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The Psychology of Boiling the Perfect Egg
Do you want the control freak's method or the laissez-faire system? They both work. A lot of cookbooks want you to think there's only one way to do something — their way, of course! In some cases, that's true. If you want to beat egg whites for an angel food cake, you can't allow even a speck of yolk to get into the whites or they won't beat up into a meringue. But more often, there are several ways to do something — like boil an egg.
The Control Freak's Method
The control freak's way is to bring the water to a boil and then place the eggs carefully into it. Cover the pan, and when the water returns to a boil, set the timer for 3 minutes for soft-boiled eggs or 13 minutes for hard-boiled. When the timer goes off, remove the pan from the stove and drain off the water. You'll probably be eating the soft-boiled egg immediately, but if you intend to refrigerate the hard-boiled ones, first run cold water over the eggs until they're no longer hot. (That will also make them easier to peel.)
The Lazy Man's Method
The easygoing laissez-faire method doesn't require a timer. Start by putting the eggs in the pan and covering them with cold water. Put the pan on a burner over high heat, and cook just until the water comes to a boil. Turn off the heat immediately and leave the eggs in the hot water, covered. After 20 minutes (you can use a timer if you want to — this is the laissez-faire way, after all) the eggs will be hard-boiled to the same degree as they are following 13 minutes of constant cooking. But you'll have helped combat global warming by saving 13 minutes of energy usage. There's nothing sacred about the 20-minute requirement; that's merely the minimum. You can let them sit in the water much longer, although eventually the whites will become a little rubbery and a greenish ring will form around the yolk. (It's harmless, but some people find it unsightly.) Eggs will be soft-boiled under this method after sitting in the hot water for 5 minutes. There's no reason for any supposed non-cook to lament, "I can't even boil an egg."
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Universal "Fragile Handle with Care" Self-Adhesive Shipping LabelsPre-Printed Labels
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$42 $60
Give her the warmth of fashionable faux fur, and the style of a model with this item! The faux fur hoodie is one of our top selling items, and is perfect for every girls wardrobe! This amazing layering piece is perfect to wear outside, and have everyone looking and asking where you got this item! Add some extra flare to this piece with a fancy headband and a skirt, or dress it down with a pair of leggings.
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Hotels in Ronchin
Hotel reservation
Hotel rooms in Ronchin
There are not so many hotel rooms in Ronchin, just 2, still everyone can find an offer to suit his choice. The most common offers in this city are 3 и 4 stars, they count 1 out of 2, and their prevalent location is central district. According to our practice, it is the most profitable to book a hotel in the south of Ronchin, as it is the location of the hotels with optimal cost and quality offers along with the cheap hotels. Taking into account the small size of the city, there is no need to dwell hotel reservation on the location: the road to the city center would take less then half an hour, so one can book a hotel in any district of Ronchin. The most populated with hotels district of Ronchin is the north, 100% of cheap hotel rooms are concentrated here. Ronchin is an ideal place for couple and solo, such travellers rate hotels there 6.9 out of 10 on average. Apart from all these, hotels in Ronchin offer their customers pets allowed, business centre, bar and low mobility guests welcome. The most appreciated amenities in Ronchin hotels are service and location. According to the visitors reviews, hotels in Ronchin has awesome vibe, fantastic service and great location. Take a look at our selection of the cheap hotel rooms in Ronchin to make a well-informed choice for your reservation!
Best hotels in Ronchin
Hotels in Ronchin are spread around the city within the distance of 0.8 kilometers. Those who are keen on antique should definitely fall in love with
As Hotel
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As Hotel
located in a 0.8 kilometers from the city center.
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On November 9 General Motors stated that its Cadillac Converj EV luxury compact has been green-lighted for production, proving that the largest US automaker is committed to following through on its pledges to produce more environmentally-friendly automobiles.
Before GM emerged from bankruptcy July 10, with about $50 billion (€34 billion) in federal aid, company executives said there were no plans in place to bring the stylish Cadillac Converj EV to production. But the company told the
Detroit Free Press newspaper that the Cadillac Converj will become GM's second EV, following its ambitious Chevrolet Volt compact car.
Cadillac did not specify a date the Converj will be available but previous estimates have targeted late 2012.
GM is clearly more committed to producing green cars than its Detroit compatriot Chrysler, which announced Monday that it is shelving its electric car program to focus instead on the new Fiat-based lineup. GM and Chrysler both stated that hybrids and EVs would play a key role in their restructuring plans earlier this year, when both were begging the federal government to bail them out of bankruptcy.
The Cadillac Converj debuted last January to rave reviews for its sleek styling, photovoltaic solar roof panel and technological advancements, such as utilizing video cameras in place of rear view mirrors to reduce wind drag and thus increase fuel efficiency. The car featured the same Voltec electric engine that is used in the Chevrolet Volt, which will hit the market late next year.
Like the Volt the Converj differs from other plug-in hybrid EVs in that it runs exclusively on electricity for the first 40 miles (64 kilometers) before switching to a gasoline engine. The top speed in electric mode is 100 miles per hour (160 kilometers per hour) and can be recharged in three hours using a 240-volt outlet.
Consumers of the luxury Cadillac marque likely won't be as put off by the price increase green technologies usually incur, a problem the brand may face with the Volt compact, which will likely carry a steep $40,000 (€27,000) price tag.
GM is positioning its Volt as a global competitor to the Nissan Leaf and other EVs arriving in the next few years, as the Volt will provide the basis for its European Vauxhall/Opel Ampera.


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By Keegan Kyle |
We've examined more than 30 bold claims surrounding San Diego's mayoral election and two more recently landed in our Fact Check queue.
During a televised debate, City Councilman Carl DeMaio said San Diego has the eighth worst roads in the nation and they are expected to get worse under the city's current maintenance plan.
At a candidates' forum, District Attorney Bonnie Dumanis claimed the racial and ethnic diversity of her employees reflects the residents they serve.


