-6 derived from liver sinusoidal endothelial cells and Kupffer cells, which are stimulated by contact with platelets during liver regeneration. Platelets also improve liver fibrosis in rodent models by inactivating hepatic stellate cells to decrease collagen production. The level of intracellular cyclic adenosine monophosphate (cyclic AMP) is increased by adenosine through its receptors on hepatic stellate cells, resulting in inactivation of these cells. Adenosine is produced by the degradation of adenine nucleotides such as adenosine diphosphate (ADP) and adenosine tri-phosphate (ATP), which are stored in abundance within the dense granules of platelets.Platelets contain three types of granules: alpha granules, dense granules, and lysosomal granules. Each granule contains various growth factors, cytokines, and other physiological substances. Platelets trigger many kinds of biological responses, such as hemostasis, wound healing, and tissue regeneration. This review presents experimental evidence of platelets in accelerating liver regeneration and improving liver fibrosis. The regenerative effect of liver by platelets consists of three mechanisms;  It is an advanced form of hepatic fibrosis in response to chronic liver injury . Up to 62.etc. [Platelets are derived from megakaryocytes (MKs). Megakaryocytes are derived from multipotent hematopoietic stem cells toward MK progenitors. Mature MK produces platelets by cytoplasmic fragmentation occurring through a dynamic and regulated process, called proplatelet formation, and consisting of long pseudopodial elongations that break in the blood flow . Plateleetc. –16. Platetc. –19. Seveetc. , wound hetc. –23, and etc. –29. On tetc. , malignaetc. ,32, and etc. –36. Platetc. –40, liveetc. , cholestetc. , viral hetc. , and theetc. .3.et al., who suggested platelet serotonin is important for liver regeneration [The proliferative effect of platelets in liver regeneration was first reported in 2006 by Lesurtel neration . Our firneration . TPO is neration . c-Mpl ineration ,47. We uneration . TPO wasneration . Thromboneration ,48, and neration . A signineration ,48. Platneration . These fneration . TPO incneration . The proneration . Cholestneration . Transmineration . The incneration . Plateleneration .4.In vivo: platelets accumulate in the liver immediately after hepatectomy. Under normal conditions, liver sinusoidal cavities show intact endothelial lining consisting of liver endothelial cells with flattened processes perforated by small pores [ll pores . These sll pores ,55. In rll pores –58. In oll pores . Platelell pores , thus IGll pores .In vitro: the signal transduction of hepatocytes, which is activated by platelets, was analyzed. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK)1/2 were analyzed in murine immortalized hepatocyte TLR2 and primary cultured murine hepatocytes stimulated by platelets [in vitro [latelets . The Aktin vitro . The Aktin vitro ,62. The in vitro .in vitro. This study revealed that direct contact between platelets and LSECs induce IL-6 release from LSECs, and IL-6 derived from LSECs accelerates hepatocyte proliferation. In addition, sphingosine-1-phosphate (S1P) in platelets plays an important role in IL-6 secretion [et al. revealed that S1P protects LSECs from alcohol-induced apoptosis via activation of eNOS [et al. revealed that S1P combined with lipopolysaccharide (LPS) activates human umbilical endothelial cells (HUVECs), famous endothelial cells, via NF-κB, ERK1/2, and p38 and activated HUVECs secrete IL-6 [Liver sinusoidal endothelial cells (LSECs) enable contact between circulating blood and hepatocytes and help to exchange various soluble macromolecules and nano-particles, such as hyaluronic acid and lipoproteins . LSECs aecretion   mice induced by CCL4 [It was reported that the increment of platelets induced by TPO administration could improve liver fibrosis in experimental studies with rodents ,84. Dime by CCL4 . An incr by CCL4 .et al. recently reported the clinical trial to investigate whether platelet transfusion improves liver function in patients with chronic liver disease and cirrhosis (Child-Pugh class A or B), who all presented thrombocytopenia . The subjects received 10 units of platelet concentrate once a week for 12 weeks. One and three months after the last transfusion, significant improvement of serum albumin was observed. Serum cholinesterase improved for nine months after the last transfusion. Serum hyaluronic acid represents liver fibrosis, and that showed a tendency toward improvement after the last transfusion [Based on these animal experiments, clinical trial was performed. Maruyama nsfusion .6.et al. reported that human platelets contributed to the suppression of HSC activation and reduction of type I collagen production in vitro [In the long-term natural history of chronic liver injury, such as viral infection, alcohol, and non-alcoholic steatohepatitis, liver fibrosis occurs. Liver fibrosis is known to be part of a dynamic process of continuous extracellular matrix (ECM) remodeling, which leads to the excessive accumulation of several extracellular proteins, proteoglycans, and carbohydrates . Among tin vitro . The levin vitro   direct effect on hepatocytes; (ii) a cooperative effect with LSECs; and (iii) a cooperation with Kupffer cells. There is significant evidence that platelets play a role in improving fibrosis. ATP and ADP inside platelets are degraded by HSCs and adenosine is incorporated into HSCs. Cyclic AMP is increased by adenosine and HSCs become inactivated by cyclic AMP. Therefore, platelet therapy,


Pediococcus pentosaceus from food and the human gastrointestinal tract have been widely identified, and some have been reported to reduce inflammation, encephalopathy, obesity and fatty liver in animals. In this study, we sequenced the whole genome of P. pentosaceus LI05 (CGMCC 7049), which was isolated from the fecal samples of healthy volunteers, and determined its ability to reduce acute liver injury. No other genomic information for gut-borne P. pentosaceus is currently available in the public domain.Strains of P. pentosaceus LI05, which was 1,751,578 bp in size and possessed a mean G + C content of 37.3%. This genome encoded an abundance of proteins that were protective against acids, bile salts, heat, oxidative stresses, enterocin A, arsenate and universal stresses. Important adhesion proteins were also encoded by the genome. Additionally, P. pentosaceus LI05 genes encoded proteins associated with the biosynthesis of not only three antimicrobials, including prebacteriocin, lysin and colicin V, but also vitamins and functional amino acids, such as riboflavin, folate, biotin, thiamine and gamma-aminobutyrate. A comparison of P. pentosaceus LI05 with all known genomes of food-borne P. pentosaceus strains  revealed that it possessed four novel exopolysaccharide biosynthesis proteins, additional putative environmental stress tolerance proteins and phage-related proteins.We obtained the draft genome of P. pentosaceus LI05 from the gut and the three other food-borne P. pentosaceus strains through genomic analyses. We have revealed the major genomic differences between these strains, providing a framework for understanding the probiotic effects of strain LI05, which exhibits unique physiological and metabolic properties.This work demonstrated the probiotic properties of  Pediococcus belongs to the family Lactobacillaceae in the order Lactobacillales. Currently, it is comprised of eleven valid published species, including Pediococcus acidilactici, P. stilesii, P. pentosaceus, P. siamensis, P. cellicola, P. argentinicus, P. parvulus, P. ethanolidurans, P. claussenii, P. inopinatus and P. damnosus[Pediococcus are used in the food and drink industry as starter and probiotic cultures as well as food spoilers [P. pentosaceus has been intensively investigated and widely employed for food preservation due to its ability to produce antimicrobial agents [P. pentosaceus have been shown to reduce inflammation, encephalopathy [P. pentosaceus for humans, the strains of P. pentosaceus adapted to the gastrointestinal tract are dissimilar from those found in food because the former may originate from sub-populations present in food at low numbers that exhibit special adaptive properties [The genus  damnosus. The majspoilers . P. pentl agents . Additioalopathy , obesityalopathy  in animaoperties ., we have isolated a potential probiotic, P. pentosaceus LI05 (CGMCC 7049), from the fecal samples of healthy volunteers. This strain is tolerant to bile and acid and possesses strong antimicrobial activities against tested enteropathogens. More importantly, the administration of P. pentosaceus LI05 during acute D-galactosamine-induced liver injury in rats was shown to reduce elevated alanine aminotransferase and aspartate aminotransferase levels, prevent the increase of total bilirubin, reduce the histological abnormalities of both the liver and terminal ileum, decrease bacterial translocation, increase the serum levels of IL-10 and result in a cecal microbiome that differ from that of the liver injury control [Previously05 CGMCC 049, fromP. pentosaceus LI05 in addition to a high-quality draft genome sequence and annotations. The probiotic properties of P. pentosaceus LI05 were analyzed using these genomic sequences combined with data from our previous study. Because the genome sequences of P. pentosaceus SL4 from kimchi [P. pentosaceus IE-3 from a dairy effluent sample [P. pentosaceus ATCC25745 from plant [In this study, we present a summary, classification and the unique characteristics of human gut-borne m kimchi , P. pentt sample , and P. om plant  are now Growth was investigated under different temperature, pH and NaCl conditions. Cell morphologies, motilities and sporulation activities were examined using transmission electron  microscopy. Phenotypic identification was achieved with API CH50 strips and the API CHL medium system according to the manufacturer’s instructions . Other physiological and biochemical tests were conducted as described previously . PhylogeP. pentosaceus LI05 strain (CGMCC 7049) was anaerobically cultured in DeMan-Rogosa-Sharpe  broth at 37°C for 24 h. Cells were obtained by centrifugation at 8,000 g for 10 min at 4°C. DNA was extracted using the QIAamp DNA Micro Kit according to manufacturer’s guidelines .After revival using standard methods, the P. pentosaceus LI05 was sequenced with the next-generation sequencing platform Illumina HiSeq 2000, and the total number of reads based on a 500-bp library database were 2 × 11,079,017 (bp). The quality of the sequencing read data was estimated by calculating the quality and GC content of each read. The draft genome sequence was assembled using SOAPdenovo2 [http://soap.genomics.org.cn/soapdenovo.html). To close the remaining gaps, reference-guided assemblies were carried out with the CLC Genomics Workbench v. 6.05 . The combination of de novo assembly and reference-guided assembly was performed manually using the microbial genome-finishing module in the CLC genomics workbench . The complete genome sequence of P. pentosaceus ATCC 25745 was used as the reference genome.The genome of Pdenovo2 , and iteP. pentosaceus LI05 genes were identified using Glimmer [P. pentosaceus ATCC reference strain from the NCBI Genome Database. After a round of manual curation, the