The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi

The Agrocybe chaxingu and Agrocybe aegerita mitochondrial apocytochrome b coding sequences are highly similar (97% of nt identity), but have highly different sizes (2312 and 4867nt, respectively), due to the presence of three large group IB introns: two (iAae1 and iAae2) in A. aegerita, one (iAch1) in A. chaxingu. All these introns encode a homing endonuclease (HE) similar to those described in introns of mitochondrial genes (cob, cox1, and nad5) from various organisms. Phylogenetic trees were built with these HE sequences. From these trees, the Agrocybe coding introns argue for recent lateral transfers, i.e., occurring after the separation of the two Agrocybe species, involving phylogenetically distant fungi such as members of the Ascomycota phylum (for iAch1 and iAae2) and, for the first time to our knowledge, a member of the Chytridiomycota phylum (for iAae1). The grouping of the HE gene (HEG) sequences according to the mitochondrial gene (cob, cox1, and nad5) where they are inserted, suggests modifications of the interactions between the HE and the recognized sequences, leading to new target genes. The largest distribution of the iAch1 HE, shared by several cob and cox1 mitochondrial genes from Ascomycota, Basidiomycota, and Chytridiomycota phyla, suggests a higher target flexibility of this HE, perhaps related to the presence of two different LAGLIDADG motifs in the catalytic site of the enzyme.     

Wide Distribution of Mitochondrial Genome Rearrangements in Wild Strains of the Cultivated Basidiomycete Agrocybe aegerita

We used restriction fragment length polymorphisms to examine mitochondrial genome rearrangements in 36 wild strains of the cultivated basidiomycete Agrocybe aegerita, collected from widely distributed locations in Europe. We identified two polymorphic regions within the mitochondrial DNA which varied independently: one carrying the Cox II coding sequence and the other carrying the Cox I, ATP6, and ATP8 coding sequences. Two types of mutations were responsible for the restriction fragment length polymorphisms that we observed and, accordingly, were involved in the A. aegerita mitochondrial genome evolution: (i) point mutations, which resulted in strain-specific mitochondrial markers, and (ii) length mutations due to genome rearrangements, such as deletions, insertions, or duplications. Within each polymorphic region, the length differences defined only two mitochondrial types, suggesting that these length mutations were not randomly generated but resulted from a precise rearrangement mechanism. For each of the two polymorphic regions, the two molecular types were distributed among the 36 strains without obvious correlation with their geographic origin. On the basis of these two polymorphisms, it is possible to define four mitochondrial haplotypes. The four mitochondrial haplotypes could be the result of intermolecular recombination between allelic forms present in the population long enough to reach linkage equilibrium. All of the 36 dikaryotic strains contained only a single mitochondrial type, confirming the previously described mitochondrial sorting out after cytoplasmic mixing in basidiomycetes.     

Mating Type Switching in the Tetrapolar Basidiomycete Agrocybe Aegerita

The study of fruiting in the basidiomycete Agrocybe aegerita has shown that some haploid homokaryotic strains can spontaneously switch their mating specificities at the two unlinked A and B mating type factors. This event causes the dikaryotisation of primary homokaryons without plasmogamy and leads to the differentiation of sporulating fruit-bodies (pseudo-homokaryotic fruiting). For each mating type factor, the genetic analyses have revealed that: (1) parental and switched mating types segregate meiotically as Mendelian markers, (2) a total of six switched mating type factors (two parental and four nonparental) were obtained from a wild strain, (3) most of the nonparental factors have specificities differing from those of a large series of wild factors, (4) strains with the same expressed mating type can generate different specificities, (5) switching is always restricted to the same mating type in a homokaryon, (6) nonparental types can switch again, and (7) meiosis fixes the specificities to which switching can occur. This suggests, for the first time in filamentous fungi, the existence of a mechanism analogous to the mating type switching in yeasts. We hypothese that both A and B mating type regions in A. aegerita are constituted of three loci, one specialized in expression and two other carrying silent information. Mating type switching in homokaryotic strains would occur by copy transposition of silent A and B information into the expression loci. Moreover, we propose that during meiosis the silent loci are substituted by copies of the expressed loci.     

