SNP discovery and genetic mapping of T-DNA insertional mutants in Fragaria vesca L.

As part of a program to develop forward and reverse genetics platforms in the diploid strawberry [Fragaria vesca L.; (2n = 2x = 14)] we have generated insertional mutant lines by T-DNA mutagenesis using pCAMBIA vectors. To characterize the T-DNA insertion sites of a population of 108 unique single copy mutants, we utilized thermal asymmetric interlaced PCR (hiTAIL-PCR) to amplify the flanking region surrounding either the left or right border of the T-DNA. Bioinformatics analysis of flanking sequences revealed little preference for insertion site with regard to G/C content; left borders tended to retain more of the plasmid backbone than right borders. Primers were developed from F. vesca flanking sequences to attempt to amplify products from both parents of the reference F. vesca 815 x F. bucharica 601 mapping population. Polymorphism occurred as: presence/absence of an amplification product for 16 primer pairs and different size products for 12 primer pairs, For 46 mutants, where polymorphism was not found by PCR, the amplification products were sequenced to reveal SNP polymorphism. A cleaved amplified polymorphic sequence/derived cleaved amplified polymorphism sequence (CAPS/dCAPS) strategy was then applied to find restriction endonuclease recognition sites in one of the parental lines to map the SNP position of 74 of the T-DNA insertion lines. BLAST search of flanking regions against GenBank revealed that 46 of 108 flanking sequences were close to presumed strawberry genes related to annotated genes from other plants.

Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle.

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant.

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily.

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.

DNA hybridization evidence for the Australasian affinity of the American marsupial Dromiciops australis.

DNA hybridization was used to compare representatives of the major groups of marsupials and a eutherian outgroup. Because of the large genetic distances separating marsupial families, trees were calculated from normalized percentages of hybridization; thermal-melting statistics, however, gave identical topologies for the well-supported clades. The most notable results were the association of the only extant microbiotheriid, Dromiciops australis, an American marsupial, with the Australasian Diprotodontia, and of both together with the Dasyuridae. Estimates of the rate of divergence among marsupial genomes suggest that the Dromiciops-Diprotodontia split occurred approximately 50 million years ago, well after the establishment of the major clades of marsupials but before deep oceanic barriers prohibited dispersal among Australia, Antarctica, and South America. Because Dromiciops is nested within an Australasian group, it seems likely that dispersal from Australia accounts for its present distribution.

DNA/DNA hybridization studies of the carnivorous marsupials. I: The intergeneric relationships of bandicoots (Marsupialia: Perameloidea).

A complete suite of comparisons among six bandicoot species and one outgroup marsupial was generated using the hydroxyapatite chromatography method of DNA/DNA hybridization; heterologous comparisons were also made with three other bandicoot taxa. Matrices of delta Tm's, delta modes, and delta T50Hs were generated and corrected for nonreciprocity, homoplasy, and, in the case of delta Tm's, normalized percent hybridization; these matrices were analyzed using the FITCH algorithm in Felsenstein's PHYLIP (version 3.1). Uncorrected and nonreciprocity-corrected matrices were also jackknifed and analyzed with FITCH to test for consistency. Finally, sample scores for delta Tm, delta mode, and delta T50H matrices were bootstrapped and then subjected to phylogenetic analysis. These manipulations were carried out, in part, to address criticisms of the statistics used to summarize DNA/DNA hybridization (especially T50H) and the method itself. However, with the exception of an unresolved trichotomy among the two Echymipera species and Peroryctes longicauda, all trees showed the same branchpoints. Except in the case of the tree generated from reciprocal-corrected delta Tm data, nodes were stable under jackknifing; and, again excepting the above-mentioned trichotomy, all nodes were supported by 95% or more of the bootstrapped trees. These results suggest that, despite arguments to the contrary, all three summary statistics can be valid for DNA/DNA hybridization data. Of taxonomic interest is the placement of Echymipera spp. and Peroryctes longicauda together and separate from the more distant Peroryctes raffrayanus; the genus Peroryctes is thus at least paraphyletic. The trees further grouped Echymipera-plus-Peroryctes as the sister group of Isoodon-plus-Perameles. Limited hybridizations with Macrotis lagotis suggest that its current position as representative of an entirely distinct family of perameloids is correct.