unannotated predicted coding sequences (CDS) were translated into amino acid sequences for a query using the NCBI non-redundant database as well as the UniProt, Pfam, COG, and InterPro databases to identify the closest existing homology annotations. Transfer RNA (tRNA) genes were detected using tRNAScanSE [http://img.jgi.doe.gov/) was used to support additional gene prediction analyses and manual functional annotations [ Glimmer  togetherNAScanSE . RibosomNAScanSE  search aNAScanSE , whereasNAScanSE . The Intotations .P. pentosaceus LI05 from the human gastrointestinal tract with three food-borne strains with available genomic sequences, including P. pentosaceus ATCC 25745, SL4 and IE-3. The P. pentosaceus LI05 genome sequences sharing low identities (<50%) with the other strains were designated as the P. pentosaceus LI05-unique regions. The proteins encoded by the genes that only existed in P. pentosaceus LI05 or that possessed sequence similarities of less than 50% with the three food-borne strains were further analyzed by BLASTp.A comparative genomic analysis using BRIG  was condP. pentosaceus LI05 is a Gram-negative, non-motile, acid-tolerant, non-sporulating, spherical, facultative anaerobe from the human gastrointestinal tract . A total of 11.05 million 100-bp paired-end reads were generated, which provided over 500-fold coverage of the reference genome. High-quality reads with Q > 30 were assembled using de novo methods to obtain a draft genome of 1.75 Mbp with 8 contigs . The G + C content of P. pentosaceus LI05 was 37.29%. For the main chromosome, 1,638 genes were predicted, 1,555 of which were protein-coding genes. A total of 1,321 protein-coding genes were assigned to putative functions, and the remainder were classified as hypothetical proteins. This genome contained 50 tRNAs and a complete 5S-23S-16S rRNA gene family. The properties and statistics of the genome are shown in Table P. pentosaceus LI05 was highly conserved compared with those of P. pentosaceus ATCC 25745, P. pentosaceus SL4 and P. pentosaceus IE-3.The genome of P. pentosaceus LI05 is resistant to gastric acidity and bile compounds [P. pentosaceus LI05 and P. pentosaceus ATCC 25745, P. pentosaceus IE-3 or P. pentosaceus sL4, but most of them showed significant divergences from other species.In a previous study, we have observed that ompounds . This waP. pentosaceus LI05 genes had predicted adhesive potentials  encoding pediocin PA-1 was detected in P. pentosaceus IE-3, but it was not identical to the prebacteriocin gene of P. pentosaceus LI05. Furthermore, genes encoding colicin V, which is a peptide antibiotic that kills sensitive cells by disrupting their membrane potentials [P. pentosaceus strains. However, the colicin V discovered in strain L105 was different from that of the other spicies. Additionally, genes encoding lysin were detected in P. pentosaceus LI05 and P. pentosaceus ATCC 25745. As an antimicrobial agent, lysin is potentially immunogenic [P. pentosaceus LI05 can achieve “competitive exclusion” not only by limiting the surface area available but also by secreting antimicrobial substances.The tentials , were founogenic . TherefoP. pentosaceus LI05, we also detected potentially beneficial properties that were not experimentally confirmed. This strain contained genes involved in the biosynthesis of not only important vitamins, such as riboflavin, folate, thiamine and biotin but also of functional factors, such as gamma-aminobutyrate  and a polysaccharide biosynthesis protein. Four of these enzymes need to be examined in further detail because they are not only potentially novel but also probably induce variations in the structures of their encoded polysaccharides that may have influenced adherence, biofilm formation and the nature of the immune response [Five putative exopolysaccharide biosynthesis proteins were detected only in response .P. pentosaceus LI05 was characterized by three extra-environmental stress tolerance proteins, including a putative ferritin-like DNA-binding protein, which maintains a steady state of iron ions and responds to stresses, such as those involving temperature, humidity, and ionizing and redox processes [rocesses , a putatrocesses .P. pentosaceus LI05. One group was comprised of twelve prophage-related proteins, including a phage integrase family site-specific recombinase, two integrases, a putative prophage repressor, two phage proteins, a replisome organizer, a terminase, two minor capsid proteins, a capsid protein and a tail protein. It is not rare for bacteria to contain multiple prophages in their chromosomes, which then constitute a sizable proportion of their total chromosomal material [P. pentosaceus LI05 included two encoding bacterial DNA type I restriction endonucleases, which are involved in prokaryotic DNA restriction-modification mechanisms that protect the bacteria against invading foreign DNA [Fourteen proteins related to the intrusion of exogenous DNA were identified in material . Pathogematerial . Phage-reign DNA .P. pentosaceus LI05. One ORF encoded DrrA, which is part of the ABC transporter complex DrrAB. The other ORF encoded DrrC, which is part of the ABC transporter permease protein. This finding partially reflects the complex interactions between drugs and gut-associated microbes [P. pentosaceus LI05.Two putative doxorubicin-daunorubicin resistance proteins existed in microbes . Both daP. pentosaceus LI05. These included a TetR family transcriptional regulator, an ABC transporter permease, an exonuclease ABC subunit A, a transposase, an acetyl xylan esterase, a PadR family transcriptional regulator, a membrane protein and a TraX family protein.Additionally, there were eight extra putative multifunctional proteins in P. pentosaceus are frequently identified in food and in the human gastrointestinal tract and are known to reduce inflammation, encephalopathy, obesity and fatty liver in animals. Therefore, it is imperative to study the probiotic ability of this organism. Future studies will focus on delineating the interactions between the host and P. pentosaceus. The genome sequences of P. pentosaceus LI05 isolated from the human gastrointestinal tract allow for a deeper understanding of its probiotic abilities, facilitating the future development of drugs for microbiota-related diseases.Strains of P. pentosaceus LI05 has been submitted to GenBank under the project accession number PRJNA237570. The project version entailing the draft assembly described herein has been deposited under the accession number JDVW00000000.The whole-genome sequencing project of The authors declare no competing interests.L-JL designed the study, interpreted the results and edited the manuscript. L-XL and Y-DL conducted the Illumina sequencing, performed the assemblies, analyzed the genome, and performed the annotations. X-JH provided advice related to the outbreak and strain features, characterized the strain and maintained it in pure cultures. H-YS contributed to the microbiology of the strain and prepared high-molecular-weight DNA for the genome sequencing. All authors read and approved the manuscript prior to submission.P. pentosaceus LI05.Scanning electron micrograph (A) and transmission electron micrograph (B) of Click here for fileP. pentosaceus LI05 to grow using specific carbohydrates.Ability of Click here for file


However, ndh genes are truncated or deleted in some autotrophic Epidendroideae orchid cp genomes. To determine the evolutionary timing of the gene deletions and the genomic locations of the various ndh genes in orchids, the cp genomes of Vanilla planifolia, Paphiopedilum armeniacum, Paphiopedilum niveum, Cypripedium formosanum, Habenaria longidenticulata, Goodyera fumata and Masdevallia picturata were sequenced; these genomes represent Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae subfamilies. Four orchid cp genome sequences were found to contain a complete set of ndh genes. In other genomes, ndh deletions did not correlate to known taxonomic or evolutionary relationships and deletions occurred independently after the orchid family split into different subfamilies. In orchids lacking cp encoded ndh genes, non cp localized ndh sequences were identified. In Erycina pusilla, at least 10 truncated ndh gene fragments were found transferred to the mitochondrial (mt) genome. The phenomenon of orchid ndh transfer to the mt genome existed in ndh-deleted orchids and also in ndh containing species.The NAD(P)H dehydrogenase complex is encoded by 11  These genes are homologs of those encoding mitochondrial NADH dehydrogenase subunits, which are involved in respiratory electron transport5Eukaryotic cells arose through the engulfment of bacterial endosymbionts and the subsequent gradual conversion of those bacteria into organelles (mitochondria and chloroplasts)1ndh genesErodium, Melianthus and orchids also lack functional ndh genes in their cp genomes10111213141516171920Phalaenopsis, Oncidium, Erycina, and Cymbidium, have been sequenced and are publicly available111416ndhB of Oncidium ‘Gower Ramsey' and ndhE, J, and C of Cymbidium have been predicted to encode functional ndh proteins14ndh gene fragments contain nonsense mutations or are truncated or absent from the plastome111416E. pusilla contains truncated versions of ndhJ, C, D, B, G, and H and lacks sequences for ndhK, F, E, A, and Indh gene fragments are common to orchid cp genomes. However, the ndh genes in the cp genome of Apostasia wallichii (Apostasioideae) are transcribed and are predicted to encode functional proteinsndh genes. So far only Epidendroideae cp genome sequences have been published111416ndh genes in their cp genomes has the potential to considerably increase understanding of photosynthetic evolution in orchids.Heterotrophic plants, which do not photosynthesize, lack functional Phalaenopsis, the ancestral ndh genes have been transferred to the genome of the nucleus or another organelle. Recently published mitochondrial genomes from different seed plant species contain 1 to 10% cpDNA originating from various regions of the plastome2829E. pusilla and O. ‘Gower Ramsey'16E. pusilla cp genomePrevious studies have demonstrated that plant cpDNA has been transferred to both the mitochondrial (mt)2312526ndh deletions from orchid cp genomes was investigated. The cp genomes of Vanilla planifolia (Vanilloideae), Paphiopedilum armeniacum, Paphiopedilum niveum, Cypripedium formosanum (Cypripedioideae), Habenaria longidenticulata, Goodyera fumata (Orchidoideae), and Masdevallia picturata (Epidendroideae) were sequenced. In addition, the mitochondrial locations of the ndh genes were investigated in E. pusilla, an Epidendroideae model orchid, by sequencing BAC clones that contained the ndh genes that were missing from the cp genome.In this report, the evolutionary timings of ndh genes in higher plant chloroplast genomes. To understand the differential expression of these genes among the orchid subfamilies, ndh transcripts were identified in 16 species from five Orchidaceae subfamilies (Supplementary Table S1 and S2). Neuwiedia malipoensis (Apostasioideae), Cypripedium singchii (Cypripedioideae), Habenaria delavayi, Goodyera pubescens (Orchidoideae), Masdevallia yuangensis and Cymbidium sinense (Epidendroideae) had all 11 ndh genes. However, Vanilla shenzhenica, V. planifolia, Galeola faberi (Vanilloideae), Drakaea elastica (Orchidoideae), and E. pusilla (Epidendroideae), had only less 5 ndh gene sequences (Supplementary Table S1). The number of ndh gene sequences in each transcriptome did not correlate with known orchid evolutionary relationships (Supplementary Table S1). The fewest ndh transcripts were found in Vanilloideae. Previous studies have reported ndh deletions in Epidendroideae species, but an analysis of the M. yuangensis transcriptome database revealed highly conserved sequences (79.4%) for all of the ndh genes. The same phenomenon also occurred in C. sinense (Supplementary Table S1). These results indicate that the loss of ndh genes from the orchid species occurred independently within the subfamilies.There are 11 ndh DNAs in the cp genome were in frame and encoded the full-length ndh gene, the genomes are classified as ndh-complete type cp genome. If any one of the 11 ndh genes had nonsense mutations, deletions, or was absent, the genomes are classified as ndh-deleted type cp genome. There were both ndh-complete- and ndh-deleted types in Cypripedioideae  and in Epidendroideae [E. pusilla (ndh-deleted) and M. picturata (ndh-complete)]. Among the only Vanilloideae species investigated, V. planifolia, was an ndh-deleted type, but this result cannot exclude the possibility of ndh-complete species in other Vanilloideae species. The two sequenced cp genomes in Orchidoideae, H. longidenticulata and G. fumata, are both ndh-complete. Based on the transcriptome results, D. elastica, an endangered native Australian orchid, has few ndh transcripts (Supplementary Table S1). This species may be an example of ndh-deleted Orchidoideae.The cp genomes of seven orchids in four subfamilies were determined . These results, therefore, indicate that non-cp ndh genes exist and may be transcribed. We therefore attempted to identify these genes in mt or nuclear genome sequences.A comparison of the cp genomic and transcriptomic data showed that only ndh gene fragments were identified in the orchid whole genomic sequences. In addition to the ndh-deleted species, the ndh-complete species  also contained non-cp ndh gene fragments (Supplementary Tables S3 and S4). Because ndh genes in the cp genomes of all plant species are clustered as ndhJ-K-C, ndhF-D-E-G-I-A-H and ndhB, the results below are presented in this order.Many non-cp E. pusilla has been established as a model for orchid genome researchndh-containing sequences, which were derived from the genomic NGS sequences, specific primers were designed to screen the E. pusilla BAC libraryndh-like gene fragments and to determine the organelle of origin. Six BAC clones that contained putative ndh gene fragments were identified: Ep-mt-ndhJC, Ep-mt-ndhC, Ep-mt-ndhDEGIAH, Ep-mt-ndhDF, Ep-mt-ndhD and Ep-mt-ndhB . In total, E. pusilla had the greatest number of ndh gene fragments transferred to the mt genome . These results showed that the number of transferred ndh gene fragments did not correlate with ndh deletions in the cp genome.Although flanking DNA sequences can provide information to identify the organelle from which they were derived, some of the sequences were too similar to cp genome sequences or were too short to identify . Therefore, it would be important to extend the sequence length in future studies. In recent studies, -mt-ndhB . An analmt-ndh regions and the original cp region, E. pusilla mt genome sequence was aligned with the corresponding C. formosanum (ndh-complete) and E. pusilla cp genome regions. This alignment showed a massive loss of DNA in the ndh copies that were transferred to the E. pusilla mt genome  mt genomes contained partial cp genome fragments (ndh DNA. The length of the mt-ndh genic regions reflected the taxonomic relationships (Supplementary Table S3). For Oryza, the presence of ndh sequences in the mt genome correlated at the genus level (Supplementary Table S3). Based on a phylogenetic analysis of their cp genomes, Bambusa oldhamii and Oryza sativa were more closely related to each other than the other Poaceae species. They have a similar ndh transfer pattern, and the presence of ndhJ, ndhK and ndhC in both their mt genomes. However, the appearance of mt genome ndh sequences at the species level was different in the Zea genus . These results indicate that the mt genomes among members of the same family or genera may show different ndh content. This phenomenon is likely due to mtDNA rearrangementsP. armeniacum and P. niveum are not complete. As more mt genomes are sequenced and completed, the taxonomic relationship between the ndh regions and the cp genome sequences can be further studied.An evolutionary snapshot of ea genus 32; transndh genes, only mt-ndhD in Vitis vinifera, mt-ndhJ in Phoenix dactylifera, B. oldhamii, and Oryza and mt-ndhB in Triticum aestivum and Zea are full length (Supplementary Table S3)mt-ndh gene fragments are in frame and their transcripts have been identified in these species, they cannot complement ndh function in ndh-deleted orchids.Of all of these mt ndh gene fragments. Some cp-like contigs were identified among the E. pusilla genomic sequences and found to be similar to other plant mt genome sequences through BLASTN. Using BAC library screening, these cp-like mt DNA clones were identified in E. pusilla . According to the transcriptome results detailed above, the ndh DNA regions in the orchid mt genome do not encode functional proteins. Interestingly, nine cp-like mt genes  contained intact gene sequences with start and stop codons but may not have required regulatory sequences.There were 60 cp-like protein-coding genes in the E. pusilla transcriptome. There were 28 cp-like mt gene transcripts (Supplementary Table S8), including three intact genes: psaJ, rpl36, and rpl22. The mt-psbI sequence shared 100% identity with the cp genome sequence; therefore, we could not conclude the derivation of the transcript. The cp-like mt transcripts could be divided into two types: those containing only cp-like gene fragments and those containing mt genes (KJ501986 and KJ501987). These results indicate that these gene fragments can be transcribed but translation requires further investigation.To determine which of the cp-like mt gene fragments could be transcribed, BLASTN was used to search the ndh genes in orchid genomes. However, transcriptome data should be interpreted with caution for several reasons. First, it is difficult to determine the source genome  from transcriptome data. Second, the transcripts identified may not be full-length, may contain untranslated portions, or may not correlate with the cp genomic sequences. Third, transcriptome profiles differed between species within a subfamily. For example, in the Cypripedioideae, all 11 ndh genes were identified in Cypripedium but only 7 were identified in Paphiopedilum. Fourth, source materials  also influence a transcription profile. Although some of the transcriptome data did not show full-length ndh genes, this does not necessarily mean that these genes were lost or truncated from these species. Because of these vagaries, results deduced from transcriptomes need to be confirmed by targeted sequencing of genomic DNA.According to our results, a whole-cell transcriptome database can provide useful information for investigation of ndh genes among orchid subfamilies. Our data also indicate that species within the same subfamily may have different ndh gene profiles.Comparison of the transcriptomes and cp genomes yielded some information; however, cp genome sequences from additional orchid species across the five subfamilies will be required to better understand the structures of ndh-deleted and ndh-complete species exist in the Orchidaceae family. Martin and Sabater hypothesized that the function of ndh genes are related to terrestrial adaptations of photosynthesisndh-deleted and ndh-complete orchids in our study in terms of biogeography or growth conditions (including light and water requirements). Leitch et al.ndh-deleted and ndh-complete orchids in terms of genome sizeAccording to the orchid cp genome data, cp ndh gene fragments exist in the mt genome of cp ndh-deleted orchids that are not translated to functional proteins. One possible reason for this phenomenon could be that these cp ndh genes were transferred to the nucleus. This phenomenon has been observed in other cases of cp gene deletion. The cp rpl22 genes have been lost in some rosid speciesrpl22 gene of these rosid species remains functional. This gene can be transcribed and translated into a protein that is targeted to the chloroplast, where it functions in protein synthesisinfA, rpl22, rpl32, rpoA), b) substitution of a nuclear encoded mitochondrial targeted gene (rps16) and c) substitution of a nuclear gene for a plastid gene ndh genes to the mitochondrial genome.Contemporary cyanobacteria encode several thousand genes, but only 20 to 200 of these genes have been retained in modern plastid genomes1ndh genes in some species of orchid. No complete and functional ndh transcripts have been identified in the ndh-deleted orchid transcriptomes2137ndh proteins play an important role in cyclic electron transport in tobacco and M. polymorpha, there are no significant growth differences between the wild type and ndh-deleted transformants63940742ndh-deleted transformants may be able to use this pathway for cyclic electron transport.Another possibility is that there are no ndh genes were found in the transcriptome, likely because these genes are expressed at low levels and/or only during specific developmental stages or under specific growth conditions. Genome sequences may be more useful because they represent complete genetic complements. We performed NGS sequencing of the E. pusilla genome and obtained 15 Gb of sequence data, which corresponds to about 10 times coverage of the genome. No nuclear ndh gene could be identified in the assembled NGS data. Nonetheless, because we have not completed the assembly of a high-quality draft genome, it is difficult to conclude whether any functional ndh genes exist in the nuclear genome. A concrete conclusion can only be made when the whole genome sequences are available for E. pusilla.Not all of the ndh-like pseudogenes could be identified. The transfer of cp DNA to mt DNA is widespread in seed plants4546474849505152Amborella mt genome has the highest amount of cp DNA of all of the ndh genes (138 kb)C. pepo C. sativus P. dactylifera mt genome, there are 74 kb of cp genomic DNA, which encompass six ndh pseudogenesAccording to our result, most of the genes transferred to the mt genome are highly similar to those within the cp genome, indicating that these DNA sequences were transferred to the mt genome more recently than the sequences that contained more insertions/deletions and mutations. The nine proteins  that are encoded by these genes are smaller than are the other cp genome-encoded proteins ; therefore, their genetic integrity may be easier to maintain. In most Tracheophyta, small cp genome fragments have been identified in mt genomes. In rice, there are 16 cp genome segments in the mt genome with sizes ranging from 32 bp to 6.8 kbE. pusilla. A previous study indicated that larger mt genomes contain more cp DNAE. pusilla mt genome may be large and complicated. A complete sequence of the mt genome of E. pusilla will increase our understanding of this phenomenon. In the near future, NGS sequencing of the BAC library