Chloroplast Introns Research Articles

MatK impacts differential chloroplast translation by limiting spliced tRNA-K(UUU) abundance.

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we used RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatK's preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for splice site selection, and we discovered alternative 5'-splice sites in three MatK target introns. The resulting alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Ribosome profiling analysis of the overexpressor revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

Optimizing efficient PCR‐amplifiable DNA extraction from herbarium specimens

AbstractPremiseThe objective of this study was to optimize an existing DNA extraction protocol for recalcitrant plant taxa to obtain high‐quality DNA from preserved herbarium tissue suitable for downstream PCR applications.Methods and ResultsLeaf tissue from 30 diverse plant species was obtained from the U.S. National Arboretum Herbarium. Our previous DNA extraction protocol (Gouker et al., 2020, Applications in Plant Sciences 8: e11403) was improved by use of 10X Tris‐EDTA buffer, addition of polyvinylpolypyrrolidone, and omission of sample heating during homogenization, and resulted in total DNA yields ranging from 60–2460 ng. Optimized PCR amplification using universal plant primers for the ITS‐p3/u4 region and the P6 loop of the trnL (UAA) chloroplast intron was successful for most specimens.ConclusionsThis protocol, which is simple, fast, and uses standard laboratory‐grade chemicals, yields DNA from herbarium specimens that is comparable in quality to that from commercially available kits, and is of sufficient quality and quantity for other applications.

Updated Progress on Group II Intron Splicing Factors in Plant Chloroplasts.

Group II introns are large catalytic RNAs (ribozymes) in the bacteria and organelle genomes of several lower eukaryotes. Many critical photosynthesis-related genes in the plant chloroplast genome also contain group II introns, and their splicing is critical for chloroplast biogenesis and photosynthesis processes. The structure of chloroplast group II introns was altered during evolution, resulting in the loss of intron self-splicing. Therefore, the assistance of protein factors was required for their splicing processes. As an increasing number of studies focus on the mechanism of chloroplast intron splicing; many new nuclear-encoded splicing factors that are involved in the chloroplast intron splicing process have been reported. This report reviewed the research progress of the updated splicing factors found to be involved in the splicing of chloroplast group II introns. We discuss the main problems that remain in this research field and suggest future research directions.

CFM6 is an Essential CRM Protein Required for the Splicing of nad5 Transcript in Arabidopsis Mitochondria.

Plant chloroplast RNA splicing and ribosome maturation (CRM)-domain-containing proteins are capable of binding RNA to facilitate the splicing of group I or II introns in chloroplasts, but their functions in mitochondria are less clear. In the present study, Arabidopsis thaliana CFM6, a protein with a single CRM domain, was expressed in most plant tissues, particularly in flower tissues, and restricted to mitochondria. Mutation of CFM6 causes severe growth defects, including stunted growth, curled leaves, delayed embryogenesis and pollen development. CFM6 functions specifically in the splicing of group II intron 4 of nad5, which encodes a subunit of mitochondrial complex I, as evidenced by the loss of nad5 intron 4 splicing and high accumulation of its pretranscripts in cfm6 mutants. The phenotypic and splicing defects of cfm6 were rescued in transgenic plants overexpressing 35S::CFM6-YFP. Splicing failure in cfm6 also led to the loss of complex I activity and to its improper assembly. Moreover, dysfunction of complex I induced the expression of proteins or genes involved in alternative respiratory pathways in cfm6. Collectively, CFM6, a previously uncharacterized CRM domain-containing protein, is specifically involved in the cis-splicing of nad5 intron 4 and plays a pivotal role in mitochondrial complex I biogenesis and normal plant growth.

