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I was looking for an appropriate picture to use as my facebook cover in case of a lockout, but I couldn't find one to accurately reflect the FHDJSKFHS I HATE EVERYTHING-ness I'll feel if/when the league doesn't come up with a new CBA by September 15th. So, I made my own. All these pictures come from the Visa Hockey Love Hurts commercial right here. Each picture is 851x467(ish) pixels, so they'll fit on facebook without weird stretching. And here they are! (Click on the image to start the slideshow, then save it for the full size)
Sunday, August 26, 2012
Lockouts hurt
Posted by gingersnowflake at 10:36 pm
3 comments:
those are good. also gives me a chance to share this video: http://www.youtube.com/watch?v=aLS0OzGAmMY&feature=plcp
My hockey league made our own version of this commercial for our end of season awards banquet 9wherei was robbed yet again)
- August 29, 2012 7:14 am
I'm also going to use that top one, thanks
- August 29, 2012 7:15 am
- gingersnowflake said...
ahaha that was awesome, loved the Sens bit
- August 29, 2012 9:04 pm
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The legend of the Enchanted Cavern continues! On each level of this mesmerizing matching game, you must eliminate groups of identically-colored jewels in order to break through obstacles and move ahead to the next level. As the levels progress, the biggest challenge is to complete each level within the allotted time. With vivid graphics, lovely music, and three game modes, Enchanted Cavern 2 offers hours of jewel-filled fun for the whole family!
|Alawar Entertainment
|71MB, about 1 minute on broadband
|Available Languages
|Deutsch, English, Español (México), Español (España), Français, Italiano, 한국어, Português
|Release Date
|November 30, 2011
|Operating System
|Windows 8, Windows 7, Vista and XP
|1.5 GHZ or better
|Video card RAM
|DirectX Version
|8.0 or above

subset: commoncrawl


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Abanol was a planet, the second orbiting the star Beshqek of the Beshqek system, within the Deep Core region of the galaxy. It was a toxic rock orbited by a single moon and neighbor to the mythical world of Byss.[1]
Behind the scenesEdit
Abanol was introduced in the Byss and the Deep Core, a Wizards of the Coast supplement for the Star Wars Roleplaying Game published in 2005.[1]
- "Byss and the Deep Core" on Wizards.com (original article link, backup link) (First mentioned)
- The Complete Star Wars Encyclopedia
- The Essential Atlas