will continue to facilitate investigations of the genomic complexity in E. pusilla.More than 130 kb of cp genomic DNA has been transferred into the mt genome in Amborella mt genome, which contains 197 foreign protein-coding genes, only 50 (25%) are full-length and contain open reading frames50NADH dehydrogenase subunit 9 (nad9)50Most cp-like mt genes cannot translate functional proteins. In the ndh genes. Using current orchid transcriptome databases and cp genomic sequences, we demonstrated that the complete ndh gene fragment set still exists in some orchid family members. During evolution, several orchids experienced ndh gene deletions, but these deletions are not correlated with orchid taxonomy. Based on the sequences and gene structures of the ndh genes and gene fragments that remain in the cp genomes, we conclude that these deletion events were independent. Because cyclic electron transport pathway is redundant with the NDH complex-dependent pathway, there is no evolutionary pressure to maintain functional copies of ndh genes in other genomes in sharp contrast to essential genes required for chloroplast protein synthesis that were transferred from chloroplast to the nuclear genome. In all of the sequenced orchid genomes, the ndh gene fragments were transferred to the mitochondrial genomes. These transfers were not directly linked to the ndh deletions in the cp genome. The ndh genes that were transferred to the mt genome have been mutated and truncated and do not complement the ndh gene function. Furthermore, the E. pusilla orchid mt genome contains the largest number of cp genome sequences among the published genomes. This result suggests that in E. pusilla, cp-to-mt gene fragment transfers are more frequent. Together, these findings indicate that the mt genomes of orchids  are unique and warrant further investigations.Previous studies have indicated that autotrophic Epidendroideae orchid cp genomes no longer encode all of the ndh genes in the current public orchid transcriptome database, a well-studied monocot species, banana (Musa acuminata), was used to blast target sequences. Eleven ndh amino acid sequences were used as templates for TBLASTN searches (Accession no. HF677508)ndh gene transcripts from public transcriptome databases2137To understand the V. planifolia, H. longidenticulata, G. fumata, P. armeniacum, P. niveum, C. formosanum, M. picturata, and E. pusilla leaves were used as the material for DNA extraction via the cetyltrimethyl ammonium bromide methodvia a limited-cycle PCR program involving 12 cycles of denaturation at 95°C for 10 s, primer annealing at 55°C for 30 s, and extension at 72°C for 30 s, during which the index primers that were required for cluster formation were also added. The amplified DNA was purified using AMPure XP beads . The fragment sizes and concentrations of the libraries were determined by a 2100 Bioanalyzer with a High Sensitivity DNA Assay Kit  and quantitative PCR . Subsequently, the libraries with insert sizes of 500 to 600 bp were denatured with NaOH and sequenced at read lengths of 250 bases from both ends using a MiSeq Personal Sequencer (Illumina).The above orchid genomic and BAC DNA fragments were sequenced by an Illumina high-throughput sequencing platform . The paired-end sequencing libraries were constructed by a Nextera XT DNA Sample Preparation Kit according to the instruction manual (Illumina). Briefly, 10 μg of purified DNA was fragmented using the Amplicon Augment Mix (Illumina). Fragmentation reactions were achieved by incubation at 55°C for 5 min, followed by neutralization in buffer at room temperature for 5 min. The neutralized DNA fragments were amplified Because the mean insert size of each library was approximately 500 to 600 bp, the ends of many reads (250 bp*2) overlapped due to the variability in the insert size. Therefore, the cleaned reads were classified into two groups: overlapping paired-end reads and non-overlapping paired-end reads. The overlapping paired-end reads were further merged into long reads (400 to 500 bp) by FLASH (version 1.2.7) before assemblyE. pusilla cp genome sequence to screen the whole genome sequences of seven orchids by BLASTNThe cp DNA contigs were identified using the ndh-coding sequences in the genomes of the nucleus and other organelles (non-cp ndh genes) have not been identified. To identify the non-cp ndh genes, 11 banana (M. acuminata) ndh amino acid sequences were used as templates for TBLASTN searches of the orchid genomic sequences (Accession no. HF677508)ndh gene fragments with the mt genome sequences in the NCBI database using BLASTN. In addition, the raw read count was used to distinguish the source of the DNA using the correlation between the number of high-throughput sequencing reads per sequence and the source  of the DNA when total DNA was used to exclude the cp genome sequenceBecause no whole nuclear genome sequence of an orchid has been published, regions containing ndh sequences which derived from genomic sequencing in E. pusilla were used to design the specific primers. These primers were used to screen the BAC library. Using this strategy, there was no need to isolate the nuclei or mitochondria or to purify the genomic DNA without cp genome contamination. The sequences were annotated by BLASTX using the Arabidopsis protein database. The figures of the 446 annotated and aligned DNA positions were drawn using PowerPoint . The flow chart is shown in Supplementary Figure S5.The non-cp rbcL, matK, psaA, and psaB nucleotide sequences were used for the phylogenetic analysis. The consensus sequences were aligned using ClustalW2  and concatenated. Subsequently, the maximum-likelihood phylogeny was estimated using a web version of PhyML 3.0 (http://www.atgc-montpellier.fr/phyml/) using the multiple sequence alignmentshttp://supfam.cs.bris.ac.uk/TreeVector/).To investigate the relationships between the orchids in this report, the combined chloroplast ndh genes in the published plant mt genomes, the 43 complete mt genomes and the cp genomes of these species or related species in the same genus were downloaded from the NCBI database. The species and accession nos. are listed in Supplementary Table S9. The cp and mt genomes were compared using BLASTN to identify the cp DNA in the mt genomes.To identify the ndh genic regions and cp-like DNA (Supplementary Table S10). The BAC clones containing the ndh regions and cp-like DNA of interest were obtained by PCR screening from the super pool, plate, row and spotO. ‘Gower Ramsey' BAC library to identify ndh clones.The specific primers were designed to identify the BAC clones carrying the M.C.S., M.T.C., H.D. and C.S.L. designed the research. J.J.W.C., Y.T.H., S.Y.L., M.C.S. and M.T.C. performed next generation sequencing. J.J.W.C., Y.T.H., S.Y.L., M.C.S., M.T.C., C.F.L. and C.Y.C. performed bioinformatics analysis. H.D., W.J.C., C.T.H., D.C.L., F.H.W. and M.L.C. performed chloroplast genome annotation and uploaded to NCBI. W.J.C., C.T.H., D.C.L. and F.H.W. performed BAC library screening and sequencing. G.K.S.W. and M.K.D. established the 1000 plants transcriptome database. M.C.S., W.J.C., H.D., M.K.D., V.A.A. and C.S.L. wrote the paper.Supplementary Information


Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus .African clawed frogs, genus  Xenopus, Pipidae, subfamily Xenopodinae) are found across sub-Saharan Africa. These frogs prefer slow moving or stagnant water as tadpoles and as adults, although migration between bodies of water occurs , coll. Reichenow; based on Bauer et al. ; 350 m; S 4.3° E 15.3°, August 1978, coll. V. Nzingula.Paratypes.—BMNH 1982.463, female, and BMNH 1982.464–65, adult males, same collection information as holotype.Diagnosis.—Xenopus epitropicalis is a tetraploid species with a biphasic-type call  sensu Salamone   Pipa fro hopping . OverallX. eysoole and X. longipes. However, a strict interpretation of phylogenetic relationships in autosomal DNA suggests an independent origin of X. eysoole and X. longipes because the α2 homeologs of X. eysoole and X. amieti are more closely related to each other than either is to that of X. longipes. Caveats to a strict interpretation of this phylogeny include that we are unable at this time to distinguish ancestral polymorphism from true orthologous relationships, that recombination between homeologs within species could introduce error into phylogenetic estimation, and that phylogenetic error and uncertainty could cause departures of the estimated phylogeny from the true phylogeny. These caveats notwithstanding, the inferences of lost ancestors argue for further fieldwork aimed at characterizing new species of Xenopus.Our summary phylogeny, which attempts to conservatively interpret nodes with weak support or phylogenetic discordance among genes , depictset al. .The parasite fauna of  anurans . This diXenopus. For example, species of Cephalochlamys occur in all tetraploid species of subgenus Xenopus so far examined, but not in octoploids, even when the octoploids co-occur with infected X. victorianus . This is consistent with the possibility that increased gene dosage or inheritance of resistance genes with complementary functions in octoploids confers parasite resistance. Parasites from the genus Protopolystoma provide a counter-example of increased susceptibility of higher ploidy levels. Most species in this parasite genus infect only one anuran host species. However, the tetraploid species X. victorianus and X. parafraseri and the octoploid species X. wittei each are infected by two Protopolystoma species  [P. fissilis occurs in X. parafraseri and in X. wittei could represent shared inheritance of an ancestral susceptibility derived from a diploid ancestor [X. victorianus and X. wittei, although two parasite species occur side-by-side in the same host populations, they never co-occur as adults within the same host individuals [In addition to parallel evolution of host and parasite , patternmplicis) , 103. Thancestor . InteresX. laevis and X. muelleri [Protopolystoma parasites and laboratory-generated F1 hybrids are also largely resistant [The effects of host interspecies hybridization (not involving genome duplication) on susceptibility to parasite infection have been investigated in muelleri . These hesistant . This stXenopus species occur in Central Africa, including the six new species described here, the resurrected species X. calcaratus, and nine other previously known species: X. amieti, X. andrei, X. boumbaensis, X. epitropicalis, X. fraseri, X. longipes, X. poweri, X. pygmaeus, X. tropicalis. This list includes representatives of both subgenera, and each species group as newly defined (but not the Ethiopian endemic X. largeni). Three of these species  have distributions centered in the Congo Basin, X. fischbergi has a large range over much of the northern Congo Basin, but the rest are probably endemic to the portion of Central Africa northwest of the Congo River.Over half of Xenopus diversity is a large number of species with high ploidy levels; specifically three of the seven octoploid species and three of the four described dodecaploid species occur in Central Africa. These species are a result of multiple independent allopolyploidization events that combined a few ancestral genomes in several unique ways. The diversity of octoploid and dodecaploid species raises the question of whether