Phylogenetic relationships in <Emphasis Type="Italic">Betula</Emphasis> (Betulaceae) based on AFLP markers

The genus Betula comprises various species in boreal and temperate climate zones of the Northern Hemisphere. The taxonomy of Betula is controversial and complicated by parallel evolution of morphological traits, polyploidization events, and extensive hybridization and introgression among species. Multilocus molecular data from AFLPs were used to provide phylogenetic information. A large number of polymorphic markers (321 variable bands) were produced in 107 Betula accessions from 23 species and 11 hybrids. The AFLP results were largely congruent with the results from previously examined nuclear DNA markers. Four distinct subgenera were identified within the genus Betula. These subgenera were partly in disagreement with the traditional (but disputed) division of the genus. In addition, the results indicated several groups of conspecific taxa. The majority of the species fell within subgenus Betula and shared a high degree of similarity with B. pendula. All hybrids were associated with this group, and the AFLP data contained signals on putative parents for some of the interspecific hybrids. Subgenus Chamaebetula and part of the Neurobetula species should be merged with Betula. The subgenera Betulenta, Betulaster, and the remaining part of Neurobetula are distinct and well supported. Although our results indicate that four major taxonomic groups can be recognized within the genus Betula, the relationship between them remains unclear. This may be due to the occurrence of hybridization and introgression, which would have a homogenizing effect on the relationships between species. Naturally occurring Betula species of hybrid origin may explain the low bootstrap values within the Betula clade. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional constraint and divergence in the G protein family in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis>

Part of the challenge of the post-genomic world is to identify functional elements within the wide array of information generated by genome sequencing. Although cross-species comparisons and investigation of rates of sequence divergence are an efficient approach, the relationship between sequence divergence and functional conservation is not clear. Here, we use a comparative approach to examine questions of evolutionary rates and conserved function within the guanine nucleotide-binding protein (G protein) gene family in nematodes of the genus Caenorhabditis. In particular, we show that, in cases where the Caenorhabditis elegans ortholog shows a loss-of-function phenotype, G protein genes of C. elegans and Caenorhabditis briggsae diverge on average three times more slowly than G protein genes that do not exhibit any phenotype when mutated in C. elegans, suggesting that genes with loss of function phenotypes are subject to stronger selective constraints in relation to their function in both species. Our results also indicate that selection is as strong on G proteins involved in environmental perception as it is on those controlling other important processes. Finally, using phylogenetic footprinting, we identify a conserved non-coding motif present in multiple copies in the genomes of four species of Caenorhabditis. The presence of this motif in the same intron in the gpa-1 genes of C. elegans, C. briggsae and Caenorhabditis remanei suggests that it plays a role in the regulation of gpa-1, as well as other loci.Electronic Supplementary Material Supplementary material is available for this article at     

Formation of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> biofilms on multiple surfaces on <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Summary A liquid-based assay was used to evaluate the ability of Yersinia pseudotuberculosis to form a bacterial biofilm on the nematode Caenorhabditis elegans. After 3 days of incubation in the liquid assay a biofilm was clearly visible by light microscopy on both the head and vulva region of the worms. At times, the biofilm formation on the vulva appeared to prevent the laying of eggs by the adult hermaphrodite; the eggs would later hatch inside of the worm. One possible explanation for the biofilm formation observed on the vulva may be the increased motion of the cuticle surrounding the vulva when the worm is immersed in a liquid culture. This is the first report of biofilm formation on the vulva of C. elegans.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

AFLP analysis for examining genetic differences in cultivated strains and their single-spore isolates and for confirming successful crosses in <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Amplified fragment length polymorphism (AFLP) analysis was used to examine genetic differences in Agaricus blazei cultivated strains and their single-spore isolates (SSIs). AFLP analysis with five primer combinations identified a total of 267 AFLP bands from nine cultivated strains (one from Brazil and eight from Japan), of which 165 were polymorphic between the nine strains. An AFLP data dendrogram grouped the eight Japanese strains, with the Brazilian strain acting as an outlier, suggesting that the Brazilian and Japanese strains are genetically quite different. Twelve SSIs derived from each of four cultivated strains were subjected to AFLP analysis. All the AFLP bands detected in the cultivated strains were also found in at least one SSI, but some unique bands were detected in SSIs. The total number of AFLP bands from individual SSIs was clearly less than those from their parental strains, and many of polymorphic AFLP bands from the parental strains segregated in SSIs at a ratio of 1 : 1, suggesting that the SSIs are homokaryotic. Distance values based on presence or absence of individual AFLP bands among SSIs from different strains were clearly higher than those among SSIs from a single strain. In addition, AFLP analysis was shown to be useful in confirming hybrid formation in crosses between SSIs.     

