Sex specific expression and distribution of small RNAs in papaya

Background

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored.

Results

We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot.

Conclusion

By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Y^h chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.     

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum

Background

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.

Results

Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination.

Conclusions

The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1517-1) contains supplementary material, which is available to authorized users.     

Testing the neutral theory of molecular evolution using genomic data: a comparison of the human and bovine transcriptome

Despite growing evidence of rapid evolution in protein coding genes, the contribution of positive selection to intra- and interspecific differences in protein coding regions of the genome is unclear. We attempted to see if genes coding for secreted proteins and genes with narrow expression, specifically those preferentially expressed in the mammary gland, have diverged at a faster rate between domestic cattle (Bos taurus) and humans (Homo sapiens) than other genes and whether positive selection is responsible. Using a large data set, we identified groups of genes based on secretion and expression patterns and compared them for the rate of nonsynonymous (dN) and synonymous (dS) substitutions per site and the number of radical (Dr) and conservative (Dc) amino acid substitutions. We found evidence of rapid evolution in genes with narrow expression, especially for those expressed in the liver and mammary gland and for genes coding for secreted proteins. We compared common human polymorphism data with human-cattle divergence and found that genes with high evolutionary rates in human-cattle divergence also had a large number of common human polymorphisms. This argues against positive selection causing rapid divergence in these groups of genes. In most cases dN/dS ratios were lower in human-cattle divergence than in common human polymorphism presumably due to differences in the effectiveness of purifying selection between long-term divergence and short-term polymorphism.     

Cytoplasmic and genomic effects on meiotic pairing in brassica hybrids and allotetraploids from pair crosses of three cultivated diploids

Interspecific hybridization and allopolyploidization contribute to the origin of many important crops. Synthetic Brassica is a widely used model for the study of genetic recombination and "fixed heterosis" in allopolyploids. To investigate the effects of the cytoplasm and genome combinations on meiotic recombination, we produced digenomic diploid and triploid hybrids and trigenomic triploid hybrids from the reciprocal crosses of three Brassica diploids (B. rapa, AA; B. nigra, BB; B. oleracea, CC). The chromosomes in the resultant hybrids were doubled to obtain three allotetraploids (B. juncea, AA.BB; B. napus, AA.CC; B. carinata, BB.CC). Intra- and intergenomic chromosome pairings in these hybrids were quantified using genomic in situ hybridization and BAC-FISH. The level of intra- and intergenomic pairings varied significantly, depending on the genome combinations and the cytoplasmic background and/or their interaction. The extent of intragenomic pairing was less than that of intergenomic pairing within each genome. The extent of pairing variations within the B genome was less than that within the A and C genomes, each of which had a similar extent of pairing. Synthetic allotetraploids exhibited nondiploidized meiotic behavior, and their chromosomal instabilities were correlated with the relationship of the genomes and cytoplasmic background. Our results highlight the specific roles of the cytoplasm and genome to the chromosomal behaviors of hybrids and allopolyploids.     

A molecular phylogeny and classification of Leptochloa (Poaceae: Chloridoideae: Chlorideae) sensu lato and related genera

Background and Aims

Leptochloa (including Diplachne) sensu lato (s.l.) comprises a diverse assemblage of C₄ (NAD-ME and PCK) grasses with approx. 32 annual or perennial species. Evolutionary relationships and a modern classification of Leptochloa spp. based on the study of molecular characters have only been superficially investigated in four species. The goals of this study were to reconstruct the evolutionary history of Leptochloa s.l. with molecular data and broad taxon sampling.

Methods

A phylogenetic analysis was conducted of 130 species (mostly Chloridoideae), of which 22 are placed in Leptochloa, using five plastid (rpL32-trn-L, ndhA intron, rps16 intron, rps16-trnK and ccsA) and the nuclear ITS 1 and 2 (ribosomal internal transcribed spacer regions) to infer evolutionary relationships and revise the classification.

Key results

Leptochloa s.l. is polyphyletic and strong support was found for five lineages. Embedded within the Leptochloa sensu stricto (s.s.) clade are two Trichloris spp. and embedded in Dinebra are Drake-brockmania and 19 Leptochloa spp.

Conclusions

The molecular results support the dissolution of Leptochloa s.l. into the following five genera: Dinebra with 23 species, Diplachne with two species, Disakisperma with three species, Leptochloa s.s. with five species and a new genus, Trigonochloa, with two species.     

Next-generation sequencing of flow-sorted wheat chromosome 5D reveals lineage-specific translocations and widespread gene duplications

Background

The ~17 Gb hexaploid bread wheat genome is a high priority and a major technical challenge for genomic studies. In particular, the D sub-genome is relatively lacking in genetic diversity, making it both difficult to map genetically, and a target for introgression of agriculturally useful traits. Elucidating its sequence and structure will therefore facilitate wheat breeding and crop improvement.