allopolyploidization conferred a selective advantage for species in Central Africa, such as those related to immune function discussed above.What could explain this high species diversity in Central Africa? Persistent forest habitat –107 coulS1 FigLabeling follows (TIF)Click here for additional data file.S2 FigLabeling follows (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 FigSilurana: X. tropicalis (BMNH 1947.2.24.83) and X. epitropicalis (BMNH 1982.462), from amieti species group: X. amieti (MHNG 2030.80), X. andrei (MHNG 2088.32), X. boumbaensis (MHNG 2080.31), X. itombwensis (MCZ A-138192), X. longipes (MHNG 2497.10), X. ruwenzoriensis (MHNG 2238.15), and X. lenduensis (MCZ A-139853), from laevis species group: X. laevis sudanensis  and X. laevis bunyoniensis , and X. fraseri (BMNH 1947.2.24.78). Scale bar is 5 mm.Images include, from subgenus (TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Amblysomus) endemic to the GMPA, to test for cryptic diversity within the genus, and to better understand diversification across the region. We sampled specimens from 50 sites across the distributional range of Amblysomus, with emphasis on the widespread A. hottentotus, to analyse geographic patterns of genetic diversity using mitochondrial DNA (mtDNA) and nuclear intron data. Molecular dating was used to elucidate the evolutionary and phylogeographic history of Amblysomus. Our phylogenetic reconstructions show that A. hottentotus comprises several distinct lineages, or evolutionarily significant units (ESUs), some with restricted geographic ranges and thus worthy of conservation attention. Divergence of the major lineages dated to the early Pliocene, with later radiations in the GMPA during the late-Pliocene to early-Pleistocene. Evolutionary diversification within Amblysomus may have been driven by uplift of the Great Escarpment c. 5–3 million years ago (Ma), habitat changes associated with intensification of the east-west rainfall gradient across South Africa and the influence of subsequent global climatic cycles. These drivers possibly facilitated geographic spread of ancestral lineages, local adaptation and vicariant isolation. Our study adds to growing empirical evidence identifying East and southern Africa as cradles of vertebrate diversity.The Greater Maputaland-Pondoland-Albany (GMPA) region of southern Africa was recently designated as a centre of vertebrate endemism. The phylogeography of the vertebrate taxa occupying this region may provide insights into the evolution of faunal endemism in south-eastern Africa. Here we investigate the phylogeographic patterns of an understudied small mammal species assemblage ( The Afromontane Region of Africa , 2 is a et al.  was used to determine the best-fit model of sequence evolution for each partition using the Bayesian Information Criterion (BIC) [MrBayes, while the GTR+G substitution model was employed in RAxML, since this program only accommodates GTR-related [Phylogenetic analyses were performed based on the combined data matrix of the three targeted gene regions in a representative sample of 17 individuals. Both a partitioned Maximum Likelihood (ML) method , as implL v7.2.6 , and Bayyes v3.2 , were ust v2.1.7 ,59 won (BIC)  to choos-related  models. -related .Tcs v1.21 [MT-ND2 sequences. Genealogical relationships between different haplotypes were inferred within a statistical parsimony framework [ArcMap v.10 [Adobe Photoshop v. 10.0.1.cs v1.21  was usedramework  reflectiMap v.10  with GLOMap v.10 , and figArlequin v3.5 [Amblysomus lineages comprising adequate MT-ND2 sample sizes. A. septentrionalis and A. robustus did not have adequate sample sizes and were therefore analysed along with A. h. longiceps as a single population, given the results of the phylogenetic analysis  [Chrysochloris arenosa (c. 6.5–5.0 Ma) [Proamblysomus antiquus (ca. 4.5–0.5 Ma) [Chrysochloris asiatica (Cape golden mole) and Microgale longicaudata (lesser long-tailed shrew tenrec) were downloaded from GenBank and used as outgroup taxa in the divergence dating analyses (accession numbers C. asiatica: AB096866.1 (MT-ND2+cyt b), AJ428944.1 (MT-ND2+cyt b); M. longicaudata: AY193410.1 (MT-ND2), AY193412.1 (MT-ND2), and AY193416.1 (MT-ND2)).Divergence dates between clades were estimated from the combined molecular dataset using ast v2.3 . Molecul[Mega v6  to deter mammals ), and a  mammals  was specago; Ma) , and two–5.0 Ma)  and Proa–0.5 Ma) . It is w–0.5 Ma) . Thus foMT-ND2: HKY+G [b: GTR+G+I [MT-ND2 dataset, comprising adequate sample sizes for population analyses. MCMC simulations ran for 30 million generations, sampling every 3000 generations. Convergence and mixing were assessed and effective sample size (ESS) values monitored using Tracer v1.6 [TreeAnnotator v1.7.2, after discarding the first 100 trees as burnin, and the tree was visualized using FigTree v1.4.2 [The best-fit substitution models were specified for each gene  and separate clearly from other Amblysomus species, consistent with the current taxonomic treatment, A. robustus and A. septentrionalis (clades A and B) instead cluster within A. hottentotus, sister to A. h. longiceps (clades C-E). Amblysomus h. meesteri (clade N) is sister to A. marleyi, and clearly distinct from A. hottentotus.While A. h. longiceps, A. septentrionalis and A. robustus , while A. h. hottentotus clusters as a poorly supported monophyletic sister clade . Samples from EL and KW cluster with A. h. hottentotus, and not with A. h. pondoliae as expected, indicating that A. h. pondoliae does not range as far south along the coast as previously suggested , sister to uggested .Amblysomus h. pondoliae (clades H-J) constitutes a monophyletic clade, sister to the abovementioned lineages . A striking finding is that samples from the northernmost localities of the currently understood distribution of A. h. pondoliae  are retrieved as a sister clade to A. h. iris (clade L), and are thus substantially divergent from A. h. pondoliae. Evidently this central coastal clade (clade K), represents a cryptic lineage more closely related to A. h. iris from Maputaland to the north than to A. h. pondoliae from the KwaZulu-Natal (KZN) Coastal Belt to the south.MT-ND2 lineages analysed were generally characterized by high haplotype diversity and low nucleotide diversity . Fu’s Fs was similarly negative and non-significant for all but the Central coastal clade , the extended A. h. longiceps clade  and A. h. meesteri . Fu’s Fs is very sensitive to population demographic expansion, which generally leads to large negative Fs values, therefore the significant negative values obtained for the latter two lineages could possibly reflect recent population expansions. Pairwise genetic distances for the various Amblysomus lineages ranged from 0.4% to 6.8%  does not represent a monophyletic clade, since the two samples from NF and ER are both more closely related to the A. robustus individual from DU than they are to each other, while the individual from SW could not be connected at the 95% confidence limit.The close phylogenetic affinity of tic tree  is borne network , revealietrieved . A. septA. h. pondoliae , but not for MT-ND2 (P = 0.9994), therefore the use of a strict clock was deemed acceptable for this partition only. The deepest split within Amblysomus was dated to the early Pliocene, c. 4.42 Ma . Several splitting events coincide with the late Pliocene, including ancestors to A. h. pondoliae and A. h. hottentotus, as well as the separation of A. h. meesteri and A. marleyi. Divergence of the unique lineage from Umtata dates to the early Pleistocene. Surprisingly, the divergence of A. robustus and A. septentrionalis from A. hottentotus is estimated at only c. 0.99 Ma, almost contemporaneous with the divergence between the Central Coast lineage and A. h. iris (1.01 Ma). Further divergence of the major lineages occurred throughout the Pleistocene, as borne out in the lineage-specific coalescent-based divergence dating trees  can be phy, BPP ) which, A. h. meesteri is a monophyletic lineage highly divergent from A. hottentotus. This ESU is a geographically isolated population that also differs morphologically [A. hottentotus, and almost certainly represents a unique species. The current taxonomic recognition of three subspecies  along the GMPA coast is challenged by our results, which conclusively show that there are four coastal ESUs. Amblysomus from the Central Coast  could therefore represent a distinct species for which the name A. natalensis [Amblysomus from the Maputaland coastal localities north of the Thugela River could similarly represent the distinct species A. iris. The apparent sympatry of these ESUs at AM requires further investigation, but may be the result of accidental anthropogenic transport of golden moles (in topsoils and sands for construction projects) further north along this highly developed coast. Samples from the KZN Coastal Belt region south of SB represent a distinct ESU (possibly worthy of species status), and samples from EL and KW, previously assigned to A. h. pondoliae, instead belong to A. h. hottentotus, which therefore ranges further north than previously hypothesized, and probably also represents a unique species (A. hottentotus) [Our results reveal that ogically  and cytoogically  from othy Durban) would beentotus) . The preA. septentrionalis and A. robustus within an A. hottentotus clade. Aside from the unexpectedly recent divergence of A. robustus  from A. h. longiceps (2n = 30) [A. h. longiceps may be warranted. Further analysis involving more geographic samples and genes will be needed to resolve taxonomic relationships within the major Amblysomus ESUs discussed here.Perhaps the most intriguing finding of this study is the clustering of 2n = 30) , 85, indWhile we present compelling evidence that some of the cryptic lineages identified by our analyses may be valid species, this remains to be corroborated. RAD-sequencing studies are currently underway to provide stronger nuclear support for such decisions, as well as to address some of the fine-scale questions raised by this study. Further sampling, as well as ecological niche modelling, could also provide additional information to support a taxonomic revision. Until then, it is important that the cryptic diversity and ESUs described here be recognised, and that possible conservation implications be addressed.Amblysomus are strikingly congruent with palaeo-ecological and geomorphological events during the Neogene and Quaternary [Amblysomus, starting c. 4.42 Ma, coincided with the epeirogenic uplift of the Great Escarpment c. 5–3 Ma [Amblysomus today exists.The major evolutionary divergences within aternary , 86, 87 . 