Influence of <Emphasis Type="Italic">UPF</Emphasis> genes on severity of <Emphasis Type="Italic">SUP45</Emphasis> mutations

Eukaryotic cells possess a special mechanism for the degradation of mRNAs containing premature termination codons (PTCs), referred to as NMD (nonsense-mediated mRNA decay). The strength of this pathway depends on the recognition of the PTCs by translational machinery and the interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins in Sachromyces cerevisiae yeast. Previously, we have shown that the decrease of eRF1 protein amounts in sup45 nonsense mutants leads to the impairment of NMD. Here we show that the deletion of UPF1 or UPF2 genes leads to an increase in the viability of sup45 mutants, while the effect of UPF3 gene deletion is allele-specific. Two-hybrid data have shown that amino acid residues 1–555 of Upf1 protein interact with eRF1. Any UPF gene deletion leads to allosupression of the adel1-14 mutation without a change in eRF1 content. The Upf1 depletion does not influence the synthetic lethality of sup45 mutations and the [PSI ⁺] prion. It is possible that the absence of Upf1 (or its activator Upf2) leads to a more effective formation of the translation termination complex and consequently to the increased viability of the cells containing mutant termination factors.     

Conservation of the MC domains in eukaryotic termination factor eRF3

Eukaryotic translation termination employs two protein factors, eRF1 and eRF3. Proteins of the eRF3 family each consist of three domains. The N and M domains vary in different species, while the C domains are highly homologous. The MC domains of Homo sapiens eRF3a (hGSPT I), Xenopus laevis eRF3 (XSup35), and Mus musculus eRF3a (mGSPTI) and eRF3b (mGSPT2) were found to compensate for the sup35-21(ts) temperature-sensitive mutation and lethal disruption of the SUP35 gene in yeast Saccharomyces cerevisiae. At the same time, strains containing the MC domains of the eRF3 proteins from different species differed in growth rate and the efficiency of translation termination.     

Viable nonsense mutants for the <Emphasis Type="Italic">SUP45</Emphasis> gene in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination factor eRF1 in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a trucated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37°C. When grown under nonpermissive conditions (37°C), cells of nonsense mutants sup45-104, sup45-105, and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.     

Evaluation of the gene encoding translation termination factor eRF3 as a possible phylogenetic marker

The region of the nuclear GSPT2 gene coding for the N and M domains of translation termination factor eRF3b was tested in Rodentia for applicability as a new molecular marker. It cannot be used as a phylogenetic marker at the intrageneric level because of insufficient variability within families and the impossibility of resolving relationships in the family Cricetidae. However, this GSPT2 region allows reliable identification of higher taxa. The phylogenetic relationships among families revealed with the proposed molecular marker is generally in agreement with current concepts. The new marker indicates a close relationship between the genus Acomys and the family Gerbillidae, which is in agreement with other molecular data but contradicts morphological data. Thus, the region of the nuclear GSPT2 gene encoding the N and M domains of eRF3b can serve as an adequate phylogenetic marker in placental mammals at the level of families or higher taxa. It can also be used in solving controversial questions of phylogeny and taxonomy.     

How does <Emphasis Type="Italic">Euplotes</Emphasis> translation termination factor eRF1 fail to recognize the UGA stop codon?

Class 1 eukaryotic release factor 1 (eRF1) recognizes all three stop codons (UAA, UAG, and UGA) in standard-code organisms. In some ciliates with variant genetic codes, one or two stop codons are used to encode amino acids and are not recognized by eRF1; e.g., UAA and UAG are reassigned to Gln in Stylonychia and UGA is reassigned to Cys in Euplotes. Stop codon recognition is due to the N-terminal domain of eRF1 in standard-code organisms. Since variant-code ciliates most likely originate from universal-code ancestors, the N-domain sequence of their eRF1 was assumed to harbor the residues that are responsible for the changes in stop codon recognition specificity. To identify the N-domain regions determining the UGA-only specificity of Euplotes aediculatus eRF1, chimeric proteins were constructed by swapping various N-domain fragments of the E. aediculatus for their human counterparts; the MC domain was from human eRF1. Functional analysis of the chimeric eRF1 in vivo revealed two regions (residues 38–50 and 123–145) restricting the E. aediculatus eRF1 specificity to UAR. The change in stop codon recognition specificity of eRF1 was regarded as the first step in the origin of the variant genetic code in ciliates.     

