Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (Ho) (0.164) and highly positive fixation indices (Fis) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an  Elaeis oleifera is a species in the oil palm genus along with the commercial Elaeis guineensis and occurs naturally in South-Central America, from Honduras to Colombia and in the Amazon region [E. guineensis × E. oleifera) breeding programs. It has attracted the attention of breeders by reason of several interesting agronomic traits: low height increment, resistance to Fusarium wilt and lethal yellowing [E. guineensis. Beside the agronomic traits, the oil from E. oleifera is highly unsaturated  with high linoleic and oleic acids, low palmitic acid and high carotene [in situ hybridization technique (GISH) using specific DNA probes to distinguish oleifera and guineensis chromosomes has been developed to assist hybrid backcross breeding programs [n region . This Amellowing , which ccarotene . A genomprograms .E. guineensis-based SSR markers have been used to construct genetic maps [In plant genetics and breeding studies, DNA-based assays, and especially molecular markers, are known to be efficient tools for genetic diversity assessment, molecular ecology studies, gene mapping as well as marker-assisted selection (MAS) . Among atic maps ,9, and atic maps .E. guineensis from Africa and E. oleifera maintained as ex-situ collections in Kluang, Johor, Malaysia. Assessing the performance and genetic diversity of the wild material is important for understanding the genetic structure of natural oil palm populations. Furthermore, the information is important for oil palm breeding programs, and also for continued ex-situ conservation of the germplasm in Malaysia. Currently, only the E. guineensis germplasm is well characterized, using various types of molecular markers, such as isozymes [E. oleifera has been limited, only involving RAPD [E. guineensis [E. oleifera has made it possible to develop SSR markers from E. oleifera and utilize them to understand the genetics of the species.The Malaysian Palm Oil Board (MPOB) has an extensive collection of germplasm from both species of oil palm. isozymes , restricisozymes , amplifiisozymes , random isozymes  and SSRsisozymes ,14. Howeing RAPD  and SSR ineensis . NeverthE. oleifera genomic SSR markers from a collection of E. oleifera genomic sequences; (b) evaluate the efficiency of these markers in assessing the genetic diversity in the MPOB E. oleifera germplasm collection; and (c) determine the transferability of E. oleifera SSR markers among selected palm genera and taxa.Thus, the objectives of this study were to (a) develop and characterize GeneThresher™ library is a comprehensive collection of gene sequences of oil palm obtained from sequencing of the hypomethylated region of the oil palm genome using methylation filtration technology [E. oleifera GeneThresher™ derived sequences used in this study, 1861 non-redundant sequences (1735 singleton and 126 consensus) were successfully assembled with CAP3 sequence assembly software [E. oleifera genomic library is a valuable resource for this genetic marker type.The chnology . As suchet al. [E. guineensis [et al. [E. oleifera is similar to that reported for E. guineensis EST-SRR [Arabidopsis thaliana [One hundred and four 22%) of the genomic sequences contained more than one SSR. Mononucleotides were the most abundant repeat type 437 = 72.4%), and showed a strong bias to the A/T repeat-motifs (97.9%) over the C/G repeat motif . Feng etet al.  reported = 72.4%,ineensis ,14, peacineensis , coffee ineensis  and rubbineensis . AG/CT S% of the  [et al. , highlig EST-SRR , Arabidothaliana , soybeanthaliana , barley thaliana  as well thaliana . The mosSorghum [E. oleifera-based gSSR primer pairs , of which 63.9% were di-repeats, 24.3% tri-repeats, 4.9% tetra and penta-repeats each, 0.7% hexa-repeats and 1.4% hepta-repeats. The failure to design primers for the remaining sequences (13.3%) was probably due to short (or absence of) flanking regions, or that the sequences submitted did not correspond to the minimum criteria required by the primer design software  and SorgSorghum , where ter pairs , represeE. oleifera-based gSSR markers in characterizing E. oleifera germplasm, 20 primer pairs (markers) were tested on a panel of 119 E. oleifera palms from the germplasm collection. Ten E. guineensis from the Nigerian collection and another 10 from the MPOB advanced breeding material population (Deli dura) were included for comparison. This allowed the study to also determine the ability of the E. oleifera-derived SSR markers to reveal the genetic diversity in the Deli dura material which had undergone several cycles of self-pollination, and also the wild Nigerian materials. This provenance is reported to be the center of diversity for E. guineensis [To ascertain the attributes of the E. oleifera gSSR markers to amplify the target sequences and detect polymorphism in both Elaeis palms.Eighteen of the 20 primers successfully produced amplicons , and 15 E. oleifera gSSRs detected 89 alleles, ranging from 1 to 13 across the Elaeis samples. Of them , 48.3% and 31.5% of alleles were specific to E. oleifera and E. guineensis, respectively, and 20.2% common in both species. Within the repeats, tri-nucleotides detected more alleles  than the other repeats. It would appear that the tri-nucleotide genomic SSRs show higher average PIC values than di-nucleotide repeats. This is most likely a reflection of the specific region of the genome targeted by the methylation filtration technique. Botstein et al. [E. guineensis-derived EST-SSRs  which were used mainly to analyze E. guineensis germplasm.The n et al.  defined E. oleifera-derived gSSRs to reveal allelic polymorphism and genetic diversity in the Elaeis genus was more efficient than by other tested marker systems. For instance, E. oleifera gSSRs generated more alleles (Ao) in both Elaeis species  than in previous studies on E. guineensis using isozymes , RFLP  and AFLP .Interestingly, the ability of E. oleifera collections, He revealed by E. oleifera SSR markers (0.262) was slightly lower than those generated by E. guineensis EST-SSR markers . As such, E. oleifera gSSR markers are also additional promising tools for characterizing E. oleifera collections, although the Ao and He values are lower than those generated by E. guineensis gSSRs . The differences revealed by both the genomic SSR markers were possibly due to the number of samples analyzed and populations evaluated. Billotte et al. [E. oleifera samples (1–2 samples per country), whereas 119 E. oleifera samples (22–34 samples per country) were analyzed in this study. Furthermore, the genomic library utilized in this study was constructed from hypo-methylated regions, the chances of the employed E. oleifera gSSR markers being closely located within the conserved coding regions are higher than in the sequences obtained from a conventional genomic library. This could also explain the lower diversity observed in this study.By focusing only on analysis carried out on Elaeis species, the heterozygosity in the E. oleifera germplasm varied from 0.102 to 0.200 (mean Ho = 0.164), while E. guineensis generated Ho from 0.118 (Deli dura) to 0.321 (Nigeria) (mean Ho = 0.220). E. oleifera generally had lower diversity, compared to E. guineensis, with higher Ho revealed by the Colombian and Panama palms . The higher Ho in both collections could be due to human assisted movement of palm samples that was probably accelerated during and subsequent to construction of the Panama Canal in 1914. Some palms could have been brought in from other South American countries, widening the genetic base of E. oleifera in these countries. Furthermore, the He value obtained for E. oleifera (mean = 0.262) was comparable to those obtained by RFLP (He = 0.225) and AFLP (He = 0.298) analyses in screening 241 E. oleifera accessions [E. guineensis, He for the Nigerian samples (0.329) was lower than that obtained with E. guineensis EST-SSR markers [He values were 0.442 and 0.534 respectively.Regarding the genetic variation between the two cessions . Among E markers ,14, wherFis values were positive at all loci in all tested collections with mean Fis ranging from 0.024 (Nigeria) to 0.546 (Deli dura)  generated by E. oleifera gSSR markers in this study, which was even lower than that revealed by E. guineensis EST-SSR markers .The li dura) . This reountries . This maE. oleifera genomic SSRs described in this study successfully grouped the six collections of oil palm into two distinct clusters: E. oleifera and E. guineensis  transferability in the tested Cocos nucifera samples. With the ornamental palms, the frequencies of transferability were Euterpe (72.7%) > Oenocarpus (63.6%) > Jessinia (54.5%) > Ptychosperma (54.5%) > Dictyosperma (45.5%) > Cyrtostachys (45.5%). Two markers: sMo00055 (di-repeats) and sMo00137 (penta-repeats), generated clear banding profiles of various sizes in all the samples analyzed, including the ornamental palms.Eleven of the eae taxa . SuccessE. oleifera SSRs averaged 75% at the genus level, comparable to Phyllostachys Pubescens (75.3%), but lower than rice (90%) [Elaeis-derived gSSR markers were able to amplify PCR products in Cocos, reflecting their capability in characterizing the three different Cocos samples tested. This also suggests the relatively close proximity of E. oleifera to coconut. The high cross-transferability of E. oleifera gSSR markers to Elaeis species and related genera suggests the potential application of these markers in comparative studies across members of the Arecaceae family. High cross-species conservation of SSR loci within genus has also been reported for Olea [Picea [Pinus [SSR primers developed for one species are known to often detect homologous sites in related species. The ability of sMo00055 and sMo00137 markers to amplify fragments with similar sizes indicates their efficiency in revealing sequence conservation among the species in the Arecaceae family. In general, cross species transferability differs highly among taxa, especially in flowering plants . The trace (90%) . Furthera [Picea  and Pinua [Pinus .E. oleifera, E. guinensis, Cocos nucifera, Jessinia bataua and Oenocarpus multicaulis). Amplified PCR fragments of these markers in selected individuals were cloned and sequenced. The amplicons of the three SSR markers were successfully cloned and sequenced. The sequences were aligned with the