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Genomic and small RNA sequencing of Miscanthus × giganteusshows the utility of sorghum as a reference genome sequence for Andropogoneae grasses
DOI: 10.1186/gb-2010-11-2-r12
© Swaminathan et al.; licensee BioMed Central Ltd. 2010
Received: 7 July 2009
Accepted: 3 February 2010
Published: 3 February 2010
Miscanthus × giganteus (Mxg) is a perennial grass that produces superior biomass yields in temperate environments. The essentially uncharacterized triploid genome (3n = 57, x = 19) of Mxg is likely critical for the rapid growth of this vegetatively propagated interspecific hybrid.
A survey of the complex Mxg genome was conducted using 454 pyrosequencing of genomic DNA and Illumina sequencing-by-synthesis of small RNA. We found that the coding fraction of the Mxg genome has a high level of sequence identity to that of other grasses. Highly repetitive sequences representing the great majority of the Mxg genome were predicted using non-cognate assembly for de novo repeat detection. Twelve abundant families of repeat were observed, with those related to either transposons or centromeric repeats likely to comprise over 95% of the genome. Comparisons of abundant repeat sequences to a small RNA survey of three Mxg organs (leaf, rhizome, inflorescence) revealed that the majority of observed 24-nucleotide small RNAs are derived from these repetitive sequences. We show that high-copy-number repeats match more of the small RNA, even when the amount of the repeat sequence in the genome is accounted for.
We show that major repeats are present within the triploid Mxg genome and are actively producing small RNAs. We also confirm the hypothesized origins of Mxg, and suggest that while the repeat content of Mxg differs from sorghum, the sorghum genome is likely to be of utility in the assembly of a gene-space sequence of Mxg.
Domesticated and wild grass species in the Andropogoneae tribe are important as sources of food, feed, fiber and fuel. Included within the Andropogoneae are major crops such as maize, Sorghum bicolor (sorghum), sugarcane (Saccharum spp.), the native North American prairie grass Andropogon gerardii (Big Bluestem) and species in the genus Miscanthus, which have recently emerged as leading candidate bioenergy crops. All species within the Andropogoneae perform C4 photosynthesis, which exhibits higher rates of carbon fixation. C4 is also associated with greater water- and nutrient-use efficiency compared to most plant species [1, 2]. These physiological properties contribute to the very high biomass productivity observed from cultivated Andropogoneae crop species, which often exceeds 20 Mg/ha. Another characteristic of the Andropogoneae is a high frequency of polyploidy, with many large and complex genomes. Though typically native to tropical and subtropical climates, Andropogoneae crop species are adapted to a wide diversity of environments and vary in both their life cycle (annual versus perennial) and primary form of harvestable carbon (sugar, grain starch or cellulose).
As a result of their global economic importance as crops, their varied biology and diversity of genome structure, Andropogoneae species have been an important focus for plant genomics research. Early comparisons of genetic maps and local genome structure helped elucidate the high degree of synteny among grass genomes [3]. These and other studies have also provided insights into the contributions of transposon content, gene duplication, and conserved noncoding sequences to plant genome evolution [4, 5]. Recently, whole-genome shotgun sequencing has produced a high coverage draft for one of the smallest known Andropogoneae genomes, sorghum [6]. In addition, a strategy to sequence the maize genome using assembled BAC contigs that are anchored to genetic and physical maps is now complete [7], and extensive EST resources exist for Saccharum officinarum [8].
The Miscanthus genus is composed of about 15 species of rhizomatous perennial grasses native to subtropical and tropical regions of Africa and southern Asia, with the range of at least four species (M. sinensis, M. sacchariflorus, M. floridulus and M. × giganteus) extending north into temperate eastern Asia [9]. Sequencing of nuclear ribosomal DNA and plastid intergenic spacers as well as amplified fragment length polymorphism genotyping has resolved some of the phylogenetic relationships among Miscanthus spp. and accessions [9–11]. Within the Andropogoneae, Miscanthus is most closely related to Saccharum spp. and interspecific hybrids of Miscanthus and Saccharum have been used in genetic improvement of sugarcane [12].
Miscanthus × giganteus [13, 14] (hereafter referred to as Mxg) is widely distributed as an ornamental and has been evaluated for more than two decades as a biomass crop in both Europe and the United States [15, 16]. Because of these attributes, Mxg has been the most extensively studied member of the Miscanthus genus. Mxg is a sterile triploid (3n = 57, x = 19) that likely originated from the hybridization of M. sinensis and M. sacchariflorus [11, 17, 18]. In addition to the potential for increased vigor because of stabilized hybridity and polyploidy, Mxg is unique among C4 grasses in performing C4 photosynthesis at temperatures as low as 5°C [19, 20], which likely contributes to a maximal 2% conversion rate of solar energy into harvestable biomass among established canopies [16]. This phenotype has been interpreted as a competitive advantage since it allows productive photosynthesis to begin earlier in spring in temperate climates. The cold-tolerant C4 photosynthesis phenotype is correlated with increased accumulation of the key C4 photosynthetic enzyme pyruvate orthophosphate dikinase under chilling conditions [21, 22].
Future studies of Mxg and other Miscanthus species, as well as genetic improvement of these species as biomass crops, will be enhanced by knowledge of their genome content and organization. The Mxg genome also offers the possibility to gain information about examples of both the M. sinensis and M. sacchariflorus genomes, and their interactions after hybridization. As a consequence of both its interesting biological properties and potential as a biomass crop, Mxg is a desirable target for whole-genome sequencing. Although the large size, complexity, and often polyploid nature of Andropogoneae genomes makes whole-genome shotgun or map-based strategies cost-prohibitive, short-read deep sequencing technologies enable rapid cost-effective approaches to survey genome content and organization [23]. Such an approach is greatly enhanced by comparisons to a closely related reference genome sequence [24, 25] and represents an efficient first step toward characterizing complex eukaryotic genomes. We reasoned that the recently completed whole-genome shotgun genome sequence for sorghum [6] would likely provide an appropriate initial reference sequence for comparative analysis with the Mxg genome, both for the characterization of conserved sequences as well as recently diverged repeats and gene content found in Mxg but not sorghum. The comparison of repeat composition between these two genomes has not yet been performed, nor is it known how similar genic sequences are likely to be between Mxg and available reference genomes. We thus evaluated this strategy by comparing a survey of the Mxg genome obtained by 454 Genome sequencer FLX system (GS-FLX) pyrosequencing to the genome sequences of sorghum, maize and rice. We also compared the repeat sequences identified using the survey results with profiles of small RNAs (sRNAs) obtained via Illumina sequencing-by-synthesis as it has been previously reported that repetitive regions of plant genomes contribute a substantial portion of the total sRNA transcriptome [26].
Genome size estimation for Miscanthusspecies
We used flow cytometry to estimate nuclear DNA contents from the exact clone used for this study, a single accession of Mxg named 'UIUC', which was originally established as an ornamental variety at the University of Illinois at Urbana-Champaign, but is also the source of material used in recent agronomic trials [16]. We obtained values ranging from 7.60 to 7.95 pg among four independent tissues samples for the UIUC accession of Mxg (Additional file 1). These values are closely consistent with those previously published for Mxg [27]. Cells in G0/G1 were identified using flow cytometry and comparison to size standards (Additional file 2) and are consistent with previous measurements [27]. Using a value of 980 Mbp per pg DNA [28], and assuming Mxg harbors three genomes of similar size, we estimate that 3C = 7.5 Gbp, and thus the three haploid genomes of Mxg are approximately 2.5 Gbp.
Survey sequencing of Mxg
We performed a whole-genome survey of the Mxg genome using the 454 sequencing platform as described previously for soybean [23]. Genomic DNA was isolated from nuclei purified from Mxg leaves to minimize possible contamination from organellar or microbial DNA. A total of 84 Mbp in 366,448 reads with an average size of 229 bp were produced from one run using the 454 LR70 FLX technology. A maximum value for chloroplast contamination (reads with a BLASTN hit to the sorghum chloroplast genome at e < 10-6) was 0.24% (901 reads). A maximum value for mitochondrial contamination (reads with a BLASTN hit to the sorghum mitochondrial genome at e < 10-6) was 0.21% (817 reads). These are maximum values because the nuclear genome may contain sequences identical or near-identical to the chloroplast and mitochondrial genomes; the 0.24% figure is similar to the proportion of the Arabidopsis nuclear genome composed of integrated chloroplast sequences [29]. The average GC content of the sequence was 44%.
Hodkinson [11] suggested that the triploid genome of Mxg is most likely derived from the cross of a diploid M. sinensis or M. sacchariflorus with a fertile M. sinensis × M. sacchariflorus allotetraploid, each of which are known to exist in Japan, where Mxg is thought to have originated. Observed patterns of meiotic chromosome pairing [17] support the view that Mxg harbors two genomes of high homology and a third with greater divergence. The component genomes of Mxg share sufficiently high similarity to prevent specific detection by genome in situ hybridization [11], but the two genomes can be distinguished by sequence variation in the internal transcribed spacer of 18-28S rDNA genes (rDNA is DNA encoding the tandem array of rRNA genes). Mxg is sterile due to frequent chromosomal mispairing during meiosis [17]. For the purpose of determining coverage, we propose that Mxg is best treated as a heterozygous diploid plus a haploid, with a total effective genome size of 7.5 Gbp. We therefore estimate that our survey sequencing generated approximately 1.2% coverage.
We interpret the broad peak of reads with lower (84 to 90%) sequence identity as the result of highly repetitive coding sequences (for example, transposase-coding genes) within the sorghum coding region dataset. The sorghum gene models are known to contain retroelement-derived sequences despite repeat masking [6]. We therefore used the Plant Repeat Databases [30], which are largely composed of repetitive sequences from related grass genomes, including sorghum, to identify 1,322 likely repeat-derived coding regions within the sorghum coding sequence dataset using BLASTN at a 10-6 e value cutoff. When these sequences were filtered from the coding region dataset, the matches with 80 to 90% identity substantially decreased, while the peak centered at 97% identity remained (Figure 1). The peak at lower levels of sequence identity is therefore likely to be the result of non-cognate matches of reads to repetitive coding regions such as the open reading frames of retrotransposons [31]. The remaining Mxg sequences matching at lower levels of sequence identity likely represent other high-copy protein-coding sequences not excluded by the filter. We consider the sequences present within the peak from 94 to 99% identity are likely non-repetitive protein coding sequences and conserved non-coding sequences. This level of similarity is consistent with the degree of nucleotide similarity observed in comparisons of the coding sequences for the C4 photosynthetic enzymes ribulose bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase, and pyruvate orthophosphate dikinase between sorghum and Mxg [21]. A similar degree of sequence similarity is observed in comparisons of Saccharum officinarum ESTs to sorghum [6]. The Mxg survey sequences were also matched to the maize genome (release 3b.50) and the rice genome (release 6). The overall results were similar to those for the sorghum genome, except for significantly fewer matches overall (Figure 1). As expected from the phylogenetic proximity of sorghum to Mxg [9–11], sorghum is the most similar fully sequenced genome to Mxg. The peak at high percentage nucleotide identity is more obvious in maize, indicating that the conserved, coding DNA is substantially similar between maize and Mxg. Rice, which is not a member of the Andropogoneae, matches substantially fewer survey reads than maize.
The reads matching the sorghum predicted protein-coding genome at between 94% and 99% identity represent 3.4% of all total survey reads. We view this as a likely overestimate of the true proportion of protein coding DNA in the Mxg genome because some of these reads overlap exon-intron boundaries and contain noncoding as well as coding DNA. Thus, the percentage of reads matching exon sequences will be higher than the percentage of base pairs in the genome that are within exons. Such splicing boundary fragments are likely to be common because the average read length from the GS-FLX 454 sequences (229 bp) is comparable to the average exon sizes of approximately 250 bp observed for rice, maize and sorghum [6, 30]. Assuming that full-length alignments of Mxg reads to the sorghum genome require at least 50 bp of overlap with sorghum exons that average 250 bp in length, then the Mxg exon space is estimated at 66% of the total sequence with significant matches to sorghum coding regions (Exon length/[Exon length + Read length - 2 × 50-bp miminum overlap] = 250/[250 + 229 -100]). Thus, our estimate of the single-copy conserved protein-coding DNA content is 2.2% of the Mxg genome. An estimated coding DNA content of 2.2% of the 7.5 Gbp Mxg genome would represent 165 Mbp. When divided by an average of 1,500 bp of coding sequence per gene, this leads to predictions of 110,000 genic loci per Mxg nuclear genome, or approximately 37,000 genes for each of the three component Mxg genomes. This number is comparable to recent estimates of gene number for sorghum and rice [6], but we note that our Mxg estimate would include any recent pseudogenes present in Mxg as these would not be distinguished by alignment of our short Mxg reads to sorghum, nor does it account for any gene loss that may have occurred after polyploidization.
Repeat content of the Mxggenome