Genetic mapping of the female mimic morph locus in the ruff

Background

Ruffs (Aves: Philomachus pugnax) possess a genetic polymorphism for male mating behaviour resulting in three permanent alternative male reproductive morphs: (i) territorial ‘Independents’, (ii) non-territorial ‘Satellites’, and (iii) female-mimicking ‘Faeders’. Development into independent or satellite morphs has previously been shown to be due to a single-locus, two-allele autosomal Mendelian mode of inheritance at the Satellite locus. Here, we use linkage analysis to map the chromosomal location of the Faeder locus, which controls development into the Faeder morph, and draw further conclusions about candidate genes, assuming shared synteny with other birds.

Results

Segregation data on the Faeder locus were obtained from captive-bred pedigrees comprising 64 multi-generation families (N?=?381). There was no evidence that the Faeder locus was linked to the Satellite locus, but it was linked with microsatellite marker Ppu020. Comparative mapping of ruff microsatellite markers against the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genomes places the Ppu020 and Faeder loci on a region of chromosome 11 that includes the Melanocortin-1 receptor (MC1R) gene, which regulates colour polymorphisms in numerous birds and other vertebrates. Melanin-based colouration varies with life-history strategies in ruffs and other species, thus the MC1R gene is a strong candidate to play a role in alternative male morph determination.

Conclusion

Two unlinked loci appear to control behavioural development in ruffs. The Faeder locus is linked to Ppu020, which, assuming synteny, is located on avian chromosome 11. MC1R is a candidate gene involved in alternative male morph determination in ruffs.

     

Diversity of root-endophytic <Emphasis Type="Italic">Trichoderma</Emphasis> from Malaysian Borneo

Trichoderma species form endophytic associations with plant roots and may provide a range of benefits to their hosts. However, few studies have systematically examined the diversity of Trichoderma species associated with plant roots in tropical regions. During the evaluation of Trichoderma isolates for use as biocontrol agents, root samples were collected from more than 58 genera in 35 plant families from a range of habitats in Malaysian Borneo. Trichoderma species were isolated from surface-sterilised roots and identified following analysis of partial translation elongation factor-1α (tef1) sequences. Species present included Trichoderma afroharzianum, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma guizhouense, Trichoderma reesei, Trichoderma strigosum and Trichoderma virens. Trichoderma asperellum/T. asperelloides, Trichoderma harzianum s.l. and T. virens were the most frequently isolated taxa. tef1 sequence data supported the recognition of undescribed species related to the T. harzianum complex. The results suggest that tropical plants may be a useful source of novel root-associated Trichoderma for biotechnological applications.     

The effects of mepiquat chloride on the lateral root initiation of cotton seedlings are associated with auxin and auxin-conjugate homeostasis

Background

Mepiquat chloride (MC) is a plant growth regulator widely used in cotton (Gossypium hirsutum L.) production to suppress excessive vegetative growth, increase root growth and avoid yield losses. To increase root growth, cotton seeds were treated with MC to increase the number of lateral root (LRs) and improve drought resistance. An increased indole-3-acetic acid (IAA) pool appeared to correlate with LR growth, and the principal source of IAA in germinating seeds is IAA conjugates. Here, the role of IAA homeostasis and signaling was investigated in cotton seedlings treated with MC.