Identification of polycistronic transcriptional units and non-canonical introns in green algal chloroplasts based on long-read RNA sequencing data

BackgroundChloroplasts are important semi-autonomous organelles in plants and algae. Unlike higher plants, the chloroplast genomes of green algal linage have distinct features both in organization and expression. Despite the architecture of chloroplast genome having been extensively studied in higher plants and several model species of algae, little is known about the transcriptional features of green algal chloroplast-encoded genes.ResultsBased on full-length cDNA (Iso-Seq) sequencing, we identified widely co-transcribed polycistronic transcriptional units (PTUs) in the green alga Caulerpa lentillifera. In addition to clusters of genes from the same pathway, we identified a series of PTUs of up to nine genes whose function in the plastid is not understood. The RNA data further allowed us to confirm widespread expression of fragmented genes and conserved open reading frames, which are both important features in green algal chloroplast genomes. In addition, a newly fragmented gene specific to C. lentillifera was discovered, which may represent a recent gene fragmentation event in the chloroplast genome.With the newly annotated exon-intron boundary information, gene structural annotation was greatly improved across the siphonous green algae lineages. Our data also revealed a type of non-canonical Group II introns, with a deviant secondary structure and intronic ORFs lacking known splicing or mobility domains. These widespread introns have conserved positions in their genes and are excised precisely despite lacking clear consensus intron boundaries.ConclusionOur study fills important knowledge gaps in chloroplast genome organization and transcription in green algae, and provides new insights into expression of polycistronic transcripts, freestanding ORFs and fragmented genes in algal chloroplast genomes. Moreover, we revealed an unusual type of Group II intron with distinct features and conserved positions in Bryopsidales. Our data represents interesting additions to knowledge of chloroplast intron structure and highlights clusters of uncharacterized genes that probably play important roles in plastids.

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

Looking Inside Non-coding Chloroplast Regions of Calophyllum brasiliense (Calophyllaceae) to Understand Its Southernmost Population Distribution

In recent years the growing interest in the conservation of Parana River’s riparian forest led to the discovery of botanical novelties for Argentina. Populations of Calophyllum brasiliense Camb. (Calophyllaceae), a typically flooded lowlands species, were identified in the remaining hygrophile forest of northeast Argentina and southeast Paraguay. Deforestation and flooding, due to the construction of dams, have caused these populations to suffer intensive fragmentation. The aim of this work was to infer phylogeographic relationships among five populations of C. brasiliense, three from Argentina and two from Paraguay, which represent the southernmost points of species’ distribution. We also compared them with samples of a C. brasiliense population from Mexico, the northernmost edge of the species distribution. The chloroplast intergenic spacers petG-trnP, psbJ-petA and the trnL-UAA chloroplast intron were amplified from leaves’ DNA. A total of 2234 bp were characterized once the three regions were analyzed. The three chloroplast regions showed nucleotide differences, represented by InDels, inversions and a few SNPs; however, only the trnL intron was selected for further phylogeographic analysis due to the amount of the information obtained for all populations. Based on trnL intron, it was possible to estimate nucleotide and haplotype diversity (π = 0.00237 and Hd = 0.29600, respectively). Three haplotypes were identified, which allowed Argentinean, Paraguayan and Mexican populations to be differentiated. Based on the three haplotypes found, we discuss and propose a model for a C. brasiliense’ geographic dispersion and historical colonization routes, including an alternative new one to the well-known of the Parana River.

OsCAF1, a CRM Domain Containing Protein, Influences Chloroplast Development.

The chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are involved in the splicing of chloroplast gene introns. Numerous CRM domain proteins have been reported to play key roles in chloroplast development in several plant species. However, the functions of CRM domain proteins in chloroplast development in rice remain poorly understood. In the study, we generated oscaf1 albino mutants, which eventually died at the seedling stage, through the editing of OsCAF1 with two CRM domains using CRISPR/Cas9 technology. The mesophyll cells in oscaf1 mutant had decreased chloroplast numbers and damaged chloroplast structures. OsCAF1 was located in the chloroplast, and transcripts revealed high levels in green tissues. In addition, the OsCAF1 promoted the splicing of group IIA and group IIB introns, unlike orthologous proteins of AtCAF1 and ZmCAF1, which only affected the splicing of subgroup IIB introns. We also observed that the C-terminal of OsCAF1 interacts with OsCRS2, and OsCAF1–OsCRS2 complex may participate in the splicing of group IIA and group IIB introns in rice chloroplasts. OsCAF1 regulates chloroplast development by influencing the splicing of group II introns.

Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation.

