A comparative analysis of plastome evolution in autotrophic Piperales.

PREMISE: Many plastomes of autotrophic Piperales have been reported to date, describing a variety of differences. Most studies focused only on a few species or a single genus, and extensive, comparative analyses have not been done. Here, we reviewed publicly available plastome reconstructions for autotrophic Piperales, reanalyzed publicly available raw data, and provided new sequence data for all previously missing genera. Comparative plastome genomics of >100 autotrophic Piperales were performed. METHODS: We performed de novo assemblies to reconstruct the plastomes of newly generated sequence data. We used Sanger sequencing and read mapping to verify the assemblies and to bridge assembly gaps. Furthermore, we reconstructed the phylogenetic relationships as a foundation for comparative plastome genomics. RESULTS: We identified a plethora of assembly and annotation issues in published plastome data, which, if unattended, will lead to an artificial increase of diversity. We were able to detect patterns of missing and incorrect feature annotation and determined that the inverted repeat (IR) boundaries were the major source for erroneous assembly. Accounting for the aforementioned issues, we discovered relatively stable junctions of the IRs and the small single-copy region (SSC), whereas the majority of plastome variations among Piperales stems from fluctuations of the boundaries of the IR and the large single-copy (LSC) region. CONCLUSIONS: This study of all available plastomes of autotrophic Piperales, expanded by new data for previously missing genera, highlights the IR-LSC junctions as a potential marker for discrimination of various taxonomic levels. Our data indicates a pseudogene-like status for cemA and ycf15 in various Piperales. Based on a review of published data, we conclude that incorrect IR-SSC boundary identification is the major source for erroneous plastome assembly. We propose a gold standard for assembly and annotation of high-quality plastomes based on de novo assembly methods and appropriate references for gene annotation.

Extreme plastomes in holoparasitic Balanophoraceae are not the norm.

BACKGROUND: Balanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling. RESULTS: Sarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae. CONCLUSIONS: For the "minimal plastomes" of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

Mining the Australian Grains Gene Bank for Rust Resistance in Barley.

Global barley production is threatened by plant pathogens, especially the rusts. In this study we used a targeted genotype-by-sequencing (GBS) assisted GWAS approach to identify rust resistance alleles in a collection of 287 genetically distinct diverse barley landraces and historical cultivars available in the Australian Grains Genebank (AGG) and originally sourced from Eastern Europe. The accessions were challenged with seven US-derived cereal rust pathogen races including Puccinia hordei (Ph-leaf rust) race 17VA12C, P. coronata var. hordei (Pch-crown rust) race 91NE9305 and five pathogenically diverse races of P. striiformis f. sp. hordei (Psh-stripe rust) (PSH-33, PSH-48, PSH-54, PSH-72 and PSH-100) and phenotyped quantitatively at the seedling stage. Novel resistance factors were identified on chromosomes 1H, 2H, 4H and 5H in response to Pch, whereas a race-specific QTL on 7HS was identified that was effective only to Psh isolates PSH-72 and PSH-100. A major effect QTL on chromosome 5HL conferred resistance to all Psh races including PSH-72, which is virulent on all 12 stripe rust differential tester lines. The same major effect QTL was also identified in response to leaf rust (17VA12C) suggesting this locus contains several pathogen specific rust resistance genes or the same gene is responsible for both leaf rust and stripe rust resistance. Twelve accessions were highly resistant to both leaf and stripe rust diseases and also carried the 5HL QTL. We subsequently surveyed the physical region at the 5HL locus for across the barley pan genome variation in the presence of known resistance gene candidates and identified a rich source of high confidence protein kinase and antifungal genes in the QTL region.

BED domain-containing NLR from wild barley confers resistance to leaf rust.

Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. hordei races, prompting the search for new sources of resistance. Here we report on the cloning of Rph15, a resistance gene derived from barley's wild progenitor H. vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that the Rph15 gene from the near-isogenic line (NIL) Bowman + Rph15 (referred to as BW719) encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc finger BED (ZF-BED) domain. A predicted KASP marker was developed and validated across a collection of Australian cultivars and a series of introgression lines in the Bowman background known to carry the Rph15 resistance. Rph16 from HS-680, another wild barley derived leaf rust resistance gene, was previously mapped to the same genomic region on chromosome 2H and was assumed to be allelic with Rph15 based on genetic studies. Both sequence analysis, race specificity and the identification of a knockout mutant in the HS-680 background suggest that Rph15- and Rph16-mediated resistances are in fact the same and not allelic as previously thought. The cloning of Rph15 now permits efficient gene deployment and the production of resistance gene cassettes for sustained leaf rust control.