Results

In the present research, MC significantly increased endogenous IAA levels in the roots, which promoted lateral root initiation (LRI) by upregulating GhARF7/19 and GhLBD18s and subsequently increasing LR quantity and elongation. The levels of IAA-amide conjugates significantly decreased in MC-treated seedlings compared with untreated control seedlings. Sixteen members of the cotton IAA amidohydrolase (IAH) gene family were identified, of which GhIAR3a, GhIAR3b, GhILR1, GhILL3 and GhILL6 were expressed during cotton seed germination. Compared with those in untreated control seedlings, the expression levels of GhIAR3a, GhIAR3b, GhILR1 and GhILL6 in the MC-treated seedlings were markedly elevated. The GhIAR3a/b and GhILR1 genes were cloned and expressed in Escherichia coli; these recombinant proteins exhibited hydrolytic activity that could cleave IAA-phenyalanine (Phe), IAA-methionine (Met), IAA-glycine (Gly) and IAA-leucine (Leu) in vitro, while only GhIAR3a hydrolyzed IAA-alanine (Ala) efficiently. The content of GhIAR3a, as detected via an established sandwich enzyme-linked immunosorbent assay (ELISA), increased in the MC-treated seedlings compared with the untreated control seedlings. In addition, the Arabidopsis iar3 mutant was less responsive to MC-induced LR growth than was wild type.

Conclusions

These findings suggested that MC application could mediate IAA homeostasis via increased IAA levels from IAA-amide conjugate hydrolysis by accelerating IAH gene expression, which might promote LRI and increase the LR quantity and elongation.

     

Haploid yeast cells undergo a reversible phenotypic switch associated with chromosome II copy number

Background

SUP35 and SUP45 are essential genes encoding polypeptide chain release factors. However, mutants for these genes may be viable but display pleiotropic phenotypes which include, but are not limited to, nonsense suppressor phenotype due to translation termination defect. [PSI ⁺] prion formation is another Sup35p-associated mechanism leading to nonsense suppression through decreased availability of functional Sup35p. [PSI ⁺] differs from genuine sup35 mutations by the possibility of its elimination and subsequent re-induction. Some suppressor sup35 mutants had also been shown to undergo a reversible phenotypic switch in the opposite direction. This reversible switching had been attributed to a prion termed [ISP ⁺]. However, even though many phenotypic and molecular level features of [ISP ⁺] were revealed, the mechanism behind this phenomenon has not been clearly explained and might be more complex than suggested initially.

Results

Here we took a genomic approach to look into the molecular basis of the difference between the suppressor (Isp^?) and non-suppressor (Isp⁺) phenotypes. We report that the reason for the difference between the Isp⁺ and the Isp^? phenotypes is chromosome II copy number changes and support our finding with showing that these changes are indeed reversible by reproducing the phenotypic switch and tracking karyotypic changes. Finally, we suggest mechanisms that mediate elevation in nonsense suppression efficiency upon amplification of chromosome II and facilitate switching between these states.

Conclusions

(i) In our experimental system, amplification of chromosome II confers nonsense suppressor phenotype and guanidine hydrochloride resistance at the cost of overall decreased viability in rich medium. (ii) SFP1 might represent a novel regulator of chromosome stability, as SFP1 overexpression elevates frequency of the additional chromosome loss in our system. (iii) Prolonged treatment with guanidine hydrochloride leads to selection of resistant isolates, some of which are disomic for chromosome II.

     