original sequence from which the primers were designed  were used to determine the sequence variability in some of the species in Arecaceae family  revealed variable numbers of repeat motifs in the SSR regions within the tested samples. This further explains the primary basis of the observed fragment length polymorphism in the Arecaceae family screened in this study. The variations were mainly due to changes in the number of repeat motifs in the SSR region, combined with indels and base substitutions. Similar results were reported by Billotte et al. [et al. [E. guineensis SSR and EST-SSR markers, respectively. However, looking specifically at sMo000555, the repeat motif observed in E. oleifera was missing in E. guineensis, and the repeat number was very low in coconut and one of the ornamental palms. Although this locus was successfully amplified in all samples, the lack of repeat conservation in some samples suggests that the amplified fragments may not represent functional SSRs in those species. Nevertheless, the ability of E. oleifera genomic SSRs (sMo00137 and sMo00138) to reveal high inter-species and inter-genera transferability (>90%) supports the close phylogenetic relationship between the species and genera.Generally, the sequence data generated by the three selected e et al.  and Ting [et al.  who emplE. oleifera gSSR was tested on three coconut (Cocos nucifera) samples and six ornamental palms . Genomic DNA was extracted and purified from each spear leaf using the modified CTAB method described by Doyle and Doyle [E. oleifera genomic library was constructed using GeneThresher™ Technology (17) and the genomic clones and sequences stored at MPOB’s Biological Resource Centre (MBRC).The oil palm germplasm collections used in this study are maintained at the MPOB Research Station, Kluang, Johor. A total 149 spear leaves  were harvested from the palms in nd Doyle . The E. E. oleifera genomic sequences were assembled using the CAP3 assembly program [et al. [A total of 2000  program  with def [et al. . The sea [et al. ; all the33p dATP  and 1U T4 polynucleotide kinase (Invitrogen™ USA) for 1 hour at 37 °C. The PCR reaction was subsequently carried out in 10 μL of 1 μL 10X PCR buffer (buffer composition-MgCl2), 15 mM MgCl2, 1 mM dNTPs, 5 μM unlabeled reverse primer, 1 μL labeled forward primer, 0.5 U Taq DNA polymerase and 50 ng template DNA. PCR was performed in a Perkin Elmer 9600 thermocycler as follows: denaturation at 95 °C for 3 min, 35 cycles at 95 °C for 30 s, 52–56 °C for 30 s (depending on the primers requirement), 72 °C for 30 s and a final extension at 72 °C for 5 min. The PCR reaction was stopped by addition of 10 μL formamide dye . A total 5 μL of the mixture was denatured at 90 °C for 3 min, chilled on ice, and separated in a 6.0% polyacrylamide gel containing 7 M urea in 0.5 X TBE buffer at constant power of 1600V for 3 hours. The gel was then dried and exposed to X-ray film (Kodak) for 3–4 days at −80 °C. The size of each allele was determined using the 100–330 bp AFLP DNA ladder (Invitrogen™ USA).The forward primer was 5′ end-labeled in 1 μL reaction containing 4.5 μM forward primer, 0.1 μL γ-P), expected and observed heterozygosity (He and Ho) in the collections used and fixation indices (Fis). Chi squared tests were performed for each locus for deviation of the genotypes from the Hardy-Weinberg equilibrium (HWE). The allelic polymorphism information content (PIC) for each gSSR marker and distance matrix [Only fragments that could be clearly scored were used in the data analysis. The genotyped data were analyzed using POPGENE version 1.32 . The gene matrix  between e matrix . The unwe matrix  dendrogre matrix  importede matrix .E. oleifera gSSR markers that produced clear banding profiles in both E. guineensis and E. oleifera samples were further used to study cross species and genus amplification within Arecaceae family. The markers were tested against three Cocos nucifera varieties and six ornamental palms  in the E. oleifera, E. guineensis, Cocos nucifera, Jessinia bataua and Oenocarpus multicaulis samples were excised from the agarose gel and purified. The purified fragments were cloned into pCR2.1-TOPO vector  and sequenced using the ABI PRISM 377 automated DNA sequencer. The sequences were aligned and compared using CLUSTALW multiple sequence alignment tool employing BIOEDIT sequence alignment editor version 7.0.0 [The amplicons generated by three selected on 7.0.0 . The seqE. oleifera gSSR markers developed were found to be valuable genetic resources for understanding the genetic diversity of E. oleifera and E. guineensis. The study indicates that E. oleifera-derived SSR markers were more efficient in revealing the genetic diversity of E. oleifera than E. guineensis EST-SSR markers. The sequence data showed their ability to amplify DNA, not only in the two oil palm species, but also in coconut and other selected ornamental palms, thus verifying the ability of SSRs to amplify across species and genera in the Arecaceae family. Furthermore, the variability in allele sizes and sequences among the species reflected the mutational processes that had taken place at both the repeat and flanking regions. An expanded study using all the available SSR markers on a larger set of samples  would provide a clearer picture on the genetic diversity of the germplasm available at MPOBA set of


Macrophage migration inhibitory factor (MIF) is a cytokine that plays a central role in immune and inflammatory responses. In the present paper, we discussed the participation of MIF in the immune response to protozoan parasite infections. As a general trend, MIF participates in the control of parasite burden at the expense of promoting tissue damage due to increased inflammation. The immune/inflammatory response triggered during infection has an essential role in eliminating the infectious agent and in promoting tissue repair . The verMacrophage migration inhibitory factor (MIF) activity was described in the sixties and it is considered one of the first cytokines to be described , 6. The 2 and cyclooxygenase via a protein kinase A and ERK-dependent pathway, the increased expression of TLRs and adhesion molecules, antagonistic effects on glucocorticoids activity, and its role as a chemoattractant and in promoting the survival of leukocytes  vary in virulence and pathology depending on the strains of Plasmodium and laboratory mouse used [P. chabaudi chabaudi AS [P. chabaudi adami DK [P. berghei ANKA or P. yoelii 17XL [P. yoelii 17XL, but not P. berghei ANKA, also induces CM when inoculated in BALB/c mice [Experimental murine models of malaria infection have provided an invaluable resource for studying the role of inflammatory and immune responses in the pathology of malaria . Infectiuse used . For exabaudi AS . For thiadami DK . This selii 17XL –56. InteB/c mice .Plasmodium strains are natural pathogens of laboratory mouse strains and the course of infection and complications observed in some mouse-parasite combinations including SMA and CM differ from the human spectrum of disease [P. c. chabaudi AS-BALB/c model correlates with a peak parasitemia of around 20%, which makes the destruction of infected erythrocytes a major contributor to the physiopathogenesis of anemia in this model [However, none of the rodent  disease . For insis model , 57. Altis model .−/−Mif mice in the BALB/c background or animals treated with anti-MIF neutralizing monoclonal antibodies are more resistant to Plasmodium chabaudi adami infection than wild-type controls presenting a significant reduction in peak and cumulative parasitemia [P. chabaudi chabaudi AS, a mouse model of SMA, revealed that elevated concentrations of MIF in the plasma are associated with severity of anemia and suppression of erythropoiesis [−/−Mif mice infected with P. c. chabaudi develop a parasitemia curve similar to that of wild-type controls but present less severe anemia, less inhibition of erythroid colony formation, and a higher survival [Studies using mouse models of malaria indicate that MIF plays a detrimental role during the infection , 44, 53.opoiesis , 53. In survival .P. c. adami but not P. c. chabaudi infection. As the development of immunity and/or anemia in mouse and human malaria result from a complex process that involves multiple factors [Plasmodium infection. For example, MIF attenuates the development of Th1 responses following P. c. adami infection in BALB/c mice by decreasing T CD4+ IFN-γ production and enhancing IL-4 [γ and TNF as inhibitors of erythropoiesis in mice [−/−Mif and wild-type mice infected by P. c. chabaudi [γ.It is not clear why blockade of MIF reduces parasitemia during  factors , 59, theing IL-4 . Neverth in mice  and seruchabaudi , indicatP. c. chabaudi-infected erythrocytes and synthetic hemozoin in a dose response manner [Production of MIF in mouse SMA seems to be triggered by hemozoin, which is an insoluble heme polymer produced by parasite catabolism of host hemoglobin , 53. Heme manner , 53. MIFe manner , 43. TakP. berghei ANKA infection followed by antimalarial treatment in C57Bl/6 mice) that present low levels of parasitemia during anemic episodes and are believed to be more closely related to the human pathology [Plasmodium infection in mice and other mechanisms known to be involved in mouse SMA such as lysis of infected erythrocytes due to schizogony and destruction of noninfected erythrocytes [Nevertheless, the role of MIF in semi-immune mouse models of SMA  is structurally similar to mammalian MIF with around 30% amino acid sequence identity and possesses some, but probably not all, activities normally attributed to the latter. pMIF expression increases with blood-stage parasite maturation: minimal in ring stage and peaking in the trophozoite and schizont stages [Plasmodium-infected mice and humans [MIF homologues have been identified in all species of lciparum –78, P. vP. vivax , P. berg berghei , 79, and. yoelii , 56. Thet stages , 55, 76.t stages , 55. Indd humans , 71. Howd humans –56. in vitro assays and animal models have shown that pMIF shares some biological properties with mammalian MIF. Indeed, both pMIF and mammalian MIF reduce AP-1 expression, interact with human CD74, induce macrophage chemotaxis, and inhibit erythropoiesis and macrophage apoptosis [In terms of functional studies,poptosis , 79, 80.poptosis , 78, a kpoptosis , indicatPlasmodium virulence by modulating host immune responses [P. yoelii 17X and P yoelii 17XL that