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns. MatK is essential for plant cell survival in tobacco, and thus null mutants have not yet been isolated. We therefore attempted to over-express MatK from a neutral site in the chloroplast, placing it under the control of a theophylline-inducible riboswitch. This ectopic insertion of MatK lead to a variegated cotyledons phenotype. The addition of the inducer theophylline exacerbated the phenotype in a concentration-dependent manner. The extent of variegation was further modulated by light, sucrose and spectinomycin, suggesting that the function of MatK is intertwined with photosynthesis and plastid translation. Inhibiting translation in the transplastomic lines has a profound effect on the accumulation of several chloroplast mRNAs, including the accumulation of an RNA antisense to rpl33, a gene coding for an essential chloroplast ribosomal protein. Our study further supports the idea that MatK expression needs to be tightly regulated to prevent detrimental effects and establishes another link between leaf variegation and chloroplast translation.

Molecular phylogenetic study of Scleria subgenus Hypoporum (Sclerieae, Cyperoideae, Cyperaceae) reveals several species new to science

Scleria subgen. Hypoporum (Cyperaceae), with 68 species, is the second largest subgenus in Scleria. Species of this pantropically distributed subgenus generally occur in seasonally or permanently wet grasslands or on shallow soils over sandstone or lateritic outcrops, less often they can be found in (open) woodlands. Previous studies established the monophyly of the subgenus, but the relationships between the species remained uncertain. In this study, DNA sequence data of 61 taxa of Scleria subgen. Hypoporum, where possible represented by multiple accessions from across their distributional range, were obtained for four molecular markers: the coding chloroplast marker ndhF, the chloroplast intron rps16 and the nuclear ribosomal regions ETS and ITS. Phylogenetic trees were constructed using Bayesian inference and maximum likelihood approaches. A species tree was constructed to summarise the results. The results indicate the existence of three sections: the monotypic, pantropically occurring, Scleria sect. Lithospermae, a new section from central and south America containing two species, and Scleria sect. Hypoporum, also pantropically distributed, containing the remainder of the species of the subgenus. Relationships in the latter section are not fully resolved. However, three or four different clades can be distinguished supported by some morphological characters. Our results indicate at least six new species in Scleria sect. Hypoporum. The new section and species are described in a taxonomical treatment. Their morphology is compared with (morphologically) closely related species.

Comprehensive analysis of chloroplast intron-containing genes and conserved splice sites in dicot and monocot plants

Despite the increasing knowledge on the importance of the intron splicing of chloroplast genes during plant growth and stress responses, identification of intron-containing chloroplast genes and determination of splice sites in chloroplast introns are still lacking. Here, we carried out a comprehensive analysis of the chloroplast genome sequences in important plants and crops, including four dicots (Arabidopsis thaliana, Coffea arabica, Nicotiana tabacum, and Panax schinseng) and four monocots (Musa acuminata, Oryza sativa, Triticum aestivum, and Zea mays). The results showed that both dicot and monocot chloroplast genomes harbor 6 intron-containing tRNAs (trnA, trnG, trnI, trnK, trnL, and trnV) and 10-12 intron-containing mRNAs (atpF, rpl2, rpl16, rps16, ndhA, ndhB, petB, petD, rpoC1, rps12, ycf3, and clpP). Notably, rpoC1 and clpP lacked introns in monocot plants, except M. acuminata. Analysis of the nucleotide sequences of chloroplast introns revealed that the 5’-splice sites, 3’-splice sites, and branch-point sites of the chloroplast introns were highly conserved among dicots and monocots. Notably, the 5’-splice sites and 3’-splice sites of the chloroplast introns were similar to those of the nuclear U12 introns, whereas the branch-point sites of the chloroplast introns were homologous to those of the nuclear U2 introns. Taken together, these results indicated that the chloroplast genomes contained strictly limited intron-containing genes with conserved splice sites, suggesting that splicing of chloroplast introns was important for chloroplast biogenesis and function in both dicot and monocot plants.