Protein synthesis meets ABC ATPases: new roles for Rli1/ABCE1

The essential NTPase Rli1/ABCE1 has been implicated in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. Two recent papers by the Krebber and Pestova groups —the former published in this issue of EMBO reports— suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes.EMBO Rep (2010) 11: 3 214–219. doi:10.1038/embor.2009.272The essential, conserved NTPase Rli1/ABCE1—a member of the ABC (ATP-binding cassette) superfamily of ATPases—has been implicated in translation initiation, ribosome biogenesis and human immunodeficiency virus capsid assembly. Two recent papers by the Krebber and Pestova groups—the former published in this issue of EMBO reports—suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes (Khoshnevis et al, 2010; Pisarev et al, 2010).Two recent papers […] suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotesProtein synthesis is divided into four phases—initiation, elongation, termination and ribosome recycling—which are catalysed by several translation factors. The fundamental reactions of protein synthesis, such as mRNA decoding, peptide bond formation and tRNA translocation, follow the same basic principles in prokaryotes and eukaryotes. However, some steps are quite different and require a larger set of factors in eukaryotes. The best-studied example of eukaryotic complexity is the initiation of protein synthesis. In prokaryotes, initiation is catalysed by only three factors—IF1, IF2 and IF3—whereas in mammals it requires at least 13. Two recent papers shed new light on termination and ribosome recycling in the yeast and mammalian systems, suggesting that these two steps are also different in eukaryotes and prokaryotes (Khoshnevis et al, 2010; Pisarev et al, 2010).…new [research] on termination and ribosome recycling in the yeast and mammalian systems [suggests] that these two steps are also different in eukaryotes and prokaryotesIn prokaryotes, translation termination is promoted by three release factors: RF1, RF2 and RF3. RF1 and RF2 recognize the three stop codons and catalyse the hydrolysis of the peptidyl-tRNA. RF3, a GTP-binding protein that is not essential in bacteria, does not participate in peptide release but, at the expense of GTP hydrolysis, promotes the dissociation of RF1 and RF2, thereby accelerating their turnover (Kisselev et al, 2003). To free the ribosome for initiation on another mRNA (a process known as recycling), the post-termination ribosome is disassembled in a step that requires ribosome recycling factor (RRF) and one of the elongation factors, the GTPase EF-G. Together, these factors promote the dissociation of the post-termination complex into subunits. The subsequent dissociation of tRNA and mRNA from the small ribosomal subunit is promoted by initiation factors, in particular IF3 (Peske et al, 2005).In eukaryotes, translation termination is mediated by only two factors: eRF1 recognizes all three termination codons and triggers the hydrolysis of peptidyl-tRNA, whereas eRF3 accelerates the process in a GTP-dependent manner (Fig 1; Alkalaeva et al, 2006). Unlike prokaryotic RF1 or RF2—which have no measurable affinity for RF3—eRF1 binds tightly to eRF3, and it is probably the complex of the two proteins that enters the ribosome. The mechanism of guanine nucleotide exchange on eRF3 is also different from that on prokaryotic RF3, suggesting that termination in eukaryotes and prokaryotes differs in almost every detail except, probably, the mechanism of peptidyl-tRNA hydrolysis itself. Nevertheless, the identification of an additional factor that facilitates termination was unexpected. In this issue of EMBO reports, Khoshnevis and colleagues use the power of yeast genetics to show that a protein named Rli1 (RNase L inhibitor 1) interacts physically with the termination factors eRF1 (known as Sup45 in yeast) and, to a lesser extent, eRF3 (Sup35; Khoshnevis et al, 2010). The downregulation of Rli1 expression increases stop codon read-through in a dual reporter system, indicating a lower efficiency of termination. Conversely, upregulation of Rli1 partly suppresses the increased read-through caused by certain mutations of eRF1. Although the mechanism by which Rli1 affects translation termination is not understood, the results of the Krebber lab provide strong evidence that Rli1 mediates the function of eRF1 and eRF3 in vivo (Fig 1).