We used a number of techniques to characterize the repeat content of the Mxg genome. First, we compared the 454 survey sequences with the repeat classifications available from the Plant Repeat Databases [30] to classify each read on the basis of sequence similarity to known repeat sequences. Because the Plant Repeat dataset does not contain Mxg sequences but does include sorghum, we compared the abundances of hits within these data to a sample of 500,000 sorghum capillary sequence reads from the sorghum genome project, which unlike the assembled genome sequence are not depleted in unassembled (primarily centromeric, pericentromeric and rDNA) repetitive sequences, and thus more closely mimic our Mxg genome survey dataset. Because the Mxg and sorghum sequences were generated using different chemistry and the sorghum sequences were generated from plasmid templates propagated in Escherichia coli, the two datasets may have different genomic sampling biases. In addition, the read length of the capillary sequences is substantially longer. However, if the comparison provides similar results, it is both an indication of the similarity of repeat content of the two genomes and an assessment of the two sequencing chemistries in terms of their ability to represent complex plant genomes. Both read sets match many types of repeats, which is expected given the overall highly repetitive nature of Andropogoneae genomes. Thirteen percent of Mxg reads have a BLAST hit to the known repeat database at 10-6 compared to 45% of sorghum reads. We attribute the large reduction in the proportion of Mxg matches to repeat sequences compared to sorghum to the shorter reads produced by the 454 technology and the absence of known Mxg repeats in the Plant Repeat Database. These results also indicate that a substantial number of Mxg sequences are not represented within known repetitive sequences.
Detection of new repetitive sequences in Mxg
We used the technique of non-cognate assembly [23] to identify novel high-copy-number repeats in the Mxg genome. As expected, a large number of Mxg reads assembled, indicating the presence of highly repetitive sequences. Based on a coverage estimate that our genome survey contains 1.2% of the Mxg genome, we calculate using the Lander-Waterman equations that three or more reads that assemble into one consensus sequence represent a multi-copy sequence present in the Mxg genome with a probability of 0.96 (thus, the null hypothesis is rejected at P < 0.05; see [23] for methods). A total of 20,774 'cluster' sequences were assembled from the Mxg survey reads that we predict are significantly repetitive (Additional file 3). These 'clusters' do not necessarily each represent an individual repeat family, since multiple clusters may be derived from different regions of a repetitive element. Also, all the repeats represented by these clusters are likely present in at least 100 highly similar copies per (total nuclear) genome in order to assemble; thus, this analysis will not detect many repeats of lower copy number. Sampling of the genome is a stochastic process; therefore, some repeats present in less than 100 copies may be detected, and some with more than 100 may not be detected. Of these assembled, repetitive sequences, 4,394 had no significant BLASTN match to the GenBank nt nucleotide database and 3,571 had no significant match to the draft sorghum genome, indicating, as with the comparison to the plant repeat database, that the Mxg genome harbors repetitive sequences not found in other sequenced plant genomes, including close relatives such as sorghum and maize.
Copy number of each assembled sequence was determined by computing the depth of coverage given by all genome survey sequences with 90% or greater similarity across their entire length. We found that 68% of all survey reads match the de novo detected repeats from the non-cognate assembly analysis. Note that in many cases, more than one repeat cluster will match a given read; in this case each read is counted only once. We thus estimate that a minimum of 68% of the Mxg genome is present in approximately 20,000 repeat families of 100 or more copies.
The ten most abundant repetitive sequence clusters detected in the Miscanthus genome
Cluster ID
Cluster length (bp)
Estimated copy number in Miscanthusgenome
Copy number in sorghum genome
Best match in GenBank nt
Best match in sorghum genome
Best match in Plant Repeat Database
8.82 × 103
2.15 × 103
AC169373 Sorghum
Chromosome 2
AF137608 Sorghum bicolor centromere-associated repetitive DNA element CEN38
1.97 × 104
7.73 × 103
No hits
Super contig 18
AY129008 Zea mays CRM centromeric retrotranposon
1.02 × 104
1.90 × 103
No hits
Super contig 20
AY828019 Oryza sativa (japonica cultivar-group) clone CRR2_CH4-2 CRR2 retrotransposon
1.32 × 104
1.27 × 103
No hits
Chromosome 10
AY129008 Zea mays CRM centromeric retrotranposon
8.27 × 103
2.05 × 102
No hits
Chromosome 2
ZRSiTERTOOT00157 giepum_438D03-1 giepum_Z438D03-1 Z438D03 retrotransposon
5.61 × 103
3.68 × 102
No hits
Chromosome 9
SRSiTERTOOT00084 leviathan_98N8-1 leviathan_98N8-1 retrotransposon
6.83 × 103
7.5 × 101
No hits
Chromosome 7
AF078902.1 Sorghum bicolor centromere element pHind12
5.56 × 103
3.30 × 102
AF11417 Sorghum
Chromosome 2
AZ922428 MRCot4C10 Sorghum bicolor MRCot Sorghum bicolor genomic similar to Sorghum bicolor centromere element pHind12
5.28 × 103
2.16 × 102
No hits
Chromosome 10
ZRSiTERTOOT00156 giepum_333J11-1hdnm giepum_Z333J11-1hdnm Z333J11 retrotransposon
7.82 × 103
1.89 × 102
No hits
Chromosome 4
ORSiTERTOOT00275 osr10 retrotransposon
Three sorghum BAC sequences were investigated from the Joint Genome Institute BAC dataset, one likely to derive from a centromeric or pericentromeric region (accession number AC169373) and the others (accession numbers AC169376 and AC169372) likely representing euchromatic regions. The euchromatic BACs show that the repetitive sequences present in these regions of the sorghum genome are almost all higher copy number in sorghum than Mxg. This is particularly notable for miniature inverted repeat transposable element (MITE) sequences. MITEs are known to undergo rapid increases in copy number [33] and this process likely has occurred for the sorghum high-copy MITEs since ancestral divergence with Mxg. Notably, the centromeric repeats from the pericentromeric BAC have the same or higher copy number in Mxg as in sorghum. This observation possibly reflects more conserved sequence in centromeric repeats than the repetitive euchromatin since the divergence of Mxg and sorghum. The slightly higher abundance of the repeat sequences is consistent with the significantly higher chromosome number of Mxg [18, 34] but could also be the result of differential selection by the two sequencing chemistries employed in this comparison (Sanger for sorghum, 454 for Mxg) [23]. The strong correlation between the sorghum and Mxg repeat content for AC169373 indicates that any such bias is not severe.
Much of the sRNA transcriptome of Mxgcorresponds to repeat sequences identified in the genome survey
Most sRNAs from cereals, including grasses, are in the 24-nucleotide size range [35] and thus are likely siRNAs. In the three sRNA libraries sequenced and analyzed for this study, between 59 and 70% of the sRNAs were 24 nucleotides in size, and 23 to 30% of the sRNAs from these libraries matched the repeat sequences identified from the genome survey. As shown in Figure 4, retrotransposons (class I transposons) produced the largest portion of sRNA (32%) followed by DNA transposons (class II transposons). Given the high abundance of retrotransposons in the genome, these results suggest that siRNAs derived from repeats are the major component of the Mxg small-RNA transcriptome. Similar findings were reported in rice and Arabidopsis [26, 36].
Correlation between number of sRNA matches and repeat copy number
Although retrotransposons constitute the largest proportion of the genome of any repeat (Figure 2) and produce the largest portion of sRNAs (Figure 4b) in the transcriptome, they have a relatively small amount of sRNA matching per kilobase of sequence. The single highest copy-number repeat, the rRNA, maintains the highest rate of sRNA matching per kilobase of repeat sequence in the genome (Figure 5), although a substantial proportion of these sRNAs may be produced as a result of ribosome degradation rather than sRNA biosynthesis. In the same context rRNA was previously reported as a major source of sRNAs in Arabidopsis [26]. The less abundant MITEs matched sRNA at a higher rate than retrotransposons and a lesser rate than rRNA (Figure 5), providing evidence for active sRNA biogenesis derived from MITEs in the Mxg genome. An exception to the correlation between copy number of a repeat and sRNA production is that few sRNAs matched retrotransposons of copy numbers higher than 200 (Figure 5). The highest copy-number retrotransposons, unlike MITEs, may therefore be relatively inactive in terms of sRNA biogenesis. Thus, small RNA production may reflect the degree of heterochromatic silencing of repeats in addition to copy number.
High-coverage survey sequencing reveals ancestral rDNA
The substantial sample of the RNA-coding genome produced by the Mxg genome survey presents an opportunity to test the hypothesis that Mxg originated from a wide cross between two parental species [11], producing a highly productive but sterile hybrid. Variants within rDNA repeats can be identified by mining shotgun genome sequence data for variant units [37]. We used the same approach to mine reads spanning an informative region of the internal transcribed spacer (ITS) [38] from the 454 genome survey of Mxg. Reads matching rDNA were filtered in order to remove those containing low quality base pairs that could cause false phylogenetic results. Reads producing an orthologous aligned block from the second intergenic spacer (ITS2) [39] were then aligned with known rDNA sequences. Since it has been proposed that the parents of Mxg are M. sinensis and M. sacchariflorus [11], sequences from these species were included in the alignment in addition to the existing Mxg rDNA sequences (accession numbers AJ426562 and AJ426563) [9] and other closely related Andropogoneae rDNA sequences. A phylogenetic tree was then generated from the ITS2 block using the Bayesian inference method [40, 41]. Thus, the 454 survey of the Mxg genome was used to sample the population of rDNA, and to explore the parentage of this complex polyploid.
We have completed a survey of the Mxg genome sufficient to give a snapshot of its overall composition and to aid planning for genome sequencing projects aimed at the large genome of Mxg and other complex, polyploid crops. The 454 technology used to sequence approximately 1.2% of the Mxg genome produced a comparable repeat profile to Sanger capillary shotgun data for sorghum, indicating that the two technologies exhibit a similar level of sampling bias and that much of the repeat content is indeed shared by the genomes of these species. Our results also indicate that GS-FLX 454 pyrosequencing is capable of producing similar datasets in terms of genome sampling to that used to successfully assemble sorghum. However, assembly of the sorghum genome was achieved using very different data: capillary sequence data with coverage of 7× to 8×, a much longer read length than that provided by the GS-FLX method, and reads in paired-end configuration from relatively large molecules. Therefore, we have demonstrated the genome sampling delivered by the 454 method is suitable for whole-genome survey work. However, greater coverage, further advances in read length and paired-end technology may be necessary before true de novo whole-genome sequencing of a genome such as Mxg is feasible using non-Sanger sequencing [42].
The combination of sRNA profiling with the genome survey extends to the grasses the earlier findings from Arabidopsis that high-copy repeats are likely to produce large amounts of siRNA [26]. In addition, this combination allowed us to determine a clear correlation between copy number of genomic repeats derived from a survey, and sRNAs matching a repeat, even when the total amount of sequence of the repeat class present in the genome is accounted for. Therefore, it may help to give insights into the sRNA production process (since sRNA levels appear in most cases to rise in proportion to both copy number and content of a sequence in the genome). A survey of sRNA adds significantly to a genomic sequence survey alone, since it provides confirmation that the repeats detected in the survey are both present and biologically active. Many of the survey reads that matched sorghum predicted coding regions also matched sRNA signatures, although the percentage was not as high as for repeats. While the known retrotransposon sequences were removed, it may be possible that repetitive sequences remain within the set of 'gene space' reads. Full characterization of the Mxg gene space is needed to fully interpret this result.
The abundance of sRNA is, when averaged over all the repeats in a copy number class, roughly in linear proportion to the content of the repeat in the genome. This is true even once the result is normalized for the number of kilobases of the repeat present in the genome, as in Figure 5. This result is consistent with previous surveys [26, 36]; however, to our knowledge it has not yet been shown that repeat copy number alone, regardless of the amount of DNA for the repeat present in the genome, is a strong indicator of likely sRNA abundance. While there are many repeats that form exceptions to this relationship, it holds true for the average of repeats in a copy number class across a range of bin sizes. The underlying biology of this relationship in plant genomes warrants further investigation.
The sRNA survey may also give insight into which classes of transposable element are the most biologically active in terms of sRNAs at the time of the survey. The MITEs present at relatively low copy number in the genome survey data produce a significant fraction of the sRNA, indicating that MITE activity is actively silenced in the vegetatively propagated Mxg [43]. The correlation between sRNA production and actual transposition activity is currently unknown. Thus, the sRNA could indicate either actively transposing elements or strong and effective silencing. Activation of an abundant class of transposons, however, has the potential to significantly impact Mxg genome function by generating somaclonal variants that, even if deleterious, may be maintained within Mxg populations because of triploid sterility and exclusively vegetative propagation. While the effect of mutagenic transposon activity is likely to be moderated by the redundancy provided by the triploid genome of Mxg, it is possible that observed phenotypic variation among Mxg accessions could derive from transposon activity (a concern in a potential fuel crop). Thus, further research is necessary to determine whether the sRNA signatures observed are indicative of active transposition of class I or II transposons.
We demonstrate that Mxg contains rDNA similar or identical to that of both presumed parents, M. sinensis and M. sacchariflorus, in approximately equal quantities. Nuclei in Mxg with both one and two nucleoli have been observed [17] with the two being less frequent. Our result is compatible with the survival in Mxg of two distinct rDNA species, which may represent the two nucleoli previously observed [17] in some cells. Ribosomal RNA genes are propagated in a manner that tends to unify all rDNA units within a hybrid species after reproductive isolation in a process known as concerted evolution [37]. While an organism such as Mxg that propagates through clonal divison may not be expected to undergo rapid homogenization of rDNA units, evidence from parthenogenetic lizard species suggests that concerted evolution can operate rapidly even within asexually reproducing hybrids [44]. Although we are not aware of any evidence from plants for rapid homogenization of rDNA during asexual propagation, our analysis of rDNA supports the view that Mxg is derived by asexual propagation of a hybridization event sufficiently recent to have not yet resulted in a complete homogenization of rDNA. Further research is necessary to identify the likely rate of homogenization, which could yield a maximum estimate for the age of this asexually propagated hybrid.