5–3 Ma , and the. 5–3 Ma , while a. 5–3 Ma . These eAmblysomus today. These conditions may have facilitated the dispersal of a stem Amblysomus that became widespread. The major uplift c. 5–3 Ma potentially isolated populations in the north of the ancestral range that gave rise to the ancestors of extant A. marleyi and A. h. meesteri, which likely represent biogeographic relicts associated with the Lebombo mountain range and the northern Drakensberg. Both of these regions are prominent in terms of narrow endemics within the GMPA [A. marleyi in the Lebombo mountain range and the northernmost coastal populations of Amblysomus, with subsequent competitive exclusion by the extant sand specialist Calcochloris obtusirostris (yellow golden mole).In the early Pliocene, relief along the eastern coast of South Africa was less steep without deep incision by rivers, and climates were warm and humid enough to support woodlands , much lithe GMPA . During the GMPA , 91. AeoAmblysomus populations may have become isolated in the developing Fynbos Biome, which was established by the late Pliocene [Amblysomus corriae may have been driven to adapt to the western winter-rainfall zone, established around the Miocene-Pliocene boundary [Southernmost Pliocene . An incrPliocene  was coupPliocene . Since gboundary .A. corriae (in developing Fynbos) and an inland Amblysomus lineage  further north. This gap, established by the end of the Pliocene, also coincides with the transition between winter/aseasonal and summer rainfall zones [Mysorex varius), which occurs sympatrically with populations of the GMPA endemic, Sclater’s Forest Shrew (Myosorex sclateri) [Myosorex cafer sensu stricto) [Strongylopus grayii) [Cordylus cordylus) [Dendrohyrax arboreus arboureus), the samango monkey (Cercopithecus mitis labiatus) and the oribi antelope (Ourebia ourebia) [Although the uplift along the southern Cape coast was less pronounced, there was a dramatic increase in relief with deep incision by river gorges and increased erosion that exposed more fertile Cretaceous sediments, leading to elevated topo-edaphic heterogeneity that facilitated diversification of numerous endemic Cape floral clades . The “Bell zones , and hasclateri) , anotherstricto) , the sou grayii) , the Capordylus) , the treourebia) .c. 2.8±0.2, 1.7±0.1 and 1.0±0.2 Ma, interspersed by warmer, humid periods [Amblysomus taxa by causing expansion and contraction of suitable habitats.Although only the most high-lying regions of southern Africa experienced the direct effects of peri-glacial conditions , 101, gl periods . It is lA. h. pondoliae, A. h. iris and the Central coastal clade) between 2.5 and 2 Ma. Cooling and aridification led to the spread of more open habitats towards the retreating coastline, and the expansion of ancestral lineages from inland refugia towards the coast owing to increased aridity in the interior. During subsequent warmer and wetter periods, ancestral populations may have moved inland owing to rising sea levels, and the spread of coastal woodland habitats [Marine regressions and transgressions associated with Pliocene climatic cycles were sufficient to change sea levels along the South African coast by 120–130 m above or below current levels, although this would have been differential along the coast due to the variation in slope and width of the continental shelf . Such chhabitats .A. h. pondoliae and the Central coastal clade) along the south-central KZN coast that are more closely related to inland A. h. longiceps than to each other. Amblysomus h. pondoliae and the Central coastal clade may therefore be descendants of two separate colonization events from an ancestral population that probably inhabited the Drakensberg foothills further inland. Gene flow between A. h. longiceps and these coastal lineages may have been (and continue to be) constrained further by the unfavourable clay soils that prevail from the Drakensberg foothills of the northeastern border of Lesotho across to the Thugela River [A. h. iris and Central coastal clade, may similarly have been mediated by the Thugela River (the largest river in KZN) which continues to act as a dispersal barrier.Such range changes, however, would have been constrained by the many perennial rivers that flow from the escarpment eastwards to the coast. This scenario is consistent with the presence of two divergent lineages  now occur. The current Grassland and Savanna Biomes comprise both arid and mesic bioregions [A. h. longiceps today occurs, are dominated by C3 grasses, reflecting persistence of more stable and moister Quaternary climates. Grasslands on the plateau of the Great Escarpment further north are instead dominated by C4 grasses, which flourished in the cooler and drier high altitude zone [Amblysomus lineages.The early Pliocene uplift raised a large area of eastern-central South Africa into cooler, higher altitudinal zones suitable for grasslands, and was followed by an increase in aridity and rainfall seasonality in the interior . Pliocenoregions . Drakensude zone . While pude zone . Mesic pAmblysomus lineages  possibly representing distinct species. We provide support for the current recognition of A. corriae and A. marleyi, and additionally for the recognition of A. meesteri, A. longiceps, A. iris, A. pondoliae, A. natalensis and A. hottentotus as valid species, but refrain from formal taxonomic revision until this hypothesis can be further corroborated by more rigorous species delimitation.In the present study we uncover substantial cryptic diversity in Amblysomus hottentotus has until now been considered widespread and abundant and, as a result, is not under formal protection. The threats posed to the cryptic taxa that have been uncovered here remain to be determined, and consequently, the conservation status of the newly recognized ESUs will need re-examination. We expect that a similar pattern of cryptic diversity may be found in other species distributed within and across the GMPA, particularly other widespread golden mole species, such as the Cape golden mole (Chrysochloris asiatica), as well as other poorly studied southern African endemics. Phylogenetic studies will be particularly important for insular populations of “widespread” species that are similarly range-restricted, and might also contain cryptic species in need of conservation attention.Amblysomus, this study provides evidence in support of our hypothesis that geomorphological changes and habitat heterogeneity primarily drove diversification in Amblysomus. Our study implicates the uplift of the Great Escarpment, as well as various palaeo-ecological and geomorphological events during the Neogene and Quaternary in shaping diversification of Amblysomus across the GMPA. The Plio-Pleistocene global climatic cycles that established the inland vegetation heterogeneity of South Africa were instrumental in establishing the deeper splits, with subsequent gene flow between coastal lineages being constrained by the many perennial rivers that flow from the escarpment towards the coast. These rivers probably served as biogeographic barriers that channeled range expansions and contractions, and continue to reinforce the genetic distinctiveness of these lineages.In addition to uncovering cryptic diversity in Amblysomus, also demonstrated rapid divergence of major lineages around 2 Ma, and similarly implicated the influence of rainfall regime and landscape heterogeneity as major drivers of this divergence [Similar patterns of divergence have been reported in a diversity of other co-distributed taxa, as we have discussed. One study focusing on southern African shrews, in particular, which are co-distributed across a highly similar geographic range to vergence . Our stuS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


AREsite2 represents an update for AREsite, an on-line resource for the investigation of AU-rich elements (ARE) in human and mouse mRNA 3′UTR sequences. The new updated and enhanced version allows detailed investigation of AU, GU and U-rich elements  in the transcriptome of Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans and Drosophila melanogaster. It contains information on genomic location, genic context, RNA secondary structure context and conservation of annotated motifs. Improvements include annotation of motifs not only in 3′UTRs but in the whole gene body including introns, additional genomes, and locally stable secondary structures from genome wide scans. Furthermore, we include data from CLIP-Seq experiments in order to highlight motifs with validated protein interaction. Additionally, we provide a REST interface for experienced users to interact with the database in a semi-automated manner. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite AU-rich elements (AREs) and GU- or U- rich elements (G/UREs) are sequence motifs found in many coding and non-coding RNAs. Upon interaction with RNA-binding proteins (RBPs) they can influence the half-life of RNA molecules. This interaction can induce RNA stabilization or destabilization, mediated by mechanisms that depend on the RBP and the genic motif context, but are otherwise not fully understood. The most prominent example is an important gene expression regulating mechanism known as AU-rich element mediated decay (AMD) .However, AMD is not the only RNA stability regulating process that depends on successful RNA-RBP interaction. RBPs interact e.g. with GU-rich elements (GRE), as well as U-rich elements (UREs) that have also been shown to modulate mRNA half-life –5.In silico methods play an important role in the identification of (novel) binding sites and the prediction of their regulatory role. Established databases like ARED , INSDC Assembly GCA_000001405.17, December 2013M. musculus, mm10: GRCm38.p3 (Genome Reference Consortium Mouse Reference 38), INSDC Assembly GCA_000001635.5, January 2012D. rerio, zv9: Zv9 (The Danio rerio Sequencing Project assembly Zv9), INSDC Assembly GCA_000002035.2, April 2010D. melanogaster, BDGP6: Berkeley Drosophila Genome Project (BDGP) assembly release 6, July 2014C. elegans, WBcel235: WS245 release of WormBase (which includes the WBcel235 version of the C. elegans reference genome) INSDC Assembly GCA_000002985.3, December 2012AREsite2 contains A/G/URE annotations for ∼60 000 genes in H. sapiens, ∼43 000 genes in M. musculus, ∼35 000 genes in D. melanogaster, ∼17 000 genes in D. rerio and ∼47 000 in C. elegans, multiplying the information content compared to version 1.Gene and transcript annotation for all genomes was retrieved from ENSEMBL  version AREsite includes only motifs ranging from the ARE core motif ATTTA to its extended 13-mer version WWWWATTTAWWWW, recent experiments ) package Gviz. The output begins with information on the genomic location of the searched gene. Figure This section explains example output from The search for more sequence patterns and parsing of the whole gene body leads to an increase in predicted motifs. Table AREsite2 presents a major update to AREsite, including three additional genomes and a high amount of newly annotated motifs. Furthermore, the new backend allows for easier integration of more genomes, other motifs, experimental and structure data. We provide the whole database as mysql-dump and all annotated motifs in bed, bed12 and gtf format for download. The RESTful service makes it easy for advanced users to retrieve information without the need to download any of these files in a semi-automatic manner. An example script for that purpose is included in the supplementary data, the most recent version can readily be downloaded from the website directly. We aim to integrate more experimental data as soon as they become available, either through CLIPdb, or directly from source if feasible.http://rna.tbi.univie.ac.at/AREsiteThe database is publicly available at: http://rna.tbi.univie.ac.at/AREsite/bulk.An example script for interaction with the REST interface, a database dump and motif annotation as bed, bed12 and gtf files are available at: Supplementary Data are available at NAR online.