…the identification [in eukaryotes] of an additional factor that facilitates termination was unexpectedOpen in a separate windowFigure 1New roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes. During termination, translating ribosomes contain peptidyl-tRNA (peptide is shown in dark blue and tRNA in dark red) in the P site and expose a stop codon in the A site. The stop codon is recognized by termination factor eRF1, which enters the ribosome together with eRF3-GTP. After GTP hydrolysis, catalysed by eRF3, the peptide is released from the peptidyl-tRNA with the help of eRF1. The point at which Rli1/ABCE1 binds to the ribosome is unknown, but the order shown is consistent with the effect of the factor on both termination and recycling. After NTP hydrolysis by Rli1/ABCE1, the 60S subunit and factors dissociate from the 40S subunit. Finally, tRNA and mRNA are released from the 40S subunit with the help of initiation factors (not shown). ABCE1, ATP-binding cassette, sub-family E member 1; eRF, eukaryotic release factor; Rli1, RNase L inhibitor 1.Surprisingly, modulating the efficiency of termination seems not to be the only function of Rli1 in translation. In a parallel study, Pestova and co-workers show that in higher eukaryotes, the homologue of Rli1—ABCE1—strongly enhances ribosome recycling (Pisarev et al, 2010). Eukaryotes lack a homologue of bacterial RRF and thus have to use other factors to disassemble the post-termination ribosome. Ribosome recycling can be brought about to some extent by eIF3, eIF1 and eIF1A (Pisarev et al, 2007), which is reminiscent of the IF3/IF1-mediated slow ribosome recycling that seems to occur in some conditions in bacterial systems. In eukaryotes, the initiation-factor-driven recycling operates only in a narrow range of low Mg²⁺ concentrations, probably because the affinity of the subunits to one another increases steeply with Mg²⁺ (Pisarev et al, 2010). By contrast, ABCE1 seems to catalyse efficient subunit dissociation in various conditions. To bind to the ribosome, ABCE1 requires the presence of eRF1, which is thought to induce a conformational change of the ribosome that unmasks the binding site for ABCE1. Subunit dissociation requires NTP (ATP, GTP, CTP or UTP) hydrolysis by ABCE1 (Fig 1). Subsequently, the dissociation of mRNA and tRNA from the small ribosomal subunit is promoted by initiation factors, which also inhibit the spontaneous reassociation of the subunits. Thus, the sequence of events during ribosome recycling in the eukaryotic system is remarkably similar to that in prokaryotes, and ABCE1 and eRF1 (possibly together with eRF3) seem to act as genuine ribosome recycling factors, similar to bacterial RRF/EF-G, despite the lack of any similarity in sequence or structure.Rli1/ABCE1 is a member of the ABC ATPases and comprises four structural domains (Karcher et al, 2008). Two nucleotide-binding domains (1 and 2) are connected by a hinge and arranged in a head-to-tail orientation. In contrast to other ABC enzymes, ABCE1 has an amino-terminal iron–sulphur (Fe–S) cluster domain, which is located in close proximity to, and presumably interacts with the nucleotide-binding loop of domain 1. Thus, there is a potential link between Fe–S domain function and NTP-induced conformational control of the ABC tandem cassette. Interestingly, although Khosnevis and colleagues map the eRF1 binding site on the second, carboxy-terminal ATPase domain, the Fe–S cluster is required for the function of Rli1/ABCE1 in termination and recycling (Khoshnevis et al, 2010). One might speculate that NTP hydrolysis is coupled to splitting the ribosome into subunits, in analogy to the prokaryotic recycling factors RRF/EF-G that couple the free energy of GTP hydrolysis and phosphate release into subunit dissociation (Savelsbergh et al, 2009). Kinetic experiments measuring single-round rates of subunit dissociation and NTP hydrolysis would be required to establish the existence and nature of such coupling.Another intriguing question is the role of the Fe–S cluster in Rli1/ABCE1. Fe–S protein biogenesis is the only known function of mitochondria that is indispensable for the viability of yeast cells (Lill, 2009). As yeast mitochondria do not contain essential Fe–S proteins, the essential character of the mitochondrial Fe–S protein assembly machinery could be attributed to its role in the maturation of extra-mitochondrial Fe–S proteins, such as Rli1/ABCE1, which is essential in all organisms tested.Another interesting finding by the Krebber group is that Rli1 can bind to Hcr1 (known as eIF3j in higher eukaryotes; Khoshnevis et al, 2010). Hcr1/eIF3j is an RNA-binding subunit of initiation factor eIF3, which is involved in initiation and required for Rli1/ABCE1-independent ribosome recycling. The fact that Rli1/ABCE1 binds to both eRF1 and Hcr1/eIF3j might indicate a functional or regulatory link between the termination, recycling and initiation machineries eukaryotes. It is unclear why eukaryotes require termination and recycling machinery that is so different from that of prokaryotes. One possibility is that Rli1/ABCE1, in contrast to its prokaryotic counterparts, not only acts in termination and recycling but also provides a platform for the recruitment of initiation factors to the ribosome, thereby acting as an additional checkpoint for translational control. Thus, the results of the Krebber and Pestova labs open a new, exciting avenue of research on eukaryotic protein synthesis.     

