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Original Research ARTICLE
Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis (Vitis vinifera L. × Vitis amurensis Rupr.)
- College of Horticulture, Shenyang Agricultural University, Shenyang, China
In this study, 149 F1 plants from the interspecific cross between 'Red Globe' (Vitis vinifera L.) and 'Shuangyou' (Vitis amurensis Rupr.) and the parent were used to construct a molecular genetic linkage map by using the specific length amplified fragment sequencing technique. DNA sequencing generated 41.282 Gb data consisting of 206,411,693 paired-end reads. The average sequencing depths were 68.35 for 'Red Globe,' 63.65 for 'Shuangyou,' and 8.01 for each progeny. In all, 115,629 high-quality specific length amplified fragments were detected, of which 42,279 were polymorphic. The genetic map was constructed using 7,199 of these polymorphic markers. These polymorphic markers were assigned to 19 linkage groups; the total length of the map was 1929.13 cm, with an average distance of 0.28 cm between each maker. To our knowledge, the genetic maps constructed in this study contain the largest number of molecular markers. These high-density genetic maps might form the basis for the fine quantitative trait loci mapping and molecular-assisted breeding of grape.
Grape (Vitis vinifera) is one of the most important fruit trees in the world and possesses high nutritional value. The demand for high-quality grape berries has been increasing in recent years. In 2013, 67 million tons grapes were produced globally over a cultivation area of 7 million hectare1. However, diseases and pest infestation as well as natural disasters adversely affect the grape growth, yield, and quality; therefore, modification of important quality characteristics and stress resistance has been a key target for grape breeders. Researchers use potential germplasms for crossing and composite-crossing to produce new cultivars with multiple favorable traits. However, the traditional cross-breeding techniques are time-consuming and require setting up many generations of hybridizations and back crosses. Recent advances in molecular biology have led to the construction of high-density molecular genetic maps and development of quantitative trait loci detection methods to identify target traits for grape breeding.
Lodhi et al. (1995) constructed the first molecular map of grapevine by using 422 random amplified polymorphic DNA (RAPD) markers, 16 restriction fragment length polymorphism (RFLP) markers and several isoenzyme markers and 60 F1 progenies of the cross between 'Cayuga White' × 'Aurore.' Since then, molecular genetic maps of grape have been constructed (Dalbó et al., 2000; Grando et al., 2003; Adam-Blondon et al., 2004; Riaz et al., 2004; Doligez et al., 2006; Lowe and Walker, 2006; Salmaso et al., 2008).
The previously published genetic maps for grape were mostly constructed using the F1 population as the mapping population; only some studies used F2 populations. Further, various types of molecular markers such as RAPD, amplified fragment length polymorphism (AFLP), single sequence repeat (SSR), sequence-related amplified polymorphism (SRAP), RFLP, and single nucleotide polymorphism (SNP) were used for the construction of the genetic maps. Of these markers, RAPD, AFLP, and SRAP markers lack repeatability and stability, and most of these markers have no sequence information; therefore, integrating information obtained using these markers with maps obtained using other markers is difficult. SSR, RFLP, and SNP markers are stable and reliable and can be used for map construction; however, SSR and RFLP marker development is a time-consuming and costly process, and only limited numbers of markers can be produced. Further, the efficiency of SSR markers is very low (Kennedy et al., 2003; West et al., 2006; Huang et al., 2009), and the uneven distribution of SSR markers in the genome hinders the construction of high-density maps. Numerous SNPs exist in the genome, and they are an ideal marker for constructing high-density genetic maps. However, detecting SNPs is difficult. Few linkage maps have been constructed using SNP markers, but the genome coverage degree of these maps was very low.
The next-generation sequencing technique can be used to detect large quantities of SNP markers in the entire genome. Several methods have been used for identifying SNPs such as restriction site associated DNA sequencing (Miller et al., 2007; Peterson et al., 2012) and genotyping-by-sequencing approach (Poland et al., 2012). By using restriction site associated DNA sequencing technique, high-density genetic maps for ryegrass (Pfender et al., 2011), barley (Chutimanitsakun et al., 2011), and grapevine (Wang et al., 2012) have been constructed; such maps have been constructed using the genotyping-by-sequencing approach for barley and wheat (Poland et al., 2012). Recently, Sun et al. (2013) developed a simplified genome sequencing technique [specific length amplified fragment sequencing (SLAF-seq)] that can efficiently explore large numbers of SNPs. The SLAF-seq technique has been used to construct high-density genetic maps for soybean (Qi et al., 2014) and sesame (Zhang et al., 2013).
In this study, we used the F1 population derived from an interspecific cross of 'Red Globe' (V. vinifera L.) × 'Shuangyou' (Vitis amurensis Rupr.) and the parent populations to construct a high-density genetic map of grape (V. amurensis). This map consists of numerous SNP and insertion-deletion (In-Del) markers (7,199 markers). It can be used for the fine quantitative trait loci mapping of important traits in grape, such as the resistance of cold and disease.
Materials and Methods
Plant Material and DNA Extraction
In the autumn of 2009, seeds were collected from the F1 population of a cross of 'Red Globe' (V. vinifera L.) and 'Shuangyou' (V. amurensis Rupr.). Between October 2009 and February 2010, the stratification treatment was performed. The hybrid seeds were sown in a greenhouse in March 2010. Young plants were transferred to an experimental field at the Shenyang Agriculture University in Shenyang (126°33′41′′E, 41°49′24′′N), Liaoning Province, China, and planted from April to June in batches. In all, 777 individuals were obtained, of which 149 individuals and their parents were used as the mapping population. Young healthy leaves from the two parents and progenies were collected, and genomic DNA was extracted using the CTAB method (Hanania et al., 2004). DNA was quantified using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and observed using electrophoresis on 0.8% agarose gels with lambda DNA as a standard.