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This howto describes how to set up and run the init replacement Upstart on Fedora.
The current init system has quite some disadvantages, therefore several distributions search for a way to extend or replace the current system. One well known replacement is initng which I already tested on Fedora. Another way to improve the system is Upstart which was introduced by Ubuntu. Since it is Open Source it can be used on Fedora as well.
You can find the newest version of this howto directly at the Upstart wiki. This post is more or less just a snapshot of the current state of the wiki howto.
Also: I used a stripped down version of Fedora 6: no X, no NFS services, no avahi, no SELinux, etc. I deactivated all that stuff to make the boot process faster – and believe me, I had to reboot quite some time till I had everything running.
And of course I used a Fedora installation inside of a virtual machine (Virtual Box in this case) because otherwise I would have gone insane.
And, last but not least: this howto can seriously damage your system! Don't try this if you not exactly know what you're doing.
Enough said, lets get down to business.
As you can imagine there are no rpms provided at the moment, so you have to compile everything by hand. Get the upstart archive and the example-jobs from the download section of the upstart wiki.
The configuration of the sources can be done in two ways: fully replacing all init files, or installing upstart to
/opt/ (see also these information). Of course we will take the second way since that is sufficient for testing. The configure line is:
./configure --prefix=/opt/upstart --sysconfdir=/etc --enable-compat=sysv
The installation afterwards is the usual
make && make install.
Configuring Upstart
First of all the set of example jobs have to be extracted to
/etc/event.d/. These examples make sure that upstart can boot your Fedora exactly like the old init does – this is called compat mode.
However, the example jobs need some work since they are all written for Ubuntu. Everything which is a comment starts with a #.
The PATH variable have to be modified so that the new init system calls the right executables:
perl -pi -e 'undef $/; s/script\n/script\n export PATH=\/opt\/upstart\/sbin:\$PATH\n/' /etc/event.d/rc*
rc sysinit script
Fedora has a very important boot rc script which should be executed only once, directly after the start. Therefore, add it to the
export PATH=/opt/upstart/sbin:$PATH # This was added in the last step
exec /etc/rc.d/rc.sysinit # ADD THIS LINE
runlevel --set S >/dev/null || true
The tty* files contain the command
/sbin/getty. Fedora uses
/sbin/mingetty, change that for all tty* files:
perl -pi -e 's/getty 38400/mingetty/' tty*
The rc* files call the executable
/etc/init.d/rc– Fedora, however, has this at
/etc/rc.d/rc. Change that for all rc* files.
perl -pi -e 's/\/etc\/init.d\/rc/\/etc\/rc.d\/rc/' rc*
There is no runlevel rcS in Fedora – therefore, change
exec /etc/init.d/rcS must be changed to exec /etc/init.d/rc S
Also, the script
/etc/event.d/control-alt-deletehas to be modified like this:
export PATH=/opt/upstart/sbin:$PATH # ADD THIS LINE
exec shutdown -r now "Control-Alt-Delete pressed" # REMOVE /sbin/ FROM THIS LINE
end script
Boot the system with Upstart
To boot with upstart you have to create a modified grub entry. Therefore, open
/boot/grub/grub.conf and create a copy of your current entry. Afterwards, change the "init" parameter of the kernel line to:
If you want to enter the debug mode, you should also add
--debug afterwards.
Benchmarking with bootstart
Of course you can also use bootchart with upstart to benchmark the init system. To do so you have to modify a normal kernel line in
grub.conf in this way:
init=/sbin/bootchartd bootchart_init=/opt/upstart/sbin/init
The first parameter tells the kernel to start bootchart instead of the default init system, and the second parameter tells bootchart to start Upstat instead of the usual init system.
I haven't done any bootchart testing (yet) because I only tested Upstart in a virtual machine. If I bring it up on my normal machine I will post some statistics about the performance.


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#Nike made a number of new product announcements at a glitzy event in New York yesterday, but perhaps the most exciting revelation was that the company is finally bringing a pair of "self-lacing" sneakers to market.
The Nike HyperAdapt 1.0 senses the presence of the wearer's heel and tightens its laces accordingly. Senior Innovator Tiffany Beers led the project and says once the shoe is on, the wearer can adjust the fit with two buttons on the side to get things perfect.
The HyperAdapt 1.0 will be available to Nike+ members "beginning Holiday 2016," according to Nike, and will come in three colors. As of now, the company hasn't given an indication of price.EN FR


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People are being urged to check their pockets, piggy-banks and down the back of the couch after a rare 50 pence piece has sold for 240 times its value.
A rare 50p coin featuring Sir Isaac Newton recently sold on eBay for £120 and people could also have one worth £245 in their spare change without even knowing it.
So it is worth checking the design of any 50p coins to see if one of them could be among the rarest and most valuable ones in circulation.
While the most sought-after 50p - the Kew Gardens design - can fetch up to £245 on the likes of eBay, other rare 50p coins usually achieve a premium of around 10 to 12 times face value, or £5-£6, when they are sold on.
It goes without saying, perhaps, but the rarer the coin the more it is worth and experts at Changechecker.org have created a scarcity index that tracks which circulating 50p coins are the most scarce and collectable.
The index uses a unique combination of mintage, collecting and swap data to give the most up-to-date picture of today's 50p coin collecting market.
The aforementioned Kew Gardens 50p tops the scarcity list. With a mintage - which means the number of coins issued - of just 210,000 it is the rarest coin and, therefore, the most in-demand.
The next nine scarcest coins are all from the 2012 Olympics.
Football, Triathlon, Judo, Wrestling 50 pences will sell for between £8 and £21 on eBay or up to £33 for a set of four.
If anyone discovers they have a rare 50p, they can either choose to sell the coin on eBay or through a specialist like ChangeChecker.org.


