text:
Contributed as many data as the first author
Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solanaceae)
Article first published online: 1 JUL 2005
DOI: 10.1111/j.1469-8137.2005.01480.x
New Phytologist
Volume 168, Issue 1, pages 241–252, October 2005
Additional Information
How to Cite
Clarkson, J. J., Lim, K. Y., Kovarik, A., Chase, M. W., Knapp, S. and Leitch, A. R. (2005), Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solanaceae). New Phytologist, 168: 241–252. doi: 10.1111/j.1469-8137.2005.01480.x
Publication History
- Issue published online: 1 JUL 2005
- Article first published online: 1 JUL 2005
- Received: 27 January 2005 Accepted: 12 April 2005
- Abstract
- Article
- References
- Cited By
- cytogenetics;
- diploidization;
- evolution;
- FISH (fluorescent in-situ hybridization);
- GISH (genomic in-situ hybridization);
- Nicotiana;
- phylogenetics;
- Repandae
- • Here, we analyze long-term evolution in Nicotiana allopolyploid section Repandae (the closest living diploids are N. sylvestris, the maternal parent, and N. obtusifolia, the paternal parent). We compare data with other more recently formed Nicotiana allopolyploids.
- • We investigated 35S and 5S nuclear ribosomal DNA (rDNA) chromosomal location and unit divergence. A molecular clock was applied to the Nicotiana phylogenetic tree to determine allopolyploid ages.
- • N. tabacum and species of Repandae were c. 0.2 and 4.5 Myr old, respectively. In all Repandae species, the numbers of both 35S and 5S rDNA loci were less than the sum of those of the diploid progenitors. Trees based on 5S rDNA spacer sequences indicated units of only the paternal parent.
- • In recent Nicotiana allopolyploids, the numbers of rDNA loci equal the sum of those of their progenitors. In the Repandae genomes, diploidization is associated with locus loss. Sequence analysis indicates that 35S and 5S units most closely resemble maternal and paternal progenitors, respectively. In Nicotiana, 4.5 Myr of allopolyploid evolution renders genomic in situ hybridization (GISH) unsuitable for the complete resolution of parental genomes.
The evolution of many, perhaps all, angiosperms, has involved polyploidy. Sequence data have indicated that even some apparently 'diploid' species are palaeopolyploids (Lagercrantz & Lydiate, 1996), for example, the 'diploid'Arabidopsis thaliana (L.) Heynh., with its small genome and only five chromosomes, may be a palaeohexaploid (Vision et al., 2000; Bowers et al., 2003). Realization of the importance of polyploidy in angiosperm evolution has stimulated considerable interest in the genetic consequences of this phenomenon, both in natural and in newly synthesized polyploids. Subsequent research has led to conflicting results, such as the rapid divergence of amplified fragment length polymorphisms (AFLPs) in newly synthesized Triticum allopolyploids, but not in Gossypium allopolyploids. In natural allopolyploids, there is evidence of sequence homogenization of 35S rDNA in Gossypium (Malvaceae), Nicotiana (Solanaceae) and Glycine (Fabaceae), but not in Brassica and Arabidopsis[both Brassicaceae, reviewed in Leitch & Bennett (1997), Liu & Wendel (2003) and Soltis et al. (2004a)].
One reason for the lack of consensus is that the genomic consequences of changes in ploidy may vary as a result of the different ages of the polyploids being studied, which may obscure changes that would otherwise be commonly associated with polyploidy. Leitch & Bennett (2004) observed that 'genome downsizing' is a general long-term response to polyploidy in many plant groups, whereby a polyploid genome may, over time, become ever more 'diploid-like' in character. The long-term genetic consequences of allopolyploidy and processes by which genomes become 'diploidized' are little understood and studied. Here we address these topics by using the polyploid Nicotiana section Repandae (Solanaceae). We take advantage of well-supported phylogenetic trees of the genus (Chase et al., 2003; Clarkson et al., 2004) to predict the closest living diploid progenitors of Repandae and ages of Nicotiana polyploids and sections. We compare our understanding of long-term genome evolution since Repandae origin with shorter-term evolutionary outcomes previously reported for allopolyploid N. tabacum (cultivated tobacco 2n = 4x = 48).
Section Repandae consists of four allopolyploid species –N. nudicaulis, N. repanda, N. stocktonii and N. nesophila (Knapp et al., 2004); all are 2n = 4x = 48. Goodspeed (1954) recognized in Repandae only the last three, with the morphologically divergent N. nudicaulis (see Fig. 3A,B in Knapp et al., 2004) in a monotypic section Nudicaules. Phylogenetic analyses based on internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (rDNA) (Chase et al., 2003) and plastid sequences (Clarkson et al., 2004) clearly show that Goodspeed's (1954) monotypic section Nudicaules is closely related to Repandae and descended from a unique common ancestor shared with the three species of Repandae. DNA sequence analysis also indicated that an ancestor of N. sylvestris (the sole member of section Sylvestres) was the maternal genome donor of Repandae (Clarkson et al., 2004) and that an ancestor of section Trigonophyllae was the other parent (Clarkson et al., unpublished). Section Trigonophyllae consists of a single species, N. obtusifolia (previously known as N. trigonophylla; Knapp et al., 2004). A second species in this section, N. palmeri, has also been described, but it is so closely related to N. obtusifolia that there were no differences detected in 4656 base pairs (bp) of plastid DNA (Clarkson et al., 2004); the type of N. palmeri falls within the range of variation of N. obtusifolia and has previously been synonymized with it (Wells, 1960). In any case, it would have been a shared common ancestor of both of these that was the parent of section Repandae. Therefore, for practical reasons we use, in the present study, N. sylvestris and N. obtusifolia as the diploid species most closely related to section Repandae.
Section Repandae has a disjunct natural distribution, with the greatest species diversity occurring in Mexico. Nicotiana repanda is the most widespread species (it is found in Texas, Mexico and Cuba). Nicotiana nudicaulis is found in north-eastern Mexico. The remaining two species (N. stocktonii and N. nesophila) are closely related and are found in the Revillagigedo Islands. These three Pacific islands (San Benedicto, Socorro and Clarion) are of volcanic origin from the deep sea Mathematician's Ridge on the Pacific Plate (Allan & Nelson, 1991) and have their origins in the Clarion Fracture Zone. They lie far from the coast, approx. 300 km west of Colima, Mexico, and 350 km south-south-west of Baja California, Mexico, and are still volcanically active. They have been called the Galápagos of Mexico because of their volcanic origin, isolation and endemic faunal and floral elements (Brattstrom, 1990). Phylogenetic analyses revealed that Repandae have their origin in a single hybridization event, subsequently followed by a radiation leading to the four extant species in the section (Clarkson et al., 2004). Nicotiana nudicaulis is sister to the rest of the section and highly genetically as well as morphologically divergent from the other three, which share a much more recent common ancestor (Chase et al., 2003; Clarkson et al., 2004).
Allopolyploids in Nicotiana range from recently formed (including artificially created crosses) to relatively ancient, natural species (Goodspeed, 1954). We aim to determine the ages of allopolyploids within the genus based on their sequence divergence from extant relatives of their progenitor diploids. Conflict exists in the literature about the age of the genus Nicotiana. Uchiyama et al. (1977) estimated that the genus originated 75–100 Myr ago, but the deeper split between Nicotiana and the genus Petunia (also Solanaceae) has subsequently been dated at only 23–25 Myr (Wikström et al., 2001).
Within Nicotiana, two potential dating points can be used to calibrate our molecular phylogenetic trees. Both rely on published dates at which a particular volcanic island rose above sea level. This type of calibration point puts a maximum age on species (Richardson et al., 2001) because speciation of an endemic species could have taken place after the island formed. Nicotiana cordifolia is endemic to the Juan Fernández Islands (or Robinson Crusoe Islands), Chile, and known only from the island of Masafuera. The two principal islands of the archipelago are Masatierra, which is approx. 3.8–4.2 Myr old and closest to the Chilean mainland, and Masafuera, which is approx. 1–2.4 Myr old (Stuessy et al., 1984; Sang et al., 1995). Divergence of N. cordifolia from its mainland (Chilean) sister taxon N. solanifolia can therefore be dated. Nicotiana stocktonii and N. nesophila are endemic to the Revillagigedo Islands, which are between recent (San Benedicto, which was devastated by an eruption in 1952, see Sanchez Rubio, 1981) and approx. 1.1 Myr old (Clarion) or slightly older (Duncan & Clague, 1985). Their divergence from the mainland (Mexican) sister taxon N. repanda can therefore also be dated.
A limited number of Nicotiana species (primarily diploid, 10 in total) have previously been compared, in a phylogenetic context using nontranscribed spacer sequences (NTS) of 5S rDNA (Kitamura et al., 2001), but no species of Repandae were included in that analysis. Here we analyze these sequences from the species of section Repandae. We also use Southern hybridization and fluorescence in situ hybridization (FISH) to determine the organization and distribution of 5S and 35S rDNA in Repandae and the modern diploid relatives of their progenitor lineages.