The SLAF-seq method was used to genotype the 149 progeny individuals and the two parents, as described previously (Sun et al., 2013) with slight modifications. The genomic DNA from each sample was treated with RsaI, HaeIII (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), and ATP (NEB), and maintained at 37°C. The restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB), and HaeIII primer for polymerase chain reaction (PCR) analyses. The PCR products were purified using E.Z.N.A. Cycle Pure Kit (Omega, London, UK). The purified PCR products were pooled and incubated at 37°C with HaeIII, T4 DNA ligase, ATP, and Solexa adapter. After incubation, the products were purified using Quick Spin column (Qiagen, Venlo, Netherlands) and electrophoresed on 2% agarose gel. Gel Extraction Kits (Qiagen) were used to isolate the SLAF products that ranged from 550 to 600 bp (including the adapter sequence indexes and adaptors). The products were then processed for a second PCR by using the Phusion Master Mix (NEB) and Solexa Amplification primer mix. According to the Illumina sample preparation guide (Illumina, Inc., San Diego, CA, USA), the PCR products were gel purified, and SLAFs of 314–414 bp were selected for paired-end sequencing on an Illumina HiSeq 2500 sequencing platform performed by the Beijing Biomarker Technologies Corporation2. DNA sequence reads were 200 bp in size.
According to the barcode sequences, raw reads were demultiplexed to individuals. Subsequently, low-quality reads (quality score < 30) were filtered out. After the barcodes were trimmed from the reads, reads of 100 bases from the same samples were mapped onto the grape genome sequence by using SOAP denovo2 software (Luo et al., 2012). SOAP2 was used with the default parameters, but r = 0, M = 4, m = 50, and x = 1,000, where r = 0 indicates that multiple matches are not reported, M = 4 detects the best hits, and m = 50 with x = 1,000 suggests that the insert size is 50–1,000 bp. Sequences mapped to the same position were defined as one SLAF loci. In each SLAF, most polymorphic loci found between the parents were SNPs. All polymorphic SLAF loci were genotyped with consistency in the offspring and parental SNP loci.
All SLAF markers were filtered four times, and the quality was assessed as described by Sun et al. (2013). The markers with less than 3 SNPs and average sequencing depth higher than 3 were treated as high-quality SLAF markers. These markers were used to construct high-density genetic maps.
Linkage Map Construction
Since next-generation sequencing data might include many genotyping errors and deletions, which could reduce the quality of the high-density linkage maps, High Map Strategy was used to order the SLAF markers and correct the genotyping errors in the linkage groups(LGs; Liu et al., 2014a). All high-quality SLAF markers were allocated to 19 LGs on the basis of their locations on chromosomes. Detaily MSTmap algorithm was used to order the SLAF markers (Wu et al., 2008), and the SMOOTH algorithm (Van et al., 2005) was used to correct the genotyping errors as per the marker ordering. All LGs were processed as follows: a primary marker was used to order the LGs by their location on chromosomes; according to the relationship between the ordered markers, genotyping errors or deletion were corrected using SMOOTH algorithm; the minimum spanning tree map was used to order the map; and SMOOTH algorithm was used to correct the newly ordered genotypes. After four or more cycles of this processing, 19 high-quality maps were obtained. The Kosambi mapping function was used to estimate the map distances (Kosambi, 1944).
Analysis of SLAF-seq Data and SLAF Markers
After the preprocessing, 41.282 Gb raw data were obtained, which consisted of 206,411,693 paired-end reads of ∼100 bp in length. Of these, 80.60% bases were of high quality, with quality scores of at least 30 (Q30, indicating a 0.1% chance of an error, and thus 99.9% confidence). The guanine–cytosine content was 37.83%. Subsequently, 115,629 SLAFs were detected, and their average sequencing depth was 41.79 for 'Red Globe'; 40.74 for 'Shuangyou'; and 5.18 for each progeny (Figure 1).
FIGURE 1. Number of markers (A) and average sequencing depth (B) expressed as genome equivalents of the 149 individuals of F1 population represented in the X-axis.
Of the 115,629 SLAFs, 42,279 were polymorphic, with a polymorphism rate of 36.56%. The number of polymorphic SLAF markers per chromosome ranged from 1,426 in chromosome 3 to 3,005 in chromosome 18 (Table 1). The genotype coding for the polymorphic SLAF tags was conducted on the basis of the genotype of the parents. In all, 27,985 tags were coded, of which, 13,738 tags could be used to construct the genetic maps, with an effective polymorphism rate of 11.88%. After filtering out the low quality SLAFs lacking parent information and the depth less than 10X, 7,199 markers were obtained that could be used to construct a genetic map by using the criteria of segregation distortion (P < 0.05); these markers were classified into five segregation patterns (Figure 2). The average sequencing depths of these 7,199 markers were 68.35-fold for 'Red Globe,' 63.65-fold for 'Shuangyou,' and 8.01-fold for each individual progeny.