Origin of new <Emphasis Type="Italic">Brassica</Emphasis> types from a single intergeneric hybrid between <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> by rapid chromosome evolution and introgression

Many novel lines were established from an intergeneric mixoploid between Brassica rapa (2n = 20) and Orychophragmus violaceus (2n = 24) through successive selections for fertility and viability. Pedigrees of individual F₂ plants were advanced to the 10th generation by selfing. Their breeding habit was self-compatible and different from the self-incompatibility of their female parent B. rapa, and these lines were reproductively isolated to different degrees from B. rapa and B. napus. The lines with high productivity showed not only a wide spectrum of phenotypes but also obvious variations in fatty acid profiles of seed oil and glucosinolate contents in seed meal. These lines had 2n = 36, 37, 38, 39 and 40, with 2n = 38 being most frequent (64.56%), and no intact O. violaceus chromosomes were detected by genomic in situ hybridization (GISH) analysis. Amplified fragment length polymorphism (AFLP) analyses revealed a high extent of variation in genomic compositions across all the lines. O. violaceus-specific bands, deleted bands in B. rapa and novel bands for two parents were detected in these lines, with novel bands being the most frequent. The morphological and genetic divergence of these novel types derived from a single hybrid is probably due to rapid chromosomal evolution and introgression, and provides new genetic resources for rapeseed breeding.     

Genetic analysis of ryanodine receptor function in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis> based on<Emphasis Type="Italic"> unc-68</Emphasis> revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca²⁺-induced Ca²⁺ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca²⁺ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.Communicated by C. P. Hollenberg     

Maternal Inheritance of Chloroplasts between <Emphasis Type="Italic">Brassica rapa</Emphasis> and F<Subscript>1</Subscript>-hybrids Demonstrated by cpDNA Markers Specific to Oilseed Rape and <Emphasis Type="Italic">B. rapa</Emphasis>

Controlled reciprocal crosses between B. rapa and F₁-hybrids (B. napus (♀) × B. rapa), giving 20 pair-crossings, were made to reveal possible irregularities in chloroplast inheritance during production of BC₁s. Despite the close relationship of chloroplasts in B. rapa and B. napus, development of PCR-based molecular markers specific to B. rapa chloroplasts and B. napus chloroplasts was successful. Offspring from each cross were investigated and among these, we found no irregular chloroplast inheritance, since their plastid genotypes in all cases were identical to that of their mother. With a certainty of 95% our data indicate that the probability that chloroplasts are being inherited paternally is less than 0.015. In oilseed rape, pollen-mediated transgene-dispersal poses a well-known risk. Our results support development of transplastomic oilseed rape as an approach to reduce transgene dispersal.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">odd-skipped</Emphasis> homologs function during gut development in<Emphasis Type="Italic"> C. elegans</Emphasis>

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, we have initiated a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family.Edited by C. Desplan     

Genes encoding putative biogenic amine receptors in the parasitic nematode <Emphasis Type="Italic">Brugia </Emphasis><Emphasis Type="Italic">malayi</Emphasis>

Filarial nematodes, such as Brugia malayi, cause major health problems worldwide. The lack of a vaccine against B. malayi, combined with ineffective chemotherapy against the adult has prompted the examination of biogenic amine receptors (BARs) as possible targets for drug discovery. We employed bioinformatics to identify genes encoding putative B. malayi BARs. Surprisingly, the B. malayi genome contains half of the genes predicted to encode BARs in the genomes of free-living nematodes such as Caenorhabditis elegans or C. briggsae; however, all of the predicted B. malayi receptors have clear orthologues in C. elegans. The B. malayi genes encode each of the major BAR subclasses, including three serotonin, two dopamine and two tyramine/octopamine receptors and the structure of orthologous BAR genes is conserved. We find that potential G-protein coupling and ligand-specificity of individual BARs may be predicted by phylogenetic comparisons. Our results provide a framework for how G-protein coupled receptors may be targeted for drug development in medically important parasitic nematodes.     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

<Emphasis Type="Italic">Caenorhabditis elegans gcs-1</Emphasis> Confers Resistance to Arsenic-Induced Oxidative Stress

Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. The amount of metal ion required to kill half of the population of worms falls in the order of wild-type/As(V)>gcs-1/As(V)> wild-type/As(III)>gcs-1/As(III). gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. These results indicate that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress.     