Mxg is sterile, and self-incompatibility is common within the Miscanthus genus. Although a low-resolution genetic map has been developed for M. sinensis [45], these resources are not yet sufficient for positional genetics. Thus, the primary use of Mxg sequence information will be to enable functional genomics efforts. These goals could be achieved with detailed knowledge of the protein-coding gene space sequence, which, in turn, could be accomplished using 454 pyrosequencing. Despite its (currently) shorter read lengths than capillary technology, 454 sequencing will likely be suitable for gene-space sequencing, since the assembly would only need to produce contigs in the range of 10 kbp, and the repeat sequences shown to be highly abundant here could be removed or reduced prior to sequencing. Purification of gene-space could be accomplished by direct 454 sequencing of mRNA [46] or by enrichment of the protein-coding fraction of the genomic DNA via methods such as methylation filtration or C0t filtration [47]. Since mRNAs vary widely in abundance and are not expressed in all tissues, it is difficult to arrive at a whole-gene-space sequence using mRNA alone, even with the effective normalization procedures now available. Detailed mRNA sequence is often, however, necessary to interpret genomic DNA sequence even in genic regions and thus makes a useful addition to gene space sequence. Thus, efforts that target the protein-coding fraction of the genome, using gene-space enrichment combined with mRNA sequencing, may represent the most promising approach in the immediate future. Such an approach has already been demonstrated for maize and sorghum [48, 49] using genomic fragments cloned into E. coli strains that restrict methylated (hence typically repetitive) inserts.
We have shown that the 454 survey and non-cognate assembly method is capable of sampling genomic repeats in detail from an uncharacterized genome. We show that by combining the resultant repeat catalog with sRNA sequencing, a detailed knowledge of both the repetitive parts of the genome and the sRNA derived from them can be obtained rapidly. The survey also gives access to rDNA sequence in a way that makes data available on the parentage of complex crosses such as Mxg. Such data are important because it may be feasible to recreate and improve upon Mxg as a biofuel crop if the fertile parents of the original hybridization event can be identified. The survey data provide sequences of several highly variable genomic regions, in addition to the rDNA sequence, that can be utilized to identify the most likely parental genotypes of M. sacchariflorus and M. sinensis, information that could be used to recreate Mxg. The high level of nucleotide similarity of the Mxg coding regions to sorghum demonstrated here promises that the sorghum genome may provide a useful template for an assembly-to-reference strategy for the Mxg gene space. Improvements in costs and efficiency offered by 454 and Illumina sequencing make obtaining gene space sequences of Miscanthus and other Andropogoneae species with similarly complex genomes using short read technologies a feasible goal.
Materials and methods
Plant material
The M. × giganteus accession 'UIUC' was obtained from a stand of Mxg originally established in the early 1990s on the Ornamental Horticultural Research Farm at the University of Illinois at Urbana-Champaign, using rhizomes obtained from the Chicago Botanical Garden. Rhizomes from this clonal stand were propagated in potted soil in a greenhouse under controlled conditions of 14 hour days and 10 hour nights at 22 to 25°C, and a brief watering each morning. Leaves from newly initiated shoots were sampled for DNA analysis.
Each flow cytometry data point shown was conducted on nuclei isolated from young leaf tissue from a single greenhouse-grown plant of the genotypes described, obtained from a collection at the University of Illinois maintained by Dr J. Juvik. Nuclei stained with propidium iodide were measured for DNA content against chicken red blood cell standards as described by Arumuganathan and Earle [50]. The analysis was done at the Flow Cytometry and Imaging Core laboratory, Benaroya Research Institute at Virginia Mason, Seattle, Washington.
M. × giganteusnuclear DNA isolation and sequencing
Nuclei were isolated from 20 g of young Mxg leaf tissue using a protocol modified from Swaminathan [23]. Instead of using a sodium lauryl sulfate and protease K to lyse the nuclei, they were lysed by an incubation at 60°C in CTAB lysis buffer (2% CTAB,100 mM Tris pH9.5, 1.4 M NaCl, 1% PEG 6000, 20 mM EDTA and 0.25% β-mercaptoethanol). The lysate was extracted twice with neutral, tris buffered, 24:23:1 phenol:chloroform:isoamyl alcohol followed by an extraction with chloroform:isoamyl alcohol (23:1). Nuclear DNA was precipitated using isopropanol. The spool of genomic DNA was washed with 70% ethanol, dried and resuspended in TE. Any contaminating RNA was removed by an incubation at 37°C with RNaseA and repurification through additional phenol:chloroform and chloroform extractions, followed by ethanol precipitation and resuspension in nuclease free water. Mxg nuclear DNA (5 μg) was supplied to the Roy J Carver Biotechnology Center at the University of Illinois at Urbana Champaign [51] for library preparation and DNA sequencing using the Roche Genome sequencer FLX system (GS-FLX).
Sorghum genome sequences were obtained from two sources. To characterize repeat content and copy number, we downloaded 500,000 whole genome shotgun sequences, the sorghum_bicolor.022 portion of the sorghum whole genome shotgun dataset available from Trace Archive. These reads are unfiltered for repeats and hence comparable to the data generated by the FLX runs.
Comparisons to sorghum used the current versions of the genome assembly and coding sequence annotation: sbi1 (genome sequence) and Sbi1-4 (coding sequences). The BAC sequences for the copy number analysis were downloaded from [GenBank: AC169372, AC169373 and AC169376]. For classification of known repetitive sequences we used the plant repeat database [30]. To match Mxg reads to the sorghum draft genome and derived sequences, BLASTN was used at expect value cutoff 10-10 and with the repeat filter on and other settings as default. For comparison between survey sequences and the Plant Repeat Database, BLASTN was used at expect value cutoff 10-6 and with repeat filter off. Matches to each class of repeat were counted and percentage of each repeat class in the genome calculated, using the codes incorporated into the IDs of the plant repetitive sequences in the Plant Repeat Database [30].
Assemblies to detect novel repeats in Mxg
Overlapping, non-cognate short reads generated by the GS-FLX run were assembled using the 64 bit manyreads build of phrap ver 1.080721 [52]. All assemblies were done on a 16-core Intel Xeon 2.93 GHz server with 128GB DDR2 RAM. Parameters for the phrap assembly were the same as those used in [23], except that the minimum overlap length for two reads to be assembled was 14 bases at 100% nucleotide identity.
Copy number estimation
N is the number of sequences that match a window size of w, L is the average length of the sequence and c the estimated coverage of the survey. For Mxg, we used the estimate of 0.012 (1.2%) coverage of the total genome. Price et al. [55] estimated the haploid genome size of sorghum to be 818 Mbp (note that the draft assembly excludes telomeric, centromeric and rDNA repeats). We thus calculated the sorghum genome coverage as coverage of the haploid genome for the 500,000 sorghum capillary sequences at 0.63 (63%). Repeats were sorted according to the estimated percentage of each in the Mxg genome, calculated from the repeat cluster sequence length, genome sequence length and estimated copy number. The ten most abundant repeats were identified by excluding all but one cluster annotated with the same top hit to the Plant Repeat Database.
sRNA isolation, sequencing and analysis
A total of 6,736,105 sRNA signatures (18 to 32 nucleotides in length) from three Solexa flow cell lane data sets produced from three libraries from greenhouse-grown Mxg 'UIUC' tissues (leaf, inflorescence, and rhizome) were mapped to 20,774 Mxg repeat clusters (derived by non-cognate assembly of genomic survey reads, above) using GMAP (Genomic Mapping and Alignment Program) [56]. The program parameters were set to report all possible hits for every sRNA signature. Only sRNA hits with coverage equal to 100% and matching identity greater than or equal to 94% were used in the subsequent analyses. These parameters allow only one mismatch between the sRNA signature and the aligned genomic locus. For the gene space survey reads, this analysis was also performed with 100% coverage required and also 100% identity.
For the analysis of sRNA matches versus copy number, sRNA signatures were matched to the repeat cluster collection using GMAP as above, with up to one mismatch per signature allowed. For each repeat sequence, the number of small RNAs matching the sequence was divided by the estimated number of kilobases of the repeat present in the Mxg genome. This estimate of kilobases per genome was arrived at by using Equation 1 to give an estimate of copy number multiplied by the length of the sequence in kilobases. The total number of sRNA signatures per kilobase in the genome for the repeat clusters was then summed across each class of repeat within copy number bins; for example, all sRNA matching all MITE sequences expected to occur between 151 and 200 times per genome was summed to create a single value for MITE sRNA matches in this repeat class.
Phylogenetic analysis
Reads that matched Mxg nuclear ribosomal sequences [GenBank: AJ426562 and AJ426563] at 90% identity were identified using BLAT. There were 114 unique reads that matched either one or both Mxg nuclear ribosomal sequences using default BLAT parameters. Additional Miscanthus, Zea, Saccharum and Sorghum nuclear ribosomal sequences previously used to construct phylogenetic trees for Miscanthus and Saccharum [9] were obtained from GenBank. The sequences and the reads with a minimum overlap of 50 bp and 80% identity to AJ426562 were assembled using Sequencher (Gene Codes, Ann Arbor, MI, USA). Regions of variations within the reads were quantified. The greatest variation lay within a 150-bp region of ITS2 (Figure 4). The alignment matrices were then trimmed by removing gaps present in more than 95% of the sequences and truncating the sequence limits to this 150-bp region, to account for poor resolution of indels, especially in homopolymeric regions, in the sequencing method, and maximize the number of survey reads used in the analysis. A total of 19 Mxg survey reads met these criteria throughout the 150-bp ITS region and were retained for this analysis. Phylogenetic relationships were estimated using Bayesian inferences conducted in MrBayes version 3.1.2. The general time reversible model taking into account the shape of gamma distribution was used, as suggested by Modeltest3.7, as the most appropriate model of sequence evolution. The analysis was run for 1.5 × 106 generations with sampling at intervals of 100 generations, thus generating 15,000 sampled trees. A 50% majority rule consensus tree was generated to assign a posterior probability for each node using the last 5,000 trees sampled. The tree was visualized in Dendroscope [57].
Data access
The 454 sequence data described in this publication have been deposited in the NCBI Sequence Read Archive [SRA:SRA010791.2]. The sRNA data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [GEO:GSE20056].
bacterial artificial chromosome
- bp:
base pair
expressed sequence tag
internal transcribed spacer
miniature inverted repeat transposable element
- Mxg :
Miscanthus × giganteus
- rDNA:
DNA encoding the tandem array of rRNA genes
- siRNA:
small interfering RNA
- sRNA:
small RNA.
We thank Dr Jack Juvik (University of Illinois at Urbana-Champaign) for providing tissue samples of Miscanthus accessions, Geoff Benn to for growth and sampling of plants used in DNA and RNA isolations, Dr Karen Hudson for critical reading of the manuscript, and the Roy J Carver Biotechnology Center at the University of Illinois at Urbana-Champaign for generating the 454 sequencing data. Funding for the genome survey work and all analysis was provided by the Energy Biosciences Institute to SPM, MEH, RM and DR, funding for sRNA data was provided by NSF DBI #0638525 to BCM and PJG.
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- Fu Y, Emrich SJ, Guo L, Wen TJ, Aluru S, Ashlock DA, Schnable PS: Quality assessment of maize assembled genomic islands (MAGIs) and experimental verification of predicted novel genes. Proc Natl Acad Sci USA. 2005, 102: 12282-12287. 10.1073/pnas.0503394102.PubMedPubMed CentralView ArticleGoogle Scholar
- Bedell JA, Budiman MA, Nunberg A, Citek RW, Robbins D, Jones J, Flick E, Rholfing T, Fries J, Bradford K, McMenamy J, Smith M, Holeman H, Roe BA, Wiley G, Korf IF, Rabinowicz PD, Lakey N, McCombie WR, Jeddeloh JA, Martienssen RA: Sorghum genome sequencing by methylation filtration. PLoS Biol. 2005, 3: e13-10.1371/journal.pbio.0030013.PubMedPubMed CentralView ArticleGoogle Scholar
- Arumuganathan K, Earle ED: Estimation of nuclear DNA amounts of plants by flow cytometry. Plant Mol Biol Rep. 1991, 9: 229-241. 10.1007/BF02672073.View ArticleGoogle Scholar
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- Green P: Phrap, SWAT, CrossMatch. 2008, University of WashingtonGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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More Videos...
Border 2 Border! Epic American Road Trip in a Toyota Tundra TRD Pro - Dirt Every Day Ep.41
On this episode of Dirt Every Day presented by 4 Wheel Parts, Fred cuts a diagonal line across the lower 48 in an epic road-trip in Toyota's new Tundra TRD Pro. From the Gulf of Mexico to the north western Pacific/Canada line Fred and his team explore a variety of off road destinations on an exciting 18-day adventure. Dirt Every Day appears every fourth Tuesday on the Motor Trend channel. http://www.youtube.com/motortrend Subscribe now to make sure you're in on all the action! http://www.youtube.com/subscription_center?add_user=motortrend Facebook - http://facebook.com/motortrendmag & https://www.facebook.com/DirtEveryDay Twitter - http://twitter.com/motortrend Google+ - https://plus.google.com/+motortrend/posts Website - http://www.motortrend.com & http://www.4wheeloffroad.com/
Trying out the TRD Supercharger
First runs with the TRD Supercharger on a 08 Tundra DC Limited 4x4 with TRD cat back duals Still trying to get use to the truck and trying like hell to not hit the rev limiter...
SDHQ Built Supercharged Long Travel Toyota Tundra TRD
SDHQ Built Long Travel Toyota Tundra test & tune session in Parker, AZ along the BITD Parker 425 course featuring front Long Travel from Total Chaos and Total Chaos rear spring under conversion with Fox shocks. A SDHQ bumper with Vision X lights shot by MWilsonPhoto.com
2015 Toyota Tundra, Quick Drive, TRD SUPERCHARGED!!!
Thanks for watching my review of my buddies 2015 Toyota Tundra. He bought the truck new and got a dealer installed TRD Supercharger. Truck makes 500ish at the crank I believe.. Enjoy :)