Basic Characteristics of the Genetic Map
In all, 7,199 genomic regions were covered. The LGs were numbered according to the chromosome numbers. The length of the genetic map was 1929.13 cm, and the average distance between two adjacent markers was 0.28 cm (Table 2, Supplementary Table S1). The average length of the 19 LGs was 101.11 cm, and the average number of markers for each LG was 397. LG10 was the shortest group with a length of 57.57 cm, and the LG5 was the longest group with the length of 177.31 cm (Table 1). LG4 was the highest density group, containing 450 markers, and the average marker density was 0.19 cm. LG5 was the longest group and contained 548 markers with an average genetic distance of 0.57 cm. LG10 was the shortest group and contained 244 markers with an average genetic distance of 0.24 cm. In these 19 LGs, the average percentage of markers having a gap distance of ≤5 cm was 98.62%, but LG8 and LG16 had no gap distance of ≥5 cm, and the largest gap was 15.14 cm, which was located on LG5 (Table 2, Supplementary Table S1).
There were 3,940 markers in the female map with a full length of 1931.91 cm. For the female population, the genetic length of LGs ranged from 16.36 cm (LG10) to 214.47 cm (LG5). LG6 was the highest density group, which contained 340 markers, and the average marker density was 0.31 cm. LG15 was the lowest density group, which contained 59 markers, and the average marker density was 3.00 cm. LG5 was the largest LG and contained 369 markers, covering a length of 214.47 cm with an average inter-marker distance of 0.58 cm. LG3 was the shortest group and contained 42 markers with the length of 16.36 cm and an average genetic distance of 0.40 cm (Table 1, Supplementary Figure S1).
There were 4,552 markers in the male map with a length of 1746.8 cm. LG2 was the shortest group with the length of 55.63 cm and contained 141 molecular markers. The average genetic distance was 0.40 cm. LG14 was the longest group with the length of 143.88 cm and contained 376 molecular markers; the average genetic distance was 0.38 cm. LG13 was the highest density group, which contained 260 markers, and the average marker density was 0.26 cm. LG10 was the lowest density group, which contained 115 markers, and the average marker density was 0.51 cm (Table 1, Supplementary Figure S2).
The Distribution of the Three Markers Types on the Genetic Map
The genetic maps constructed using SLAF sequencing contained three types of molecular markers: 7124 'SNP_only'-type, 7 'Indel_only'-type, and 68 'SNP&Indel' markers. The 'SNP_only' was the major marker type and occupied 98.96% of the markers. 'Indel_only' markers were present only in LG1, LG6, LG7, and LG19; two 'Indel_only' markers each were present in LG1, LG6, and LG7 and only one existed in LG19. 'SNP & InDel' markers existed in all the 19 LGs. The number of 'SNP & InDel' markers in each LG ranged from 1 to 9, and only LG1 had 9 'SNP & InDel' markers.
Visualization and Evaluation of the Genetic Map
The quality of the genetic map was evaluated using haplotype maps and heat map. Haplotype map can be used to detect double crossover populations, suggesting genotyping errors. It can also show the reorganization of each individual. The 7,199 SLAF markers were used to construct haplotype maps for each of the 149 progenies and parental controls, as described by West et al. (2006). Haplotype maps directly reflected the recombination events of each individual. Most of the recombination blocks could be easily identified in the haplotype maps. These maps suggested that the double crossover and missing ratio on the genetic maps were low (<1.5% had heterozygous fragments and <0.6% were missing). All LGs were distributed uniformly. Therefore, the population used in this study was suitable for the construction of genetic maps and performing genetic analysis.
In each LG, the relationship of recombination between all the markers was reflected by the heat map; this was subsequently used to detect ordering errors. This was also used to evaluate the quality of genetic maps by using pair-wise recombination values for the 7,199 SLAF markers. Most of the LGs had good visualization in general.
The Feasibility and Advantages of SLAF Sequencing in Marker Development
The key step for high-density map construction is to develop numerous stable and reliable molecular markers. In this study, SNP and InDel markers were used to construct a high-density genetic map for grape.
Specific length amplified fragment sequencing is a high throughput sequencing technique based on bioinformatics. It can be used for large-scale genotyping, which plays an important role in genetic linkage analysis. Unlike traditional molecular marker sequencing techniques, SLAF sequencing can provide high marker density and good uniformity. It also has many advantages (Sun et al., 2013).
Thus far, several high-density genetic maps have been constructed using SLAF-seq. Zhang et al. (2013) developed the first high-density genetic map in sesame; Qi et al. (2014), in soybean; and Xu et al. (2015) in cucumber. Chen et al. (2013) and Huang et al. (2013) conducted such genetic analysis in kiwi fruit and wheat, and the marker number produced by SLAF-seq was more than 1,000, which was considerably higher than the numbers of markers used in conventional maps.
In this study, the total quantity of SLAF-seq data generated from the parents and progenies was 41.282 Gb, including 115,629 high-quality SLAF tags, of which 42,279 were polymorphic. The final high-density genetic map contained 7,199 molecular markers. SLAF-seq has been able to detect numerous high-quality markers, especially in species with low polymorphism (Xie et al., 2010). In this study, the percentage of valid polymorphic markers between two parents was 11.88%. This percentage was not very high, but all the chromosomes were covered by polymorphic tags, whose number ranged from 1,426 to 3,005 on each chromosome. Therefore, SLAF-seq technology can be considered as a cost-effective technique to successfully develop chromosome-specific molecular markers for fruit trees, with high specificity and stability.