constitutively overexpress P. yoelii MIF (PyMIF) [P. yoelii 17X and prolonged course of infection with a reduction in overall mortality rate in animals infected with the lethal strain P yoelii 17XL [P. berghei wild-type or P. berghei MIF knockout parasites [Plasmodium MIF in the pathogenesis of malaria.The role of pMIF during malaria infection is also not completely understood. Although counterintuitive, studies in mouse models indicate that pMIF attenuates esponses –56. C57B (PyMIF)  or when lii 17XL , 56. On arasites . Once agarasites . Thus, fToxoplasma gondii is an intracellular parasite of the phylum Apicomplexa that is highly adapted to infect different cell types and tissues. T. gondii enters its host via the gastrointestinal tract and the innate immune response in the intestine is triggered by the recognition of parasite molecules by enterocytes, macrophages, and dendritic cells (DCs) [γ, and IL-1β promote resistance against T. gondii in part due to the generation of NO by macrophages, an important mechanism responsible for parasite elimination.ls (DCs) . The estls (DCs) . The proT. gondii through the intraperitoneal route demonstrated an increased susceptibility of−/− Mif mice when compared to wild-type mice [−/−Mif mice presented higher parasite burden in brains and peritoneal macrophages and reduced plasma concentrations of IL-12, TNF, IFN-γ, IL-1β, and nitrite during infection [T. gondii infection in the BALB/c background also demonstrated an increased lethality and tissue parasitism with reduced IL-12 production and DC activation on −/−Mif mice compared to wild-type mice [−/−Mif mice orally infected with T. gondii had impaired maturation, with decreased expression of CD80, CD86, CD40, and MHC class II [T. gondii infection is apparently related to the production of proinflammatory cytokines, the activation of DC, and the better control of parasite burden.A model of systemic infection with ype mice . Mif−/− nfection . These fype mice . DCs obtclass II . Thus, tT. gondii, while those of C57BL/6 are highly susceptible displaying intestinal inflammation especially in the ileum [−/−Mif mice have reduced intestinal and systemic inflammation and survive longer compared to wild-type mice, despite an increase in intestinal parasite burden [γ, and IL-23 and an increased expression of IL-22 in ileal mucosa. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in −/−Mif mice compared to wild-type mice [T. gondii infection, these findings demonstrated a pathogenic role of MIF in natural T. gondii infection in susceptible hosts. This dichotomy seems to depend on the route of infection and the genetic background of the host. Thus, MIF is necessary to control parasite burden in resistant and susceptible hosts, but it increases intestinal tissue damage causing death in susceptible hosts while it is essential for survival in resistant hosts.BALB/c mice are naturally resistant to oral infection with he ileum –84. Thise burden . Lack ofype mice . AlthougT. gondii-infection is the severe congenital malformations when the primary infection occurs in the first trimester of pregnancy. A series of studies demonstrated a putative role of MIF on placental biology upon infection with T. gondii. Infection or stimulation of chorionic explants with molecules of T. gondii, IFN-γ, and IL-12 evoked the secretion of MIF [T. gondii infected monocytes, could facilitate the dissemination of the infection into the deep placental tissues or increase the tissue damage due to inflammation. The same group recently demonstrated, however, that MIF is important for control of placental T. gondii infection in first trimester of pregnancy [A major consequence of human n of MIF , 86. MIFn of MIF . These rLeishmania, comprises a large spectrum of clinical manifestations including benign ulcer, destructive muco-cutaneous lesions, disseminated cutaneous lesions, and systemic visceral forms [Leishmania sp. is an obligatory intracellular parasite infecting mainly macrophages. Parasite killing requires macrophage activation with ensuing NO and ROS production [Leishmania major causes skin lesions, which in general parallel the parasite load. A highly polarized Th1 response is effective against L. major, activating macrophages to produce NO and resulting in resolving skin lesions. Addition of MIF to macrophage cell cultures results in increased L. major elimination [L. major burden in macrophages is high , this concentration is within the range reached in inflammatory conditions. The MIF-induced leishmanicidal effect requires the production of TNF and NO by infected macrophages, and can be reversed by the addition of IL-10, TGF-β or IL-13, indicating that it depends on an M1 activation status [L. major footpad inoculation in popliteal lymph node, but the kinetics of its expression compared to that of MIF secretion by T cells upon antigenpresentation suggests that lymph node MIF comes from another cellular source [Salmonella typhimurium transfected with MIF reduces the size of skin lesions [−/−Mif mice are highly susceptible to L. major, developing severe skin lesions late after infection [L. major infection, as indicated by the similar IFN-γ and IL-4 production among T cells from −/−Mif and wild-type mice. However, IFN-γ-activated macrophages from −/−Mif mice infected in vitro with L. major have slightly decreased parasite clearance [γ or MIF production is partially required as an intermediary step to IFN-γ-induced leishmanicidal activity. The contribution of MIF produced by CD4+ lymphocytes to protective immunity against cutaneous leishmaniasis was demonstrated using a model of vaccination with the L. pifanoi antigen P-4 [γ mRNAs than the adjuvant controls. Moreover, blockage of MIF with anti-MIF antibody significantly reduced the leishmanicidal ability of macrophages cultured with CD4+ lymphocytes obtained from P-4-immunized mice.Leishmaniasis, caused by the protozoan parasites from the genus al forms . In the oduction . Infectimination . Though n status . The expr source . Consist lesions , while Mnfection . MIF doelearance , indicatigen P-4 . BALB-c L. donovani presented CD4+ lymphocytes expressing low amounts of CD2, IFN-γ, and MIF [L. chagasi have increased plasma concentrations of MIF [ Patients with visceral leishmaniasis due to infection with  and MIF . Antileis of MIF . The MIFL. major revealed two genes with significant sequence similarities to human MIF (22% identity) [L. major orthologue proteins adopt trimeric ring architecture. Lm1740MIF binds to CD74, the MIF receptor, indicating a putative role of L. major MIF affecting host immunity [L. major MIF orthologues to induce ERK1/2 phosphorylation, to cause the reduction of Ser15-p53 in the cytoplasm, and to protect macrophages from NO-induced apoptosis [Leishmania, the ability of L. major MIF to increase the survival of macrophages might represent an important selective advantage that guarantees more efficient amastigote replication.The complete genome sequencing of dentity) . Cloningdentity) , 94. Thedentity) , 94. Simimmunity . In factpoptosis . Since tTrypanosoma cruzi is an intracellular protozoan that can infect many cell types, including macrophages. The effective response to T. cruzi comprises innate activation of macrophages to induce NO production and, ultimately, the establishment of antigen-specific Th1 CD4 and CTL CD8 responses [Mif also are more susceptible to Trypanosoma cruzi infection [γ along with acute infection and also decreased IL-12 and IFN-γ production by splenocytes stimulated with T. cruzi antigens early in the acute phase, indicating that in contrast to the trypanosomatid, L. major, MIF participates in Th1 polarization in T. cruzi infection. This deficient Th1 polarization is reflected by decreased titers of anti-T. cruzi IgG2a (but not IgG1). Also, −/−Mif mice have decreased plasma concentrations of TNF, IL-1β, and IL-18, suggesting that decreased production of proinflammatory cytokines underlies their susceptibility to T. cruzi infection. The deficient Th1 polarization, specific IgG and pro-inflammatory cytokine secretion are all highly compatible with susceptibility to T. cruzi infection, but there is currently no functional data to support this hypothesis. In fact, IFN-γ-activated macrophages have a prominent role in T. cruzi clearance through NO production, a function that can be enhanced by TNF production. As MIF controls TNF production by macrophages in a number of cases and, along with TNF, enhances production of NO by macrophages and the elimination of trypanosomatid L. major [T. cruzi infected BALB/c mice and positively correlated with parasite burden and myopathic alterations [esponses . Mice genfection . This inL. major , it seemerations .T. cruzi-infected mice are highly susceptible to systemic inflammation, which can be caused by infection itself in mice lineages that develop severe inflammatory response or by administration of TNF, anti-CD3 [T. cruzi infection and LPS inoculation likely results from redundant lethal pathways induced by TNF and MIF: although both −/−Mif and −/−Tnfr1 infected mice succumb to LPS administration, treatment with anti-MIF rescues Tnfr1-deficient mice from lethal shock [A prior intracellular infection can sensitize the organism to septic shock by priming monocytes to overreact in the presence of very low amounts of TLR ligands, as happens in influenza , VSV 9898, LCMV anti-CD3 , SEB [98anti-CD3 . The letal shock . HoweverT. cruzi infection.Almost no information is available on MIF biology in Chagasic patients. The only study that addressed this issue demonstrated that the MIF-173G/C polymorphism confers susceptibility to Chagas disease in two cohorts from Colombia and Peru . Future In this paper we described the involvement of MIF in several models of protozoan infections, considering common themes and certain peculiarities specific to each parasite. In general, MIF seems to participate in the control of parasite burden but, in many cases, with the cost of promoting tissue damage due to increased inflammation. The essential role of MIF in the pathogenesis of infectious diseases and, consequently, the concept that it might be used as therapeutic target still require extensive clinical studies. Thus, for the years to come, several aspects of the biology of MIF and its participation in the response to infectious diseases, including parasitic diseases, need to be addressed opening up new highways of research and, possibly, novel therapeutic strategies.


Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. However, global identification of Pinus densata miRNAs has not been reported in previous research.MicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. The biogenesis and regulatory functions of miRNAs have been intensively studied in model angiosperms, such as P. densata mRNA transcriptome database using local BLAST and MIREAP programs. The primary and/or precursor sequences of 29 miRNAs were further confirmed by RT-PCR amplification and subsequent sequencing. The average value of the minimal folding free energy indexes of the 34 miRNA precursors was 0.92. Nineteen (58%) mature miRNAs began with a 5' terminal uridine residue. Analysis of miRNA precursors showed that 19 mature miRNAs were novel members of 14 conserved miRNA families, of which 17 miRNAs were further validated by subcloning and sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 miRNAs were more than 2-fold higher in needles than in stems. In addition, 72 P. densata mRNAs were predicted to be targets of 25 miRNA families. Four target genes, including a nodal modulator 1-like protein gene, two GRAS family transcription factor protein genes and one histone deacetylase gene, were experimentally verified to be the targets of 3 P. densata miRNAs, pde-miR162a, pde-miR171a and pde-miR482a, respectively.Here, we report the identification of 34 conserved miRNAs belonging to 25 miRNA families from a Pinus densata. These results lay a solid foundation for further studying the regulative roles of miRNAs in the development, growth and responses to environmental stresses in P. densata.This study led to the discovery of 34 conserved miRNAs comprising 25 miRNA families from  MicroRNAs (miRNAs), generally 21-24 nt in length, spatiotemporally regulate gene expression at transcriptional and/or posttranscriptional level in most eukaryotes . Most miIn silico prediction of miRNAs includes searching genomic or EST databases for orthologous sequences of known miRNAs and analyzing their pre-miRNA hairpin structures [Triticum aestivum, tomato and Oryza sativa [Identification of conserved and novel miRNAs usually relies on two approaches: bioinformatic prediction and experimental sequencing. ructures . The lima sativa -25. For Arabidopsis, rice and poplar [Pinus taeda have been identified and the expression of 10 miRNA families was significantly repressed in the galled stem [P. taeda were demonstrated to play vital roles during embryogenesis and seed development [Pinus contorta [Taxus chinensis cell line, Qiu et al. found that the expression levels of 14 miRNAs were down-regulated whereas that of 3 miRNAs were up-regulated after treatment with methyl jasmonate [Pinus abies, 24 novel and 4 conserved miRNAs were identified, and 7 conserved and 9 novel miRNAs were found participating in epigenetic regulation [Hundreds of miRNAs have been identified and characterized in model angiosperms, such as d poplar -29. Howeled stem . Stage-selopment . Morin econtorta . By sequasmonate . In Pinugulation .Pinus densata is an ecologically important conifer. It represents a highly successful case of homoploid hybrid speciation with far-reaching evolutionary consequences [Pinus densata miRNAs has not been reported in previous research. To gain the knowledge of its mRNA transcriptome, we recently performed a high-throughput sequencing of mRNAs isolated from P. densata needles. The present study was designed to identify P. densata miRNAs from the mRNA transcriptome database. Subcloning and sequencing were conducted to further confirm the pri- and/or pre-miRNA sequences. Meanwhile, using real-time RT-PCR, the expression profiles of 10 miRNAs in P. densata seedling tissues were examined. We have also predicted miRNA targets and 4 target mRNAs were experimentally validated by 5' RACE. Our study is the first comprehensive investigation of miRNAs in P. densata, which not only forms a solid base for further study of regulative functions of miRNAs in the development and growth, but also expands our knowledge of conifer miRNAs.equences . But to P. densata mRNA library. To identify conserved miRNAs, we used 3,968,794 sequences as query against mature and precursor sequences in the public microRNA database (miRBase version 16), which contains 2952 miRNAs across 43 plant species [P. densata miRNA precursors ranged from 76 to 526 nt, with a majority of them (80%) ranging from 67 to 150 nt. It is consistent with that observed in Arabidopsis and rice [P. densata miRNA precursors varied from 0.54 to 1.28, with an average value of 0.92. It is similar to that of other plant miRNAs, such as Arabidopsis, rice, Glycine max, Medicago truncatula, Saccharum officinarum, Sorghum bicolor and Zea mays [P. densata miRNA precursors predicted by MFOLD are shown in Additional file P. densata mature miRNAs varied from 19 to 22 nt, with 21 (38%) and 22 (41%) nt ones as the two major size classes , while the antisense strand of which harbored the precursor sequence of pde-miR3704a  in needles and stems of two-month-old seedlings, using real-time RT-PCR  [Mature miRNAs can direct RISC complexes to slice target mRNAs or inhibit their translations through nucleotide complementarity . The cleMethods) .P. densata  tails and thus can be isolated and sequenced as mRNAs, it allows us to identify candidate pri-miRNA sequences from the P. densata mRNA transcriptome database by bioinformatics tools [P. densata. The primary and/or precursor sequences of 29 miRNAs were further confirmed by subcloning and sequencing. In addition, 19 novel mature miRNAs belonging to 14 conserved miRNA families were found through the analysis of their precursor sequences. The 34 pre-miRNA sequences could form hairpin structures as predicted by MFOLD. They had an average MFEI value of 0.92  were only found in P. densata [P. taeda miRNA families, including pta-MIR319, pta-MIR398 and pta-MIR408, were not found in P. densata. It could be due to the simplicity of tissue used for Illumina sequencing.Among the 21 miRNA families conserved between dicots and monocots, 8 of which were identified in iosperms . Compare densata ,34. ThreP. densata.We also found a few contigs highly homologous to 12 conserved miRNAs. They were excluded for further analysis because they were not able to form canonical hairpin structures. With whole genome sequences and larger EST databases, more miRNAs will be identified from tomato leaf curl New Delhi virus (ToLCNDV) agroinfection in tomato cv Pusa Ruby [P. densata needles. Meanwhile, Singletons59617, coding for a protein highly homologous to Class III HD-Zip protein HDZ33, was predicted to be the target of pde-miR166a. These results suggest that the two miRNAs may also involve in needle polarity establishment and antiviral reaction in P. densata.In the present study, we found the expression levels of 7 conserved miRNAs were more than 2-fold higher in needles than in stems, using real time RT-PCR. It suggests that these conserved miRNAs may play specific roles in a variety of physiological processes in needles. Previous publications have shown that many miRNAs have important functions in the establishment of leaf polarity and virus-induced leaf curling. Zma-miR166a is an important miRNA known for establishing the adaxial/abaxial  leaf polarity by repressing the expression of class III homeodomain leucine zipper (HD-ZIPIII) transcription factors in maize . The accusa Ruby . In our P. densata using an online miRNA target search program, psRNATarget [P. densata miRNA targets encoded not only indispensable transcription factors, but also non-transcriptional factor proteins involving in diverse physiological processes. For example, HD-ZIP and GRAS family transcription factors, which are important to root and nodule development in Medicago truncatula and nutrient homeostasis in maize, were predicted to be targets of pde-MIR166 and pde-MIR171, respectively [P. densata.To define and elucidate the putative functions for a miRNA in plant, a necessary step is to predict and validate its target mRNAs. Currently, the most efficient tool available for this purpose is the bioinformatics approach, which is based on perfect or near perfectly complementarity between miRNAs and their targets. In this study, we predicted miRNA targets in NATarget . Our resectively ,51. Non-ectively . DCL1 isP densata, we performed a modified RLM-5' RACE experiment. In the present study, 4 miRNA targets, including singletons10015, singletons83401, singletons11093 and singletons7264 were confirmed to be targets of pde-miR171a, pde-miR162a and pde-miR482a, respectively. These results suggest that miRNAs can regulate the expression of their targets by cleavage in P. densata.To validate the miRNA targets in P. densata. A total of 34 conserved miRNAs comprising 25 miRNA families were identified. The primary and/or precursor sequences of 29 miRNAs were confirmed by subcloning and sequencing. Analysis of miRNA precursors revealed 19 pre-miRNA sequences harboring novel mature miRNAs belonging to 14 conserved miRNA families. Seventeen of the novel miRNAs were validated by sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 P. densata miRNAs were more than 2-fold higher in needles than in stems. Utilizing a Web-based program psRNATarget, 72 mRNAs were predicted to be targets of 25 miRNA families. Four mRNAs were experimentally validated to be targets of 3 P. densata miRNAs by RLM-5' RACE. These results suggest that regulative miRNAs exist in ecologically important conifer, P. densata, and may play key roles in P. densata development, growth and response to environmental stress.In summary, we performed a transcriptome-wide identification and characterization of miRNAs from P. densata needles were harvested from a mature Pinus densata tree in Linzhi city, Tibet, China and stored at -80°C until use. P. densata seedlings were grown under standard greenhouse conditions.Using RNAiso-mate for plant tissue and RNAiso plus , total RNAs were purified from needles and treated with RNase-free DNase I for 30 min at 37°C (Promega) to remove residual DNA. Total RNA of needles was used for Illumina sequencing of mRNA transcriptome, which was performed at Beijing Genomics Institute (BGI), Shenzhen, China. After removing reads containing only 3' sequencing adapters and reads of low quality (reads containing Ns > 5), transcriptome de novo assembly was carried out with short read assembling program - SOAPdenovo .P. densata against the mature and precursor sequences of known plant miRNAs deposited in miRBase version 16 http://www.mirbase.org/, using local BLASTN and MIREAP programs http://sourceforge.net/projects/mireap/[Pinus taeda and Picea abies were downloaded and used in the analysis as described above [E-value of lower than -2 or a score > 32 were processed for further analysis, allowing for a maximum of 2 nt mismatches. Overlapping contig sequences were used to form longer sequences according to their alignments to known precursor sequences in the miRBase. MFOLD was employed to predict hairpin structures with default parameters http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi[In order to find conserved