Phylogenetics, ancestral state reconstruction, and a new infrageneric classification of Scleria (Cyperaceae) based on three DNA markers

AbstractThe genus Scleria (Cyperaceae), with ca. 250 species, is placed in the monotypic tribe Sclerieae. It has a primarily pantropical distribution. Previously, infrageneric classifications have been proposed but none has been based on molecular phylogenetic evidence and most studies have treated the genus from only one geographical region. In this study, DNA portions from 101 species of Scleria from across its distributional range and all infrageneric groups were extracted and amplified for three molecular markers: the coding chloroplast marker ndhF, the chloroplast intron rps16 and the nuclear ribosomal region ITS. Phylogenetic hypotheses were constructed using Bayesian inference and maximum likelihood approaches. Additionally, ancestral states of four morphological characters were reconstructed at important nodes using the program BayesTraits. A new infrageneric classification based on molecular evidence and supported by morphology is presented. Scleria is confirmed as monophyletic and sister to tribe Bisboeckelereae. A subdivision of Scleria into four subgenera (S. subg. Browniae, S. subg. Hypoporum, S. subg. Scleria, S. subg. Trachylomia) is supported by our results. In this paper, twelve major clades are recovered within Scleria subg. Scleria. Clear morphological diagnostic features match these clades. Ancestral state reconstruction indicates that androgynous spikelets, a zoniform hypogynium, a paniculate inflorescence with staminate and subandrogynous spikelets, and normal linear leaves are ancestral in Scleria. Androgynous spikelets are found in the three first‐branching subgenera, while in S. subg. Scleria an evolutionary shift towards subandrogynous and strictly unisexual spikelets is noted. Hypogynia are generally well developed with the exception of S. subg. Hypoporum, where the hypogynium is reduced or even lost. Inflorescences in Scleria vary from short, densely paniculate and pyramidal to subcapitate, only in S. subg. Hypoporum a glomerate‐spicate inflorescence is found. The pseudopremorse leaf habit arose only once within S. subg. Scleria. Scleria sect. Scleria shows a reversal to normal leaf tips.

Streptocarpus redefined to include all Afro‐Malagasy Gesneriaceae: Molecular phylogenies prove congruent with geographical distribution and basic chromosome numbers and uncover remarkable morphological homoplasies

AbstractThe taxonomy of the African, Madagascan and Comoro Island (Afro‐Malagasy) Gesneriaceae attracts a large amount of interest given the horticultural importance of Cape Primroses (Streptocarpus) and African Violets (Saintpaulia). Earlier studies indicated that the Afro‐Malagasy genera form a strongly supported clade, and recent classifications have included some of the genera within an expanded Streptocarpus. Given the global importance of this group, we carried out a comprehensive molecular phylogenetic analysis of all Afro‐Malagasy genera in subfamily Didymocarpoideae, tribe Trichosporeae, subtribe Streptocarpinae, to investigate species relationships in these genera as the basis for a new classification. Phylogenetic analyses of the nuclear ribosomal spacer (ITS, 5S NTS) and chloroplast intron and spacer regions (rpl20‐rps12 spacer, trnL intron, trnLF spacer) of 226 samples were performed, including all Streptocarpinae genera, except the monotypic Nodonema. The molecular phylogenies demonstrate that the genera with non‐twisted fruits are nested within Streptocarpus which has twisted fruits. Two main clades were found, one comprising herbaceous caulescent Streptocarpus that also included Saintpaulia, the caulescents Hovanella and Schizoboea, and the unifoliates Acanthonema and Trachystigma. The second clade comprises the woody caulescents and acaulescent Streptocarpus, Colpogyne and Linnaeopsis. Altogether, twelve well‐supported subclades can be recognized, each with a combination of distinct morphological characteristics. A new classification of tribe Streptocarpinae, de facto Streptocarpus, is presented, retaining the two subgenera, Streptocarpus and Streptocarpella, and dividing them into five and seven sections respectively. Nodonema is attributed to subg. Streptocarpus for morphological reasons. The former genus Saintpaulia is classified as Streptocarpus subg. Streptocarpella sect. Saintpaulia with ten species recognized.

Was Myriophyllum spicatum L. (Haloragaceae) recently introduced to South Africa from Eurasia?