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mcu@home Banking Terms and Conditions
MCU@Home Banking Terms and Conditions
This MCU@Home banking disclosure sets forth the terms and conditions of Missouri Credit Union's internet banking product. These terms may be changed from time to time. By using or allowing another person to use MCU@Home, you are agreeing to the terms of this agreement.
Transactions Available
You may use MCU@Home to perform the following transactions:
- Obtain account balances and account history (including copies of cleared checks);
- Transfer funds between your accounts.
- Transfer funds to another member.
- Transfer funds in and out of Missouri Credit Union
- Perform self-service account maintenance such as re-ordering checks, requesting a check from your account, stopping payment on a check or changing address and phone number.
- Sending us secure online messages.
Additional online banking services may be introduced from time to time. By using the service when they become available, you agree to be bound by the rules that will be made available to you concerning these services. Different features and services available through our website may be subject to separate terms and conditions in addition to the terms of this Agreement.
It is your responsibility to keep your user name, password, security phrase and security picture confidential. You agree not to give your user name, password, security phrase and security picture to any person not authorized to have access to your account. Also, you agree that you will not use or allow anyone else to use your password for any transaction that is illegal under applicable federal, state, or local law.
In order to provide important information related to your on-line security, automatic alerts will be sent to your primary email address when certain changes have been made to your online account, such as a change in your email, home address or telephone number. We will do our best to provide the alerts in a timely manner, but alerts may be delayed or prevented for a variety of factors beyond our control. You agree that we are not liable for any delays or failures to deliver.
Notify us immediately if you believe your user name and password have been stolen, used without your consent, or compromised in any way. Also, if your statement shows transactions you didn't make, notify us immediately. If you have authorized someone else to use your password, you are responsible for all transactions that person(s) initiates at any time, even if the amount or transaction exceed what you may have authorized.
If you believe your password has been stolen or someone has accessed your account without permission, call us at:
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or write us at:
P.O. Box 1795, Columbia, MO 65205-1795.
For recurring internal or external transfers, if the scheduled date is on a non-business day, then the transfer may be done on the business day prior to your scheduled date. (On-line banking business days are Monday through Friday, excluding federal holidays.) A one-time immediate transfer to another Missouri Credit Union account cannot be cancelled after it has been submitted.
External transfers are to be used to move money to and from your accounts at other financial institutions. You agree that you are authorized to perform transactions on all accounts involved in the external transfer. You are responsible for having enough money in the indicated accounts one day prior to the scheduled transfer date. If the account does not have sufficient funds available on the scheduled date of the transfer, the transfer may not be made. If we elect to initiate the transfer, it may cause an overdraft in your account in which case you shall be liable for the overdraft and any fees as set forth in your membership agreement. You will be responsible for all fees and charges should your ACH transfer be returned for any non-payment reason. Also, your external transfer privileges may be canceled if ACH transfers are returned for non-payment or any other fraudulent or suspicious activity occurs on the account.
External transfers can only be made to and from domestic accounts in the United States. It may take up to 5 business days to process an external transfer. MCU does offer same day external transfers to other institutions. In order to use this service, the account at the other institution must already be set up and verified in the external transfers section. These same day transfers must be done by 12:00 (noon) and an applicable fee will apply.
Missouri Credit Union cannot guarantee the timely delivery or return of funds as a result of the failure of another financial institution to act in a timely manner.
External Transfer limits are $2,500 from or into your account per month.
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To use MCU@Home, internet access and internet browser software are required. Your internet browser must support 128- bit encryption, and be at these minimum versions:
Internet Explorer – 8 or greater
Safari – 6 or greater
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Credit Union Liability
Missouri Credit Union assumes no liability due to your inability to access mcu@home for any reason including, but not limited to, communication problems or equipment/software failures.
MCU@Home internet banking is free for all members. Any fees for special services provided through MCU@Home are described in your Truth in Savings Fee Disclosure.
You may terminate this agreement with us at any time. Missouri Credit Union reserves the right to terminate this agreement and/or your use of MC@Home at any time. We may do so immediately if the following occurs:
- You or any authorized user of your account breaches this or any other agreement with the credit union;
- We have reason to believe that there has been or might be an unauthorized use of your account;
- You or any authorized user of your account requests us to do so;
- You cause a loss to the credit union.