These data are also compared with patterns of divergence in more recent allopolyploid species, N. tabacum, N. rustica and N. arentsii, which, like Repandae, are all 2n = 4x = 48 [reviewed in Kovarik et al. (2004) and in Lim et al. (2004)]. These three more recently formed allopolyploids (based on their lower levels of sequence divergence in analyses of plastid DNA data) all show some or complete sequence homogenization of 35S rDNA sequences, and each has 5S and 35S rDNA loci numbers that are equal to the sum of their progenitor diploids. Nicotiana tabacum is formed from a cross involving something close to N. sylvestris, the maternal S-genome donor (like Repandae) and N. tomentosiformis, the T-genome donor (Goodspeed, 1954; Kitamura et al., 2000; Chase et al., 2003; Murad et al., 2005). Genomic in situ hybridization (GISH) clearly identifies the parental S and T genomes (Parokonny et al., 1992; Kenton et al., 1993; Lim et al., 2000b). In synthetic tobacco, concerted evolution of 35S rDNA occurs rapidly and involves thousands of units that become altered in only four generations (Skalicka et al., 2003). These young synthetic tobacco plants also show evidence for preferential loss of repetitive sequences from the paternal genome (Skalicka et al., 2005). A similar phenomenon was previously reported from restriction fragment length polymorphisms (RFLPs) in synthetic Brassica allotetraploids. In Brassica, the preferential loss of RFLPs of paternal origin was ascribed to adverse interactions of the paternal genome in a maternal cytoplasm (the nuclear cytoplasmic interaction hypothesis). We compare some of these data with the long-term consequences of allopolyploidy on genome evolution in Repandae.
Materials and Methods
DNA sequencing
For DNA extractions, 1 g of fresh tissue or 0.3 g of silica-gel dried material was used. All accessions are vouchered at the Natural History Museum, London (BM; Table 1).
|Voucher details
|GenBank accession
|Nicotiana alata Link & Otto
|Helgason & Monro 501, 518 BM
|Nicotiana attenuata Torrey ex S. Wats.
|Helgason & Monro 621 BM
|Nicotiana bonariensis Lehm.
|Helgason & Monro 622 BM
|Nicotiana cordifolia Phil.
|Saikia 008 BM
|Nicotiana linearis Phil.
|Helgason & Monro 609 BM
|Nicotiana miersii J. Rémy
|Clarkson 003 BM
|Nicotiana nesophila I.M. Johnst.
|Saikia 011 BM
|Nicotiana noctiflora Hook.
|Lim 005 BM
|Nicotiana nudicaulis S. Wats.
|Saikia 004 BM
|Nicotiana obtusifolia M. Martens & Galeotti
|Lim 004 BM
|Nicotiana otophora Griseb.
|Nicotiana palmeri A. Gray
|Helgason & Monro 631 BM
|Nicotiana petunioides (Griseb.) Millán
|Lim 001 BM
|Nicotiana repanda Willd.
|Clarkson 019 BM
|Nicotiana stocktonii Brandegee
|Lim 003 BM
|Nicotiana sylvestris Speg. & Comes
|Helgason & Monro 628 BM
|Nicotiana tomentosiformis Goodsp.
|Nicotiana wigandioides Koch & Fintelm.
|Nee et al. 51764 BM
DNA was extracted by using a modified 2× cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1987). DNA was precipitated in chilled ethanol (−20°C) for at least 24 h and then resuspended in 1.55 g ml−1 cesium chloride/ethidium bromide. Samples were then purified using a density gradient, followed by removal of the ethidium bromide and cesium chloride with butanol/dialysis and then storage in Tris–EDTA (TE).
Target regions were amplified in a Gene Amp™ 9700 polymerase chain reaction (PCR) system (ABI; Applied Biosystems, Inc., Warrington, Cheshire, UK), using ReddyMix PCR Mastermix, at 2.5 mm MgCl2 (ABgene, Epsom, Surrey, UK). Details of primers were as described previously (Matyasek et al., 2002). The PCR program consisted of 4 min at 94°C followed by 28 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C. Dimethylsulphoxide (DMSO) (5%, v/v) was used in both PCR and cycle sequencing reactions to remove the effects of secondary structure caused by a high GC content, which makes primer annealing and sequencing difficult (see Chase et al., 2003 for more details). PCR products were cleaned on Nucleospin® miniprep columns (Macherey–Nagel, Duren, Germany) following the manufacturer's protocols. Samples were sequenced on an ABI 3100 capillary DNA sequencer using Big Dye terminator v3.1 chemistry, following the manufacturer's protocols (ABI). For cleaning, we used precipitation in ethanol, etc.
The raw sequences were edited and assembled using sequencher version 4.1 (Gene Codes, Inc., Ann Arbor, MI, USA). They were aligned by eye following the guidelines provided by Kelchner (2000). Gaps were coded as missing data; they were thus excluded from the analysis. We did, however, survey their distribution after the analysis to determine whether their inclusion would provide improved resolution or bootstrap support, but in no case were they useful in this way; they routinely marked clades for which bootstrap support was already high, based simply on analyses of nucleotide substitutions alone.
Parsimony analysis
paup, version 4.0b (Swofford, 2001), was used for parsimony analysis. Eighteen accessions were included in searches (16 of them new in this study), and 180 bp had to be excluded from the sequences as a result of ambiguous alignment. Branch-and-bound searches were performed, and all character transformations were treated as equally likely and unordered (Fitch, 1971). DELTRAN character optimization was used to illustrate branch lengths throughout (owing to reported errors with ACCTRAN optimization in paup version 4.0b). Nicotiana tomentosiformis and N. otophora (representatives of section Tomentosae) were designated as outgroups. Section Tomentosae has consistently been shown to be a sister to the rest of Nicotiana (Chase et al., 2003; Clarkson et al., 2004). These two sequences were downloaded from GenBank (see Table 1 for accession numbers). To assess internal support, 100 bootstrap replicates (Felsenstein, 1985) were performed by using the branch-and-bound algorithm with equal weights. We follow the new sectional classification of Nicotiana throughout (Knapp et al., 2004), rather than that of Goodspeed (1954).
Molecular clock methods
Our previously published combined plastid and ITS data set (Clarkson et al., 2004) was used for the dating exercise because this is the most robust analysis obtained, to date, for the genus (judged by bootstrap percentages). This data set encompassed only diploid taxa and so we had to add the four allopolyploids of Repandae and N. tabacum. Both data sets are congruent for the phylogenetic position of the Repandae species because the ITS region has been converted in all species to the maternal type (the same parent as the plastid donor). However, for N. tabacum, the ITS sequence had to be excluded because the two data sets are incongruent. Nicotiana tabacum inherited its plastid genome from its maternal parent (N. sylvestris), and the ITS has been converted to that of its paternal parent (N. tomentosiformis; Lim et al., 2000a; Chase et al., 2003). As N. tabacum and N. tomentosiformis have identical ITS sequences, the removal of these data will not affect the overall estimation of its time of origin. After the addition of the five extra allopolyploids to the combined data set, analysis produced just a single most-parsimonious tree (the same number as yielded by the original published analysis). The relative rates test (Felsenstein, 1981) showed that divergence was not proportional to time. Therefore, nonparametric rate-smoothing (NPRS) (Sanderson, 1997) was used to smooth out rate heterogeneity within this tree. Trees were calibrated using 2.4 Myr for the split between N. solanifolia and N. cordifolia because this is the more divergent, and therefore presumably the more reliable, of the two potential calibration points (see Richardson et al., 2001). Scaling the tree according to the longer of the two available branches reduces the effect of errors. By using this approach, one calibration point can be used to see how well it corroborates the other.
Plant material and chromosome preparations
Root tips of N. repanda, N. stocktonii, N. nudicaulis, N. nesophila, N. sylvestris and N. obtusifolia (Table 1) were used for FISH. Root tips from pot-grown plants were analyzed following pretreatment with a saturated aqueous solution of Gammexane® (hexachlorocyclohexane; Sigma Aldrich, Poole, UK) for 4 h. All root-tips were fixed in absolute ethanol : glacial acetic acid (3 : 1, v/v) for longer than 1 wk. Chromosome squashes were prepared following the enzymatic softening of material, as described previously (Leitch et al., 2001).
Probes for FISH
- 1Nicotiana sylvestris total genomic DNA was labeled by nick translation with digoxigenin-11-dUTP (Roche Biochemicals, Lewes, UK), and N. obtusifolia total genomic DNA was labeled by nick translation with biotin-16-dUTP (Sigma Aldrich), according to Leitch et al. (2001).