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Member Since: March 4, 2008
Country: United States
Electronics (duh), Aviation/Aerospace, Metal working, things that fly, things with blinky lights, flying things with blinky lights, etc etc.
Edit: description says black, everything else says clear. Assuming copy/paste typo.
I need something like this that then automatically rotates through around 8 single cells. Round-robining a top off/state check on each battery. ideally a channel expansion board, maybe 4 each, so you could just add as many ports as you needed. I2C controlled or such.
Hookup guide is 404.
Hmm, any relation to Conner Kuhns?
That aside, look forward to seeing the build progress!
Pressurized propane doesn't need a pump. A regulator might be good though…
Depending on the dimensions, some plaster of paris and a popsicle tray might make perfect sensorsicles. Not sure how much that helps with corrosion though.
Does anybody know if one can bodge this together with lcdproc without herculean effort? I expect to be using a usb/serial adapter, motherboards being sans serial these days.
I'd really like to see an FTDI alternative serial/usb board.
After recent cluelessness by FTDI, I'm no longer comfortable with them as a source, and there seems to be a few other options available.
Would pairing this with the mechanum wheels be appropriate, or does it need gearing down?
Parts for a wearable pulsemeter for aerobic fitness goals.


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The following testcase asserts on TI revision a3eeee8f7803 (run with -m -n -a),
tested on 64 bit:
var o = {
w: 2,
x: 3
var o = 430717;
o.x = 4;
Created attachment 525968 [details] [diff] [review]
lhs->isTypeKnown() returned true after the call to frame.shimmy, so we did not rejoin from the OOL call to stubs::SetName and marched right into PutActivationObjects.
*** Bug 649936 has been marked as a duplicate of this bug. ***
Ah, noticed this and fixed it in rev dca50d9a5047, we also did the same thing in jsop_getprop (internet blackout since yesterday afternoon, sorry). I'll add the testcase.
A testcase for this bug was automatically identified at js/src/jit-test/tests/jaeger/bug649824.js.