miRNAs, we aligned 3,968,794 unique sequences from the mRNA transcriptome database of s/mireap/,54. To ded above ,34. Sequform1.cgi. Sequencform1.cgi.Actin was used as a positive control. Thirty-eight pairs of primers for P. densata primary or precursor sequences were designed  and 1 μg random nonamer, according to the manufacturer's protocol. The housekeeping gene m of a mature miRNA was < 60°C, it would be adjusted by adding Gs or Cs to the 5' end and/or As to the 3' end of the miRNA sequence  following the manufacturer's recommendations. The resulting cDNA was diluted 10 times with sterile water. Quantitative real-time PCR was performed in triplicate reactions using the MX3000P detection system . Ten forward primers were designed based on mature miRNA sequences. If the TP. densata miRNAs, we used 38 mature miRNAs as custom miRNAs and 3,968,794 sequences in the P. densata mRNA database as custom mRNAs to search for complementary hits using the psRNATarget program http://bioinfo3.noble.org/psRNATarget/ with default parameters. The score system was applied according to Zhang [E-value of less than e-10 were considered a hit.To identify putative targets of to Zhang . SequencTo identify cleavage sites in the target mRNAs, a modified RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was performed using a 5' RACE kit (Takara) . Total RAGO: Argonaute; DCL: Dicer-like; GO: Gene Ontology; MFEI: Minimal folding free energy index; miRNA: MicroRNA; pre-miRNA: Precursor miRNA; pri-miRNA: Primary miRNA; RISC: RNA-induced silencing complex; RT: Reverse transcription.LW designed and carried out the study and drafted the manuscript. HZ and LZ participated in the bioinformatics analysis. SL, ZQ and YZ helped to draft the manuscript. QZ and JL conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.P. densata conserved miRNAsPrecursor sequences of .Click here for fileP. densata conserved miRNAsPrimary sequences of .Click here for fileP. densata miRNA precursorsPredicted hairpin structures of . The hairpin structures of P. densata miRNA precursors were predicted by MFOLD. Mature miRNAs were marked in yellow.Click here for fileP. densata microRNA sequences (red and blue)Predicted stem-loop structures of precursors containing .Click here for fileP. densataConserved mature miRNAs without hairpin structure in .Click here for fileP. densata miRNAs and their putative functionsPredicted targets of .Click here for fileP. densata miRNAs and their targetsPrimers used for amplifying .Click here for file


Vibrionaceae family are widespread in the marine environment. Today, 128 species of vibrios are known. Several of them are infamous for their pathogenicity or symbiotic relationships. Despite their ability to interact with eukaryotes, the vibrios are greatly underexplored for their ability to produce bioactive secondary metabolites and studies have been limited to only a few species. Most of the compounds isolated from vibrios so far are non-ribosomal peptides or hybrids thereof, with examples of N-containing compounds produced independent of nonribosomal peptide synthetases (NRPS). Though covering a limited chemical space, vibrios produce compounds with attractive biological activities, including antibacterial, anticancer, and antivirulence activities. This review highlights some of the most interesting structures from this group of bacteria. Many compounds found in vibrios have also been isolated from other distantly related bacteria. This cosmopolitan occurrence of metabolites indicates a high incidence of horizontal gene transfer, which raises interesting questions concerning the ecological function of some of these molecules. This account underlines the pending potential for exploring new bacterial sources of bioactive compounds and the challenges related to their investigation.Bacteria belonging to the  Vibrionaceae are Gram-negative, rod-shaped γ-proteobacteria that are usually motile and possess a chemoheterotrophic metabolism . Th. Th4) (4. (2005)  revealedEnterobacter sp. from the planthopper Nilaparvata lugens  are are27) [rophores .29)   69] is  is 29)  [V. vulnificus.Siderophores are used as therapeutic deferration agents to treat iron overload in chronically transfused thalassemia patients. A stereochemically modified version of fluvibactin efficiently removed iron without increasing microbial growth . It has . (2009)  made a cVibrionaceae is the kahalalides. These cyclic depsipeptides were originally isolated from the herbivorous mollusc Elysia refescens and its diet, the green algae Bryopsis sp. In particular, kahalalide F (30)   has an a31)  (31) , also knnalogues . Also, Vuced TTX . Vibriosicularis , suggesticularis . The rolicularis .P. halotolerans two novel depsipeptides (32–33) that interfere with QS regulated virulence genes in S. aureus were isolated [hla encoding α-hemolysin and RNAIII, while increasing expression of spa encoding Protein A. This suggested that the depsipeptides interfere with agr, the global virulence regulator in S. aureus. High structural similarity of the solonamides to the natural autoinducers of the agr system suggested that they might be competitive inhibitors. Interestingly, the solonamides had a pronounced effect on virulence gene expression in S. aureus strain USA300, which is the predominant community-acquired MRSA (CA-MRSA) strain in the USA [17–18) found in an unidentified Photobacterium sp. (Section 2.3.1) [Vibrios produce compounds that interfere with the quorum sensing system of Gram-positive bacteria. From a strain related to peptides , solonamisolated . In part the USA . The soln 2.3.1) . Thus, icyclo (34), cyclo (35), and cyclo (36), DKPs commonly isolated from vibrios [et al. (2001) [37) (P. angustum (V. angustum) S14 with the ability to mediate expression in two AHL-regulated systems, inducing bioluminescence in V. harveyi and the AHL reporter system in Agrobacterium tumefaciens.Several small molecules isolated from vibrios induce Gram-negative quorum sensing systems. That includes various diketopiperazines (DKP) . For exa vibrios , modulat vibrios . It is s vibrios . However vibrios . De Nys . (2001)  isolated01) [37)  from P. P. halotolerans S2753, we isolated a series of cyclic tetrapeptides  (38), cyclo (39), cyclo (40), and cyclo(l-Leu-l-Ile-l-Leu-l-Ile) (41) . These types of peptides are often found in marine culturable bacteria [Vibrios also produce numerous compounds for which no biological activity has been reported so far. That includes small-molecule by-products, for example some nitro-substituted compounds such as 3-nitroindazole and 3-nitro-4-hydroxycinnamic acid . From P.peptides ; cyclo  [Many of compounds isolated from vibrios are suspected to be artifacts generated from media components during work-up procedures ,144,148., Bio249 . An exam249 [42)  that couolyticus . From thine (43) . This waVibrionaceae makes this group of bacteria very apt to resist various environmental changes, for example through the acquisition of biosynthetic genes linked to the production of antibiotics or siderophores. So, rampant horizontal gene transfer occurs in these bacteria. As a reflection, most compounds isolated from vibrios have also been found in other types of bacteria, in many cases from distantly related taxa [The versatility and widespread occurrence of vibrios can be ascribed to different characteristics such as their superior nutrient utilization, their excellent biofilm formation, and their genetic construction. High genomic flexibility in ted taxa . Sometimted taxa .N-containing compounds produced by NRPS-independent pathways. Despite this narrow structural span compared to metabolites produced by other marine bacteria, vibrios produce compounds with a broad range of interesting biological activities. For example the solonamides, cyclic depsipeptides from P. halotolerans were found to attenuate virulence in a CA-MRSA strain [V. medierranei [Production of secondary metabolites in vibrios has been linked to antagonism, intraspecies communication, and pathogenicity. The compounds produced by vibrios are mainly non-ribosomal peptides or hybrids hereof, with examples of A strain  and the ierranei  that is ierranei .V. coralliilyticus were found to produce andrimid in high yields, while pathogenic related strains did not have the ability to produce the antibiotic [Many vibrios have multiple lifestyles, including a planktonic (free swimming), sessile (attached to zooplankton or other surfaces), and a pathogenic form . As prodtibiotic .Vibrionaceae metabolites. Of the 227 vibrio genomes sequenced so far [V. cholerae strains [The cosmopolitan occurrence of several vibrio metabolites raises the question whether there are unique d so far , only a d so far , mainly  strains , and non


AbstractTillandsia capillaris Ruiz & Pav., which belongs to the subgenus Diaphoranthema is distributed in Ecuador, Peru, Bolivia, northern and central Argentina, and Chile, and includes forms that are difficult to circumscribe, thus considered to form a complex. The entities of this complex are predominantly small-sized epiphytes, adapted to xeric environments. The most widely used classification defines 5 forms for this complex based on few morphological reproductive traits: Tillandsia capillaris Ruiz & Pav. f. capillaris, Tillandsia capillaris f. incana (Mez) L.B. Sm., Tillandsia capillaris f. cordobensis (Hieron.) L.B. Sm., Tillandsia capillaris f. hieronymi (Mez) L.B. Sm. and Tillandsia capillaris f. virescens (Ruiz & Pav.) L.B. Sm. In this study, 35 floral and vegetative characters were analyzed with a multivariate approach in order to assess and discuss different proposals for classification of the Tillandsia capillaris complex, which presents morphotypes that co-occur in central and northern Argentina. To accomplish this, data of quantitative and categorical morphological characters of flowers and leaves were collected from herbarium specimens and field collections and were analyzed with statistical multivariate techniques. The results suggest that the last classification for the complex seems more comprehensive and three taxa were delimited: Tillandsia capillaris (=Tillandsia capillaris f. incana-hieronymi), Tillandsia virescenss. str. (=Tillandsia capillaris f. cordobensis) and Tillandsia virescenss. l. (=Tillandsia capillaris f. virescens). While Tillandsia capillaris and Tillandsia virescenss. str. co-occur, Tillandsia virescenss. l. is restricted to altitudes above 2000 m in Argentina. Characters previously used for taxa delimitation showed continuous variation and therefore were not useful. New diagnostic characters are proposed and a key is provided for delimiting these three taxa within the complex. Tillandsioideae comprises 10 genera , Diaphoranthema (21 spp.), Phytarrhiza (7 spp.), and Allardtia (3 spp.)  . TillandTillandsia capillariss. l. is distributed from southern Ecuador to central Argentina and Chile, between altitudes from 300 m to 4000 m.  and the variety: Tillandsia propinqua var. saxicola Hieron. Mez published in Tillandsia hieronymi Mez and Tillandsia dependens Hieron. ex Mez, the later with two varieties Tillandsia dependens var. perusneoides Mez and Tillandsia dependens var. percordobensis Mez; and named new varieties: Tillandsia capillaris var. incana Mez, Tillandsia capillaris var. lanuginosa Mez. Tillandsia williamsii Rusby from Bolivia. Finally, Tillandsia permutata A. Cast. and new varieties and forms: Tillandsia hieronymi var. lichenoides (Hieron.) A. Cast., Tillandsia virescens var. sanzinii (Hicken) Castell., Tillandsia dependens f. perusneoides (Mez) Castell., Tillandsia dependens f. percordobensis (Mez) Castell.In the past two centuries several species that are currently included in the Tillandsia capillaris with 5 forms: Tillandsia capillaris Ruiz & Pav. f. capillaris, Tillandsia capillaris f. incana (Mez) L.B. Sm., Tillandsia capillaris f. cordobensis (Hieron.) L.B. Sm., Tillandsia capillaris f. hieronymi (Mez) L.B. Sm. and Tillandsia capillaris f. virescens (Ruiz & Pav.) L.B. Sm. The separation of these forms is based on only four characters: the number of nerves in the floral bracts, the length of the peduncles, the indument of the floral bracts and the size of the leaves.The currently accepted classification in Argentina  is basedDiaphoranthema in South America was done by Tillandsia capillaris  with a distribution area ranging from southern Ecuador to central Argentina, reaching altitudes of 3500 m; and Tillandsia virescens (including Tillandsia capillaris f. virescens and Tillandsia capillaris f. cordobensis) with a similar distribution area , but thriving up to 4300 m. This proposal is also based on a few traits: mainly on differences in the connation of the sepals, the indument of the floral bracts, and the architecture of the sepal veins. Although this classification  from northern and central Argentina, therefore the results are valid for Argentina but not for the whole range of the complex . Each specimen was treated as a taxonomical operational unit (OTU), and 35 floral and vegetative morphological characters were registered, including 12 continuous variables, 7 discontinuous (or discrete) variables, 11 binary variables and 5 multistate variables  were run for all the variables among the taxa considered. Box-plots were made for continuous variables.A two-steps analysis was carried out to detect the most informative characters. First, a Principal Component Analysis (PCA) was run using all characters , obtainiH=4.68; P= 0.24) among the putative taxa but with unclear trends  (results not shown). Analyzing the variables individually, only 19 variables were selected to explain the variance among taxa , considering the ones which showed values up to 0.20 . A seconPageBreakof the variability using the 19 characters previously selected in the PCA with coefficient >0.20  (Tillandsia capillaris f. cordobensis (=Tillandsia virescenss. str. sensu Till) (Tillandsia capillaris f. virescens (=Tillandsia virescenss. l. sensu Till)  has the smaller sizes of the fertile shoot, scape, leaf blade, fruit, seed, and embryo. This last form showed statistically similarities in some of the characters  , Tillandsu Till)  and Tillsu Till) . For exat groups .Bromeliaceae currently prevails in the literature to resolve different complex groups (The criteria used in previous classifications  are not x groups  stressinx groups .Tillandsia capillaris f. virescens and Tillandsia capillaris f. hieronymi). Within the non-informative qualitative characters, those referred to the peltate trichomes  were cited in previous classifications . The quantitative characters are significant to separate groups, while the categorical characters were useful when the taxa had similar plant size .For example, Tillandsia capillaris f. incana described by Tillandsia capillaris f. hieronymi was previously circumscribed by the 3 nerves joined together at the apex of the floral bract ; a second group with adaxial sepals more fused (50-90%) (Tillandsia virescenss. str. and s. l.=Tillandsia capillaris f. cordobensis and Tillandsia capillaris f. virescens). Data for the material studied here showed values of 40-70% fusion for the adaxial sepals in the first group (Tillandsia capillaris=Tillandsia capillaris f. incana- hieronymi), and 60-90% in the second group (Tillandsia virescenss. str. and s. l.=Tillandsia capillaris f. cordobensis and Tillandsia capillaris f. virescens). Although the trend described by Tillandsia virescens as one species, and Tillandsia cordobensis as a taxonomic synonym of Tillandsia virescens  comprise “group 1: Tillandsia cordobensis” and “group 2: Tillandsia cordobensis “var.” tucumanensis nom. nud.”. Otherwise, Tillandsia virescenss. l. (=Tillandsia capillaris f. virescens) include “group 4: Tillandsia propinqua “var.”” ((50-90%) , and aba “var.”” . Till  in future taxonomical work for the complex, since we saw that the characters he used  were useful to defined these groups. We expect to propose conclusive nomenclatural changes, however, only after gathering additional data. Taxonomic resolution of complex groups, ideally, should be done combining morphological data from the whole range of distribution.Summarizing, our results partially support the classification of Tillandsia capillaris (=f. incana- hieronymi) and Tillandsia virescenss. str. (=f. cordobensis), co-occur in almost the same sites and altitude levels. Tillandsia virescenss. str. was mentioned by Tillandsia capillaris. On the other hand, Tillandsia virescenss. l. (=f. virescens), with saxicolous habit, was found restricted to higher altitudes in the central Argentina , or in the western foothill of the Andes (between 2000-3500 m). It is interesting to note that in some regions and at higher altitudes , the three forms can co-occur, but at lower altitudes  only two of these forms can be found (Tillandsia capillaris and Tillandsia virescenss. str.).All the forms analyzed are distributed in the central and northern Argentina, in the southern distributional range of the complex. The taxa Tillandsia capillaris complex using a relatively large sample of material with a multivariate perspective. This methodological approach allowed us to define three taxa in Argentina , MINCyT and BMFW.


Dekkera (Brettanomyces) bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs) at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs). Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits.Despite its industrial importance, the yeast species  Dekkera (Brettanomyces) bruxellensis is predominantly associated with wine, beer, and cider, but has also been detected in kombucha (sweet tea) and sourdough fermentations The yeast species D. bruxellensis is Dekkera anomala, a species that has been found to co-exist with D. bruxellensis in some niches, such as cider fermentation D. bruxellensis is well adapted to this environment. Some of these adaptations; osmotolerance, ethanol tolerance, acid tolerance, are shared with Saccharomyces cerevisiae - the ubiquitous fermentation yeast that precedes D. bruxellensis in population ecology of the industrial fermentation systems mentioned above. In addition, D. bruxellensis is both petite and crabtree positive, and has convergently evolved the make-accumulate-consume lifestyle in parallel with S. cerevisiaeD. bruxellensis is capable of surviving and proliferating for extended periods of time after S. cerevisiae has completed alcoholic fermentation and its population declined The nearest phylogenetic neighbour of It is well accepted that D. bruxellensis is the key wine microorganism that converts hydroxycinnamic acids present in grape must and wine into the volatile phenols 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG) and 4-ethylcatechol (4-EC). Wine containing above-threshold concentrations of these compounds exhibits ‘Brett’ character D. bruxellensis CBS 2499 genome D. bruxellensis strains in the same study, leading the authors to question existence of a sexual cycle for this species.Partial survey sequencing of the D. bruxellensis, we have sequenced the prevalent wine spoilage strain, AWRI1499 D. bruxellensis AWRI1499 has a triploid genome, that relative to phylogenetically related species is enriched in genes that may aid survival in the challenging environment of wine.Despite its importance in food and beverage industries, very little is known regarding the genetics of this microorganism. Next-generation genomic sequencing is providing the means of fast tracking genetic analysis of intractable, non-model organisms, by enabling direct access to genetic make-up, and subsequent functional analysis. To gain insight into the genome features and gene content of D. bruxellensis was estimated at between 20 and 30 Mb by pulsed-field electrophoresis of several European strains D. bruxellensis CBS 2499 D. bruxellensis cell.De-novo auto-assembly of sequence reads yielded 26,043 contigs, with N50 of 7 kb at 9.7-fold coverage, and an assembly length of 40.2 Mb. The genome size of D. bruxellensis AWRI1499 predicted proteins.RNA samples pooled from cultures grown under standard laboratory conditions, and in the presence of 2 mM ferulic acid S. cerevisae ORFs YGR181w, YOR103c, YGL231c, YGL047w) were detected at low identity or had only partial matches in the scaffold consensus. A further 11 CEGs with AWRI1499 gene model tblastn hits at worse than >1e-20 were confirmed by tblastn of non-S.cerevisiae fungal orthologs of each protein against the scaffold consensus. This analysis suggested the genomic assembly of AWRI1499 was approximately 98% complete.To test the AWRI1499 genome assembly for completeness, the core eukaryote gene (CEG) set of 248 proteins from six reference species Pichia angusta the closest relative to D. bruxellensis among those fungi for which sequenced genomes are available. Along with Pichia pastoris, these species, while not closely related, form a grouping distinct from the whole genome duplication and CTG clades, respectively.The 20 fungal species most represented as blastp top-hits for AWRI1499 gene models  were selSignificant heterozygosity was observed in the assembly, with a median of 27 single nucleotide polymorphisms (SNPs) per 1000 nucleotides - a total of 342,900 sites across the genome. However, this density was not uniform across the assembly with some regions displaying substantially reduced rates of heterozygosity . In partD. bruxellensis ORFs. During assembly it was noted that in many regions more than two distinct sequences were evident amongst sequence reads. To investigate the distribution of coding region SNPs across multiple alleles, a subset of ORFs (60 of 4969) with very high SNP density (6%), high SNP density (4%), and moderate SNP density (2%) were randomly sampled across the genome, and manually separated into haplotypes. Of the 60 ORFs sampled, 37 could be resolved into three distinct haplotypes. ORFs from which two haplotypes were resolved did not originate only from regions with low SNP density, such as scaffold 3, but were dispersed across the assembly (data not shown). The probability of resolving three haplotypes from ORFs with 2% SNP density was, however, significantly lower . Pair wise nucleotide sequence alignments . Regions of reduced SNP density, such as scaffold 3, would therefore most likely reflect a