- 2The entire plasmid containing pTa71 [isolated by Gerlach & Bedbrook (1979)], which includes the 18-5.8-26S rDNA subunits and the intergenic spacer isolated from Triticum aestivum, was labeled with digoxigenin-11-dUTP by nick translation.
- 3The coding portion of the 5S region (approx. 120 bp) was amplified from N. rustica by following the PCR program and primers described by Fulnecek et al. (1998). Biotin-16-dUTP was used to PCR label the sequence, according to the methods of Schwarzacher & Heslop-Harrison (2000), by using ×10 concentrations of dNTPS and dUTP. Nicotiana sylvestris was probed with pTZ-19R [5S rDNA; Venkateswarlu et al. (1991)].
FISH was carried out as described in Leitch et al. (1994), with modifications as described in Lim et al. (1998). Briefly, slides were pretreated with 100 µg ml−1 RNase A for 1 h and then with 0.25 µg ml−1 pepsin for 5 min, followed by denaturation in 70% (v/v) formamide in ×2 SSC (0.3 m sodium chloride, 0.03 m sodium citrate), at 70°C for 2 min. The hybridization mix contained 50% (v/v) formamide, 10% (w/v) dextran sulphate and 0.1% (w/v) sodium dodecyl sulphate in ×2 SSC. For GISH, the hybridization mixture contained 8 µg ml−1 digoxigenin-labeled N. sylvestris DNA and 8 µg ml−1 biotin-labelled N. obtusifolia DNA. A concentration of 4 µg ml−1 DNA was used in the mixture when using digoxigenin-labelled pTa71 and biotin-labelled 5S rDNA PCR product (or pTZ19-R). After overnight hybridization at 37°C, slides were washed in 20% (v/v) formamide in 0.1 × SSC at 42°C at an estimated hybridization stringency of 80–85%. Sites of probe hybridization were detected by using 20 µg ml−1 fluorescein-conjugated anti-digoxigenin IgG (Roche Biochemicals) and 5 µg ml−1 Cy3-conjugated avidin (Amersham Biosciences, Little Chalfont, UK). Chromosomes were counterstained with 2 µg ml−1 4′,6-diamidino-2-phenylindole (DAPI) in 4 × SSC, mounted in Vectashield medium (Vector Laboratories, Peterborough, UK), examined using a Leica DMRA2 epifluorescence microscope (Wetzlar, Germany) and photographed with an Orca ER camera (Hamamatsu Photonics, Welwyn Garden City, UK). Images were processed for colour balance, contrast and brightness uniformity.
Restriction endonuclease digestion and Southern hybridization
Genomic DNA was extracted from the young leaves of these plants by using the method of Saghai-Maroof et al. (1984) with modifications according to Kovarik et al. (1996). The methods used were those described in Sambrook et al. (1989). DNA samples were digested to completion with excess restriction endonuclease and fractionated in 1% (w/v) agarose by gel electrophoresis. DNA was transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech., Little Chalfont, UK). Southern hybridization was carried out under high-stringency conditions (Fulnecek et al., 2002) by using a heat-denatured 32P-labeled rDNA probe that was either (i) a 1.7-kb EcoRI fragment of the 18S rRNA gene subunit from Solanum lycopersicum L. (tomato, accession number X51576; Kiss et al., 1989a) or (ii) a 220-bp fragment of the 26S rRNA gene subunit from S. lycopersicum (accession number X13557; Kiss et al., 1989b) obtained by PCR amplification of the region between +2901 (5′-GAATTCACCCAAGTGTTGGGAT-3′) and +3121 (5′-AGAGGCGTTCAGTCATAATC-3′) with respect to the start of the 26S rDNA subunit (Kiss et al., 1989b). Labeling of DNA probes was carried out by a random primer method using [32P]dCTP (Dekaprime™ kit; Fermentas, Vilnius, Lithuania).
NTS sequences
The NTS region of 5S rDNA has a high frequency of informative positions, which more than makes up for its short length (358 bp included in analysis), producing well-resolved trees (Fig. 1). The total number of characters was 358, of which 188 (53%) were variable and 128 (36%) were potentially parsimony informative. The percentage of parsimony-informative characters (36%) is favorable when compared to other data sets for Nicotiana, for example 27% for ITS (Chase et al., 2003) and only 8.8% for the plastid data set (Clarkson et al., 2004). Analysis produced one most-parsimonious tree [length 277 steps; consistency index (CI) 0.87; retention index (RI) 0.88]. The tree presented in Fig. 1 has branch lengths above (DELTRAN optimization) and bootstrap percentages (BP) below the branches.
Sections Tomentosae (BP 100), Trigonophyllae (BP 100), Repandae (BP 67), Noctiflorae (BP 100), Alatae (BP 100), and Petunioides (BP 89) all form clades. Sections Paniculatae and Undulatae together form a moderately supported clade (BP 85). Section Sylvestres is sister to section Noctiflorae (BP 86). Section Tomentosae was specified as the operational outgroup, based on other evidence (Chase et al., 2003; Clarkson et al., 2004).
Molecular clock
The node that separates N. stocktonii and N. nesophila from N. repanda (second potential calibration point – see Fig. 2) was dated at 1.0 Myr using the rate-smoothed molecular data set. This is consistent with the geological data that predicted it to be approx. 1.1 Myr. The divergence time between Repandae and its maternal progenitor (N. sylvestris) is dated at 4.5 Myr. Nicotiana tabacum originated within the last 0.2 Myr. The genera Nicotiana and Symonanthus (tribe Anthocercideae) are sister groups (Clarkson et al., 2004) that split approx. 15.3 Myr ago (data not shown). Dates from the rate-smoothed tree not pertaining to the discussion of evolution in Repandae will be dealt with elsewhere (J. Clarkson et al., in preparation).
35S and 5S rDNA chromosome distribution
FISH analyses of mitotic metaphases of N. sylvestris reveal three subtelomeric rDNA loci on the short arms of chromosomes 10, 11 and 12, and one 5S rDNA locus interstitial to the long arm of chromosome 8 (Fig. 3a; Lim et al., 2000b). There is a similar distribution of signal in N. obtusifolia (2n = 24), although the 5S DNA locus has an interstitial position on a short-armed chromosome (Fig. 3b). We might therefore have predicted that all members of section Repandae (2n = 4x = 48) would have all of these FISH signals, but N. nudicaulis (Fig. 3c), N. nesophila (Fig. 3e) and N. stocktonii (Fig. 3f) have two and N. repanda (Fig. 3d) has three 35S rDNA loci subtelomeric to the short arm, a situation that resembles many of the diploid species within the genus. Likewise, on a separate chromosome, all four Repandae species have only a single 5S rDNA locus on the short arm, although its precise chromosomal location varies among these species (Fig. 3).
Southern blot hybridization
Southern hybridization to BstNI-restricted DNA revealed that all section Repandae species had one predominant 5S rDNA gene family (Fig. 4a). Three of the species of the group (N. stocktonii, N. repanda, and N. nudicaulis) and N. sylvestris have an indication of a minor band that may represent a low-abundance family of 5S rDNA units. Sizes of the predominant 5S rDNA family were 430 bp for N. sylvestris, as reported by Fulnecek et al. (2002), 600 bp for section Trigonophyllae and 500 bp for all species in section Repandae.
Southern hybridization analysis of BstNI-restricted DNA, by using the 26S rDNA probe, a method that predominantly reveals polymorphism in the intergenic spacer (IGS), demonstrates that there are three families of 35S rDNA units in N. sylvestris with bands of 2.4, 2.7 and 3.0 kb (Fig. 4b), as reported previously (Lim et al., 2000a). Similarly, restricted DNA of N. obtusifolia (and plants identified as N. palmeri) generates two or three bands that represent distinctive families of 35S rDNA units. No bands in section Repandae species precisely match those from the extant descendents of the progenitor diploids (Fig. 4b).
GISH was conducted on all four species in section Repandae by using labeled N. sylvestris and N. obtusifolia genomic DNAs as probes. In N. repanda, N. stocktonii and N. nesophila, there was no discrimination of the parental origin of chromosomes, although there were scattered dots with the N. sylvestris probe and an interstitial band on a large metacentric chromosome with the N. obtusifolia probe (shown for N. nesophila in Fig. 3g). However, there was some discrimination in N. nudicaulis (Fig. 3h). We attempted to construct a karyotype of GISH-labelled N. nudicaulis metaphase chromosomes in order to differentiate chromosomes of potential N. sylvestris origin and of N. obtusifolia origin. This proved impossible for at least 10 of the 48 chromosomes of the metaphase set owing to blending of the signal. Nevertheless, the majority of the chromosomes did appear to have distinctive genome-specific labeling.