There is debate over the native or exotic status of Myriophyllum spicatum L. (Haloragaceae) in South Africa, which has important implications for developing and implementing management strategies. The aim of this study was to determine if M. spicatum was recently introduced from Eurasia by reconstructing the genetic relationships between South African and Eurasian M. spicatum using both a nuclear ribosomal (ITS1-5.8S-ITS2-26S) and a chloroplast intron (trnQ-rps16) sequence from 40 populations. For both these DNA markers, the South African populations were distinct from Eurasian populations, but always stemmed from a European origin. The data suggest that South African and European M. spicatum share a common ancestor, however the divergence of both markers are characteristic of a long period of isolation rather than a recent introduction from Europe. The genetic data from this study suggest that M. spicatum has not been introduced recently, but is most likely a native component of the South African flora.

Chloroplast DNA analysis of Tunisian pistachio (Pistacia vera L.): Sequence variations of the intron trnL (UAA)

The trnL (UAA) intron sequences of Pistacia vera ecotypes were employed to reconstruct the phylogeny and to elucidate molecular evolution. A single band of approximately 500pb length was amplified from each cultivars. Nucleotides sequences were aligned using the ClustalW package in the Bioedit program and analyzed with MEGA program version 4. The average of GC content was 35.8% to 39.6%. High values of A+T contents were detected in data sets and may explain the high proportions of transitions founded. Sequence alignment revealed the existence of 50 informative sites and 25 different haplotypes among the 39 cultivars studied yielding to a haplotype diversity of 0.8758 and a level of nucleotide diversity of 0.018. Furthermore, an average value of pairwise nucleotide of 8.641 differences was found, showing a relatively high diversity in the chloroplast intron. The neighbor joining dendrogram shows the presence of fairly high chloroplasts diversity, structured independently from the plant's sex but structured according to geographical origin. The trnL intron mismatch distribution is multimodal; characteristic of a stable population. This was also supported by non-significant values of all neutrality test parameters (Tajima's D and Fu and Li's). Although, the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate pistachio cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local pistachio germplasm.

Exon primed intron-crossing (EPIC) markers reveal natural hybridization and introgression in Actinidia (Actinidiaceae) with sympatric distribution

Interspecific hybridization is widespread among plants. Understanding the phylogenetic relationships among species is necessary for revealing the potential hybridization events. Actinidia, best known as kiwifruit genus found throughout a wide range in eastern Asian from Indonesia to Siberia. In this study, phylogenetic relationships of Actinidia species with sympatric distributions were investigated using three chloroplast introns (trnL-F, atpB-rbcL and rpl32-trnL) and three Exon primed intron-crossing (EPIC) markers. Chloroplast phylogeny supports non-monophyly of the five species studied excluding Actinidia fulvicoma var. lanata. The non-monophyly was also revealed by EPIC markers. Our results showed EPIC markers are more variable and informative for phylogenetic inference than that of chloroplast markers. The incongruences between loci from the plastid and nuclear DNA phylogenic trees may stem from incomplete lineage sorting or historical introgression hybridization. Incomplete lineage sorting may explain the non-monophyly between Actinidia chrysantha (section Maculatae) and other four species (section Stellatae), and introgression hybridization and high level of interspecific gene flow may explain the non-monophyly among the species of sect. Stellatae. Thus, natural hybridization and introgression may be common in Actinidia with sympatric distribution.

The Australasian genus Schizacme (Loganiaceae): new combinations and new species in the New Zealand flora

Schizacme Dunlop (Loganiaceae) is a genus of five perennial subshrubs of predominantly alpine and montane habitats in south eastern Australia and in New Zealand. Schizacme was described as a segregate from Mitrasacme Labill. but combinations in the genus were not made for two New Zealand taxa, Mitrasacme montana Hook.f. var. helmsii Kirk and M. novae-zealandiae Hook.f., thought to be congeneric with Schizacme. By using nucleotide sequence data from the nuclear ribosomal ETS and the chloroplast intron petD, we confirm that New Zealand Mitrasacme and Australian Schizacme form a monophyletic group. New combinations are created in Schizacme and species limits are reviewed, with an emphasis on the New Zealand taxa. Mitrasacme montana var. helmsii is here elevated to species rank, as S. helmsii (Kirk) K.L.Gibbons. Schizacme ciliata K.L.Gibbons is here described as a segregate from S. novae-zelandiae (Hook.f.) K.L.Gibbons. The morphology of Schizacme, including the New Zealand species, is discussed.