The Agrocybe aegerita mitochondrial genome contains two inverted repeats of the nad4 gene arisen by duplication on both sides of a linear plasmid integration site

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains two disrupted genes with plasmid origin Aa-polB P1 and a small ORF homologous to a small gene described in two basidiomycete linear plasmids. The phylogenetic analyses argue in favor of a same plasmid origin for both genes but, surprisingly, these genes were separated by a mitochondrial tRNA-Met. Both strands of the complete region containing the two nad4 inverted copies and the tRNA-Met appear to be transcribed on large polycistronic mRNAs. A model summarizing the events that would have occurred is proposed: (1) capture of the tRNA by the plasmid before its integration in the mtDNA or acquisition of the tRNA gene by recombination after the plasmid integration, (2) integration of the plasmid in the mtDNA, accompanied by a large duplication containing the nad4 gene and (3) erosion of the plasmid sequences by large deletions and mutations.     

The Agrocybe aegerita mitochondrial genome contains two inverted repeats of the nad4 gene arisen by duplication on both sides of a linear plasmid integration site.

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains two disrupted genes with plasmid origin Aa-polB P1 and a small ORF homologous to a small gene described in two basidiomycete linear plasmids. The phylogenetic analyses argue in favor of a same plasmid origin for both genes but, surprisingly, these genes were separated by a mitochondrial tRNA-Met. Both strands of the complete region containing the two nad4 inverted copies and the tRNA-Met appear to be transcribed on large polycistronic mRNAs. A model summarizing the events that would have occurred is proposed: (1) capture of the tRNA by the plasmid before its integration in the mtDNA or acquisition of the tRNA gene by recombination after the plasmid integration, (2) integration of the plasmid in the mtDNA, accompanied by a large duplication containing the nad4 gene and (3) erosion of the plasmid sequences by large deletions and mutations.     

Multiple levels of single-strand slippage at cetacean tri- and tetranucleotide repeat microsatellite loci.

Between three and six tri- and tetranucleotide repeat microsatellite loci were analyzed in 3720 samples collected from four different species of baleen whales. Ten of the 18 species/locus combinations had imperfect allele arrays, i.e., some alleles differed in length by other than simple integer multiples of the basic repeat length. The estimate of the average number of alleles and heterozygosity was higher at loci with imperfect allele arrays relative to those with perfect allele arrays. Nucleotide sequences of 23 different alleles at one tetranucleotide repeat microsatellite locus in fin whales, Balaenoptera physalus, and humpback whales, Megaptera novaeangliae, revealed sequence changes including perfect repeats only, multiple repeats, and partial repeats. The relative rate of the latter two categories of mutation was estimated at 0.024 of the mutation rate involving perfect repeats only. It is hypothesized that single-strand slippage of partial repeats may provide a mechanism for counteracting the continuous expansion of microsatellite loci, which is the logical consequence of recent reports demonstrating directional mutations. Partial-repeat mutations introduce imperfections in the repeat array, which subsequently could reduce the rate of single-strand slippage. Limited computer simulations confirmed this predicted effect of partial-repeat mutations.     

Genetic evidence for nonrandom sorting of mitochondria in the basidiomycete Agrocybe aegerita.