The evolution of polyploid genomes
There is considerable interest in the genetic consequences of polyploidy on gene expression, genome evolution and speciation. Research, to date, has concentrated on three aspects: (1) the immediate consequences of allopolyploidy, with work derived from synthetic allopolyploids resembling natural species (Skalicka et al., 2003; Adams et al., 2004; Joly et al., 2004; Levy & Feldman, 2004; Lukens et al., 2004; Wang et al., 2004); (2) the role of polyploidy in the establishment and evolution of young (less than 150-yr-old) species (Abbott & Lowe, 2004; Ainouche et al., 2004; Soltis et al., 2004b); and (3) the comparison of established polyploid species (thousands or millions of years old) in relation to the closest living diploids (cf. Leitch & Bennett, 1997; Liu & Wendel, 2003; Osborn et al., 2003; Levy & Feldman, 2004; Soltis et al., 2004a). Here we examine a fourth aspect – the divergence of polyploid taxa long after an ancient (c. 4.5 Myr ago) polyploid event.
Finding genetic events that are a unifying response to de novo allopolyploidy is proving difficult, and possibly no event is ubiquitous to all cases, but the existing data point to a number of important features: (1) genetic change in polyploids can occur rapidly (within one or a few generations); (2) changes can influence a large range of sequences (from genes to repeats), but no type of sequence can be expected to change; (3) patterns of change are usually variable between sibling lines; and (4) changes can appear stochastic (e.g. Arabidopsis suecica) or targeted (e.g. N. tabacum, T. aestivum).
In established polyploid species (thousands of years old), genetic change generated by polyploidy is almost certainly fixed, and genetic patterns observed are a combination of this fixed change and subsequent genome divergence. Distinguishing between the two is difficult and requires knowledge of phylogenetic divergence in the diploids and polyploids alike. We aimed to be able to distinguish allopolyploid-induced change from genome divergence by taking advantage of ancient polyploidy and speciation in Repandae. Those genetic patterns that are in common between species represent features of the genome in the common polyploid ancestor. Those features that are different reflect divergence since allopolyploid fixation.
Phylogenetic relationships between diploid Nicotiana species
The Nicotiana species chosen in this analysis were selected to represent at least one species from each of the diploid sections (sensuKnapp et al., 2004). In addition, all species in allopolyploid section Repandae were analyzed. In previous analyses, it has been difficult to establish the relationship between N. sylvestris (the sole member of section Sylvestres) and other diploids owing to a lack of informative variation in the DNA regions analyzed. The most robust 'diploids-only' phylogenetic analysis to date, which combined both plastid and ITS sequences (Clarkson et al., 2004), weakly supported N. sylvestris as sister to Alatae (BP 72). However, N. sylvestris has the same chromosome number as section Noctiflorae (n = 12), not Alatae (n = 9,10). Our NTS data resolve N. sylvestris as sister to section Noctiflorae (BP 86). Sections Noctiflorae, Alatae and Petunioides all form a well-supported clade (BP 85), as in the combined ('diploids only') analysis (Clarkson et al., 2004). However the inter-relationships between the three sections are different here, with sections Petunioides and Alatae being sister taxa (see Fig. 1), rather than Noctiflorae and Alatae (see Fig. 3 of Clarkson et al., 2004).
Node ages in Nicotiana divergence
After rate smoothing, our data set is internally consistent: one independent calibration point corroborates the other. The age of the split between Nicotiana and Symonanthus (15.3 Myr ago) is in conflict with the results of Uchiyama et al. (1977), who estimated the age of the genus Nicotiana to be 75–100 Myr, based on protein composition analysis. However, these data are, in general, consistent with the more recent findings of Wikström et al. (2001), who dated the split between Nicotiana and Petunia at 23–25 Myr by using DNA sequence data calibrated with one fossil calibration point. These two dates fit together well because Anthocercideae are sister to Nicotiana, and Petunia is a yet more-distant relative (Olmstead et al., 1999). These data show that the hybridization event leading to the formation of N. tabacum occurred less than 0.2 Myr ago. Okamuro & Goldberg (1985) estimated that N. tabacum was 6 Myr old, based on differences between stretches of single-copy DNA in N. tabacum and its two parents. This date seems an extreme overestimation of the age of the species, in view of the fact that we observed no differences between the ITS sequence of N. tabacum and its paternal progenitor (Chase et al., 2003) and only one substitution between N. tabacum and its maternal progenitor in over 4 kb of plastid DNA (Clarkson et al., 2004). In cases in which single-copy nuclear genes have been sequenced for N. tabacum and its progenitor diploids, copies corresponding almost exactly to both progenitors are found in N. tabacum, supporting its recent formation [e.g. putrescine N-methyltransferase (Riechers & Timko, 1999) and heterotrimeric GTP-binding protein (Takumi et al., 2002)]. In addition to this, the chromosome characteristics of N. tabacum are still physically similar to those of its two progenitor diploids, including repeat sequences and integrated viral elements (Lim et al., 2000b; Murad et al., 2002).
Genome evolution in Repandae
Glutamine synthetase (J. Clarkson et al., unpublished) and plastid DNA sequences (Clarkson et al., 2004) have revealed that the closest living relatives of the species of section Repandae are N. obtusifolia (section Trigonophyllae) and N. sylvestris (section Sylvestres), the latter being the maternal genome donor.
GISH labeling of N. nudicaulis with N. sylvestris and N. obtusifolia total genomic DNA revealed some discrimination in label distribution, but entire genome sets could not be readily distinguished. Nevertheless, it was clear that there is discrimination as to genome origin for some chromosomes (Fig. 3). In contrast, no other species in Repandae revealed discrimination between parental chromosome sets and only hybridize weakly across all chromosomes with the N. sylvestris genomic DNA probe and at a large interstitial locus with the N. nudicaulis probe (see Fig. 3). The likely maximum age of section Repandae can be calculated by using the calibrated node on the rate-smoothed tree that separates the mainland N. repanda from its island relatives. We estimate the age of section Repandae to be approx. 4.5 Myr (see Fig. 2). Our results show that these species are old enough to be beyond the point at which GISH is useful in Nicotiana as a method to distinguish parental genomes of allopolyploids.
In more recently formed allopolyploids of Nicotiana (e.g. N. tabacum, N. rustica and N. arentsii), GISH is a highly effective way to discriminate parental genomes (reviewed in Lim et al., 2004). Loss of GISH discrimination over 4.5 Myr of Repandae evolution has presumably arisen as a consequence of diploidization processes that might include genome downsizing (Leitch & Bennett, 2004), sequence mobility (e.g. retroelement, transposon mobility) and sequence divergence, in both allopolyploids and extant diploid species derived from progenitor lineages. All allopolyploid species of Repandae have a common origin, but they differ in the effectiveness of GISH, with N. nudicaulis retaining the greatest overall similarity to progenitor diploids. This differential response among the species of section Repandae indicates that divergence in the allopolyploids has played more of a role in the efficacy of GISH than divergence of the diploid progenitor lineages. At least in Nicotiana, 4.5 Myr appears to be beyond the point at which clear resolution is possible.
5S rDNA evolution in Repandae
Direct NTS sequencing from PCR products confirmed that sections Repandae (BP 67) and Trigonophyllae (BP 100) are sister groups (BP 96; Fig. 1). However, direct sequencing in allopolyploids cannot detect rare classes of rDNA types if present at less than 5% of the predominant type (Rauscher et al., 2002). To confirm that we had good representation of the whole NTS population, we cloned and sequenced a further 10 NTS units per Repandae species (data not shown). All sequences showed the highest similarity to N. obtusifolia, the closest extant relative to the paternal genome parent. In N. sylvestris, the 5S rDNA locus was localized to an interstitial region on the long arm of a small metacentric chromosome. In N. obtusifolia, the locus occurs in subtelomeric positions on the short arm of a metacentric chromosome. In Repandae, the 5S locus occurs in subtelomeric positions, consistent with their N. obtusifolia origin (see Fig. 3 and Table 2). It is likely that one locus of maternal genome origin has been in lost in the species of Repandae. Perhaps reduction in locus number and loss of N. sylvestris-like units is associated with genome diploidization processes.
|Nicotiana species
|Haploid chromosome number (n)
|Number of loci
|N. sylvestris
|N. obtusifolia
|N. stocktonii
|N. nesophilla
|N. repanda
|N. nudicaulis
Southern hybridization was used to reveal the entire population of 5S rDNA units. There is a near-identical hybridization pattern in all species of section Repandae with a predominant 5S rDNA band at 0.50 kb (Fig. 4). All species of section Repandae except for N. nudicaulis also have a faint minor band at 0.60 kb. This band is of similar size to the predominant band in section Trigonophyllae (N. obtusifolia, including N. palmeri). It is therefore possible that a few, unaltered Trigonophyllae units remain in section Repandae. The predominant N. sylvestris band is at 0.43 kb. The major family of 5S rDNA in section Repandae is dissimilar in size to both species likely to be the closest extant descendents of the two parents. However, the sequence of the predominant NTS type does indicate similarity to section Trigonophyllae.