Assessment of within-population genetic structure in <Emphasis Type="Italic">Quercus crispula</Emphasis> and <Emphasis Type="Italic">Q. dentata</Emphasis> by amplified fragment length polymorphism analysis

The spatial genetic structure was assessed by amplified fragment length polymorphism (AFLP) technique for Quercus crispula Blume (37 individuals) and Q.dentata Thunberg (54 individuals) growing along a 550-m transect in Ishikari, Hokkaido, northern Japan. The simple Mantel test revealed significant negative correlation between genetic similarity and geographic distance in Q.dentata and negative but insignificant correlation in Q.crispula. Spatial autocorrelation analysis revealed that Mantels r generally decreased from positive to negative values with the increase of spatial distance in both oak species with significant deviation from zero for the 50–100-m (positive) and 250–300-m classes (negative) in Q.dentata. Thus, significant spatial genetic structure was revealed at least in Q.dentata     

Concomitant reiterative BAC walking and fine genetic mapping enable physical map development for the broad-spectrum late blight resistance region,<Emphasis Type="Italic"> RB</Emphasis>

The wild potato species Solanum bulbocastanum is a source of genes for potent late blight resistance. We previously mapped resistance to a single region of the S. bulbocastanum chromosome 8 and named the region RB (for "Resistance from S. Bulbocastanum "). We now report physical mapping and contig construction for the RB region via a novel reiterative method of BAC walking and concomitant fine genetic mapping. BAC walking was initiated using RFLP markers previously shown to be associated with late blight resistance. Subcontig extension was accomplished using new probes developed from BAC ends. Significantly, BAC end and partial BAC sequences were also used to develop PCR-based markers to enhance map resolution in the RB region. As they were developed from BAC clones of known position relative to RB, our PCR-based markers are known a priori to be physically closer to the resistance region. These markers allowed the efficient screening of large numbers of segregating progeny at the cotyledon stage, and permitted us to assign the resistance phenotype to a region of approximately 55 kb. Our markers also directed BAC walking efforts away from regions distantly related to RB in favor of the 55-kb region. Because the S. bulbocastanum genotype used in BAC library construction is heterozygous for RB (RB/rb), codominant PCR-based markers, originally developed for fine-scale mapping, were also used to determine homolog origins for individual BAC clones. Ultimately, BAC contigs were constructed for the RB region from both resistant (RB) and susceptible (rb) homologs.Communicated by R. Hagemann     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Comparative study of interspecific genetic divergence and phylogenic analysis of genus <Emphasis Type="Italic">Jatropha</Emphasis> by RAPD and AFLP

Genus Jatropha with 172 species having significant economic importance belongs to the family Euphorbiaceae. There are no reports on molecular characterization and phylogenetic relationship among the species of Jatropha. Hence, the present study was undertaken to assess the extent of genetic variability that exist and also to establish phylogenetic relationship among Jatropha curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica and J. tanjorensis using RAPD and AFLP. The percentage of loci that are polymorphic among the species studied was found to be 97.74% by RAPD and 97.25% by AFLP. The mean percentage of polymorphism (PP) was found to be 68.48 by RAPD and 71.33 by AFLP. The phylogram generated with RAPD and AFLP data showed maximum similarity. With the generated data maximum relatedness was found between J. curcas and J. integerrima this may be the reason for the success of inter hybrid crosses between these two species. Neither RAPD nor AFLP data generated in this study supports the view of J. tanjorensis, a natural interspecific hybrid between J. curcas and J. gossypifolia. The present study concludes that both RAPD and AFLP techniques are comparable in divergence studies of Jatropha species. The markers generated by RAPD and AFLP can be employed efficiently for interspecific hybrids identification, marker assisted selection and genetic resource management.