partial replacement of the divergent haploid by the diploid genome, a phenomenon observed in Saccharomyces interspecific hybrids D. bruxellensisD. bruxellensis does not by default infer sterility D. bruxellensis lacks a fully functional sexual cycle.Within predicted open reading frames there were fewer SNPs than seen for the entire genome, with an average of 1.9%. The majority of these coding region SNPs were synonymous Nonetheless 0.66% of all nucleotides are predicted to produce non-synonymous amino acid substitutions across ignments  revealedD. bruxellensis, the predicted proteome of AWRI1499 was submitted to OrthoMCL for assignment to orthologous clusters  and are hypothesised to have undergone convergent evolution of several phenotypic traits D. bruxellensis were 198 predicted proteins. Seventy-seven of these proteins were not annotated by Blast2go which has a more stringent blastp cut-off (E<1e-6). A further 64 were found to have a blastp match to a protein from one of the other three species (assigned to a different OrthoMCL cluster) and there were 10 dubious assignments of short predicted proteins (<100 amino acids). Excluding four transposon-like proteins, there were no significantly enriched GO-terms for the remaining 43 proteins . Nonetheless, some of these may contribute to the ‘scavenging’ lifestyle of this yeast. Several proteins involved in carbon source utilisation , were detected. Interestingly, most genes required for chitin/N-acetylglucosamine catabolism were found to be present in a single cluster of five genes, which were also found to be present in M. guilliermondii, although separated in two blocks of synteny  and expansion driven by convergent evolution . D. bruxellensis proteins corresponding to expanded clusters were then examined for GO-term enrichment using Blast2go, with significantly over-represented terms  summarised in D. bruxellensis.To detect instances of conserved gene family expansion during the evolution of D. bruxellensis were highly enriched in GO terms corresponding to the cell membrane. OrthoMCL cluster OG5_126579 was quantitatively the most expanded in D. bruxellensis. This cluster, to which S. cerevisiae ORFs FIG2, FLO1, FLO5, FLO9, HKR1, HPF1, MSB2, MUC1, PRM7, YIL169C, and YMR317W, were assigned, encompasses divergent plasma membrane and cell wall proteins involved in cell wall budding, adhesion, and pseudohyphal growth. D. bruxellensis has been shown to form biofilms D. bruxellensis. Amino acid permeases were expanded compared to all three species, while the D. bruxellensis/Pichia lineage expansion was enriched in transmembrane drug transporters, and D. bruxellensis/S. cerevisiae expanded clusters were enriched in nucleobase transporters. Survey sequencing of D. bruxellensis strain CBS 2499 also revealed an enrichment of transporters, consistent with adaptation to relatively low nutrient environments like wine Proteins corresponding to OrthoMCL clusters expanded in D. bruxellensis AWRI1499 has only one ORF (g80.t1) that encodes a protein with homology to the S. cerevisiae sulfite efflux transporter, Ssu1p. This protein confers sulfite tolerance S. cerevisiae populations exposed to sulfite S. cerevisiae, SSU1 is duplicated, and strains with high levels of sulfite tolerance harbor one or more SSU1-R (resistant) alleles SSU1-R allele was recently described for the wine yeast S. cerevisiae 71B D. bruxellensis strains will be required to determine whether molecular mechanisms conferring sulfite tolerance are similar for the two species.Despite exposure to the common wine preservative, sulfite, D. bruxellensis contains eight oxidoreductases corresponding to the OrthoMCL cluster of alcohol dehydrogenase proteins +. proteins , and fivproteins , while aS. cerevisiae, alcohol and aldehyde dehydrogenases, along with other oxidoreductase enzymes, contribute to the formation of aroma compounds through the Ehrlich pathway D. bruxellensis has been reported to produce the latter compound, which has a ‘rancid’ aroma thought to contribute to the sensory impact of D. bruxellensis on wine D. bruxellensis proteins were found that each have homology to isoamyl-acetate hydrolysis enzymes  or NADP(H) binding domains, thus it seems questionable that DbVPR indeed performs the VPR function. Godoy et al. D. bruxellensis, which exhibited very different kinetic properties. Finding the gene that encodes this critical enzyme in D. bruxellensis may be possible by functional analysis of the oxidoreductase OrthoMLC clusters expanded in D. bruxellensis.Tchobanov et al. S. cerevisiae Pad1p, and the bacterial phenolic acid decarboxylases (IPR008729). No homolog of the S.cerevisiae protein Pad1p was found in the partial sequence of D. bruxellensis CBS 2499 D. bruxellensis genome sequence for potential PAD-encoding genes using characterised bacterial proteins from Bacillus pumulis, Bacillus subtilis, Pediococcus pentosaceus and Lactobacillus plantarum. No sigificant hits were found amongst the D. bruxellensis ORFs, however a significant tblastn hit (E<1e-30) was found in contig 3340 of scaffold 100. A hypothetical protein, DbPad, was generated by fusing two frameshifted (due to assembly sequence errors) translations, and aligned against fungal proteins with homology to DbPad or S. cerevisiae Pad1p. The resultant maximum likelihood tree  which includes ood tree  shows thD. anomala was attributed to a protein with homology to S. cerevisiae Pst2p, a member of the flavodoxin family D. bruxellensis AWRI1499 ORFs encode Pst2 homologs, exhibiting 99% (g3584.t1), 67% (g3585.t1) and 57% (g797.t1) amino acid similarity to the D. anomala Pst2 protein. These proteins do not share homology with the phenylacrylic acid decarboxylase, or phenolic acid decarboxylase families. Nonetheless, it is possible that through duplication of Pst2 in D. bruxellensis AWRI1499 and subsequent functional divergence, PAD activity can be attributed to one of the three proteins. On the other hand, it is interesting to note that M. guilliermondii was the only budding yeast found to contain a DbPad homolog  than 4-ethylcatechol D. bruxellensis spoilage of wine are a consequence of DbPad substrate specificity. Availability of the AWRI1499 genomic assembly and annotation will facilitate functional characterisation of the DbPAD gene, and others that may confer industrially relevant phenotypic traits.PAD activity in  homolog . An enzyD. bruxellensis biology, both from an evolutionary perspective and in terms of its role in fermentation processes.This study sheds light on an industrially important microorganism which, because of its intractable nature, has been poorly understood. Analysis of gene content points to an organism that is metabolically well equipped to survive in challenging environments such as wine. The genomic assembly described in the current study reveals a triploid genome, consistent with speciation through inter-specific hybridisation and an asexual lifestyle. Availability of the genomic assembly and annotation will facilitate advancement in our knowledge of D. bruxellensis strain AWRI1499 was obtained from The Australian Wine Research Institute Microorganisms Culture Collection. Genomic DNA was prepared using a standard zymolyase and phenol-chloroform extraction from cultures grown under standard conditions. RNA was extracted from cultures grown under standard conditions and incubated for 2 h in the presence of 2 mM ferulic acid to induce a stress response MIRA (v3rc2) Regions of copy number variation were determined by calculating the per-base sequencing coverage across each sequencing contig with median smoothing . The ratio between coverage at each genomic location and overall median genomic coverage was calculated to determined the level of over- or under-representation for each location. Large scale aneuploidies were detected by screening for regions where median ratio for a contiguous stretch of at least 101 individual segments differed from the overall genomic median by either 1.25- or 1.5-fold.S. cerevisiae and P. pastoris gene models built in Augustus were compared for prediction of D. bruxellensis ORFs, with the S. cerevisiae based model resulting in a higher number of predicted ORFs and therefore preferred. ORFs were visualised in IGV v2.03 Open reading frame prediction was performed using Augustus v2.5.5 D. bruxellensis predicted proteins. The proteome for each species was used in pairwise blastp against D. bruxellensis, and reciprocal top-hits identified as putative orthologs. A table of putative orthologs across all 21 species is available in supplementary data Click here for additional data file.Figure S2GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Biological process GO-terms enriched in comparison to all three spp.(PDF)Click here for additional data file.Figure S3GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Cell component GO-terms enriched in comparison to all three spp.(PDF)Click here for additional data file.Figure S4GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Molecular function GO-terms enriched in comparison to all three spp.(PDF)Click here for additional data file.Figure S5GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Biological process GO-terms enriched in comparison to P. pastoris and P. angusta, but not S. cerevisiae.(PDF)Click here for additional data file.Figure S6GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Cellular component GO-terms enriched in comparison to P. pastoris and P. angusta, but not S. cerevisiae.(PDF)Click here for additional data file.Figure S7GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Molecular function GO-terms enriched in comparison to P. pastoris and P. angusta, but not S. cerevisiae.(PDF)Click here for additional data file.Figure S8GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Biological process GO-terms enriched in comparison to S. cerevisiae, but not P. pastoris and P. angusta.(PDF)Click here for additional data file.Figure S9GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Cellular component GO-terms enriched in comparison to S. cerevisiae, but not P. pastoris and P. angusta.(PDF)Click here for additional data file.Figure S10GO-term enrichment analysis for OrthoMCL clusters common to D. bruxellensis, P. pastoris, P. angusta, and S. cerevisiae. Molecular function GO-terms enriched in comparison to S. cerevisiae, but not P. pastoris and P. angusta.(PDF)Click here for additional data file.Table S1Contig statistics. Average sequence coverage and length of contigs ordered by position within scaffolds.(XLS)Click here for additional data file.Table S2D. bruxellensis open reading frame ortholog group assignments. OrthoMCL assignments and sequence ID of best hit for all predicted protein coding open reading frames.(XLS)Click here for additional data file.Table S3D. bruxellensis proteins across 20 fungal species. The proteomes of species frequently assigned as top blastp hit for D. bruxellensis proteins in Blast2Go were used in pairwise blastp against the D. bruxellensis proteome, and reciprocal top-hits identified as putative orthologs.Putative orthologs of (XLS)Click here for additional data file.