An mTERF domain protein functions in group II intron splicing in maize chloroplasts

The mitochondrial transcription termination factor (mTERF) proteins are nucleic acid binding proteins characterized by degenerate helical repeats of ∼30 amino acids. Metazoan genomes encode a small family of mTERF proteins whose members influence mitochondrial gene expression and DNA replication. The mTERF family in higher plants consists of roughly 30 members, which localize to mitochondria or chloroplasts. Effects of several mTERF proteins on plant development and physiology have been described, but molecular functions of mTERF proteins in plants are unknown. We show that a maize mTERF protein, Zm-mTERF4, promotes the splicing of group II introns in chloroplasts. Zm-mTERF4 coimmunoprecipitates with many chloroplast introns and the splicing of some of these introns is disrupted even in hypomorphic Zm-mterf4 mutants. Furthermore, Zm-mTERF4 is found in high molecular weight complexes that include known chloroplast splicing factors. The splicing of two transfer RNAs (trnI-GAU and trnA-UGC) and one ribosomal protein messenger RNA (rpl2) is particularly sensitive to the loss of Zm-mTERF4, accounting for the loss of plastid ribosomes in Zm-mTERF4 mutants. These findings extend the known functional repertoire of the mTERF family to include group II intron splicing and suggest that a conserved role in chloroplast RNA splicing underlies the physiological defects described for mutations in BSM/Rugosa2, the Zm-mTERF4 ortholog in Arabidopsis.

Spliceosome twin introns in fungal nuclear transcripts

The spliceosome is an RNA/protein complex, responsible for intron excision from eukaryotic nuclear transcripts. In bacteria, mitochondria and plastids, intron excision does not involve the spliceosome, but occurs through mechanisms dependent on intron RNA secondary and tertiary structure. For group II/III chloroplast introns, “twintrons” (introns within introns) have been described. The excision of the external intron, and thus proper RNA maturation, necessitates prior removal of the internal intron, which interrupts crucial sequences of the former. We have here predicted analogous instances of spliceosomal twintrons (“stwintrons”) in filamentous fungi. In two specific cases, where the internal intron interrupts the donor of the external intron after the first or after the second nucleotide, respectively, we show that intermediates with the sequence predicted by the “stwintron” hypothesis, are produced in the splicing process. This implies that two successive rounds of RNA scanning by the spliceosome are necessary to produce the mature mRNA. The phylogenetic distributions of the stwintrons we have identified suggest that they derive from “late” events, subsequent to the appearance of the host intron. They may well not be limited to fungal nuclear transcripts, and their generation and eventual disappearance in the evolutionary process are relevant to hypotheses of intron origin and alternative splicing.

Fast and efficient DNA-based method for winter diet analysis from stools of three cervids: moose, red deer, and roe deer

Effects of cervid browsing on timber production, especially during winter, lead to economic losses in forest management. The aim of this study was to present an efficient DNA-based method which allows qualitative assessment of the winter diet from stools of moose (Alces alces), roe deer (Capreolus capreolus), and red deer (Cervus elaphus). The preliminary results of the diet composition of the three cervids from Poland were also presented with a special emphasis on moose. The electropherograms of the chloroplast intron trnL (UAA) P6 loop amplification products using g (fluorescence-labeled) and h primers revealed differences in the length of PCR products among various plant species eaten by these herbivores. In addition, the usage of species-specific primers allowed unambiguous identification of different gymnosperms and angiosperms. The preliminary moose diet analysis, based on winter fecal samples from the entire range of moose occurrence in Poland, revealed the presence of 15 to 24 tree, shrub, and herbaceous species. This fast, cost-efficient, and simple method proved also to be reliable for the diet analysis of red deer and roe deer. It may be a valuable tool in forest and conservation management, as well as a way of enhancing ecological studies focusing on the impact of herbivores on the ecosystems and their possible food niche overlap.Electronic supplementary materialThe online version of this article (doi:10.1007/s13364-013-0146-9) contains supplementary material, which is available to authorized users.