Results

We generated shotgun sequences from each arm of flow-sorted Triticum aestivum chromosome 5D using 454 FLX Titanium technology, giving 1.34× and 1.61× coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. By a combination of sequence similarity and assembly-based methods, ~74% of the sequence reads were classified as repetitive elements, and coding sequence models of 1314 (5DS) and 2975 (5DL) genes were generated. The order of conserved genes in syntenic regions of previously sequenced grass genomes were integrated with physical and genetic map positions of 518 wheat markers to establish a virtual gene order for chromosome 5D.

Conclusions

The virtual gene order revealed a large-scale chromosomal rearrangement in the peri-centromeric region of 5DL, and a concentration of non-syntenic genes in the telomeric region of 5DS. Although our data support the large-scale conservation of Triticeae chromosome structure, they also suggest that some regions are evolving rapidly through frequent gene duplications and translocations.

Sequence accessions

EBI European Nucleotide Archive, Study no. ERP002330

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1080) contains supplementary material, which is available to authorized users.     

A strategy for generation and balancing of autosome: Y chromosome translocations

We describe a method for generation and maintenance of translocations that move large autosomal segments onto the Y chromosome. Using this strategy we produced (2;Y) translocations that relocate between 1.5 and 4.8 Mb of the 2^nd chromosome.. All translocations were easily balanced over a male-specific lethal 1 (msl-1) mutant chromosome. Both halves of the translocation carry visible markers, as well as P-element ends that enable molecular confirmation. Halves of these translocations can be separated to produce offspring with duplications and with lethal second chromosome deficiencies . Such large deficiencies are otherwise tedious to generate and maintain.     

More reliable estimates of divergence times in Pan using complete mtDNA sequences and accounting for population structure

Here, we report the sequencing and analysis of eight complete mitochondrial genomes of chimpanzees (Pan troglodytes) from each of the three established subspecies (P. t. troglodytes, P. t. schweinfurthii and P. t. verus) and the proposed fourth subspecies (P. t. ellioti). Our population genetic analyses are consistent with neutral patterns of evolution that have been shaped by demography. The high levels of mtDNA diversity in western chimpanzees are unlike those seen at nuclear loci, which may reflect a demographic history of greater female to male effective population sizes possibly owing to the characteristics of the founding population. By using relaxed-clock methods, we have inferred a timetree of chimpanzee species and subspecies. The absolute divergence times vary based on the methods and calibration used, but relative divergence times show extensive uniformity. Overall, mtDNA produces consistently older times than those known from nuclear markers, a discrepancy that is reduced significantly by explicitly accounting for chimpanzee population structures in time estimation. Assuming the human–chimpanzee split to be between 7 and 5 Ma, chimpanzee time estimates are 2.1–1.5, 1.1–0.76 and 0.25–0.18 Ma for the chimpanzee/bonobo, western/(eastern + central) and eastern/central chimpanzee divergences, respectively.     

Erratum to: genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 83 extant, fully sequenced Bacillus phages revealed 10 distinct clusters, 24 subclusters and 15 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,442 protein families (phams) of which only 894 (20%) had a predicted function. In addition, 2,583 (58%) of phams were orphams (phams containing a single member). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

When parasitic wasps hijacked viruses: genomic and functional evolution of polydnaviruses

The Polydnaviridae (PDV), including the Bracovirus (BV) and Ichnovirus genera, originated from the integration of unrelated viruses in the genomes of two parasitoid wasp lineages, in a remarkable example of convergent evolution. Functionally active PDVs represent the most compelling evolutionary success among endogenous viral elements (EVEs). BV evolved from the domestication by braconid wasps of a nudivirus 100 Ma. The nudivirus genome has become an EVE involved in BV particle production but is not encapsidated. Instead, BV genomes have co-opted virulence genes, used by the wasps to control the immunity and development of their hosts. Gene transfers and duplications have shaped BV genomes, now encoding hundreds of genes. Phylogenomic studies suggest that BVs contribute largely to wasp diversification and adaptation to their hosts. A genome evolution model explains how multidirectional wasp adaptation to different host species could have fostered PDV genome extension. Integrative studies linking ecological data on the wasp to genomic analyses should provide new insights into the adaptive role of particular BV genes. Forthcoming genomic advances should also indicate if the associations between endoparasitoid wasps and symbiotic viruses evolved because of their particularly intimate interactions with their hosts, or if similar domesticated EVEs could be uncovered in other parasites.     