The homologous identification of the stem rust resistance genes <Emphasis Type="Italic">Rpg5</Emphasis>, <Emphasis Type="Italic">Adf3</Emphasis> and <Emphasis Type="Italic">RGA1</Emphasis> in the relatives of barley

The barley genes Rpg5, RGA1 and Adf3, which provide a strong resistance to many pathotypes of stem rust, were cloned a few years ago, but it was still unclear whether their homologues were represented in wheat and in related species. The paper describes the results of a bioinformatic research to determine the homologues of Rpg5, RGA1 and Adf3 in the genomes of Triticum aestivum and several wild grasses, which breeders usually use as sources of stem rust resistance, and which are available in the genome databases. It was found that the Th. elongatum sequence Q9FEC6 and T. aestivum sequence Q43655 were the highly identical homologues of the Adf3 sequence. T. urartu M8A999 sequence and T. aestivum W5FCU1 sequence were found to be the closest homologues of Rpg5 complete protein sequence, but the identity of their kinase domains was not as clear as that of the other domains. The separate Rpg5 kinase part analysis did not provide the strong evidences that its orthologs were present in our corn species. T. urartu M7ZZX9 sequence and T. aestivum W5FFP0 and W5FI33 sequences were shown to be the homologues of RGA1. The analysis of the predicted active sites allowed finding out the difference between sequences of Rpg5, RGA1, Adf3 protein and their homologues.     

Association between expression levels and growth trait-related SNPs located in promoters of the <Emphasis Type="Italic">MC4R</Emphasis> and <Emphasis Type="Italic">MSTN</Emphasis> genes in <Emphasis Type="Italic">Spinibarbus hollandi</Emphasis>

Melanocortin 4 receptor: (MC4R) and Myostatin (MSTN) are two important growth trait-related genes in animals. In this study, we showed that two SNPs, MC4R-719A>G and MSTN-519C>T, found in the promoters of the MC4R and MSTN genes, respectively, are both associated with growth traits in Spinibarbus hollandi. Furthermore, we observed that there were significant associations between the expression levels of the MC4R and MSTN genes and these two growth trait-related SNPs. The expression level of MC4R gene in brain was lower in GG genotype fish with extremely high growth performance than that in AA genotype fish with extremely low growth performance. Expression level of the MSTN gene in muscle was lower in TT genotype fish with extremely high growth performance than that in CC and CT genotype fish with lower growth performance. The results indicated that these SNPs located in the promoters of MC4R and MSTN are associated with growth-related traits through modification of gene expression levels. The MSTN and MC4R SNPs may have useful application in effective marker-assisted selection aimed to increase output in S. hollandi.     

Evolution of blue-flowered species of genus <Emphasis Type="Italic">Linum</Emphasis> based on high-throughput sequencing of ribosomal RNA genes

Background

The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n?=?30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes.

Results

High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH.Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n?=?30) could originate either as the result of hybridization of two diploid species (2n?=?16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n?=?16) and a diploid ancestor of modern L. narbonense (2n?=?14).

Conclusions

High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the phylogeny of blue-flowered flax species and also reveal intra- and interspecific divergence of the rRNA gene sequences.