Evaluation and Usage of Genetic Maps
In this study, SLAF-seq technique was used to construct a molecular genetic map for grape fruit; this map contained the largest number of molecular markers compared to the published genetic maps for grape. The quality of the genetic map was evaluated by creating a single source figure and heat map, or by comparing the colinearity between the genetic map and genome. The origin of the major segment in each chromosome was consistent. Further, the ratio of double recombination for markers and marker deficiency pattern were 1.5 and 0.6%, respectively, indicating that the marker order was accurate in each LG. The heat map of the marker exchange relationship was used for evaluating the linkage relationship among the markers. The trend of recombination change between the markers was consistent with the marker order in each LG. The colinearity between the genetic map and genome was higher, indicating the high quality of the map. The order of most markers was consistent with those in the reference genome, indicating a good colinearity. Thus, the result of recombination rate was accurate.
In this study, grape (V. vinifera L. cv. Red Globe) was used as the maternal parent. It is the main cultivar and accounts for over 20% of the total grape area in China. It has a good berry quality, large cluster size, large berry size, low acid content, late-ripening, firm flesh, and good endurance of storage and transportation, whereas it has poor cold and pathogen resistance such as elsinoe anthracnose, downy mildew and so on (Liu et al., 2014b). The paternal parent used was 'Shuangyou' (V. amurensis Rupr.), which is a bisexual cultivar native to China; this cultivar is extensively cultivated in northeast China. It has high cold resistance and can withstand a temperature of -40°C. It also has high disease resistance such as elsinoe anthracnose, white rot, powdery mildew, and so on (Liu et al., 2014b). However, this plant bears small berries, has high content of acid, and is an early ripening variety. In China, it is used for making amurensis wine and grape juice. For over several years, we have been collecting the phenotypic data, including sugar content, acid content, cluster weight, berry weight, cold hardiness, and pathogen resistance, of 'Red Globe,' 'Shuangyou,' and their progenies. Remarkable segregation was found in these characters. In the future, we intend to map the quantitative trait loci for these traits on the genetic map; the markers associated with the agronomic traits and cold and disease resistance will also be determined. This information might be used for maker-assisted breeding and gene cloning based on the map developed in the study. Considering the distant hybridization between the parents, there might be a possibility of obtaining hybrid offspring that have the favorable traits of both the parents.
This project is supported by the Research Fund for the National Natural Science Funds of China (grant number, 31372021, 31000894); the China Agriculture Research System (grant number, CRAS-30-yz-6); Specialized Research Fund for the Doctoral Program of Higher Education (20102103120003); the Research project in Liaoning Province Science and Technology Department (2014204004); the Foundation of Liaoning Educational Committee (L2010492); and the Shenyang Science and Technology Development Funds (201207). The funding bodies have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fpls.2015.00393/abstract
Table S1 | Information of the marker on the19 LGs.
Figure S1 | Grape high density genetic map of the female parent. The map was constructed based on SLAF markers.
Figure S2 | Grape high density genetic map of the male parent. The tructed based on SLAF markers.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Keywords: grape, SLAF-seq, SNP, genetic map, linkage group
Citation: Guo Y, Shi G, Liu Z, Zhao Y, Yang X, Zhu J, Li K and Guo X (2015) Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis (Vitis vinifera L. × Vitis amurensis Rupr.). Front. Plant Sci. 6:393. doi: 10.3389/fpls.2015.00393
Received: 10 March 2015; Accepted: 16 May 2015;
Published online: 04 June 2015
Edited by:Tiziana Pandolfini, University of Verona, Italy
Reviewed by:Fernando Martinez, University of Seville, Spain
Raffaele Testolin, University of Udine, Italy
Copyright © 2015 Guo, Shi, Liu, Zhao, Yang, Zhu, Li and Guo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yuhui Zhao and Xiuwu Guo, College of Horticulture, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning, China, email@example.com; email@example.com


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Once Rahina was technically able to access benefits, the temporary accommodation was withdrawn, leaving her and her children without anywhere to live. She was then forced to seek help from Southend council who up until Monday 18 May had provided emergency accommodation in a hostel. Concluding that, having lost her job, she had therefore made herself 'voluntarily homeless', she was asked to leave.
Rahina's story was featured local newspaper the Hackney Citizen, see here - http://hackneycitizen.co.uk/2014/08/07/hackney-mot...For more background on the problems facing people with 'No Recourse to Public Funds' status see here - http://hackneycitizen.co.uk/2015/04/07/charities-u...

subset: commoncrawl


text:
Granville Island Tea CoEdit Company
Popular Teas from Granville Island Tea CoSee All 81 Teas
Recent Tasting Notes
So since I am home today, I have been trying to make my way through the box so I can pass it on in a timely manner. That means I have tried a lot of tea and since most of it is caffeinated, I have gotten a little jittery. So, I decided to balance out all that caffeine with some caffeine free options. This one has a faint berry and maybe a little vanilla but mostly it is bland to me. It's not bad but not really memorable either. Nonetheless, thank you Arby for the chance to try something new.
GCTTB (round 6) entry
mmm minty and delicious. I did find this a tiny bit herby, but in a good way. Delicious spearmint and peppermint, slightly floral (chamomile?). The herbs are a bit sweet and extremely fragrant. This is a fine herbal, I'm quite impressed.
Unlike another tasting note, there is no licorice or fennel that I can taste.
EDIT: steeped it longer and there is definitely licorice root, it was just hidden with a soirt steep time
Flavors: Flowers, Herbs, Mint, Peppermint, Spearmint, Sweet
GCTTB tea 7/8 that I picked.
Similar to the Evening lights tea I picked, I did not find this tea to have a prominent licorice taste like other reviewers did (and once again, that's prob due to my shorter steep time). about 2tsp, 200F water, 5 min. I found this had a mint taste, followed by a mix of floral, then maybe a bit of licorice? Altogether it's alright. I'll use it up no problem.