Fine mapping of leaf rust resistance gene Rph13 from wild barley.

KEY MESSAGE: Fine mapping of the barley leaf rust resistance locus Rph13 on chromosome 3HL facilitates its use in breeding programs through marker-assisted selection. Barley leaf rust (BLR-caused by Puccinia hordei) is a widespread fungal disease that can be effectively controlled by genetic resistance. There is an ongoing need to both diversify and genetically characterise resistance loci to provide effective and durable control given the ongoing threat of rapidly evolving P. hordei populations. Here, we report on the molecular genetic characterisation of the Rph13 locus, originally derived from wild barley and transferred to barley accession Berac (then referred to as PI 531849). The 2017 reference genome of cv. Morex was used as a road map to rapidly narrow both a genetic and physical intervals around the Rph13 resistance locus. Using recombination-based mapping, we narrowed the physical interval to 116.6 kb on chromosome 3H in a segregating population of a cross of the Rph13 carrying resistant line PI 531849 with the leaf rust-susceptible cultivar Gus. We identified two nucleotide-binding leucine-rich repeat genes as likely candidates for the Rph13 resistance. Sequences from the candidate genes enabled the development of a KASP marker that distinguished resistant and susceptible progeny and was found to be predictive and useful for MAS.

Can systemically administered antibiotics be detected in wound tissues and surfaces under negative pressure wound therapy?

In this study, we evaluated a new aspect of negative pressure wound therapy (NPWT) as an analytical tool for pharmacokinetic studies. Twenty-one patients with soft tissue defects scheduled to receive NPWT were included in this study. Concomitant to NPWT, all patients received intravenous moxifloxacin (MX). At different time intervals, blood plasma levels of MX were sampled and compared with synchronous concentrations of MX in the exudate obtained from the NPWT drainage system. Serial measurements were performed upon initiation of the therapy as well as in the steady state (after 5 days). At steady state, wound tissue was obtained intraoperatively. High-performance liquid-chromatography (HPLC) was used for analysis. At 1 hour post-administration, the exudate/plasma levels (mg/L) were 1.92/3.07; at 12 hours, 0.80/1.14; at 24 hours, 0.26/0.43; and at 120 hours (steady state), 0.42/0.47. There was a correlation between exudate and plasma levels reaching approximately 0.75. Until now, methods for pharmacokinetic studies concerning interstitial fluid are difficult to apply in the clinical context. The presented method showed limitations, but we believe that, after methodological improvements, measurements of substances in the interstitial fluid by means of NPWT are feasible.

A Homolog of Blade-On-Petiole 1 and 2 (BOP1/2) Controls Internode Length and Homeotic Changes of the Barley Inflorescence.

Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement. We analyzed the recessive mutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected.

The Mitochondrial Genome of the Holoparasitic Plant Thonningia sanguinea Provides Insights into the Evolution of the Multichromosomal Structure.

Multichromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum, and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multichromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multichromosomal structure of 18 circular chromosomes of 8.7-19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome; however, no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multichromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination, and repeat degradation.

Plant and pathogen genomics: essential approaches for stem rust resistance gene stacks in wheat.

The deployment of disease resistance genes is currently the most economical and environmentally sustainable method of crop protection. However, disease resistance genes can rapidly break down because of constant pathogen evolution, particularly when they are deployed singularly. Polygenic resistance is, therefore, considered the most durable, but combining and maintaining these genes by breeding is a laborious process as effective genes are usually unlinked. The deployment of polygenic resistance with single-locus inheritance is a promising innovation that overcomes these difficulties while enhancing resistance durability. Because of major advances in genomic technologies, increasing numbers of plant resistance genes have been cloned, enabling the development of resistance transgene stacks (RTGSs) that encode multiple genes all located at a single genetic locus. Gene stacks encoding five stem rust resistance genes have now been developed in transgenic wheat and offer both breeding simplicity and potential resistance durability. The development of similar genomic resources in phytopathogens has advanced effector gene isolation and, in some instances, enabled functional validation of individual resistance genes in RTGS. Here, the wheat stem rust pathosystem is used as an illustrative example of how host and pathogen genomic advances have been instrumental in the development of RTGS, which is a strategy applicable to many other agricultural crop species.

Phylogenetic and comparative analyses of Hydnora abyssinica plastomes provide evidence for hidden diversity within Hydnoraceae.