We studied mitochondrial transmission in the homobasidiomycete Agrocybe aegerita during plasmogamy, vegetative growth, and basidiocarp differentiation. Plasmogamy between homokaryons from progeny of three wild-type strains resulted in bidirectional nuclear migration, and the dikaryotization speed was dependent on the nuclear genotype of the recipient homokaryon. Little mitochondrial migration accompanied the nuclear migration. A total of 75% of the dikaryons from the fusion lines had both parental mitochondrial haplotypes (mixed dikaryons), and 25% had only a single haplotype (homoplasmic dikaryons); with some matings, there was a strong bias in favor of one parental haplotype. We demonstrated the heteroplasmic nature of mixed dikaryons by (i) isolating and subculturing apical cells in micromanipulation experiments and (ii) identifying recombinant mitochondrial genomes. This heteroplasmy is consistent with the previously reported suggestion that there is recombination between mitochondrial alleles in A. aegerita. Conversion of heteroplasmons into homoplasmons occurred (i) during long-term storage, (ii) in mycelia regenerated from isolated apical cells, and (iii) during basidiocarp differentiation. Homokaryons that readily accepted foreign nuclei were the most efficient homokaryons in maintaining their mitochondrial haplotype during plasmogamy, long-term storage, and basidiocarp differentiation. This suggests that the mechanism responsible for the nonrandom retention or elimination of a given haplotype may be related to the nuclear genotype or the mitochondrial haplotype or both.     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans

Abstract We describe the identification of polymorphic microsatellite loci in the pathogenic yeast, Candida albicans . A search for all coding-region microsatellites with more than four repeats that can be found in Candida sequences in GenBank was conducted. Nine such microsatellite sequences consisting of trinucleotide motifs were found. Three of these were perfect microsatellites while the remaining six sequences were found in one imperfect microsatellite and two compound microsatellites. Because of the close proximity of some of these repeats, all could be assayed with six PCR primer pairs. All of these microsatellite sequences were found in five nuclear genes, ZNF1, CCN1, CPH1, EFG1 , and MNT2 . Except for a single (CTT)₅ serine tract, all coded for polyglutamine tracts. Another locus with seven alleles, a region of the ERK1 protein kinase gene, was also examined, and may be a representative of a new class of highly polymorphic ‘clustered’ microsatellites. Such loci, in which several non-contiguous but closely linked microsatellites are clustered together, may be a useful source of DNA polymorphisms in microorganisms in which long microsatellite sequences are unavailable. All seven regions amplified were polymorphic, having between two and seven variable length alleles in the 11 strains of Candida albicans examined. The results of this and similar searches will facilitate epidemiological and evolutionary studies of Candida and other microorganisms.     

PRIMER NOTE. Using the incomplete genome of the ectomycorrhizal fungus Amanita bisporigera to identify molecular polymorphisms in the related Amanita phalloides

We describe the cross‐genomic isolation of 13 single nucleotide polymorphisms (SNPs) and one variable microsatellite from five loci for the death cap mushroom Amanita phalloides. Microsatellite repeats were identified by searching the partial Amanita bisporigera genome. Flanking primers were designed for 25 of these microsatellite loci and tested for cross‐amplification in A. phalloides. One locus contained an interrupted, compound microsatellite, and four loci contained one to six SNPs. These results demonstrate the usefulness of even an incomplete genome to identify molecular markers for population studies in nonmodel organisms.     

Microsatellite Loci of the Cattle Tick Boophilus Microplus (Acari: Ixodidae)

This brief communication reports the identification of microsatellite loci in the economically important tick species Boophilus microplus. The data are potentially useful in distinguishing different strains of B. microplus. Eight polymorphic loci were isolated in larvae, male and female adults analysed individually from 12 field isolates and laboratory strains from Australia (n = 8), Brazil, Mexico, Papua New Guinea and Zimbabwe. Nucleotide sequencing of alleles at these microsatellite loci revealed that non-repeat bases interrupted dinucleotide and tetranucleotide repeats in some loci. Loci with non-repeat bases interrupting them were shorter compared with loci that were not interrupted. Thus the presence of non-repeat bases in a repeated sequence seems to constrain the evolution of additional repeats by slip-strand misparing at these loci.     