     

Genetic polymorphisms in the 5'-flanking region of the melanocortin 1 receptor (<Emphasis Type="Italic">MC1R</Emphasis>) gene in foxes

The one of the key pigment genes, the melanocortin 1 receptor (MC1R) gene, plays a fundamental role in the determination of coat color in a variety of mammals. However, so far there has been no report regarding the genetic variants of the MC1R promoter region and the potential association of its mutations with coat color in foxes. This work aimed to characterize 5'-flanking region of the MC1R gene and its mutations associated with coat color variations in foxes. A total of 76 individuals including 64 red foxes (Vulpes vulpes), representing 11 color morphs, and 12 arctic foxes (Vulpes lagopus), representing 2 color morphs were studied. To explore the potential cause of coat color variation in foxes, an 1105 bp region located upstream of the MC1R gene coding region was sequenced in 76 foxes. In the present study, a 1267 bp 5'-flanking region of fox MC1R gene was obtained using a PCR-mediated chromosome-walking technique and a 1105 bp segment was sequenced. A total of 8 novel SNPs and an insertion/deletion of 4 nucleotides were detected. The results of mutations analysis indicated that SNPs g.-52G>A, g.-266A>G, g.-297T>C, g.-300G>A and the insertion/deletion spaning positions g.-382~-379 were important in distinguishing V. vulpes and V. lagopus. This work, for the first time, described and confirmed the different variants existed in the 5'-flanking region of MC1R gene between red foxes and arctic foxes. These findings may be extremely helpful for further exploring the alternative splicings or promoter activity of MC1R gene for different coat-colored foxes.     

Multilocus phylogeny of <Emphasis Type="Italic">Clonostachys</Emphasis> subgenus <Emphasis Type="Italic">Bionectria</Emphasis> from Brazil and description of <Emphasis Type="Italic">Clonostachys chloroleuca</Emphasis> sp. nov.

Phylogenetic analyses based on protein-encoding gene exons and introns of ATP citrate lyase (ACL1), beta tubulin (TUB), the largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-α (TEF1) are used for inferring the existence of a new Clonostachys species from the Cerrado biome in Brazil, described here as C. chloroleuca. The species produces dimorphic, primary, and secondary conidiophores that form consistently greenish conidial masses on artificial media. It resembles therefore C. rosea f. catenulata although it differs from this species by less adpressed branches in the secondary conidiophores. The new species is also phylogenetically related to C. byssicola and C. rhizophaga. Our inventory suggests that C. byssicola, C. chloroleuca, C. pseudochroleuca, C. rhizophaga, C. rogersoniana, and C. rosea commonly occur in native and agriculturally used soils of the Cerrado and Amazon Forest. Using sequences available from two genome-sequenced strains employed as biological control agents, we confirm the identity of the European strain IK726 as C. rosea and identify strain 67-1 from China as C. chloroleuca.     

Species diversity of <Emphasis Type="Italic">Pseudocercospora</Emphasis> from Far East Asia

This study reflects on the monophyly of, and species diversity within, the genus Pseudocercospora in Far East Asia. Morphological characteristics and phylogenetic analyses of Pseudocercospora species were based on type specimens and ex-type cultures, which were collected from Japan and Taiwan. A phylogenetic tree was generated from multi-locus DNA sequence data of the internal transcribed spacer regions of the nrDNA cistron (ITS), partial actin (actA), and partial translation elongation factor 1-alpha (tef1), as well as the partial DNA-directed RNA polymerase II second largest subunit (rpb2). Based on these results, Pseudocercospora amelanchieris on Amelanchier and Ps. iwakiensis on Ilex were newly described from Japan, and a further 22 types (incl. two neo-, five lecto-, and 15 epitypes), were designated. The genus Pseudocercospora as presently circumscribed was found to be monophyletic, while the secondary barcodes, actA, tef1, and rpb2 were shown to be well suited to delimitate species within the genus.