Flavors: Floral, Mint
After trying Movie Night from DAVIDsTEA, I figured I black popcorn tea would be equally as good. Like Movie Night, this tea steeps super light, but that is about all these two teas share.
The tea has a strange taste. It's like it's trying to be sort of sweet and kind of caramel-ish, but has no taste of popcorn or butter or even toffee.
I hate wasting tea, but I ended up throwing this one out because I don't think I could have pawned it off on anyone.
Flavors: Apple, Caramel, Toasty
I like this tea so much, I went through two 100g bags in two months. I don't live very close to Granville Island, so I probably should have savoured it longer.
Like the other reviewer mentioned, there is no "pastry" taste, so if that's what you're expecting then you will be disappointed. Rather, it tastes like a sweet black tea with a slight hint of chocolate and a light raspberry flavour. It doesn't steep super strong, even at the 5 minute mark.
I just add milk and no sweetener because I find it's sweet enough on its own.
Flavors: Chocolate, Raspberry, Sweet
The fluffy white bits in this tea were a bit odd, and I am not sure if I tasted them or not. I am glad that I had used a tea bag though because they made a mess.
I cold brewed this overnight.
It was really refreshing and just a little sweet but not overwhelming. The mango was fairly natural tasting. This was a pretty good one!
Pulled out a bunch of rooibos samples from my cupboard that I haven't had in ages which included this one, which is oft forgotten about. It didn't taste too bad; a sort of grapefruit and lemongrass mixture with hints of spice and coconut. It's not my favourite but it's not unpleasant either – very much like Zeste Eclair from Camellia Sinensis. However, the coconut is definitely not very fresh and while this is still drinkable it's clear that it's on the cusp of not being pleasant for much longer…
So, my mom is currently on vacation in British Columbia right now and like every four or so days she calls me up and is like 'Hey, at a new tea store! What do you want?' – so when she gets back this week, I'm going to have quite the tea haul!
That's great – but the big issue is where is it all going to go!?
So, I'm working on sipping down some stuff in tins right now including this tea, which I actually got from her last time she went on vacation to BC. This was one she blind picked for me, and actually I've liked it a fair bit overall considering I wouldn't have chosen it for myself. It's very, very grapefruit-y. However, I know one of the places she loaded up was one where I picked another rooibos blend, so I definitely need the tin space for that one when she brings it back!
So… sip sip sip.
Roommate's sick, so I made him some tea last night.
Of course, if I'm gonna make for him then I have to make for myself as well. So we both had a cup of this with some honey. Meh; I wasn't feeling it so much last night. Too much spice notes and not enough citrus, imo. However he liked it and found it soothing/relaxing and I suppose that was the point in the first place.
This morning's commute tea.
I was craving something fruity but also comforting and slightly spicy. I thought the warming spice notes in this with the brighter citrus would PERFECTLY hit the spot. It wasn't bad, necessarily, but it also didn't quite taste like how I was wishing it would. The spice notes WERE wonderful and comforting exactly like how I'd wanted: lots of cardamom and cinnamon with a nice tickle from the peppercorn in the finish. The problem area of this tea was the citrus notes though. You know the medicinal lemon flavour of Neo Citron? Yeah; that's what it tasted like. Eck.
This is a queued tasting note.
Tried this one hot, and I can confidently say that I found it like 100 times better than the cold brew I halfheartedly drank a while back. For starters, the citrus notes really delivered and made me feel like I was tasting a well defined trio of lemon, orange and grapefruit flavours. The finish was definitely more on the warming spices side with soothing, soft notes of cinnamon, ginger and allspice and then a light, tickly peppercorn finish. The transition between the contrasting flavours was really smooth as well.
And so I couldn't help but pair the tea, which made the perfect before bed companion, with this new Two Door Cinema Club song that got released this month:
It might not seem like the best pairing, but the smooth citrus and transitional period between top notes and finish seemed to perfectly match the smooth vocals of the song. I'm honestly still not 100% in love with this song; it's just so different from the rest of TDCC's music but I like the 80s vibes so I'm trying to embrace it.
Anyway, it's nice to know that this tea was capable of making a more lasting second impression after a really forgettable first.
This is a queued tasting note.
I guess thanks to my mom for picking this tea out for me a few months ago when she was on vacation in BC? Though, personally, I don't think I'd have picked it for myself. It seems very all over the place – probably 'cause there are like a dozen added ingredients. And that's not an exaggeration. Quantity of ingredients isn't the same as quality…
Mostly; I just remember this cold brew tasting REALLY muddled. Really the only flavours that stick out in my memory are ginger, cinnamon and generic citrus? I'm definitely feeling really meh towards all that bullshit.
The thought was there; but I think this will take me a LONG time to finish.
While I really love the fruity apricot flavour, I find this tea to become bitter and tannic very quickly. You can only leave it steeping for two minutes at most.It is nice with milk in it, but is sweet enough you don't need to add sweetener.
Flavors: Apricot, Fruity, Peach, Stonefruits, Tannic


text:
The median home value in Freehold, NJ is $308,000.
This is
lower than
the county median home value of $350,000.
The national median home value is $209,892.
The average price of homes sold in Freehold, NJ is $308,000.
Approximately 51% of Freehold homes are owned,
compared to 42% rented, while
7% are vacant.
Freehold real estate listings include condos, townhomes, and single family homes for sale.
Commercial properties are also available.
If you like to see a property, contact Freehold real estate agent to arrange a tour today!
Learn more about Freehold.