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Invaluable cannot guarantee the accuracy of translations through Google Translate and disclaims any responsibility for inaccurate translations.
Show translation options
Lot 182: ANTIQUE OAK TABLE W GLASS BALL & CLAW FEET
Charleston Antiques & Fine Art- October 23rd
by Charleston Estate Auctions
October 23, 2016
Mr. Pleasant, SC, USALive Auction
Looking for the realized and estimated price?
Description: Antique Round Oak Occasional Table with Glass Ball and Claw Feet. Measures 30"x30"x29". PROVENANCE: A Charleston SC Private Estate. *Pickup only or Shipping on this item offered through On the Go Transport 843-360-7472, UPS 843-202-0141, Eshipify 702-715-0168 or A.J. Williams (SC-local) 843-442-0462. Please contact them or your own choice of shipper directly.**
Condition Report: good
Browse more items


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Hammer falls on raft of cut-price homes
First-time buyers and investors alike were treated to yet another rock bottom house clearance sale in Belfast on Thursday night.
Dozens of cut-price properties went under the hammer — with several selling for just over £20,000 — and many going for only 20% of their original value during the boom.
As the latest housing price survey shows the market is still on a downward spiral, dozens of buyers had their catalogues in hand and bidding cards held high at Wilson's Auctions in Mallusk.
One lucky young first-time buyer — Ross Irwin — picked up a three-bedroom house in Newtownabbey for a measly £26,000, less than a quarter of the original asking price at the height of the property boom.
"I'm a first-time buyer and it's just for myself," he said.
"I've been to these before, my father spotted this one and I wasn't able to get to it so I'm taking his word.
"I'm happy as Larry, though — you couldn't beat it."
The bulk of the some 30 lots which went under the hammer sold on the night, with the crowded auction house heaving with eager bidders.
The man holding the gavel was auctioneer Gareth Semple, who said the large number of keen bidders showed just how busy the market has become with eager investors.
"Out of 30 we have sold approximately 25 outright. Things are down at the moment but conditions are now right again for investors to start buying properties.
"You have terraced houses that are now going for £30,000; we also had a few first-time buyers, too, and that's good to see," he said.
Several other terraced houses sold for a fraction of their original market value, including a three-bedroom property in Belfast which went for £24,000.
Those in the market for something a bit bigger could have landed yet another bargain — snapping up a massive five-bedroom house in Portavogie which went for a mere £49,000, £90,000 less than previously valued.
Further north one lucky bidder grabbed a four-bedroom detached home in Ballymoney, Co Antrim, once valued at a quarter of a million pounds, for only £88,000.
And along with a tranche of budget homes going under the hammer, several low cost office properties also sold for significantly less than they were once worth.
According to the latest quarterly survey of property prices, the average cost of a home is down 3.6% on last year to just under £139,000.
Belfast Telegraph


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Action Mahjong
by: GAMBLIC • 1.6K
All Android applications categories
All Android games categories
by: GAMBLIC • 1.6K
"Action Mahjong" is similar to traditional mahjong, but is played in a different way.
The objective of this simple yet addictive game is to eliminate pairs of identical tiles. As long as they are separated by two kinks or less, tap on matching tiles to eliminate them as fast as you can!
★ over 400 maps!
★ 10 adorable characters to choose from!
★ fun and unique touchscreen actions during game-play!
★ 20 types of avatars available!
★ over 200 missions to conquer!
★ wide variety of items to redeem!
★ crisp touch action~~
★ explosive and exciting effects~~~~
★ smart touchscreen effects: cut, rub, hit~!!!
Tags: action majong , action mahjong
Users review
from 1.589 reviews
Download Action Mahjong
Free - V1.0 - 14M
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Solange Looks Glorious on Interview's Powerful February Cover
Interview hasn't been on our forum's radar much since Willow and Jaden Smith's joint cover back in September. This month, the magazine reminds us that it's still a contender with its newly-unveiled cover for February. Solange Knowles makes for a surprising yet welcome cover star, serving modern-day Diana Ross vibes in the powerful and arresting image by Mikael Jansson.
The cover became an instant sensation in our forums. "OMG this is sublime. Cannot wait to buy this issue. A Seat At The Table is one of the best albums last year. Solange is on fire!" declared happycanadian within moments of the cover dropping.
"What a beautiful cover! Just stunning. The inside shoot is amazing too, Mikael can do no wrong in my eyes and Solange is fantastic. I haven't gotten round to picking up Interview in a while but I'll definitely be getting this," said an equally as impressed LastNight. (more…)


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"I always believe that a journey to a thousand miles, starts with a single step". I am student of IT. My major motto is to be transparent with clients and to provide them best of my services. Client satisfaction is my first priority.
I am expert in :-
* Deep internet research
* Data entry
* Document conversion
* Amazon Product Research
* Microsoft Csv
* Video Editing
*LinkedIn Lead generation


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Cooks Oasis Garden Villa 6
- Airport Shuttle
- Breakfast
- Gym
- Parking
- Pets Allowed
- Swimming Pool
- Wifi
- Smoking Allowed
This Garden Villa is a great free standing family holiday villa within a small and relaxed complex offering the benefit of privacy but also reception services when and if required.
It offers its own deck area with outdoor furniture for that relax in the sun, and has a dining table and chairs, and comfy chairs. Convenience of an in -room electronic safe.
Bedding is a super king size bed (that can be split to singles). There's a ceiling fan for your comfort, and air-conditioning available. It also has a well set up kitchenette, and a spacious bathroom with a shower, toilet , and vanity. Hairdryer and toiletries for convenience.
The kitchenette comes with an electric hob, microwave, fridge, electric jug, toaster, coffee plunger, cooking equipment, well supplied utensils, crockery, and kitchen equipment along with basics such as tea, coffee, sugar, salt and pepper.
Air-conditioning is offered if required, at an extra $20 per day.
You also have your own flat screen TV and DVD player and can access books and / or DVDs from the family room.
You can use our kayaks and snorkeling gear, beach towels. Wander to restaurants nearby at night, BBQ at the complex, or cook in the privacy of your own villa. Any which way, you're going to love your holiday for the relaxed , sun filled, fun filled times spent here.
Special conditions:
Smoking allowed outside only.
Extras: Cleaning (included); Linen (included);