It is likely that the 5S units in section Repandae are of Trigonophyllae origin, but there has been divergence in Repandae and Trigonophyllae since allopolyploid formation. This divergence must have been associated with inter- and intralocus homogenization because the vast majority of units in each species is identical in size. The uniformity of the 5S rDNA units across the four species of section Repandae is a surprise given that divergence of the diploid species of section Tomentosae is associated with the evolution, amplification and deletion of an array of 5S rDNA families (Matyasek et al., 2002). We suggest that the common ancestor of Repandae already possessed a repeat of this size before speciation took place or that it was inherited from a parental lineage that has subsequently diverged. It is unlikely that all four species could have independently developed repeats of the same size.
The patterns of 5S rDNA evolution in the allopolyploid section Repandae are different from those of the similarly allopolyploid N. tabacum (c. 0.2 Myr) in which 5S units of both N. sylvestris and N. tomentosiformis origins remain intact and occur at their expected chromosomal loci (Matyasek et al., 2002). Perhaps the different ages of the allopolyploids are an important factor. The differences between N. tabacum and the species of section Repandae are reflected elsewhere. For example, no 5S rDNA loci loss or interlocus sequence homogenization is observed in the 5S arrays of Gossypium allopolyploids (Cronn et al., 1996). These allopolyploids are approx. 1.5 Myr old and formed from parental diploids that are separated by 6.7 Myr of evolution (Senchina et al., 2003). However, in Glycine, Krishnan et al. (2001) found 5S rDNA locus loss in putatively recently formed polyploids. They showed that, as in Repandae, Glycine allotetraploids [e.g. G. tabacina (Labill.) Benth.] have been reduced to the same locus number as their 'diploid' progenitors.
It has been proposed that sequences located near chromosome ends are more prone to genetic change (e.g. gene conversion, elimination, translocation) than sequences at other positions (Kovarik et al., 2004). However, here in Repandae this does not explain the observed locus loss because it is the subtelomeric 5S rDNA of N. obtusifolia origin that has been retained, whereas the more proximal locus of N. sylvestris origin has been lost.
35S rDNA evolution in section Repandae
Three 35S rDNA loci can be detected in each of the two extant species most closely related to the progenitor diploid species of section Repandae. Therefore, we might expect six loci in allopolyploid Repandae, but species have either two or three 35S rDNA loci (Table 2). Chase et al. (2003) previously published a phylogenetic tree based on nuclear ITS sequences of Nicotiana. Here the tree is reproduced for the species studied in this work (Fig. 5). As noted by Chase et al. (2003), ITS sequences of Repandae are similar to those of N. sylvestris, which is the species most closely related to their maternal diploid progenitor. Southern hybridization reveals the gross unit structure for all populations of 35S rDNA. All species examined had a unique profile of bands, with the exception of N. stocktonii and N. nesophila, which are closely related species (i.e. they have only a few differences in their DNA sequences). However, the sequence similarity of repeats in each Repandae species indicates that there has been sequence divergence in sections Repandae and/or Sylvestres, sequence homogenization and locus loss. Previously we have shown in the allopolyploids N. tabacum, N. rustica and N. arentsii that the parental origin of the 35S rDNA loci does not seem to influence the direction of sequence homogenization (Kovarik et al., 2004). In section Repandae, inheritance/maintenance of 5S unit types that are similar to the paternal Trigonophyllae parent and of 35S units typical of the maternal Sylvestres parent (Fig. 5) demonstrate that inheritance patterns of these two different rDNA loci are not linked.
The more recently formed allopolyploids –N. tabacum, N. arentsii and N. rustica– have the sum and location of the 35S units that would be expected by direct combination of their progenitor genomes. However, these units have been overwritten, at least to some extent, by one parental type (concerted evolution), summarized previously (Kovarik et al., 2004). In Nicotiana, this situation can be viewed as the 'first level' of diploidization of 35S rDNA loci. In section Repandae, the number of rDNA loci is reduced, and this can be considered as a 'second level' of rDNA diploidization.
We would like to thank Linda Jenkins and John Sitch for the cultivation of plants used in this study. Thanks to Martyn Powell, Dion Devey and David Springate for technical support in the laboratory. This research was funded by the Natural Environmental Research Council and Grant Agency of the Czech Republic (award 521/04/0775).
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Mickey took turns to greet and hug the children and their families the hospital was transported in to the magical world of Disney.

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The yearlong celebration of "Opening Doors" will conclude with a finale event on Jan. 28 from 4:30 to 6 p.m. in the Hogan Campus Center Ballroom that will commemorate the strong legacy of women — past, present and future — at the College of the Holy Cross. The keynote address will be given by Ann McElaney-Johnson '79, Ph. D., president of Mount St. Mary's College and member of the Holy Cross Board of Trustees. The program will feature performances by the Delilahs, an all-female a cappella group, and the Rhythm Nation Steppaz, a co-ed step team, as well as recognition of alumnae from the previous four decades.
"When Holy Cross opened its doors to women in 1972, the College not only created opportunities for women to access the superior education that has always been the hallmark of the College, it also changed the lives of the men who sat in class with those women," said McElaney-Johnson. "We are always better when diverse minds come together as a community. I am proud of this commitment to an inclusive learning environment that educates all those who pass through these doors to be 'men and women for others, ' and I am excited and honored to be part of this celebration of coeducation at Holy Cross."
The College's 40th anniversary of coeducation celebration featured a robust lineup of events throughout the 2012-13 academic year. Students, faculty, staff, and alumni/ae have had — and will continue to have — extensive opportunities to reflect on, acknowledge, and commemorate the moment in 1972 when the College opened its doors to women students for the first time. Events spanned all academic disciplines — from the arts to the sciences — and explored the impact of Holy Cross alumnae in fields ranging from business to medicine to civic life.
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A man in a wheelchair detonated a homemade bomb at Beijing International Airport, injuring himself but no one else, China's state media says.
The explosion occurred at about 6:24 p.m. Beijing time on Saturday. State-run China Central Television says the wounded man was taken to a hospital after setting off the device and that no one else was injured, no flights were affected and order had been restored at the airport.
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Department Product Management Job Overview The Business Analytics Product Manger is responsible to translate business needs into end capabilities that differentiate Pentaho and provide the highest value to clients. The role will lead the success of Pentaho Analysis,3/10/2015 Orlando, FL Save This Job
About the Role Balihoo?s Customer Success team is evolving into a top-notch, customer-focused organization that embodies customer service, excellence in business-to-business program and relationship management, and strategic program design. This team plays a central role in our3/4/2015 Chicago, IL Save This Job
About ed? Mission: To transform shopping & saving Consumers love trying new things and saving money. But the world of saving is cluttered and impersonal ?from coupons to daily deals to mobile wallet technology -- deals lack relevancy and redemption takes a lot of work for both3/4/2015 Chicago, IL Save This Job
Job Description The Project Manager will have a dual role at HealthHelp. The Project manager will help manage projects for the data team, the implementation team, the software development team, and the infrastructure team. This includes coordinating the efforts of team members2/25/2015 Houston, TX Save This Job
Position Type: Full Time Position Location: San Francisco Job Description Insight products form the foundation of RadiumOne?s relationships with clients, partners, and the industry. We seek an experienced Product Manager to own all aspects of insight reports. Stakeholders2/22/2015 San Francisco, CA Save This Job
Position Type: Full Time Position Location: San Francisco Job Description Insight products form the foundation of RadiumOne?s relationships with clients, partners, and the industry. We seek an experienced Product Manager to own all aspects of insight reports. Stakeholders2/22/2015 San Francisco, CA Save This Job
General Overview: The Email Marketing Manager is responsible for managing execution of the Gaiamemail program. Primary responsibilities include all aspects of email operations from start to finish including analysis of the email program. The Email Marketing Manager will serve as2/22/2015 Boulder, CO Save This Job
Job Description Insight products form the foundation of RadiumOne?s relationships with clients, partners, and the industry. We seek an experienced Product Manager to own all aspects of insight reports. Stakeholders throughout the organization use these tools to analyze user2/22/2015 San Francisco, CA Save This Job
Job Description At Bullhorn, we put the world to work. Our international recruitment software delivers best-of-breed technology solutions for the world?s leading recruitment firms. With over 300,000 users, we work at a rapid pace to deliver an incredible customer experience. As a2/20/2015 Boston, MA Save This Job
With over 2 million verified small businesses and 20 million unique monthly visitors, Manta is the largest online community dedicated to small businesses. We allow small businesses to improve their online presence, gain visibility with customers and prospects, and increase sales.2/15/2015 Columbus, OH Save This Job
COMPANY BACKGROUND Welocalize, Inc., founded in 1997, offers innovative translation and localization solutions helping global brands to grow and reach audiences around the world in more than 125 languages. Our solutions include global localization management, translation, supply2/14/2015 Portland, OR Save This Job
Here is your opportunity to be part of one of the hottest upstarts in the Smart Grid industry. "EcoFactor answers one of the biggest concerns in the growing Smart Grid" ? GreenBeat ?The air conditioners have spoken: EcoFactor looks like the winner.? - Forbes EcoFactor is2/8/2015 Redwood City, CA Save This Job
Human Resources Manager Charlotte, North Carolina About Agility Agility Fuel Systems is the leading designer and producer of alternative fuel storage and delivery systems for heavy duty trucks, buses and specialty vehicles. Our systems can be fueled by compressed and liquid2/3/2015 Charlotte, NC Save This Job
Description The Manager, Support Services (MSS) provides exceptional end user support to all clients by managing the Help Desk functions and personnel. Exemplary client service, support, and management skills are essential for success in this position. The Support Service12/14/2014 Cleveland, OH Save This Job
Location:NE-Omaha Job Code:817 Description Ellie Mae (NYSE:ELLI) is a leading provider of enterprise on-demand solutions, including an online network, software and services for the residential mortgage industry. Ellie Mae is leading the mortgage industry into the future,3/25/2015 Omaha, NE Save This Job
Location:Chicago, IL - BMW Position #:800 DESCRIPTION JOB SUMMARY Logistics Manager responsible for the operational oversight and client relationships of multiple high profile global accounts. Manages the complete operation of complex global supply chains for the accounts3/23/2015 Chicago, IL Save This Job
CityOakland StateCA Description The Production Manager is responsible for the production of the 80,000+ meals a day that we provide to local schools. The Production Manager reports to the General Manager, and will lead a team of Cooks, Prep Cooks, Dishwashers and the Porter.3/23/2015 Oakland, CA Save This Job


text:
PARIS (AP) -What started as a replay of a U.S. Open nightmare turned into something much better for Roger Federer: a trip to the French Open semifinals.