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Open House:
Sunday, 1/26
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Sunday, 1/26
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text:
This section provides information on DVA's work with the ex-service organisation (ESO) community relating to the National Consultation Framework and its constituent forums.
National Consultation Framework
Working with the ex-service community is an important objective of the Department and Government.
The National Consultation Framework is a formal consultative structure designed to facilitate effective communication between the veteran and ex-service community, the Repatriation and Military Rehabilitation and Compensation Commissions, and the Department of Veterans' Affairs (DVA).
The National Consultation Framework comprises national state and territory Forums. More information on each of the forums, including membership and Terms of Reference, can be found through the links on the menu to the left. Special interest groups or industry specific groups required to address particular needs may be formed to complement the framework.


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Volume 90 Issue 40 | p. 26 | Concentrates
Issue Date: October 1, 2012
Issue Date: October 1, 2012
Linde Purchases Chinese Gas Unit
Through its joint venture Linde LienHwa, Linde has acquired Zhenjiang Xinhua Industrial Gases, a producer of specialty nitrous oxide gas in eastern China. Manufacturers of high-resolution three-dimensional displays use high-purity N2O to make metal oxide transistors. The acquisition brings to nine the total number of N2O plants that Linde and Linde LienHwa already operate or are in the process of commissioning in Asia. Linde LienHwa is a joint venture of Linde and Taiwan's MiTAC International, a maker of electronic devices.
- Chemical & Engineering News
- Copyright © American Chemical Society


text:
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Warminster is situated in the county of Wiltshire, approximately 20 miles from Bath and Salisbury and close to the border with Somerset.
For those arriving by public transport, Warminster Rail Station is on the Cardiff-Bristol-Portsmouth rail line which operates approximately every hour, with connections available from London and the West Country at Westbury and Salisbury and also from Melksham, Chippenham and Swindon at Westbury – please see the Great Western Railway website for timetables and further details. There are also a few direct trains from London Waterloo to Warminster each day – for more information, please visit the South Western Railway website .
If you would prefer to travel by bus, the following bus services run to places served by Imberbus on Saturdays:
- First service D1 runs to Warminster every 30 minutes from Bath, Bradford on Avon, Trowbridge and Westbury, and every 2 hours from Salisbury;
- Frome Minibuses service 53 runs from Frome to Warminster every hour;
- Faresaver service X72 runs every hour from Bath, Melksham and Devizes to West Lavington and Market Lavington;
- Salisbury Reds service 2 runs approximately every hour from Salisbury to Shrewton, Tilshead, Gore Cross and West Lavington.
On Sundays, The only bus service to Warminster is First bus service D1 which runs from Bath, Bradford on Avon,Trowbridge and Westbury every 2 hours.
Please visit the Traveline website during the summer for more details of these services.
For those travelling by car, Warminster is located at the junction of the A36 and A350 and is near to the infamous A303 ! You should find plenty of parking available Warminster's Central Car Park which is off station road, so round the corner from the rail station – see map below (for those of you using SatNav, the postcode for Warminster's Central Car Park is BA12 9BX). Other public car parks are also available (including a small one at the station itself) but please note that normal weekday charges apply on all the public car parks on Saturdays.
Imberbus operations mean there is hardly any car parking available in Imber village itself except for those with disabilities. We therefore ask you to travel to Imber by bus instead on the Saturday, but if you do decide to drive all the way, please use the new Park and Ride site to the East of the village, which will be clearly signposted from both directions. On Saturday, a frequent bus service will then take you from the site to St Giles's Church, and other destinations on the Plain.
A small amount of roadside parking is also available in Chitterne and West Lavington but please ensure that you leave plenty of space for the Imberbuses to get past and (in the case of Chitterne) turn round!
Thinking of stopping overnight ? Then the following websites may help you:
- Bristol – Visit Bristol website, Tripadvisor Bristol page
- Bath – Visit Bath website, Tripadvisor Bath page
- Warminster, Salisbury and other towns in Wiltshire: Visit Wiltshire website, Warminster Information Centre, Tripadvisor Wiltshire page
Remember that buses and trains operate to Warminster from many of the surrounding towns, so you don't have to stay in Warminster – please email us if you need further advice.


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[ ĕk′tō-thrĭks′ ]
A fungal parasite, such as certain dermatophytes, forming a sheath of spores on the outside of a hair as well as growing within the hair shaft.
The American Heritage® Stedman's Medical Dictionary Copyright © 2002, 2001, 1995 by Houghton Mifflin Company. Published by Houghton Mifflin Company.
Examples from the Web for ectothrix
Of this latter there are two main subvarieties—endothrix and ectothrix.Essentials of Diseases of the Skin|Henry Weightman Stelwagon


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FAQ - Social Networks
Here you will find our extensive tutorials on how to use our Content Management System. You can also look by keyword using the search facility at the bottom of this page.
Add a Facebook Like button
Learn how to add a facebook like button to your website
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- Promoting your site
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Please note that IST Research Explorer no longer supports Internet Explorer versions 8 or 9 (or earlier).
We recommend upgrading to the latest Internet Explorer, Google Chrome, or Firefox.