BACKGROUND: To date, plastid genomes have been published for all but two holoparasitic angiosperm families. However, only a single or a few plastomes represent most of these families. Of the approximately 40 genera of holoparasitic angiosperms, a complete plastid genome sequence is available for only about half. In addition, less than 15 species are currently represented with more than one published plastid genome, most of which belong to the Orobanchaceae. Therefore, a significant portion of the holoparasitic plant plastome diversity remains unexplored. This limited information could hinder potential evolutionary pattern recognition as well as the exploration of inter- and intra-species plastid genome diversity in the most extreme holoparasitic angiosperms. RESULTS: Here, we report the first plastomes of Kenyan Hydnora abyssinica accessions. The plastomes have a typical quadripartite structure and encode 24 unique genes. Phylogenetic tree reconstruction recovers the Kenyan accessions as monophyletic and together in a clade with the Namibian H. abyssinica accession and the recently published H. arabica from Oman. Hydnora abyssinica as a whole however is recovered as non-monophyletic, with H. arabica nested within. This result is supported by distinct structural plastome synapomorphies as well as pairwise distance estimates that reveal hidden diversity within the Hydnora species in Africa. CONCLUSION: We propose to increase efforts to sample widespread holoparasitic species for their plastid genomes, as is the case with H. abyssinica, which is widely distributed in Africa. Morphological reinvestigation and further molecular data are needed to fully investigate the diversity of H. abyssinica along the entire range of distribution, as well as the diversity of currently synonymized taxa.

De novo Assembly and Comparative Analyses of Mitochondrial Genomes in Piperales.

The mitochondrial genome of Liriodendron tulipifera exhibits many ancestral angiosperm features and a remarkably slow evolutionary rate, while mitochondrial genomes of other magnoliids remain yet to be characterized. We assembled nine new mitochondrial genomes, representing all genera of perianth-bearing Piperales, as well as for a member of the sister clade: three complete or nearly complete mitochondrial genomes from Aristolochiaceae and six additional draft assemblies including Thottea, Asaraceae, Lactoridaceae, and Hydnoraceae. For comparative purpose, a complete mitochondrial genome was assembled for Saururus, a member of the perianth-less Piperales. The average number of short repeats (50-99 bp) was much larger in genus Aristolochia than in other angiosperm mitochondrial genomes, and approximately 30% of repeats (<350 bp) were found to have the capacity to mediate recombination. We found mitochondrial genomes in perianth-bearing Piperales comprising conserved repertories of protein-coding genes and rRNAs but variable copy numbers of tRNA genes. We identified several shifts from cis- to trans-splicing of the Group II introns of nad1i728, cox2i373, and nad7i209. Two short regions of the cox1 and atp8 genes were likely derived from independent horizontal gene transfer events in perianth-bearing Piperales. We found biased enrichment of specific substitution types in different lineages of magnoliids and the Aristolochiaceae family showed the highest ratio of A:T > T:A substitutions of all other investigated angiosperm groups. Our study reports the first mitochondrial genomes for Piperales and uses this new information for a better understanding of the evolutionary patterns of magnoliids and angiosperms in general.

A pathogen-induced putative NAC transcription factor mediates leaf rust resistance in barley.

Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley. The barley leaf rust resistance locus Rph7 has been shown to have unusually high sequence and haplotype divergence. In this study, we isolate the Rph7 gene using a fine mapping and RNA-Seq approach that is confirmed by mutational analysis and transgenic complementation. Rph7 is a pathogen-induced, non-canonical resistance gene encoding a protein that is distinct from other known plant disease resistance proteins in the Triticeae. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor with a zinc-finger BED domain with structural similarity to the N-terminal DNA-binding domain of the NAC transcription factor (ANAC019) from Arabidopsis. A global gene expression analysis suggests Rph7 mediates the activation and strength of the basal defence response. The isolation of Rph7 highlights the diversification of resistance mechanisms available for engineering disease control in crops.

Plastome phylogenomics reveals an early Pliocene North- and Central America colonization by long-distance dispersal from South America of a highly diverse bromeliad lineage.