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1882 – Berkeley Square Houses, London
Architect: J.T. Wimperis
The Examination Schools were designed by Thomas Graham Jackson, Oxford's most famous 19th century architect, in 1876 and their construction was completed in 1882.
Perspective View to St. Aldate's Street including ground & 1st floor plans. Selected Design by H.T.
A conspicuous and imposing feature when entering Newark from the old Great North Road,


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I've had 4 starters replaced in as many months. What could be making them go bad. I when I return them and have them tested they work. I've checked for 12 volts going to the starter and have that. Solenoid replaced already.
Asked by balance for the 1996 Ford Bronco


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Jay Tucker net worth: Jay Tucker is an electrical engineer who has a net worth of $500 thousand dollars. Jay Tucker recently married comedienne, actress and host, Niecy Nash. The two met in the fall of 2009, and became engaged a year later. They married at the Church Estate Vineyards with 200 guests in attendance. Ms. Nash starred on the comedy series, "Reno 911!", as well as guest starring in a number of other television shows and films, such as "The Bernie Mac Show" and "That's So Raven". She served as an entertainment correspondent for "The Tonight Show with Jay Leno", and also hosted "Clean House" on TLC until 2010. She and Mr. Tucker were the subjects of a wedding special that aired on TLC in 2011, and her family is now participating in a reality show called, "Leave It to Niecy", about their life together. She has three children from a previous marriage, and Mr. Tucker has one.
Jay Tucker Net Worth
Jay Tucker
More Actors
All net worths are calculated by applying a proprietary algorithm. The results are then fact checked and confirmed by a team of editors. We scan all publicly available data and resources and work diligently to ensure that our numbers are the most accurate and up to date net worth data you will find anywhere on the internet.


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Works from my home now too.
Read the rules before posting.
Do you have an unsolved issue? Give us more informations about your problem.
Do you have problems with compiling or installing TrinityCore? Unknown96's Tutorial will help you.
Visit our other helpful sites:
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There are currently 1 users browsing this thread. (0 members and 1 guests)


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This area is part of Plandora project. Here you will find articles and tutorials about features and tool usability.
How a project customer could do to create a new request? (pt-BR demo)
How project leader should do to handle a new request? (pt-BR demo)
How project resource (worker) could do to update the task status? (pt-BR demo)


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112 Trans Canada Hwy., Gander, NL, Canada, A1V 1P8
Phone: (709) 256-3535 Fax: (709) 256-9302
Choice Preferred Platinum Award
Hotels that achieve the prestigious Platinum Award represent the top 3% of hotels within each Choice brand. The awards are based on a ranking system that measures guest satisfaction, product quality, room condition and cleanliness, and staff service. These properties have a proven track record of providing exceptional guest service and facilities.
This Comfort Inn® hotel is set close to many of Gander`s attractions, including the North Atlantic Aviation Museum, Cobb's Pond Rotary Park, the Commonwealth War Graves Commission Cemetery, Gander Mall, Fraser Mall, Gander Lake and Gander International Airport.
All tastefully decorated guestrooms at this pet-friendly Gander hotel are air-conditioned and come equipped with hair dryers, ironing equipment and cable television. This 100% non-smoking hotel also features kitchenettes. Jungle Jim's Family Restaurant is located on the premises.
Other hotel features include an exercise room and guest laundry facilities.
High-value amenities of this Comfort Inn hotel include:
Check-In Time: 15:00 Check-Out Time: 12:00 Guest Rooms: 79


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Free Responsive, Multipurpose Business and Corporate Theme perfect for any business.Read More
How to better recruit and promote the best person for every role. Selecting the right person is critical for the organisation's success and is based on Job Eli...Read More
Developing technical, soft skills & attitudes and improve performance. Top executives and management understand that they cannot win, without the right peo...Read More
Learn how to better align your team to the organisation's objectives. Organisations are continually challenged to identify ways to increase employee engageme...Read More
Discover how you can strengthen your staff's "Whole Life Balance". We all have a stake in our staff's wellbeing or what we term Whole Life Balance. In b...Read More


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Marshall Thundering Herd Tickets
One of America's greatest collegiate football franchises, Marshall Thundering Herd is all set to compete against the fiercest competitors this season. To watch your favorite football team play live grab your Marshall Thundering Herd tickets that are now up for sale.
Marshall Thundering Herd Venues
- Marshall Thundering Herd Cam Henderson Center
- Old Dominion Monarchs vs. Marshall Thundering Herd Constant Convocation Center
- Marshall Thundering Herd FAU Stadium
- North Carolina Charlotte 49ers vs. Marshall Thundering Herd Halton Arena
- Marshall Thundering Herd Joan C. Edwards Stadium
Marshall Thundering Herd Cities
- Marshall Thundering Herd Boca Raton FL
- Marshall Thundering Herd Charlotte NC
- Marshall Thundering Herd Huntington WV
- Marshall Thundering Herd Norfolk VA
Marshall Thundering Herd Schedule 2017-18
- Old Dominion Monarchs vs. Marshall Thundering Herd, Constant Convocation Center tickets for 02/23 08:00 PM at Constant Convocation Center, Norfolk, VA
- Thu Feb 23 2017
- North Carolina Charlotte 49ers vs. Marshall Thundering Herd, Halton Arena tickets for 02/25 06:00 PM at Halton Arena, Charlotte, NC
- Sat Feb 25 2017
- Marshall Thundering Herd vs. North Texas Mean Green, Cam Henderson Center tickets for 03/04 07:00 PM at Cam Henderson Center, Huntington, WV
- Sat Mar 04 2017
- Marshall Thundering Herd vs. Kent State Golden Flashes, Joan C Edwards Stadium tickets for 09/16 03:30 AM at Joan C Edwards Stadium, Huntington, WV
- Sat Sep 16 2017
- Marshall Thundering Herd vs. Old Dominion Monarchs, Joan C Edwards Stadium tickets for 10/14 03:30 AM at Joan C Edwards Stadium, Huntington, WV
- Sat Oct 14 2017
- Marshall Thundering Herd vs. Florida International Golden Panthers, Joan C Edwards Stadium tickets for 10/28 03:30 AM at Joan C Edwards Stadium, Huntington, WV
- Sat Oct 28 2017
Formed in 1895, the Marshall Thundering Herd football team is one of the fifteen varsity franchises that play from the Marshall University. Apart from football the institution also supports sports' programs such as baseball, basketball, cross county, football, soccer, golf, volleyball, track and field, swimming and diving, softball and tennis. The Marshall University's football program currently competes in National Collegiate Athletic Association's Eastern division in the USA Conference. The team plays its homes games at the Joan C. Edwards Stadium. The open air stadium located in Huntington, West Virginia offers a seating capacity of thirty nine thousand people including expandable space that can cater to audiences as much as fifty thousand.
The Marshall Thundering Herd football team currently holds the record of the best overall score by a football franchise at its home stadium. With a score of 118-19, the Herd surpasses renowned teams from University of Alabama and West Virginia University. With Marco the Buffalo as its official mascot, the Herd currently supports the colors green and white at all their games. Coached by Doc Holliday with Mike Hamrick as its athletic director, the Herd is a force to reckon with.
Over more than a decade of games, the Herd has been able to establish an all-time record score of 542-520-47. Their postseason bowl record is 7-3 after making their way to ten bowl games. Over their career, the Herd has won two National Collegiate Athletic Association titles, twelve Conference titles and forty four Consensus All-Americans titles. If this wasn't enough the football franchise has also gathered six MAC Championships from 1998 till 2002. The team also supports long established rivals with teams namely the West Virginia Mountaineers, East Carolina Pirates, Ohio Bobcats and UCF Knights.
Four of the team's players have won individual awards such as the Walter Payton Award, Fred Biletnikoff Award, Sammy Baugh Trophy and William V. Campbell Trophy. These awards have been given to players including Michael Payton, Randy Moss and Chad Pennington respectively. Players who have been inducted to the NCAA Hall of Fame include Mike Barber, Harry Young, Jackie Hunt, Troy Brown, Jim Donnan and Frank Gatski.
The Herd has also produced players that have been drafted into the National Football League. Marshall Thundering has being invited to play with teams such as Tennessee Titans, Baltimore Ravens, Indianapolis Colts, Philadelphia Eagles, New England Patriots, Oakland Raiders and Buffalo Bills. Watch this successful football team as it prepares to compete with a tough competitor yet again. Grab your set of Marshall Thundering Herd tickets immediately to watch the adrenaline pumping game.
Marshall Thundering Herd Events Tickets Prices
The average price for Marshall Thundering Herd Tickets start from $9. The minimum get in price is $9 for Marshall Thundering Herd Tickets at the Constant Convocation Center, Norfolk. For a detailed look at ticket prices and amazing discounts, visit our website.
|Average Ticket Price
|Get In Price
(Minimum Price)
|Old Dominion Monarchs vs. Marshall Thundering Herd
|North Carolina Charlotte 49ers vs. Marshall Thundering Herd
|Marshall Thundering Herd vs. North Texas Mean Green
|Marshall Thundering Herd vs. Kent State Golden Flashes
|Marshall Thundering Herd vs. Old Dominion Monarchs
|Marshall Thundering Herd vs. Florida International Golden Panthers
|Florida Atlantic Owls vs. Marshall Thundering Herd
|Boca Raton
|Marshall Thundering Herd vs. Western Kentucky Hilltoppers
|Marshall Thundering Herd vs. Southern Miss Golden Eagles
* Ticket Prices vary on hourly basis.