2535 Publications
2010 | Journal Article | IST-REx-ID: 3867
K. Chatterjee, L. Doyen, and T. A. Henzinger, "Expressiveness and closure properties for quantitative languages," Logical Methods in Computer Science, vol. 6, no. 3, pp. 1–23, 2010.View | Files available | DOI
2010 | Conference Paper | IST-REx-ID: 3855
K. Chatterjee, L. Doyen, and T. A. Henzinger, "Qualitative analysis of partially-observable Markov Decision Processes," presented at the MFCS: Mathematical Foundations of Computer Science, Brno, Czech Republic, 2010, vol. 6281, pp. 258–269.View | Files available | DOI
2010 | Journal Article | IST-REx-ID: 2409
A. Kupczok, H. Schmidt, and A. Von Haeseler, "Accuracy of phylogeny reconstruction methods combining overlapping gene data sets ," Algorithms for Molecular Biology, vol. 5, no. 1, 2010.View | Files available | DOI
2010 | Journal Article | IST-REx-ID: 3774
H. Senn, G. Swanson, S. Goodman, N. H. Barton, and J. Pemberton, "Phenotypic correlates of hybridisation between red and sika deer (genus Cervus)," Journal of Animal Ecology, vol. 79, no. 2, pp. 414–425, 2010.View | DOI | PubMed | Europe PMC


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Majesco: Fiscal 2Q Earnings Snapshot
MORRISTOWN, N.J. (AP) _ Majesco (MJCO) on Tuesday reported fiscal second-quarter net income of $908,000.
The Morristown, New Jersey-based company said it had net income of 2 cents per share.
The insurance software developer posted revenue of $34.1 million in the period.
Majesco shares have increased 28% since the beginning of the year. In the final minutes of trading on Tuesday, shares hit $9.10, a rise of 25% in the last 12 months.
This story was generated by Automated Insights (http://automatedinsights.com/ap) using data from Zacks Investment Research. Access a Zacks stock report on MJCO at https://www.zacks.com/ap/MJCO


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Getting a book into the ancient Library of Alexandria was a big deal back in the day. A manuscript was discovered – sometimes even, er, "borrowed" – then copied out by hand, letter by letter. It took days, at the least. It's a bit easier to get a new book added to the Lennoxville Library's collection. We buy them, ready made. Or, this week, we ask you to buy them.
The Adopt-A-Book campaign is an important annual fundraiser. Volunteers have been reading and researching to find the best books that are out there, and we present them to you this Thursday, October 1, 2015, from 1 p.m. to 7 p.m. Each book sports a price tag. There are expensive reads, and inexpensive books. Some are novels, some are children's books, some are non-fiction. We ask our patrons – you! – to pop by and see what we have on offer. When you donate the price of a book, you ensure that it becomes a part of our permanent collection.
You get to read it first, then it's placed on our shelves for everyone to read. Your book becomes a gift to our community.
And while the Lennoxville Library isn't quite as grand as Alexandria's, it's a fun, cozy space for all, located in a beautiful building. It was created to make reading accessible to all (we have audio books, as well), to jump-start the imagination, and to expand horizons. Oh, and because reading is fun.
By the time the just-a-tiny-bit-obsessive Ptolemy III was in power, in 246 BCE, the Library of Alexandria was truly massive. "Ptolemy III ordered that all ships in the Alexandria harbor be searched. If books were found, they were copied, the copies returned to the owners, the originals stored in the library," notes author Steve Berry in the 2007 thriller, The Alexandria Link (filed on our shelves in Adult Fiction). This is one of the Cotton Malone series, featuring a retired American spy whose efforts at a quiet life selling rare books in Copenhagen are constantly thwarted. This time, because his son is kidnapped by bad guys seeking something called The Alexandria Link (as in the library).
That library, in real life, no longer exists. It was, as Berry notes, perhaps accidentally destroyed by the conquering Julius Caesar when a fire he'd ordered in another part of the Egyptian city went out of control. Another theory blames Christians seeking to destroy pagan artifacts. Yet a third theory credits Muslims seeking to destroy pagan artifacts. We may never know what really happened.
But where there's a mystery, there's an author happy to have some controversial fun with it. The Alexandria Link is, it turns out, a man with some sort of secret knowledge connected to the Arab-Israeli conflict. Only Malone knows where he lives, but unscrupulous others want that guy. And they'll stop at nothing to find him.
This is a fun read that forces a squabbling divorced couple back together to ensure the safety of their son, then folds in a complex issue (Middle Eastern politics), the Old Testament, and a global conspiracy involving the US government and a cabal of evil capitalists. Things blow up, and spies lose their lives. Phew!
The Lennoxville Library is much less complicated, and a rather great place to visit. We offer membership to Sherbrooke residents for free, sponsor reading groups for adults (in both official languages), and hold regular (free) children's activities. Plus wifi, and a couple of computers. The worst that might happen is a paper cut. (We're very sorry, but I might have a plaster in my bag; hold on a minute.)
We'd love for you to come by on Thursday, October 1, between 1 p.m. and 7 p.m., and Adopt A Book. If you'll forgive the forthrightness: We accept cheques, and we'll put a book (or more) aside for you if you want to come by a bit later and make your donation to the Lennoxville Library.
Can't make it Thursday? Books will still be on display Friday and Saturday, October 2 and 3. Come on by! We're at the corner of Queen and College. Give the gift of reading.
— Eleanor Brown, September 30, 2015


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We love this time of the year because restaurants all over Miami create special menus for Miami Spice. It lasts from August 1 to September 30, so get ready to experience the best restaurants in Miami. And for the record "Miami Spice" has become a verb: "Where are you Miami Spicing this weekend?"
What is Miami Spice?
Miami Spice Months showcases the very best of Greater Miami and the Beaches' diverse cuisine featuring three-course meals at the top eateries in the city reasonably priced at $23 for lunch/brunch and $39 for dinner. For two whole months, explore signature dishes at reduced prices by world renowned chefs every summer.