Understanding the spatial and temporal frameworks of species diversification is fundamental in evolutionary biology. Assessing the geographic origin and dispersal history of highly diverse lineages of rapid diversification can be hindered by the lack of appropriately sampled, resolved, and strongly supported phylogenetic contexts. The use of currently available cost-efficient sequencing strategies allows for the generation of a substantial amount of sequence data for dense taxonomic samplings, which together with well-curated geographic information and biogeographic models allow us to formally test the mode and tempo of dispersal events occurring in quick succession. Here, we assess the spatial and temporal frameworks for the origin and dispersal history of the expanded clade K, a highly diverse Tillandsia subgenus Tillandsia (Bromeliaceae, Poales) lineage hypothesized to have undergone a rapid radiation across the Neotropics. We assembled full plastomes from Hyb-Seq data for a dense taxon sampling of the expanded clade K plus a careful selection of outgroup species and used them to estimate a time- calibrated phylogenetic framework. This dated phylogenetic hypothesis was then used to perform biogeographic model tests and ancestral area reconstructions based on a comprehensive compilation of geographic information. The expanded clade K colonized North and Central America, specifically the Mexican transition zone and the Mesoamerican dominion, by long-distance dispersal from South America at least 4.86 Mya, when most of the Mexican highlands were already formed. Several dispersal events occurred subsequently northward to the southern Nearctic region, eastward to the Caribbean, and southward to the Pacific dominion during the last 2.8 Mya, a period characterized by pronounced climate fluctuations, derived from glacial-interglacial climate oscillations, and substantial volcanic activity, mainly in the Trans-Mexican Volcanic Belt. Our taxon sampling design allowed us to calibrate for the first time several nodes, not only within the expanded clade K focal group but also in other Tillandsioideae lineages. We expect that this dated phylogenetic framework will facilitate future macroevolutionary studies and provide reference age estimates to perform secondary calibrations for other Tillandsioideae lineages.

Single amino acid change alters specificity of the multi-allelic wheat stem rust resistance locus SR9.

Most rust resistance genes thus far isolated from wheat have a very limited number of functional alleles. Here, we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) are characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation are used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1 and Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differs from SR9B by a single amino acid. SR9B and SR9G resistance proteins are also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences. Together, our results show that functional diversification of allelic variants at the SR9 locus involves single and multiple amino acid changes that recognize isolates of wheat stem rust.

Structural Plastome Evolution in Holoparasitic Hydnoraceae with Special Focus on Inverted and Direct Repeats.

Plastome condensation during adaptation to a heterotrophic lifestyle is generally well understood and lineage-independent models have been derived. However, understanding the evolutionary trajectories of comparatively old heterotrophic lineages that are on the cusp of a minimal plastome, is essential to complement and expand current knowledge. We study Hydnoraceae, one of the oldest and least investigated parasitic angiosperm lineages. Plastome comparative genomics, using seven out of eight known species of the genus Hydnora and three species of Prosopanche, reveal a high degree of structural similarity and shared gene content; contrasted by striking dissimilarities with respect to repeat content [inverted and direct repeats (DRs)]. We identified varying inverted repeat contents and positions, likely resulting from multiple, independent evolutionary events, and a DR gain in Prosopanche. Considering different evolutionary trajectories and based on a fully resolved and supported species-level phylogenetic hypothesis, we describe three possible, distinct models to explain the Hydnoraceae plastome states. For comparative purposes, we also report the first plastid genomes for the closely related autotrophic genera Lactoris (Lactoridaceae) and Thottea (Aristolochiaceae).

New plastome structural rearrangements discovered in core Tillandsioideae (Bromeliaceae) support recently adopted taxonomy.

Full plastome sequences for land plants have become readily accessible thanks to the development of Next Generation Sequencing (NGS) techniques and powerful bioinformatic tools. Despite this vast amount of genomic data, some lineages remain understudied. Full plastome sequences from the highly diverse (>1,500 spp.) subfamily Tillandsioideae (Bromeliaceae, Poales) have been published for only three (i.e., Guzmania, Tillandsia, and Vriesea) out of 22 currently recognized genera. Here, we focus on core Tillandsioideae, a clade within subfamily Tillandsioideae, and explore the contribution of individual plastid markers and data categories to inform deep divergences of a plastome phylogeny. We generated 37 high quality plastome assemblies and performed a comparative analysis in terms of plastome structure, size, gene content and order, GC content, as well as number and type of repeat motifs. Using the obtained phylogenetic context, we reconstructed the evolution of these plastome attributes and assessed if significant shifts on the evolutionary traits' rates have occurred in the evolution of the core Tillandsioideae. Our results agree with previously published phylogenetic hypotheses based on plastid data, providing stronger statistical support for some recalcitrant nodes. However, phylogenetic discordance with previously published nuclear marker-based hypotheses was found. Several plastid markers that have been consistently used to address phylogenetic relationships within Tillandsioideae were highly informative for the retrieved plastome phylogeny and further loci are here identified as promising additional markers for future studies. New lineage-specific plastome rearrangements were found to support recently adopted taxonomic groups, including large inversions, as well as expansions and contractions of the inverted repeats. Evolutionary trait rate shifts associated with changes in size and GC content of the plastome regions were found across the phylogeny of core Tillandsioideae.

Discordant Phylogenomic Placement of Hydnoraceae and Lactoridaceae Within Piperales Using Data From All Three Genomes.