He overcame a two-set deficit for the seventh time in his career Tuesday for a 3-6, 6-7 (4), 6-2, 6-0, 6-3 win over Juan Martin del Potro, who famously beat Federer in the final at Flushing Meadows in 2009, snapping his 40-match U.S. Open winning streak.
The third-seeded Federer's victory Tuesday pushed him to his record-tying 31st Grand Slam semifinal. He'll play Novak Djokovic or Jo-Wilfried Tsonga in the semifinals.
No. 9 seed del Potro said he'd need a nearly perfect day to reprise his upset over Federer from 2009, and for two sets, he did it. But Federer loosened up and rallied - including a steamroller fourth set in which he hit 13 winners and no unforced errors - and earned his record-extending 237th Grand Slam match win.
"Letting go helped me," Federer said. "I pushed harder at the end of the second set."
Del Potro, meanwhile, played with a heavily taped left knee and appeared to slow as the match went on.
A day he started by hitting out, forcing the action, pushing Federer from side to side, ended with the Argentine on defense, trying simply to hang in there and hope for something to change the momentum - like, say, a game-stopping dose of rain, which was merely sprinkling on Court Suzanne Lenglen when Federer served out the match.
Federer beat del Potro for the seventh straight time since that U.S. Open loss. One of those wins came earlier this year in the Australian Open quarterfinals and there have been three more since, but all have come in straight sets without much drama.
This time, it seemed like a real possibility that Federer could lose. He looked tired and a bit sloppy (14 unforced errors) in the first set. In the second, Federer was serving down 5-4 in the tiebreaker but del Potro drove him backward, off the court and forced misses off Federer's forehand side two times to wrap up the set.
Things changed dramatically and quickly, though. The third and fourth sets took a total of 55 minutes - only four fewer than entire second set.
Federer got a break right away in the fifth set, then served it out.
Earlier in the day, in the women's quarterfinals, No. 6 Samantha Stosur defeated No. 15 Dominika Cibulkova 6-4, 6-1 and No. 21 Sara Errani made her first Grand Slam semifinal with a 6-3, 7-6 (2) victory over 10th-seeded Angelique Kerber.


text:
on-demand software deliveryA type of software delivery service where the software is provided to the customer through a network (such as the Internet) as a service. On-demand software delivery is also called software on-demand. The terms on-demand software and Software as a Service (SaaS) are often used interchangeably.
Perceptual computing is the ability for a computer to recognize what is going on around it. More specifically, the computer can perceive the... Read More »Apple Pay Promises to Strengthen Payment Security
Experts believe that Apple Pay and other competitive payment systems will be far more secure than cards, even cards equipped with EMV chips. Read More »The Great Data Storage Debate: Is Tape Dead?
Tape clearly is on the decline. But remember, legacy systems can hang for a shockingly long time. Read More »
A network is a group of two or more computer systems or devices, linked together to share resources, exchange files and electronic communications.... Read More »Computer Architecture Study Guide
This Webopedia study guide describes the different parts of a computer system and their relations. Read More »Webopedia Polls
The trend for the past two years has been for shoppers to spend more online during the holiday season. How do you typically shop for holiday... Read More »
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Samsung has a new 7-inch tablet, the Galaxy Tab 3, which will compete with Apple, Google and Amazon's smaller models.
Click here to read Bob's blog.
CDC Public Health Image Library
Vikings sack Paris, the Hollywood Walk of Fame gets its first star,…
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Click on each site below to learn more about the flyways, byways, stopovers and residences of our two-winged and four-footed friends in the Tuscarawas County Section of the Canalway. Your journey can wander along the Towpath Trail, through Zoar Woods and through the beautiful sights and sounds of the 82-acre Zoar Wetland Arboretum. Any day out in the Canalway offers a wealth of viewing opportunities – including those to be found along the Towpath Trail.
The region boasts a number of Important Bird Area (IBAs) — and some of Ohio's best bird-watching opportunities.
Stay up to date on all things Ohio & Erie Canalway by subscribing to our newsletter. You'll receive periodical updates on Canalway happenings, special offers and the latest information. Learn more »
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A Towpath was a Towing Path Bicyclists and walkers on the Ohio & Erie Canal Towpath Trail today follow the same path that in the 1800s was used by mules and mule-drivers to pull canal boats—with some exceptions. Canal Fever , KSU Press, 2009 More »


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The San Diego Bluegrass Society is a 501(c)(3) nonprofit corporation dedicated to the preservation and promotion of bluegrass music in the San Diego area.
Looking for bluegrass music in San Diego? You're in the right place!
The San Diego Bluegrass Society sponsors a wide variety of bluegrass music events, including regular Tuesday events with open mics and jam sessions for San Diegans and visitors who desire to play bluegrass in a group setting, with a featured band on the 4th Tuesday. SDBS also sponsors bluegrass concerts by nationally recognized bands touring through our region, along with workshops and other educational activities.
Check out our Events+Calendar page for details. Better yet, sign up for our email list and we'll keep you informed of special events. Best of all, join the SDBS for our monthly e-newsletter, special member discounts, and more, You'll enjoy becoming part of our family-friendly community, and knowing that your support keeps bluegrass music alive and well in San Diego.
SDBS Presents Front Porch Music Preservation Society as Featured Band March 24, 2015 at the Boll Weevil
SDBS welcomes back for its second Featured Band appearance, the Front Porch Music Preservation Society or FPMPS as they are often called. This relatively new North County band brings to SDBS a combination of seasoned bluegrass pickers along with new blood, which goes right to the heart of the bluegrass tradition - young and old sharing the music and keeping it alive.
The action starts at 6:30 pm with open mic and pick up bands. Sign up in advance by email to: email@example.com, or sign up on-site, space permitting (preference given to those signing up in advance).
Schedule: Open mic and Pick-up bands 6:30-8; FPMPS 8-9 pm Location: Boll Weevil Restaurant, 9330 Clairemont Mesa Blvd. San Diego, CA 92123.Admission: Free; donations suggested.
Summergrass: August 14-16, 2015.
With our sister organization in San Diego, The North San Diego County Bluegrass and Folk Club, we co-produce Summergrass, a premier 3-day bluegrass festival held each August. Summergrass was a blast in 2014 and looks to be even better this year! Check out the linieup and all the details here: Summergrass. Check out our summergrass video to get ecited!
Perennial Summergrass favorites Bluegrass Etc. (Photo courtesy of Dennis Andersen)
Free Music Outreach with Emma's Gut Bucket Band
SDBS has an active music outreach program with Emma's Gut Bucket Band, bringing bluegrass music and instruction to public and private schools, senior centers, assisted living facilities, homeless centers and the like at no cost. We also offer a lending library of instructional materials, plus an instrument lending Library, to our members. An instrument donation program is also available. Send us an email for info: email@example.com.
Upcoming events
See a full listing of upcoming events on our Events+Calendar page.