How it Works: New to Miami Spice? There is no membership or sign-up required to enjoy Miami Spice. Simply visit the individual webpage of the restaurant you want to visit and make sure to check the days of the week that the restaurant offers Miami Spice lunch/brunch or dinner. Reservations are strongly recommended but not required.
Miami Spice Participating Restaurants
To check out the full list of participating restaurants, please visit Miami Spice. The Miamism Sales Team always has recommendations. Among our favorites are:
- El Cielo by Chef Juan Manuel Barrientos (a culinary experience you can't miss)
- Kyu in Wynwood by Chef Michael Lewis
- Chotto Matte on Lincoln Road
- Mignonette in Miami's Art & Entertainment District
- Stiltsville Fish Bar by culinary duo Jeff McInnis & Janine Booth
Buen Provecho and please let us know about your favorites!


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Holiday Cottages in Penmon, Isle of Anglesey
Find a great selection of self catering holiday cottages in Penmon, Isle of Anglesey.
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Cottage Favourites in PenmonSimply run your mouse over the images to find out more.
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Graig, Penmon
Self catering in Isle of Anglesey, North Wales
Graig is tucked away in the beautiful town of Beaumaris, Anglesey's best kept secret. At the end of a winding country lane and close to the rocky shoreline, it is set in a cluster of properties and is surrounded by six acres of fascinating ... more


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Call for info: (888) 347-0346
332 Ferrule, Kyle TX 78640
At Creekside
From $212,426 Why 'From' pricing?
New homes can be personalized to your needs and taste.
Builders pricing can vary greatly. Builders display base pricing because you have not selected your options and upgrades yet and some homes may indicate that the land cost is not included.
Elevation B 1/8Previous Next
This charming 1-story home features 3 bedrooms including an owner's suite with its own private bathroom and large walk-in closet. Kitchen opens to the living room and includes an island with room for bar stools; perfect for a quick meal or entertaining. A large dining room off the kitchen offers plenty of natural light. An optional "intergenerational suite" is perfect for extended families.
Builder Community Sales Office Map
332 Ferrule, Kyle, TX 78640
Sales Agent: Charlene Epps
Hours: Mon & Tues 11am - 6pm; Thurs & Fri 11am - 6pm; Sun 12pm - 6pm
Announcing our newest community in Kyle! Walking trails throughout the community as well as connecting to the trails of the Plum Creek Preserve and Lake Kyle. Premium locations available over looking neighborhood parks and Lake Linebarger. With a home in Creekside, residents will also enjoy the close proximity of dining, shopping, parks, and attractions. Whether you find time to enjoy the trails along the creek or spend your time at the nearby amenities, having a home in Creekside is an ideal location for everyone.Visit the website Free brochure
Actual schools may vary. Contact the builder for more information.
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Hi. Great and very sleek looking app. Miles ahead of the original Domoticz app, and my favourite by far.
I have transmitters in my Domoticz that are showing both temp and humidity, however, when selecting gagues in HomeHabit, I only get humidity part of the sensor. If I add the same sensor as a temperature feedback for a thermostat, I do get the temperature. Is it possible to fix this parsing in the coming betas? Either to be able to select the temperature part of the device (as for thermostat feedback), or to add a gague with both temp and hum.
Also, It would be awesome to be able to add selector switches (presemce, mode etc.)
(I have currently bypassed the issue by creating new virtual devices that are copying only temperature, and addit the new device in HomeHabit)
Much obliged


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Tel: 01443 830 750 / 07528322502
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Telephone01443 830 750 / 07528322502
AddressBargoed Sight Centre26 Hanbury RoadBargoedCF81 8QT
Tel: 01443 830 750 / 07528322502
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Boundaries between the physical and the digital world are today getting fuzzier and fuzzier. Everything is becoming data: from our social interactions to the measurements of all the different-purpose sensors and IoT devices surrounding us.
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Greg passed his first block test on the latest portion of his Utiliesman training. He has 16 days left to "A" school and then he will get to come home. I am so looking forward to seeing him again. I've missed him so much. He keeps talking about all the places he wants to go with us and where he wants to go out to eat when he gets home. LOL! He is just doing so well and we are so proud of him.
Last night when I got home from work Scott was out walking Amelia and I called to find out where they were so that I could walk to meet them. We met up down by the power line right away behind our house they were coming down from the community pool and I up toward it. As we walked together a gust of wind blew a cloud of pollen right into our faces. It's definitely Spring here now.... UGH pollen! No wonder our heads are all stopped up. After we got back to the house we washed our faces and hands to remove some of the pollen that had rained down upon us and went to the grocery store for dog food and odds and ends - $70 later we came home and cooked a dinner together - pork chops, steamed broccoli and cauliflower and some refried rice with onion and bell pepper. YUM! We had enough leftovers for lunches today... yeah no junk for me or Scott!
Scott cleaned out the largest of the raised beds in the garden and he plans to plant out the potatoes that have sprouted here in the house. We also bought some "fresh" seeds - peas, spinach, squash, peppers, okra and a spicy mesculen mix. Perhaps Sunday we will be able to build a couple new raised beds to replace the ones that have broken down.
I bought Greg a book called Dr BBQ's All Year Long Cookbook - by Ray Lampe. It has some absolutely delicious sounding recipes in it and since he loves to cook on the grill I thought it would make a wonderful welcome home gift. I'm thinking about putting together a whole gift basket of things for him to enjoy once he is home again.
My Mom called to say she has been laid off too. It seems to be going around these days - lay offs.