Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.
Recent phylogenetic studies on the members of the Diaporthales have shown that the order includes a number of distinct phylogenetic groups. These groups represent the Gnomoniaceae, Melanconidaceae, Valsaceae, Diaporthaceae and Togniniaceae. New groups representing undescribed families also have emerged and they have been referred to as the Schizoparme, Cryphonectria-Endothia and Harknessia complexes. In this study we define the new family Cryphonectriaceae (Diaporthales) to accommodate genera in the Cryphonectria-Endothia complex. These genera can be distinguished from those in other families or undescribed groups of the Diaporthales by the formation of orange stromatic tissue at some stage of their life cycle and a purple reaction in KOH and a yellow reaction in lactic acid associated with pigments in the stromatic tissue or in culture.
Cryphonectria havanensis is a fungus associated with Eucalyptus species in Cuba and Florida (U.S.A.). Until recently, there have been no living cultures of C. havanensis and it has thus not been possible to assess its taxonomic status. Isolates thought to represent this fungus have, however, emerged from surveys of Eucalyptus in Mexico and Hawaii (U.S.A.). Results of this study showed that these isolates represent C. havanensis but reside in a genus distinct from Cryphonectria sensu stricto, which is described here as Microthia. Isolates of an unidentified fungus occurring on Myrica faya in the Azores and Madeira also grouped in Microthia and were identical to other M. havanensis isolates. Cryphonectria coccolobae, a fungus occurring on sea grape (Coccoloba uvifera) in Bermuda and Florida, was found to be morphologically identical to Microthia and is transferred to this genus, but as a distinct species. Surveys for M. coccolobae on sea grape in Florida, yielded a second diaporthalean fungus from this host. This fungus is morphologically and phylogenetically distinct from M. coccolobae and other closely related taxa and is described as Ursicollum fallax gen. et sp. nov. Phylogenetic analyses in this study have also shown that isolates of C. eucalypti, a pathogen of Eucalyptus in South Africa and Australia, group in a clade separate from all other groups including that representing Cryphonectria sensu stricto. This difference is supported by the fact that Cryphonectria eucalypti has ascospore septation different to that of all other Cryphonectria species. A new genus, Holocryphia, is thus erected for C. eucalypti.
Cryphonectria includes important tree pathogens as well as species believed to be saprophytes. Recent phylogenetic studies have concentrated on North American and southern hemisphere Cryphonectria spp., but little is known about Asian and European taxa. In this study we identify and differentiate among the species occurring in Europe, China, and Japan using morphological and phylogenetic comparisons among the Cryphonectria species. Phylogenetic comparisons were based on sequence data from the ribosomal ITS operon and two regions in the β-tubulin gene. Unknown Japanese and Chinese isolates showing different cultural features than those of Cryphonectria parasitica (Murrill) M.E. Barr from Japan and the USA, grouped with isolates of Cryphonectria nitschkei (G.H. Otth) M.E. Barr from Quercus spp. and Rhus javanica L. Isolates of Cryphonectria havanensis (Bruner) M.E. Barr from Quercus grosseserrata Blume, Castanopsis cuspidata Schottky, Pyrus sinensis Lindl., and Eucalyptus globulus Labill. also grouped in this phylogenetic clade. We propose that Cryphonectria nitschkei and the fungus that has been referred to as Cryphonectria havanensis in Japan should be treated as a single taxon. Phylogenetic and morphological data also suggest that there are two species currently representing Cryphonectria radicalis (Schwein.: Fr.) M.E. Barr in Europe. One of these species is similar to the type specimen of Cryphonectria radicalis, while the other species probably is new.Key words: Cryphonectria parasitica, Cryphonectria radicalis, Cryphonectria havanensis, Cryphonectria macrospora, Cryphonectria nitschkei, Diaporthales.
Cryphonectria cubensis (Bruner) Hodges is the causal agent of an important stem canker disease of Eucalyptus. Previous phylogenetic studies based on sequence data have shown that C. cubensis is distinct from other species of Cryphonectria but that C. cubensis isolates reside in two distinct groups, consistent with geographical origin. Thus, isolates of C. cubensis from South America and South Africa grouped together but apart from those originating from Southeast Asia and Australia. These results were in contrast with the symptoms of Cryphonectria canker in South Africa, which are different from those observed elsewhere in the world. The aim of this study was to use more variable regions of the fungal genome to test whether South African isolates of C. cubensis are genetically distinct from those from other parts of the world. For this comparison, β-tubulin and histone H3 gene sequences were used. Specimens from South America, Southeast Asia, Australia, and South Africa were also compared morphologically. The phylogram emerging from the analysis indicated that South American and Southeast Asian Australian isolates resided in two well-resolved but closely related clades. However, isolates from South Africa were distinct from other groups. This is consistent with ecological aspects of the South African fungus, although no obvious morphological differences between the fungi from the various regions could be found. Our results suggest that the South African fungus represents a species distinct from C. cubensis occurring elsewhere in the world.Key words: β-tubulin, histone H3, molecular phylogeny, Cryphonectria cubensis.
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