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Liquid Force Men's S4 Wakeboard w/ Transit Bindings '13
The Liquid Force Men's S4 Wakeboard w/ Transit Bindings is the board that will help you get to the top! The S4 is super quick from rail to rail and features a progressive 3-stage rocker for explosive wake hits. The high end performance is there for those in need but don¹t let it scare you away. This board has what we call a ³user friendly² performance feel. Even the top pros need a bit of forgiveness on the wake to help land their toughest tricks, so whether your tough trick is a 1080 or a 180, you¹ll be digging the extra love this board gives. Step into a pair of 2013 Transit bindings and you'll be ready to take your riding to the next level. The Transit is the perfect binding to transition from an entry to mid-level weekend warrior to the dedicated "core" rider. This year they upgraded the Cuff Stretch Zone to Injection molded TPU allowing the upper to wrap even better. Loaded with performance features such as the dual zone lacing and control liner you can customize the Transit to your perfect fit.
- Brand
- Liquid Force
- Features
- Wakeboard: Progressive, Three Stage Rocker Quad V Hull, Double Concave-Faster rail to rail, extra lift, softer landings Low Volume Rails- Low volume equals, faster, better holding rails. Rail flex at end for more drive out of turns. Center Line Single Vent- The vent detaches water flow across center line at the end of the board for increased speed Deep Molded Long Base Side Fins / Shallow Molded Inside Blade Fins- Long base for superior hold at speed... cutaway trailing edge for quicker release off the wake. Shallow blades at the top of inside V, control water flow through inside concave for more control. Bindings: IPX Chassis- Injection molded Nylon plastic chassis with metal teeth Impact EVA- Takes the shock of landings and displaces it from your foot EVA Foot-Bed- 3D molded foot-bed with ergonomic contours Control Liner- Seamless molded tongue design and integrated J bars Zonal Lace System- Lacing system allows you to adjust lace tension Cuff Stretch Zone-Stretch zone that allows flex with no restrictions
- Age
- Adult
- Gender
- Male


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The Nigerian Communications Commission, NCC, has warned telecom operators to desist from f sale of pre-registered SIM cards or face stiff sanctions.
The Commission accused major telecom operators and their distributing agents of selling pre-registered sims, despite the ban.
According to NCC's Director of Public Affairs, Mr Tony Ojobo, "Despite regulations and directives against the sale, operators are still selling pre-registered SIM cards in several parts of the country".
Sections 19 and 20 of NCC's Registration of Telephone Subscribers Regulations, 2011, says the commission would not hesitate to mete out appropriate sanctions for policy violations.
Read Also: The economy is not in recession — Vice President Osinbajo.
Section 19 of the Act states that:
(1) Any licensee who fails to capture, register, deregister or transmit the details of any individual or corporate subscribers to the Central Database as specified in these Regulations or as may be stipulated from time to time by the Commission is liable to a penalty of N200,000.00 for each subscription medium.
(2) A licensee who activates any Subscription Medium without capturing, registering and transmitting the personal information to the Central Database commits an offence and shall on conviction be liable to a fine of N200, 000.00 for each unregistered activated Subscription Medium. 20.
(1) Any licensee who activates or fails to deactivate a subscription medium in violation of any provision of these Regulations is liable to pay a penalty of N200,000.00 for each unregistered but activated subscription medium.
(2) Where the Commission is satisfied that a body corporate is culpable, the Director, Chief Executive Officer, Manager or Secretary shall also be liable to pay a fine of N200, 000.00 unless, having regard to the nature of his functions in that capacity and to all the surrounding circumstances, he proves that-
(i) the offence was committed without his knowledge, consent or connivance; and (ii) he took all reasonable precautions and exercised due diligence to prevent the Commission of the breach.


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Event: LONDON PARIS NEW YORK actor Ali Zafar strums guitar and sings at the Kala Ghoda Festival 2012. Actress Aditi Rao Hydari also joined Ali on the stage
Guest List: Ali Zafar, Aditi Rao Hydari
Bollywood celebs Kangana Ranaut, Vishal Bhardwaj, Randeep Hooda, Aanand L Rai, Vikas Bahl, Vidhu Vinod Chopra, Anupama Chopra, Prasoon Joshi among others were snapped at the special screening of RANGO...
Bollywood celeb Adah Sharma promoted her upcoming film COMMANDO 2.
Bollywood celebs Riya Sen, Kyra Dutt, Gulshan Nain, Vivek Vaswani among others were snapped at the special screening of LONELY GIRL.
Bollywood celebs Ranveer Singh, John Abraham, Kiran Rao, Manyata Dutt, Poonam Dhillon, Baba Siddiqui, Shekhar Suman comes out to vote.
Bollywood celebs Varun Dhawan and Alia Bhatt were snapped at Mehboob studio.


text: 300 FXUS65 KCYS 240037 AFDCYS Area Forecast Discussion National Weather Service Cheyenne WY 537 PM MST Thu Feb 23 2017 .SHORT TERM...(Tonight) Issued at 244 PM MST Thu Feb 23 2017 Latest water vapor loop was showing a very elongated upper level trof stretching from the intermountain west east towards the high plains. This has created a large area of broad scale diffluence over much of the region this afternoon. Latest surface analysis was showing surface low continuing to weaken across southwest Carbon county in response to the jet energy pushing east into the high plains. Meanwhile, the best low level cyclogenesis has shifted into southwest Kansas. As this surface low moves to the northeast this will cause the high pressure ridge to nose down the lee slope of the Rockies. The main question will be how much more snowfall will occur tonight. Based on the latest satellite trends it appears like the majority of the heavier snowfall was still in east central Wyoming and the northern Nebraska panhandle. This snowfall is showing some signs of diminishing with the visibility now starting to climb a bit in Torrington and Chadron. However, we cannot rule out another 2 to 4 inches in this region before the night is finished due to plenty of upper level divergence. We have cancelled the Blizzard Warning in southwest Carbon county due to that area being centered near the surface low with lack of snow and lower wind speeds. We may also have to drop temperatures even more tonight due to the influence of the surface high nosing down the lee slope of the Rockies. Otherwise, current forecast looks to be in good shape with the snowfall diminishing during the course of the night. .LONG TERM...(Friday through Thursday) Issued at 244 PM MST Thu Feb 23 2017 The upper low deepens over the central Plains Friday with snow rates diminishing east of the Laramie Range mid to late morning while persisting through the afternoon out west across Carbon and Albany counties. Models consistently are diminishing snow for the lower elevations west of the Laramie Range by Friday night and diminishing in the higher elevations of the Sierra Madre and Snowy Ranges Saturday evening. After that, next chance for snow comes out west in the mountains Monday as a low amplitude fast moving shortwave moves east across the central Rockies. As for temperatures, expect Friday and Saturday to be a bit colder than guidance due to the snowpack, with all areas remaining below freezing until Monday. Slight warmup expected next week with 30s and 40s for highs east onto the plains and 20s to lower 30s west of the Laramie Range. && .AVIATION...(For the 00Z TAFS through 00Z Friday evening) Issued at 537 PM MST Thu Feb 23 2017 IFR to LIFR will prevail at most airports through tonight with MVFR at Laramie closer to the surface to midlevel low pressure circulation and in downslope flow. Will steadily see snow lighten in intensity overnight, although llvl moisture should keep fog in place and thus low vis. Conditions will slowly improve through the day, with MVFR/VFR returning to the area by late afternoon as the snow ends. && .FIRE WEATHER... Issued at 244 PM MST Thu Feb 23 2017 No fire weather concerns are expected due to widespread snowfall. && .CYS WATCHES/WARNINGS/ADVISORIES... WY...Winter Storm Warning until 11 AM MST Friday for WYZ101>103- 105>108-110-112>119. Blizzard Warning until 11 AM MST Friday for WYZ104-109. NE...Winter Storm Warning until 11 AM MST Friday for NEZ002-003- 019>021-054-055-095-096. && $$ SHORT TERM...REC LONG TERM...JG AVIATION...RJM FIRE WEATHER...JG


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Children who were overweight or obese at age 10 were more likely to have higher mean heart rates and systolic blood pressure than normal-weight peers, but an effect on vascular function was not found, according to a study in the Journal of the American College of Cardiology. "Our findings suggest that there may be a window of opportunity in early childhood to target and potentially reverse obesity before adverse vascular consequences manifest," the investigators wrote.
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Montreal Canadiens head coach Michel Therrien is really a cry baby... Therrien did nothing but cry and complain during the second round series between the Boston Bruins and Canadiens as well. He confronts Ulf Samuelsson during the Canadiens team practice. Seriously, what are the Habs going to give away during their practice?
SNYRangers Blog -- Therrien's quote on the subject was "There is always a gentleman's agreement between two teams and the general manager that coaches are not allowed to attend practices between games. Game day is different. So when we saw those assistant coaches there, they were not supposed to be there, so we let them know." (via the NHL)
#canadiens Michel Therrien was upset at Ulf Samuelson who was watching practice. A no-no when it's not a morning skate. #tvasports
— Renaud Lavoie (@renlavoietva) May 24, 2014