Phylogenetic relationships within the magnoliid order Piperales have been studied extensively, yet the relationships of the monotypic family Lactoridaceae and the holoparasitic Hydnoraceae to the remainder of the order remain a matter of debate. Since the first confident molecular phylogenetic placement of Hydnoraceae among Piperales, different studies have recovered various contradictory topologies. Most phylogenetic hypotheses were inferred using only a few loci and have had incomplete taxon sampling at the genus level. Based on these results and an online survey of taxonomic opinion, the Angiosperm Phylogeny Group lumped both Hydnoraceae and Lactoridaceae in Aristolochiaceae; however, the latter family continues to have unclear relationships to the aforementioned taxa. Here we present extensive phylogenomic tree reconstructions based on up to 137 loci from all three subcellular genomes for all genera of Piperales. We infer relationships based on a variety of phylogenetic methods, explore instances of phylogenomic discordance between the subcellular genomes, and test alternative topologies. Consistent with these phylogenomic results and a consideration of the principles of phylogenetic classification, we propose to exclude Hydnoraceae and Lactoridaceae from the broad circumscription of Aristolochiaceae, and instead favor recognition of four monophyletic and morphologically well circumscribed families in the perianth-bearing Piperales: Aristolochiaceae, Asaraceae, Hydnoraceae, and Lactoridaceae, with a total of six families in the order.

Molecular assays for the detection of prostate tumor derived nucleic acids in peripheral blood.

BACKGROUND: Prostate cancer is the second leading cause of cancer mortality in American men. Although serum PSA testing is widely used for early detection, more specific prognostic tests are needed to guide treatment decisions. Recently, the enumeration of circulating prostate epithelial cells has been shown to correlate with disease recurrence and metastasis following definitive treatment. The purpose of our study was to investigate an immunomagnetic fractionation procedure to enrich circulating prostate tumor cells (CTCs) from peripheral blood specimens, and to apply amplified molecular assays for the detection of prostate-specific markers (PSA, PCA3 and TMPRSS2:ERG gene fusion mRNAs). RESULTS: As few as five prostate cancer cells were detected per 5 mL of whole blood in model system experiments using anti-EpCAM magnetic particles alone or in combination with anti-PSMA magnetic particles. In our experiments, anti-EpCAM magnetic particles alone exhibited equivalent or better analytical performance with patient samples compared to a combination of anti-EpCAM + anti-PSMA magnetic particles. Up to 39% of men with advanced prostate cancer tested positive with one or more of the molecular assays tested, whereas control samples from men with benign prostate hyperplasia gave consistently negative results as expected. Interestingly, for the vast majority of men who tested positive for PSA mRNA following CTC enrichment, their matched plasma samples also tested positive, although CTC enrichment gave higher overall mRNA copy numbers. CONCLUSION: CTCs were successfully enriched and detected in men with advanced prostate cancer using an immunomagnetic enrichment procedure coupled with amplified molecular assays for PSA, PCA3, and TMPRSS2:ERG gene fusion mRNAs. Our results indicate that men who test positive following CTC enrichment also exhibit higher detectable levels of non-cellular, circulating prostate-specific mRNAs.

The First Plastid Genome of the Holoparasitic Genus Prosopanche (Hydnoraceae).

Plastomes of parasitic and mycoheterotrophic plants show different degrees of reduction depending on the plants' level of heterotrophy and host dependence in comparison to photoautotrophic sister species, and the amount of time since heterotrophic dependence was established. In all but the most recent heterotrophic lineages, this reduction involves substantial decrease in genome size and gene content and sometimes alterations of genome structure. Here, we present the first plastid genome of the holoparasitic genus Prosopanche, which shows clear signs of functionality. The plastome of Prosopanche americana has a length of 28,191 bp and contains only 24 unique genes, i.e., 14 ribosomal protein genes, four ribosomal RNA genes, five genes coding for tRNAs and three genes with other or unknown function (accD, ycf1, ycf2). The inverted repeat has been lost. Despite the split of Prosopanche and Hydnora about 54 MYA ago, the level of genome reduction is strikingly congruent between the two holoparasites although highly dissimilar nucleotide sequences are observed. Our results lead to two possible evolutionary scenarios that will be tested in the future with a larger sampling: 1) a Hydnoraceae plastome, similar to those of Hydnora and Prosopanche today, existed already in the most recent common ancestor and has not changed much with respect to gene content and structure, or 2) the genome similarities we observe today are the result of two independent evolutionary trajectories leading to almost the same endpoint. The first hypothesis would be most parsimonious whereas the second would point to taxon dependent essential gene sets for plants released from photosynthetic constraints.