A Phylogenetic Perspective on Sequence Evolution in Microsatellite Loci

We examined the evolution of the repeat regions of three noncoding microsatellite loci in 58 species of the Polistinae, a subfamily of wasps that diverged over 140 million years ago. A phylogenetic approach allows two new kinds of approaches to studying microsatellite evolution: character mapping and comparative analysis. The basic repeat structure of the loci was highly conserved, but was often punctuated with imperfections that appear to be phylogenetically informative. Repeat numbers evolved more rapidly than other changes in the repeat region. Changes in number of repeats among species seem consistent with the stepwise mutation model, which is based on slippage during replication as the main source of mutations. Changes in repeat numbers can occur even when there are very few tandem repeats but longer repeats, especially perfect repeats led to greater rates of evolutionary change. Species phylogenetically closer to the one from which we identified the loci had longer stretches of uninterrupted repeats and more different motifs, but not longer total repeat regions. The number of perfect repeats increased more often than it decreased. However, there was no evidence that some species have consistently greater numbers of repeats across loci than other species have, once ascertainment bias is eliminated. We also found no evidence for a population size effect posited by one form of the directionality hypothesis. Overall, phylogenetic variation in repeat regions can be explained by adding neutral evolution to what is already known about the mutation process. The life cycle of microsatellites appears to reflect a balance between growth by slippage and degradation by an essentially irreversible accumulation of imperfections. Received: 13 April 1999 / Accepted: 8 September 1999     

High incidence of nonslippage mechanisms generating variability and complexity in eurasian badger microsatellites

The use of microsatellites in population genetics is hindered by a lack of understanding of the pattern and origin of mutations, the need to develop more specific and better computational models, and a paucity of information about specific taxa and loci. We analyzed between 4 and 10 allele sequences from 10 different microsatellites in Eurasian badgers in order to determine the compliance of the sequences with stepwise mutation models and the origin of that variability which cannot be detected through standard genotyping procedures. All microsatellite loci exhibited imperfections and/or substitutions and indels in the flanking region, as well as additions or deletions of repeat units. Our data set of sequences showed a higher number of imperfect repeats than other published badger and carnivore sequences. This could be attributed to the process of loci isolation because when genetic variability is low, researchers may be more likely to use imperfect loci if these are variable in the population being studied. Locus Mel15 had 2 repetitive arrays: one was part of a polypyrimidine region of a carnivoran short interspersed nuclear element (CAN-SINE) and the other was located in an A-rich region typical of these insertions. In spite of this complexity, heterozygosity was correlated with the maximum number of repeats. Thus, although new theoretical models are being evolved to cover complex patterns of microsatellite mutation, sequencing electromorphs is needed to identify microsatellites or portions of them whose evolution can be modeled under simple models.     

Contrasting patterns of gene diversity between microsatellites and mitochondrial SNPs in farm and wild Atlantic salmon

Levels of genetic variability at 12 microsatellite loci and 19 single nucleotide polymorphisms in mitochondrial DNA were studied in four farm strains and four wild populations of Atlantic salmon. Within populations, the farm strains showed significantly lower allelic richness and expected heterozygosity than wild populations at the 12 microsatellite loci, but a significantly higher genetic variability with respect to observed number of haplotypes and haplotype diversity in mtDNA. Significant differences in allele- and haplotype-frequencies were observed between farm strains and wild populations, as well as between different farm strains and between different wild populations. The large genetic differentiation at mitochondrial DNA between wild populations (F_ST = 0.24), suggests that the farm strains attained a high mitochondrial genetic variability when created from different wild populations seven generations ago. A large proportion of this variability remains despite an expected lower effective population size for mitochondrial than nuclear DNA. This is best explained by the particular mating schemes in the breeding programmes, with 2–4 females per male. Our observations suggest that for some genetic polymorphisms farm populations might currently hold equal or higher genetic variability than wild populations, but lower overall genetic variability. In the short-term, genetic interactions between escaped farm salmon and wild salmon might increase genetic variability in wild populations, for some, but not most, genetic polymorphisms. In the long term, further losses of genetic variability in farm populations are expected for all genetic polymorphisms, and genetic variability in wild populations will be reduced if escapes of farm salmon continue.     

Mitochondrial microsatellite variability in common wheat and its ancestral species

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.     

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

Isolation and Characterization of Nine Microsatellite Loci from the Hawaiian Grouper <Emphasis Type="Italic">Epinephelus Quernus</Emphasis> (Serranidae) for Population Genetic Analyses

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin–coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.     

High-throughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae

Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.