Reproduction,Chromosome Number,and Postembryonic Development of Panagrellus redivivus (Nematoda: Cephalobidae)

Panagrellus redivivus (L.) T. Goodey reproduced amphimictically; the sexual primordia of males had nine chromosomes, those of females had ten. Eggs contained five chromosomes, sperm four or five. There were four molts, all after hatching. The sexes could be separated at the second molt by development of a lobe of somatic cells in the gonad, anteriorly in males, posteriorly in females. The lobe in males reflexed posteriorly at the third molt and joined the rectum at the fourth molt. Third molt females had a thickened vaginal primordium and at the fourth molt the spermathecal and uterine primordia were evident. The uterus elongated enormously in the adult. The 15 ventral chord nuclei between esophagus and rectum in the first stage increased to approximately 63 during the first molt; specialized nuclei, not evident until the third molt, participate in vaginal lining formation in fourth molt females. Sperm were first produced at the late fourth molt. Eggs, not produced until after copulation, hatched within the uterus.     

Ancyronyx Erichson, 1847 (Coleoptera,Elmidae) from Mindoro,Philippines, with description of the larvae and two new species using DNA sequences for the assignment of the developmental stages

Ancyronyx buhid sp. n. and Ancyronyx tamaraw sp. n. are described based on adults and larvae, matched using their cox1 or cob DNA sequence data. Additional records of Ancyronyx schillhammeri Jäch, 1994 and Ancyronyx minerva Freitag & Jäch, 2007 from Mindoro are listed. The previously unknown larva of Ancyronyx schillhammeri is also described here, aided by cox1 data. The new species and larval stages are described in detail and illustrated by SEM and stacked microscopic images. Keys to the adult and larval Ancyronyx species of Mindoro and an updated checklist of Philippine Ancyronyx species are provided. The usefulness as bioindicators, the phylogenetic relationships and biogeographic aspects affecting the distribution patterns are briefly discussed.     

Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.     

Phylogeny and biogeography of Allium (Amaryllidaceae: Allieae) based on nuclear ribosomal internal transcribed spacer and chloroplast rps16 sequences, focusing on the inclusion of species endemic to China

Background and Aims

The genus Allium comprises more than 800 species, placing it among the largest monocotyledonous genera. It is a variable group that is spread widely across the Holarctic region. Previous studies of Allium have been useful in identifying and assessing its evolutionary lineages. However, there are still many gaps in our knowledge of infrageneric taxonomy and evolution of Allium. Further understanding of its phylogeny and biogeography will be achieved only through continued phylogenetic studies, especially of those species endemic to China that have often been excluded from previous analyses. Earlier molecular studies have shown that Chinese Allium is not monophyletic, so the goal of the present study was to infer the phylogeny and biogeography of Allium and to provide a classification of Chinese Allium by placement of Chinese species in the context of the entire phylogeny.

Methods

Phylogenetic studies were based on sequence data of the nuclear ribosomal internal transcribed spacer (ITS) and chloroplast rps16 intron, analysed using parsimony and Bayesian approaches. Biogeographical patterns were conducted using statistical dispersal–vicariance analysis (S-DIVA).

Key Results

Phylogenetic analyses indicate that Allium is monophyletic and consists of three major clades. Optimal reconstructions have favoured the ancestors of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum as originating in eastern Asia.

Conclusions

Phylogenetic analyses reveal that Allium is monophyletic but that some subgenera are not. The large genetic distances imply that Allium is of ancient origin. Molecular data suggest that its evolution proceeded along three separate evolutionary lines. S-DIVA indicates that the ancestor of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum originated from eastern Asia and underwent different biogeographical pathways. A taxonomic synopsis of Chinese Allium at sectional level is given, which divides Chinese Allium into 13 subgenera and 34 sections.     

The genotoxic and anti-genotoxic effects of <Emphasis Type="Italic">Stachys petrokosmos</Emphasis> leaf extract in human lymphocytes using microsomal fractions

The genotoxic and anti-genotoxic effects of Stachys petrokosmos leaf extracts (Sp) were investigated in human lymphocytes. The cells were treated with 1.5, 3.0 and 6.0 μL/mL concentrations of Sp leaf extracts for 24 and 48 h treatment periods in the absence and presence of metabolic activator (S9mix). In the absence of S9mix, Sp alone did not induce chromosome aberrations and formation of micronucleus while inducing the mean sister chromatid exchange at the highest concentration. In addition, Sp decreased the mutagenic effect of mitomycin-c. Sp alone showed a cytotoxic effect determined by a decrease in the proliferation index, mitotic index and nuclear division index. On the other hand a mixture of Sp and mitomycin-c resulted in a higher cytotoxic effect especially for 48 h treatment period. In the presence of S9mix, Sp was not genotoxic and cytotoxic however, it showed an anti-genotoxic effect by decreasing the effects of cyclophosphamide.