Zostera muelleri and Zostera marina, a new model for hormone studies.An aquatic life genome sequencing suggests a complete loss of genes for ethylene biosynthesis and signalling pathways in the seagrasses,  Zostera muelleri were generated and the unassembled data were compared with the annotated genes of five sequenced plant species . Genes which are conserved between Z. muelleri and the five plant species were identified, together with genes that have been lost in Z. muelleri. The effect of gene loss on biological processes was assessed on the gene ontology classification level. Gene loss in Z. muelleri appears to influence some core biological processes such as ethylene biosynthesis. This study provides a foundation for further studies of seagrass evolution as well as the hormonal regulation of plant growth and development.Seagrasses are flowering plants which grow fully submerged in the marine environment. They have evolved a range of adaptations to environmental challenges including light attenuation through water, the physical stress of wave action and tidal currents, high concentrations of salt, oxygen deficiency in marine sediment, and water-borne pollination. Although, seagrasses are a key stone species of the costal ecosystems, many questions regarding seagrass biology and evolution remain unanswered. Genome sequence data for the widespread Australian seagrass species  Seagrasses are a polyphyletic group of monocotyledonous plants which are capable of living underwater in marine environments , Musa acuminata (banana), Phoenix dactylifera , and Spirodela polyrhiza (duckweed). Through the identification of genes that are either lost in Z. muelleri or remain conserved, insights can be gained into Z. muelleri evolution and adaptation to the marine environment.Previous genomic analysis of seagrasses has been performed using expressed sequence tags (ESTs)  according to the manufacturer’s instructions. Prior to DNA extraction, the plant material was ground to a fine powder in liquid nitrogen with a pre-chilled mortar and pestle. Genomic DNA was sequenced using an Illumina HiSeq 2000-SBS v3.0 sequencer with 100bp paired-end (PE) technology and an insert size of 304bp at the Ramaciotti Centre at the University of New South Wales . The libraries for genome sequencing were prepared with the Illumina Tru-seq DNA-seq kit.Zostera marina sequencing reads were downloaded from the NCBI Short Read Archive (SRA: SRR494397). A subset of the reads  totalling ~30× coverage was used for analysis. Furthermore, the coding sequences  for A. thaliana , M. acuminata .TAIR 10; , O. sati MSU v7; , S. polyITAG2.3; , P. dactZ. muelleri and Z. marina were estimated using k-mer analysis . The percentage of bases not sequenced at a given coverage was calculated according to the following probability function: P(Y=y)=Cy∙e–C/y!, y=0   A. thaliana coding sequences (RvsCDS comparison). In the second, full-length tomato CDSs were compared with A. thaliana CDSs. Short read sequence data were simulated using the tomato genome as the reference using wgsim  (Solanum lycopersicum (tomato) CDSs were compared with A. thaliana CDSs using dc-megaBLAST  and only best hits were retained.The two BLAST results were used to evaluate the feasibility of using short read mapping as replacement for the whole-length CDS comparison. To evaluate the robustness of the proposed approach, discrepancy rates (DRs) between RvsCDS and CDSvsCDS results were calculated according to the following formulae:DR genes lost [DRL]=/DR genes conserved [DRC]=/The DR values were plotted against different horizontal coverage cut-off values and used to define optimal horizontal coverage values for further analysis.Z. muelleri and Z. marina were compared with the CDSs from five plant species: A. thaliana, O. sativa, P. dactylifera, M. acuminata, and S. polyrhiza using dc-megaBLAST . Only best hits were retained, and CDS coverage was determined as previously described in the section on short read discontiguous (dc-) megaBLAST mapping evaluation.Sequencing reads from O. sativa, P. dactylifera, M. acuminata, and S. polyrhiza; and dicot: A. thaliana, S. lycopersicum, and N. nucifera) was identified using reciprocal best BLAST  with an e-value 1e-5 threshold between A. thaliana genes and genes from the remaining six species. If an A. thaliana gene had more than one RBB hit within the same species , bit scores were considered; the gene with the highest bit score and all the genes within 10 points of the highest bit score were included as RBB hits. Genes were assigned into clusters, each containing one A. thaliana gene and its putative orthologues in the six remaining species. A subset of OGCs including only clusters which contained at least one gene originating from a monocot species was extracted and termed OGCsM.A set of genes conserved between the seven species . Therefore, to be considered as a lost or conserved gene it had to be present in A. thaliana and at least one of the four remaining species . The approach used allows identification of genes lost and conserved using unassembled genomic data. However, it is important to note that while this approach will identify genes which are lost or conserved relative to the existing reference gene set, it will not discover newly evolved genes.For each gene in the OGCsM, previously calculated CDS coverages were inspected across A. thaliana orthologue from each identified cluster was used to derive gene ontology (GO) annotation  were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/). UCLUST . Genes present were identified using the same strategy as described above for Z. muelleri and Z. marina genomes. Mirroring the threshold of <2% to consider a gene as lost, a threshold of ≥2% was used to consider a gene as present.Assembled transcriptomes of Z. marina and Z. noltii EIL1 orthologues, identified from the Dr Zompo EST database, EIN3/EIL1 orthologues in the seven monocot and dicot species identified in OGCs, and EIN3 orthologues identified in A. andraeanum and Z. aethiopica transriptomes was performed using T-coffee . The longest open reading frame (ORF) was chosen for further analysis.Multiple sequence alignment between Z. muelleri was conducted, yielding a total of 39 Gbp of sequence data (191 141 643 read pairs), with an average coverage of ~43×. Additionally, ~30× genome coverage Z. marina sequence data were used for comparative analysis and genome size estimation. The estimated genome size of Z. muelleri based on the K-mer distribution  and core monocots . In the second comparison, full-length tomato CDSs were compared with A. thaliana CDSs (CDSvsCDS comparison). The DRs between RvsCDS and CDSvsCDS search results were calculated using different horizontal coverage cut-off values and are presented in In order to evaluate the feasibility and robustness of using short read mapping as a replacement for full-length CDS comparison as a gene loss/conservation detection tool, two BLAST comparison searches were performed. Tomato was chosen for simulations because of a similar estimated divergence time (~120–130 million years) between tomato and Supplementary Table S1 at JXB online. A total of 4091 genes conserved and 2147 genes lost between Z. muelleri and the five other plant species  were identified, respectively. Lists of genes conserved and lost can be found in Supplementary Tables S2 and S3 at JXB online, respectively.A total of 17 522 OGCs were identified. A total of 16 007 clusters which contained at least one orthologue representing a monocot species (OGCsM) were found. A list of OGCs genes can be found in P<0.05) biological process terms within the conserved gene data set. The 39 GO terms with the lowest P-values are presented in Supplementay Table S4 at JXB online. Biological processes significantly enriched in genes conserved include: glucose metabolic processes, ribosome biogenesis, translation, photosynthesis, and response to salt stress.GO analysis identified 175 significantly enriched . The GO terms and associated confidence values are presented in Z. muelleri. Additionally out of 10 genes involved in the regulation of ethylene-mediated signalling, six are lost in Z. muelleri.The list of Z. muelleri. Therefore, a detailed analysis was performed on 27 core genes associated with ethylene biosynthesis and signalling  and its genome was recently sequenced  synthase (ACS) and ACC oxidase (ACO) (S. polyrhiza orthologue of both ACS (Spipo24G0002100) and ACO (Spipo23G0011700) was identified in the OGCsM. Additionally, orthologues of ethylene receptors (Spipo6G0049300 and Spipo1G0021500), CTR1 (Spipo0G0009700), and EIN2 (Spipo8G0029200) were also identified in S. polyrhiza.A. andraeanum, Z. aethiopica, and A. bulbifer. In A. andraeanum, transcripts corresponding to orthologues of ACO, ethylene receptors, EIN2, and EIN3 were detected. In Z. aethiopica, transcripts corresponding to orthologues of ACO, ethylene receptors, CTR1, EIN2, EIN3, and EIL3 were found, while in A. bulbifer transcripts corresponding to orthologues of ACO, ACS, ethylene receptors, CTR1, EIN2, EIN3, EIL1, and EIL3 could be detected.Genes associated with ethylene synthesis and signalling are present in the transcriptomes of three terrestrial Alismatales species: Z. muelleri include all orthologues of the genes found in OGCsM encoding ACS  and all orthologues of the genes found in OGCsM encoding ACO . Additionally, orthologues of OGCsM genes associated with the regulation of ethylene signalling are lost in Z. muelleri. The list includes orthologues of four well characterized ethylene signalling molecules . The orthologue of an ethylene signal transducer protein EIN2 is also missing in Z. muelleri. Orthologues of transcription factors EIN3, EIL1, and EIL3 appear to be present in Z. muelleri  at the N-terminus, a proline-rich domain (PRD) in the central region, and a protein degradation domain (PDD) at the C-terminus . The two key DNA binding residues Pro216 and Lys245 . SAM is converted to ACC by ACS, and ACC is then oxidized by ACO to form ethylene  have a large deletion in the N-terminal region (~130 amino acids). It could be hypothesized that the loss of the ethylene signalling network has relaxed the selective pressure of all genes involved in this pathway (considering that they are not involved in other functions). This could have offered the opportunity to recruit some of these genes in new functions. Such genes could have been under transient positive selection favouring amino acid changes or deletions . Further analysis of these transcription factors, their sequences, regulators, and downstream targets may shed more light on the effects of loss of ethylene signalling.Analysis of both genomic and transcriptomic data points to the conservation of some of the downstream targets of the ethylene signalling cascade in seagrasses. Orthologues of EIN3/EIL transcription factors appear to be present in JXB online.Supplementary data are available at Figure S1. K-mer distribution for the Z. muelleri genome.Figure S2. K-mer distribution for the Z. marina genome.Figure S3. EIL1 multiple sequence alignment.Table S1. Complete list of genes found in OGCs.Table S2. Complete list of genes conserved with corresponding GO annotation.Table S3. Complete list of genes lost with corresponding GO annotation.Table S4. Complete list of significantly enriched GO terms associated with genes conserved in Z. muelleri.