Featured Articles
History of the SDBS in Words & Pictures, by Dwight Worden
SDBS (originally the San Diego Bluegrass Club) has been a force for bluegrass music in San Diego since the 1960s.
For a fascinating glimpse into its storied past, illustrated with lots of photos and historical images, and chock-full of interesting anecdotes and memories, download A Brief History of the San Diego Bluegrass Society (in print-friendly PDF format).
So Long Doc, Earl & Doug, by Dan Crary
Click here to read the full article.
A Tribute to Mike Auldridge
It is with great sadness that we report that Mike Auldridge passed away on Dec. 29, 2012, one day before his 74th birthday. Visit the Mike Auldridge Tribute site to learn more about MIke: his 2012 National Endowment for the Arts National Heritage Fellowship; his 2007 IBMA Distinguished Achievement Award; and his significant contributions to the Dobro and to bluegrass music.. Rob Ickes' moving eulogy is also posted there, along with a guestbook with hundreds of tributes from his friends and fans.
Looking for a bluegrass band for your event? Let us know, and we'll assist you in locating a band that meets the particular needs of your party or event. Contact: firstname.lastname@example.com.


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Random things of happiness from this past week. This is something new I'll be doing as I saw it on another blog and agree that it's a good idea! So, here are my five!
1.) Kat has her 6 week post operation check up at Shriners. Her x-ray looks awesome and she gets cleared for swimming, and doing chores. Woo! She's doing fantastic, and is pain free most of the time.
2.) Krispy Kreme offers mini-donuts, so we get two of them while getting donuts on the drive home from Shriners. Risi's super thrilled, but ends up mostly just ripping it to pieces and falling asleep. Ah well, her cuteness here made all the cleaning up worth it.
3.) Beautiful rainbow cloud on the drive home after the Shriners appt and getting donuts. How pretty right? This really made Kat so happy to see, and I have to admit getting to see the pictures (I didn't take them, I was driving!) I wish I could have been sitting in the car and able to see this instead of drive!!
4.) Kat wears her dress and looks happy. I love seeing her smile! Plus she's a vision in white, even if my iPhone isn't so great at taking pictures.
5.) Nacho time!! I managed to make dinner, lol. Sometimes lately… I just am not doing well. So Nacho time and Family Feud!!
Risi ended up eating so much of my avocado I ended up needing more! 2lbs of pork in the crock pot with 3/4 cup of water and taco seasoning on low for about 6 hrs. Yummy! So happy to have a crock pot and then a toaster oven.
Kisa Johnson
Owner at Ohayo Okasan
Blogger, Illustrator, Reviewer, Voice Actress, Mom of 5 humans, 7 cats and 5 mice! I do a little bit of it all. Crafting, singing, sewing, baking, cooking, and driving myself a little crazy every day. :) I have ADHD, Anxiety and Depression that I battle with daily. But I'll make it! Someway, somehow.
Latest posts by Kisa Johnson (see all)
- Male Cats and The Importance Of Good Cat Food - March 24, 2015
- Chococurb Review and Unboxing - March 21, 2015
- 10 Delicious Nutella Recipes For You - March 19, 2015


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Centralized Contact Sharing for Exchange Hybrid
Our customers continue to rave about our
Exchange Hybrid
offering. It gives them the flexibility to purchase Microsoft Exchange mailboxes for power users, while purchasing budget-friendly Rackspace Email mailboxes for everyone else—all on a single domain.
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on Aug. 31, 2010 by Robert McAden
Three-Column View: Now in Rackspace Webmail
With monitors getting wider and laptops/netbooks becoming more prevalent, we're excited to introduce our latest Rackspace Webmail feature: Three-Column View. Now, users with limited vertical space can quickly read more of their email messages without scrolling. This new feature is LIVE now – so give it a try.
Filed in
on Aug. 5, 2010 by Manny DeJesus
Hybrid Exchange Calendars now Available in Rackspace Webmail
We've just released an exciting new enhancement to our
Exchange Hybrid
offering that allows Rackspace Email customers to view Hosted Exchange calendars. So regardless of the platform, Hybrid users can now view or share their business calendars more effectively.
Filed in
on Jun. 16, 2010 by Nell-Marie Colman
Sharing Rackspace Email Calendars
If you use our
Exchange Hybrid
, you might not realize that Rackspace Email users can share their calendars with co-workers who use Exchange, even though they're on different platforms. Here's all you have to do . . .
Filed in
on Feb. 19, 2010 by Matt Pfeil
Group Scheduling with Rackspace Email
Being a Project Manager, I schedule a lot of meetings with members all across our company. Before the new "Group Scheduling" feature, I would have to type in each member's email address in order to add them to the invite list. This is fine for a handful of people, but when you are consistently inviting 10+ members to meetings, it can really feel like a waste of time.
Filed in
on Feb. 3, 2010 by Rex Card
Never Forget an Attachment Again!
Every week, if not every day, I forget to attach a file to an email. So like everyone else, I quickly send a follow-up with the famous "Oops!" in the subject. Annoying for all involved; I'm sure.
Filed in
on Nov. 23, 2009 by Manny DeJesus
Rackspace Email: New Features in Webmail
Rackspace Webmail
releases have themes or a singular focus. For example, this past summer we worked on significant speed improvements and had a "Speed Release."
Filed in
on Nov. 16, 2009 by Manny DeJesus
One Email Outage Pays for a Year of Rackspace Email Hosting
Steven Ng, Vice President of Professional Services for
came to Rackspace after a hard drive failure took their email down for a week and reallocated Steven's time from billable work to fixing email. Due to a failed backup, they also lost a week's worth of email. By outsourcing their email to
, they now have dedicated support when they have email issues from our team of email experts.
Filed in
on Oct. 8, 2009 by Cameron Nouri
New Features in Beta for Rackspace Email
For those of you who don't already know, we have a beta version of our webmail product for
Rackspace Email
. We use this version, affectionately known as simply "beta," to get our newest features into the hands of customers as soon as possible, so we can get valuable feedback and work out any kinks before going live.
Filed in
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Launch: Auto Delete Invitations
I receive several event invitations a week—sometimes even several a day. Every time I get an invitation, I either accept or decline, and then I delete the email. The way I see it, if I can't attend the event, I don't need to keep the invitation. And if I accept the invitation, the event information is transferred to my calendar.
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text:
Alawar Coupons & Promo Codes
2 verified offers in December, 2013
Check out our 2 Alawar offers including 2 sales. Latest offer: 'Check Out the New and Popular Games Today!' The last time an offer was used was March 27, 2015.
Check Out the New and Popular Games Today!
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Dr. Kristin M. Farrell, MD
Pediatric Emergency Medicine
- 4401 Penn Ave Ste 2400
Pittsburgh, PA 15224
- 1 Location
- (412) 692-7692
Dr. Kristin M. Farrell graduated from the University of Massachusetts Medical School in 2003. She works in Pittsburgh, PA and specializes in Pediatric Emergency Medicine. Dr. Farrell is affiliated with Childrens Hospital Of Pittsburgh Of UPMC. She speaks English and Spanish.
Gender: Female
Languages Spoken: English, Spanish
Pediatric Emergency Medicine
Medical School: University of Massachusetts Medical School; Graduated 2003
4401 Penn Ave Ste 2400
Pittsburgh, PA 15224
(412) 692-7692
Fax: (412) 692-7464
Practice Website: Not Available
Accepting New Patients: Yes
Medicare Accepted: Yes
Medicaid Accepted: Yes
Not Available


text:
Any Black Woman in the South Florida area?
Hi, Im a Black gentleman looking for a cruise mate late March or anytime in April. A 4 or 5 day Carnival cruise to the Caribbean, ocean view cabin
Im a neat, clean cut professional gentleman who needs a fun break away from a demanding job. I prefer to meet face to face for coffee first in the Miami-Fort Lauderdale area. Ive done this before and its worked out very well.