MicroRNAs (miRNAs) are small (~19-24nt) non-coding RNAs that play important roles in various biological processes. To date, the next-generation sequencing (NGS) technology has been widely used to discover miRNAs in plants and animals. Although evolutionary analysis is important to reveal the functional dynamics of miRNAs, few computational tools have been developed to analyze the evolution of miRNA sequence and expression across species, especially the newly emerged ones,Drosophila melanogaster and Arabidopsis, respectively.We developed miREvo, an integrated software platform with a graphical user interface (GUI), to process deep-sequencing data of small RNAs and to analyze miRNA sequence and expression evolution based on the multiple-species whole genome alignments (WGAs). Three major features are provided by miREvo: (i) to identify novel miRNAs in both plants and animals, based on a modified miRDeep algorithm, (ii) to detect miRNA homologs and measure their pairwise evolutionary distances among multiple species based on a WGA, and (iii) to profile miRNA expression abundances and analyze expression divergence across multiple species . Moreover, we demonstrated the utility of miREvo with Illumina data sets from http://evolution.sysu.edu.cn/software/mirevo.htm under the GNU/GPL license.This work presents an integrated pipline, miREvo, for exploring the expressional and evolutionary dynamics of miRNAs across multiple species. MiREvo is standalone, modular, and freely available at  MicroRNAs (miRNAs) are small non-coding RNAs about 19–24 nt in length that regulate gene expression post-transcriptionally . Extensihttp://www.mirbase.org, Version 18.0).In the last few years, the next-generation sequencing (NGS), an inexpensive and high-throughput sequencing method with high sensitivity and specificity, has been widely used to measure the abundance of small-RNAs in multiple species -15. AddiDrosophila[Besides the conserved miRNAs, NGS has identified many new miRNA genes, which are highly divergent between closely-related species, such as miR310 cluster in rosophila,24. New rosophila and may rosophila. Furtherrosophila, which hrosophila and thusDrosophila[Arabidopsis[Meanwhile, whole genome sequencing has been completed for a great number of species. The multiple-species whole genome alignments (WGAs) provides us a luxuriant resource for comparative genomic analysis and evolutionary studies -29, and rosophila and Arababidopsis. Such inabidopsis,25, and abidopsis.Here, we reported miREvo, an integrated miRNA evolutionary analysis platform for NGS experiments by using miRDeep2  as core MiREvo is committed to provide an efficient and easy-to-use solution for miRNA analysis based on NGS experiments. To achieve this goal, we have implemented miREvo with both command-line interface (CLI) and Graphical User Interface , according the coordinate of the miRNA precursor located in the reference. Compared with the methods that directly align miRNAs against the reference genome using BLASTN, our WGA-based method is more credible in screening the orthologs of highly divergent miRNAs.One challenge in homology identification is to produce a single best orthologous match for a given miRNA/sequence. To reduce the interference by tandem duplication sequences, WGA is well-adjusted and optimized for orthologous alignment extension and specificity . miREvo http://genome.ucsc.edu) has built multiple genome alignments for most sequenced animals, such as vertebrates or inserts. For the plants, we have constructed two MAF files. One represents the alignment of 11 green plant genomes  genomes of Japonica cv. Nipponbare and Indica cv. 93–11  or NCBI . The expression of mature miRNAs from 5’ and 3’ arms of the hairpin precursors is estimated by counting the mapped reads and normalized by the total number of mapped reads, respectively. Then, a Poisson regression model  and likehttp://www.ncbi.nlm.nih.gov/geo/). One was from Drosophila simulans embryo (GSM343915) and the other was from Arabidopsis thaliana leaves (GSM707679). As a working example, we only presented the results of Drosophila melanogaster here. The example of Arabidopsis thaliana was provided in the additional files. The full demo data can be downloaded from our website (http://evolution.sysu.edu.cn/software/mirevo.htm). After a full run of analysis, we provided the following results  database (mbryo GSM43915 andD. simulans (miRbase V18.0), 101 were observed with a total of 1,746,656 reads  mapped to the corresponding loci. In addition, another 43 putative new miRNAs with 5,957expressed reads were predicted using default criteria of miRDeep2[Drosophila species by crosschecking with miRBase. For example, the novel miRNA dsi_22_3.9 has been annotated as miR-1009 in D. melanogaster  Reads Statistics of miRNAs: A total of 4,609,104 reads were sequenced, representing 863,547 unique signatures. A table summarized the reads count for each miRNA was provided. Among the 136 characterized miRNA genes of  miRDeep2. Among tDrosophila species were parsed out from the 12 Drosophila species WGA produced by UCSC (http://hgdownload.cse.ucsc.edu/goldenPath/dm3/multiz15way/). A multiple alignment of these orthologous sequences were constructed. To evaluate the RNA secondary structure conservation, the alignments of minimum free energy (mfe) structure were also displayed in bracket notation. Finally, the pairwise genetic distances  FASTA sequences and multiple alignments: The orthologous sequences of the predicted new miRNAs in the other 11 er model ) and seq(3) The minimum energy and the miRNA hairpin folding. The hairpin folding of individual pre-miRNA was graphed with detailed information of folding free energy. The pre-miRNA sequence was underlined as color-coded according to the reads’ coverage pre base of the hairpin.D.melanogaster (miRBase V18) in genome of D.pseudoobscura using both methods. Considering miRNA precursors that have at least 90% identity out of a coverage length greater than 50nt between the two species, miREvo identified 93 (39%) orthologs with at least ten sequencing reads at the embryonic stage in D. pseudoobscura. By contrast, the BLASTN-based search (Options: -e 0.01 -W 7 -b 3 -v 3 -q −1 -F F) only identified 66 (28%) orthologs. Therefore, the WGA-based method is significantly more effective .Another application of miREvo is to identify the homologous sequences of a given miRNA. To do so, MiREvo requires input files including 1) the precursor miRNA sequence, 2) the mature miRNA sequence, 3) the reference genome, and 4) the MAF files containing the WGA of species of interests; the output file presents the best-fitted results. Taking the advantage of synteny information of WGA, miREvo is particularly useful in identifying orthologs of highly divergent miRNAs, or paralogous members of old miRNA families. To demonstrate that the WGA-based method has better performance than BLASTN, we conducted a genome-wide screening for all the orthologs of the 240 known miRNA of Drosophila, which has accumulated a large number of nucleotide substitutions since the split of D. melanogaster and D. pseudoobscura. The orthologous sequence of dme-miR-310 in D. pseudoobscura can be clearly identified with miREvo  of dme-miR-310 between the two species is 0.5, which is half of the averaged genome-wide synonymous divergence [Arabidopsis ath-miR-169 family was given in Additional file Arabidopsis ath-miR-169 family can be individually distinguished  and D. pseudoobscura (GSM343916). Using D. melanogaster miRNAs as references, orthologs from the other two species were extracted. The expression of orthologs for each library was analyzed separately, cross-species expression profiles were constructed : Unix/Linux based· Programming language: Bash, Perl 5 and Perl/TK· Other requirements: RNAfold, Bowtie, BioPerl, mafsInRegion and BLAT· License: GNU GPL v3· Any restrictions to use by non-academics: specified by GNU GPL v3· Before the application can be started, the following open source packages must be installed:http://www.perl.org/get.html1. Perl 5 – http://search.cpan.org/~ni-s/Tk-804.027/pod/UserGuide.pod2. Perl-TK – http://www.bioperl.org/wiki/Getting_BioPerl3. BiolPerl – 4. The following free available packages are also needed by miREvo, which has been deposited into miREvo package, or users can install them independently:http://www.tbi.univie.ac.at/~ivo/RNA/(a) Vienna RNA Package – http://bowtie-bio.sourceforge.net/index.shtml(b) Bowtie – http://genomewiki.ucsc.edu/index.php/Kent_source_utilities(c) masfsInRegion – http://genome.ucsc.edu/FAQ/FAQblat.html#blat3(d) BLAT – http://www.imagemagick.org/script/index.php)e) ImageMagick (http://evolution.sysu.edu.cn/software/utilities.tar.gz. The documentation also illustrates the usage of each individual functions, including the case-oriented help session that demonstrate the examples described in the paper.Such information is given in the user manual accompanied with miREvo. All these packages can also be downloaded from miRNA, microRNA; NGS, Next-generation sequencing; WGA, whole genome alignment.The authors declare that they have no competing interests.MW and YS developed miREvo and carried out the analysis. YS and TT conceived and directed the project. MW, YS, SSH and TT wrote the manuscript. All authors have read and approved the final manuscript.Supplementary Materials.doc. The Supplementary Material for the paper.Click here for filePlant Parameters.doc. The modification of miRDeep2 parameters that are used for the prediction of novel plant miRNAs in miREvo.Click here for fileSupplementary Figure S2.pdf. Supplementary Figure S2, which is too large to include in the text.Click here for file