Bacillus amyloliquefaciens subsp. plantarum UCMB5033 is of special interest for its ability to promote host plant growth through production of stimulating compounds and suppression of soil borne pathogens by synthesizing antibacterial and antifungal metabolites or priming plant defense as induced systemic resistance. The genome of B. amyloliquefaciens UCMB5033 comprises a 4,071,167 bp long circular chromosome that consists of 3,912 protein-coding genes, 86 tRNA genes and 10 rRNA operons. Bacillus amyloliquefaciens is a plant-associated species belonging to the family Bacillaceae. The members of the genus Bacillus are ubiquitous in nature and include biologically and ecologically diverse species, ranging from those beneficial for economically important plants, to pathogenic species that are harmful to humans. B. amyloliquefaciens UCMB5033 is a plant growth promoting bacterium (PGPB) that was isolated from a cotton plant [B. amyloliquefaciens UCMB5033 is an important tool for studies of plant-bacteria associations, has potential to confer protection against soil borne pathogens, and to stimulate growth of oilseed rape (Brassica napus) [B. amyloliquefaciens UCMB5033 and its annotation.on plant . Studiesa napus) . Such trB. amyloliquefaciens group based on phenotypic analysis [B. amyloliquefaciens UCMB5033 shares 99% identity with many Bacillus species including Bacillus atrophaeus (CP002207.1) and Bacillus subtilis subsp. spizizenii str. W23 (CP002183.1). B. amyloliquefaciens UCMB5033 with other species within the genus Bacillus. The tree highlights the close relationship of UCMB5033 with the B. amyloliquefaciens subsp. plantarum type strain FZB42. The other B. amyloliquefaciens type strain DSM 7T representing subsp. amyloliquefaciens, displayed less taxonomic relatedness and strain UCMB5033 can thus be regarded as belonging to the subsp. plantarum also in line with its plant associated characteristics [Strain UCMB5033 was identified as a member of the analysis . The coms CP00220.1 and BaB. amyloliquefaciens UCMB5033 is a Gram-positive, rod shaped, motile, spore forming, aerobic, and mesophilic microorganism (B. amyloliquefaciens UCMB5033 has properties as a plant growth promoting rhizobacterium (PGPR) [B. amyloliquefaciens UCMB5033 an advantage in developing a symbiotic relationship with plants in competition with other microorganims in the soil microbiota.organism . Strain m (PGPR) . The abiB. amyloliquefaciens UCMB5033 was grown in LB medium at 28°C for 12 hours (cells were in the early stationary phase). The genomic DNA was isolated using a QIAmp DNA mini kit (Qiagen).B. amyloliquefaciens UCMB5033, originally isolated from cotton plant, was selected for sequencing on the basis of its ability to promote rapeseed growth and inhibit soil borne pathogens. Genome sequencing of B. amyloliquefaciens UCMB5033 using Illumina multiplex technology and Ion Torrent PGM systems was performed by Science for Life Laboratory (SciLifeLab) at Uppsala University. The genome project is deposited in the Genomes On Line Databases [atabases  and the B. amyloliquefaciens UCMB5033 was assembled using 21,919,534 Illumina paired-end reads (75bp) and 1,922,725 single-end reads (Ion Torrent). The chromosome of size 4,071,167 bp was assembled by providing paired-end reads to MIRA v.3.4 [B. amyloliquefaciens UCMB5036 (accession no. HF563562) [de novo assembly method. Both forms of assemblies were compared after alignment to identify indels and cover gap regions using Mauve genome alignment software [The genome of F563562) . Whereassoftware .via the Magnifying Genome (MaGe) Annotation Platform [The genome sequence was annotated using a combination of several annotation tools Platform . Genes wPlatform  and AMIGPlatform  as part Platform  was usedPlatform .B. amyloliquefaciens UCMB5033 genome consists of a circular chromosome of size 4,071,168 bp. The genome having G+C content of 46.19% were predicted to contain 4,095 predicted ORFs including 10 copies each of 16S, 23S, and 5S rRNA; 86 tRNA genes, and 3,912 protein-coding sequences with the coding density of 87.51% (The f 87.51% . The majf 87.51% . The disComparative genome analysis might reveal mechanisms by which UCMB5033 mediates plant protection and growth promotion, will further enable the investigations of the biochemical and regulatory mechanisms behind the symbiotic relationship, and will shed light on the activity of PGPR in different environments.


Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.Strain-specific genomic diversity in the  Genetic variation in Mycobacterium tuberculosis complex (MTBC) bacteria is responsible for differences in factors such as virulence and transmissibility. Here, the authors analyse the genomes of 1,601 MTBC isolates from diverse geographic locations and identify 62 SNPs that may be used to resolve lineages and sublineages of these strains. Mycobacterium tuberculosis complex (MTBC) results in a variety of outcomes including latent infection and/or progression to pulmonary or extra-pulmonary manifestations of disease. Such diversity has been historically attributed to host and environmental factors, and the MTBC was previously considered genetically monomorphic in natureMycobacterium africanum I, 6 West Africa or M. africanum II), distinct from a M. bovis clade. Lineages 1, 5 and 6 are considered ‘ancient’, and 2 to 4 ‘modern’. A novel phylogenetic lineage of MTBC that appears to be intermediate between the ancient and modern has been described in Ethiopia and the Horn of Africa5Infection with bacteria of the M. africanum strains16171819Lineage is of importance to tuberculosis control as it has been shown that strain type may play a role in disease outcome, variation in vaccine efficacy1112n=1,601) from a diverse geographic spread. We identify a single panel of 62 SNPs that may be used to resolve all seven lineages and a total of 55 sublineages.In this study, we have sought to provide an improved system by examining whole-genome data from a large number of strains , as well as M. bovis  and sublineages were subsequently identified based on the spoligotype and regions of difference (RD) composition of the clades in the SNP-based phylogeny (see Methods). The phylogeny revealed the presence of new clades for which RDs do not discriminate. In particular, there were gaps in the Euro-American lineage for which molecular fingerprint classifications are less accurateA genomic analysis was performed on whole-genome sequences of 1,601 MTBC isolates from eleven independent sequencing studies from different areas of the world, with representation of all seven major lineages  and 6 (π=0.0093), and the least within lineage 2 (π=0.0039) and 3 (π=0.0040) strains . The distribution of functional categories of genes containing the 91,648 and 6,915 SNP sets did not differ , there was some evidence of difference in the distribution of functional categories between lineages, namely a greater proportion of lipid metabolism non-synonymous polymorphism in lineage 2  robust SNPs in essential genes with mutations that lead to synonymous amino acid changes, therefore less likely to be under selective pressure . RedundaM. bovis, when it was found to predict 100% their reported strain types  that can be used to construct high resolution and reproducible phylogenies, which can be incorporated in diagnostic assays and assess genotype–phenotype associations. Future work should focus on other types of lineage-specific polymorphisms , which are less common than SNPs, but may have major functional consequences. The proposed system has the flexibility to incorporate novel strain types should they be reported. Incorporating anti-tubercular drug-resistance loci will further enhance the usefulness of the barcode as an important tool for tuberculosis control and elimination activities worldwide.http://www.ebi.ac.uk/ena/). The dataset consists of nine independent whole-genome sequencing studies, available under ENA accessions ERP000192, ERP000276, ERP000520, ERP001731, ERP000111, SRP002589, ERP002611 and ERP000436 (ref. SRA065095 (ref. ERP001885 (ref. ERP001567 (ref. NC_000962.3) using BWASAMtools/BCFtoolsGATKn=1,601).The raw sequence data of 1,804 MTBC isolates available in the public domain were downloaded from the European Nucleotide Archive  using SpolPred softwarede novo assembled using VelvetAll samples were N=100, x=12,345) combined with maximum likelihood search was chosen to construct the phylogenetic tree. The resulting tree was rooted on M. canettii (Genbank accession number: NC_019950.1) and nodes were annotated. Subsequently, the ancestral sequence at all internal nodes was computed using DnaPars from the Phylip packageRAxML trees were constructed for the 1,601 samples from alignments using other proposed lineage-informative SNPs . Similarly, the ancestral reconstructed sequence for the clade-defining node was compared with its closest ancestral node, and the SNP differences derived. A high-confidence set of clade-specific SNPs was obtained by selecting those at the clade-defining internal node and having FST values of >0.99 in between group comparisons. To ensure that clade-specific SNPs were also suitable markers for their use in strain typing assays, we applied filtering criteria: (1) only synonymous SNPs were retained as they are generally under lower selection pressure; (2) SNPs at non-coding regions were discarded since insertions or deletions (indels) are usually more frequent. The density of small indels and large deletions is five and 17 times smaller, respectively, in coding regions of the genome compared to non-codingwww.tbdreamdb.com) and recent studieswww.iedb.org). The gene functional categories were extracted from Tuberculist (tuberculist.epfl.ch).For each lineage and sublineage, the dataset was split into two populations: one containing all samples descending the clade-defining node and the other with remaining samples. The F.C. and T.G.C. conceived the project. J.A.G.-A., J.R.G., J.P., M.V., I.P. and A.P. contributed to the construction of data. F.C. analysed the data. R.M., N.M. and T.G.C. jointly supervised the research. F.C., R.M. and T.G.C. wrote the paper.How to cite this article: Coll, F. et al. A robust SNP barcode for typing Mycobacterium tuberculosis complex strains. Nat. Commun. 5:4812 doi: 10.1038/ncomms5812 (2014).Supplementary Figures 1-6, Supplementary Tables 1-7 and Supplementary ReferencesMycobacterium tuberculosis complex lineages and sub-lineages