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Sakura Live Wallpaper New by Wallpaper qHD Sakura Live Wallpaper will make you feel like a part of marvelous lands of Far East where the Sun rises and beautiful ch … 5+ downloads, Rating 0.00 (0) Free
Jackson-Pace Pharmacy New by RxWiki, Inc. We've been serving the communities of Cantonment, Beulah, Seminole Alabama, Molino, Pensacola since 1981. Our pharmacist … 0+ downloads, Rating 0.00 (0) Free
Kids Writing Numbers Lite by Magmentis The eco-friendly counting and writing application KIDS WRITING NUMBERS is the latest edition of this very popular edu … 1,000+ downloads, Rating 3.17 (6) Free
EM Cal by CrossFact This is a simple calendar memo. You can registered memo and icons. Please use this calendar, such as when you manage yo … 500+ downloads, Rating 4.67 (6) Free
Cullen Care Pharmacy New by RxWiki, Inc. We've been serving the communities of Houston since 2004. Our pharmacist has 20 years of experience and our friendly sta … 0+ downloads, Rating 0.00 (0) Free
Small Comandas - Smallsoft by Smallsoft Comanda eletrônica para restaurantes Smallsoft. O aplicativo interage com o software Small Commerce. Através do Small Co … 1,000+ downloads, Rating 3.96 (23) Free
Baking Pound Cake by Axis Entertainment Top Cooking Games shows how to Bake Pound Cake. Traditional Pound Cakes are made with a Pound each of the following ing … 50,000+ downloads, Rating 3.91 (305) Free
Tamilnadu Cities by VIJAYARAJAN RAMANATHAN Tamilnadu Cities App Provides info such as Places to Visit, Map View and getting there for major cities in tamilnadu suc … 10,000+ downloads, Rating 4.08 (166) Free
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Champagne Express by mPaySecure CHAMPAGNE EXPRESS - PARIS Champagne Express livre du Champagne en 3 heures à Paris, de 9h à 19h du lundi au vendredi. … 10+ downloads, Rating 4.33 (3) Free
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Torrnado - Transmission Remote by Gábor Demkó Torrnado allows you to remotely access your Transmission© BitTorrent client running on your desktop or server. It connec … 1,000+ downloads, Rating 4.68 (194) $2.49
Foundation Maths by Read Write Phonics Foundation Maths supports the UK School Curriculum Early Years Foundation Stage and Key Stage 1. Suitable for children a … 1,000+ downloads, Rating 4.50 (4) Free
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text:
u guys will like this: Sonny was unimpressed by the elite xbox lauch
this is why they had to say:
Originally Posted by sony
u guys will like this: Sonny was unimpressed by the elite xbox lauch
this is why they had to say:
Originally Posted by sony
i think sony had to have some kind of response to it's release, and of course they had to do their best to degrade their competition, and what they said is true, so yeah. =\Originally Posted by dollar
I've read a quote in a magazine from one of the head honchos at Epic Games. Apparently a lot of game developers have been complaining to him that they can't get the Unreal 3 engine running nearly as fast or as pretty on the PS3 as they could with the Xbox 360. I'll give them a year though, to learn how to actually use the Cell processor before I make a proper judgement.
EDIT: What the hell? I'm seeing more spelling and grammar mistakes in this one post than I've made in the past 10 years. FIXED.
Last edited by MadDogMike; 03-30-2007 at 12:45 AM.
if its the same price... eh... might not be too bad of an idea. I hope it stays rumor though, it would be such a cheese move to up the ps3 hdd just because the 360 did it too.
damnit i wanna know what the ps3's capabilities with exhdds are!!! >.<
I'd like to die with the songs I love stuck in my head. I hope to make the most of these hollow bones we become.
I raise a toast to the the souls that sang all along. I've been gathering friends to just to make some sounds,
before the ship goes down, I've been making amends by making the rounds before the whole world ends
[Chit Chat Specific Forum Rules] // Last Update - Friday March 13, 2009
It's here Xbox 360 Elite, they come with HDMI (1.2) and more storage with black finish.
I was wondering how much graphic and sound quality can be better with HDMI on Xbox 360 compared to the VGA connection?
To everyone who thinks that the PS3 is more powerful than the X360:
I would like some facts and figures showing how exactly it's more powerful and beneficial to games.
Essentially, the RSX is a 7900GT and the Xenos is like an HD2600XT.
Let's slap the 8600GTS into the equation.
From an architectural point of view (G71 vs G84 vs R580 vs R630)
The G71 and R580 use the legacy/traditional pixel shader + vertex shader combo for rendering.
The G84 and R630 are equipped with stream processors/unified shaders.
Let's take Derek's take on some video cards:
As you can see, in older games that are not shader-heavy, the X1900XT is much more powerful than slower clocked and crippled GPUs equipped with stream processors.
From that graph, I can safely say that the RSX is somewhere between the X1950 Pro and the 7950GT. The performance of the Xenos is somewhere between the 7950GT and X1900XT.
Here's a shader-heavy game:
The gap between the 8600GTS and the higher-end cards is dramatically reduced.
GPUs like the G71 and G73 are totally left behind.
Here's what I intend to show with these facts:
The RSX has lower shader processing capabilities than the Xenos to begin with in older games.
The Xenos itself is very interesting. It has 48 unified shaders, more than the 32 of the 8600GTS. Its performance is visibly better in older games like FEAR (due to the greater amount of shaders) and a more powerful GPU itself (X1900XT>7950GT) and in newer games that require lots of shader processing capabilities from the graphics processing solution, the unified shaders help A LOT and sets the pace against non unified-shader video cards.
Let's look at some engines that are/will be used for the new generation of video games. Offset Engine, Unreal Engine 3, Renderware, Gamebryo's next iterations as well as the Source Engine (unreleased build number).
The Offset Engine is particularly shader intensive with the per-pixel motion blur that utilizes a specific shader function for that. NVIDIA developed their Lumenex Engine based on that.
The Unreal Engine speaks for itself.
Gamebryo and Renderware will perform almost the same on both the RSX and Xenos due to the fact that they don't really take advantage of the more advanced capabilities of the Xenos. These two are mostly tweaked and enhanced versions of their predecessors rather than complete revisions and updated tools that Unreal Engine 3 and Offset are.
The Source Engine's orientation will be focused on the processor and will be multithreaded efficiently but requires the cores to be very similar.
You guys are smart enough to conclude this on your own. The differences in architecture between the graphics processing capabilities of both consoles is obvious and the limitations are there.
Last edited by Jyuu; 04-24-2007 at 07:48 AM.
Blow my brains out? Or were you just using my quote to support what you were saying? I already understand that the Xbox 360's Xenos GPU has better shader processing capabilities than the RSX. Those are pretty much the distinguishing features of the two consoles, the PS3 has plenty of raw processing power but the Xbox 360 has greater graphical capabilities.
Actually, has anyone else noticed this trend with Sony? With all 3 generations of the Playstation console, they've focused almost solely on raw power and big numbers to impress people, but have always featured less than impressive graphical capabilities (looking from a technical viewpoint).
PS1: Could render more polygons than any of the other competing consoles, but pretty much used software rendering.
PS2: 128-bit processing turned out to be a load of hype with no noticeable benefits, and I don't even know if the PS2 could use pixel shading at all. If it did it was very minimal, because I never really noticed it.
PS3: Explained above.
Last edited by MadDogMike; 04-24-2007 at 08:23 AM.
Why is it so difficult to believe?
MS Gaming Divison has been making massive loses and it's not like Windows and Mr. Gates aren't the gaming world's perennial monopoly/new world order whipping boys.
Initially the PS3 will be slow until it gets the game support in the same way as the orginal X-Box vs PS2 situation.
But frankly consoles and the X box in particular are just PCs in a nice box which you plug into your TV. Hell they're even giving the X Box 360 a keyboard.
Consoles seem to be growing into PC gaming dressed for people who are still scared of computers. It'd be interesting to see how the consoles stack up against Online PC gaming revenue.
Nintendo did the sensible thing, came out with something radical, fun and aimed for a different market.
Last edited by Warhamster; 04-24-2007 at 08:25 AM. Reason: Spelling
What a load of garbage.
In the original Xbox v.s. PS2 situation, the PS2 won due to its huge head-start. Since the roles have reversed this time, what you said made no sense at all.
The Xbox 360 will most likely never get a keyboard for gaming, because Microsoft expects people like you to make silly comparisons like you just did. They're just releasing a tiny keyboard attachment for controllers to save people from having to type with the controller when Windows Live Messenger gets released for the Xbox 360 in the near future.
Comparing any console to a PC is stupid. Of course all consoles are computers, they're just made specifically with games and home-entertainment in mind. The user interface however (both graphically and physically) is very different, which is what defines gaming consoles and differentiates them from the average PC. How is the PS3 any different to the Xbox 360 in these regards? I thought all the people who were silly enough to make that comparison were shut down by people who actually knew what they were talking about during the last generation.
Just because the original Xbox used retail PC components doesn't make it any different from the rest of the consoles, and it also has no effect on the Xbox 360.
I also fail to see why any of these things would fuck over Xbox fans.
Last edited by MadDogMike; 04-24-2007 at 08:39 AM.
I'd like to die with the songs I love stuck in my head. I hope to make the most of these hollow bones we become.
I raise a toast to the the souls that sang all along. I've been gathering friends to just to make some sounds,
before the ship goes down, I've been making amends by making the rounds before the whole world ends
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text:
Combine all ingredients in blender container.
Blend until smooth.
Serve in icy cold glasses.
Note: Many of the canned fruits today are available without added sugar.
Look for labels that say lite,diet, or packed in its own fruit juice.
Some have no sugar, others have reduced the amount by 50%.