text: Microsoft announced Friday that it is providing Windows Server 2008 R2 Hyper-V, the company's newest enterprise virtualization software, to the OpenStack cloud-computing platform. The partnership with Cloud.com includes technical guidance, and the open-source company will write its own code to support Hyper-V. OpenStack was launched with code from Rackspace and the NASA Nebula cloud project, Microsoft said. More info is here.


text:
Press releases
Melton Local Plan to be submitted to GovernmentPublished on Thursday, 28th September 2017 in Planning press releases
Following a full council meeting held on 27th September 2017, councillors agreed to submit the Melton Local Plan to the Government for independent examination.
First anniversary of Melton Community LotteryPublished on Monday, 20th November 2017 in Families, communities and living press releases
The Melton Community Lottery is getting ready to celebrate by giving away three great local prizes on its first birthday bonanza prize draw!
Crime & community safetyPublished on Friday, 1st September 2017 in Crime prevention press releases
Council Leader Joe Orson comments on the recent issues reported in Melton Mowbray
Melton to Fly the Flag for Merchant Navy DayPublished on Thursday, 17th August 2017 in Events press releases
Fly the red ensign for Merchant Navy Day - 3 September 2017
Local Plan to be discussed at council meetingPublished on Monday, 26th June 2017 in Planning and building control press releases
Local Plan to be discussed at council meeting
- More articles in the press release archive


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As a fairly young director (he is currently 37), Bryan Singer has a pretty impressive, if short, set of films to his credit. His first major Hollywood film, The Usual Suspects, was followed by the dark and creepy Apt Pupil. Then he was on to comic book history with the first X-Men movie and now its sequel, X2: X-Men United. At a recent series of roundtable press interviews in Los Angeles, the director chatted with reporters about all things X-Men, including editing for the ratings, the characters &#Array; old and new, the ease of doing a sequel, the new Wolverine videogame, and the eerie relationship of current events to the war in the film. Oh, and he also shares a bit about the unusual credit on his resume &#Array; his time spent in uniform as a Starfleet Bridge Officer.
Singer defended his decision not to reintroduce the characters at the beginning of the sequel. "I call it the Lost Horizon concept. You make a film, as they did with Lost Horizon, and they made it three hours long and they took an hour to establish all the characters aboard a plane, and the plane crashes and they go off on an adventure. And then when they looked at the picture, they said, 'This is a really long picture,' so they cut the first hour off. But, because they had that first hour for the characters to develop and the relationships to evolve, when they finally crashed and discovered the lost city and whatnot, you really felt that you knew the characters. I think, this film X2, is designed to stand on its own as a film, as a separate entity. It's kind of like the movie I would have, could have, should have made the first time, but could not have made without having made the first film. I think now that those characters are so defined, even when they move into X2 you know where they're coming from, the universe is somewhat established, you see them use their powers very early on. There's a few things I put in there, like with Lord of the Rings, he opens the picture with the dream of [Ian McKellen's character, Gandalf] fighting the dragon. Likewise, there's certain gifts for the audience so they can catch up to speed, but ultimately there seemed no point in it. The characters were already moving forward, and I didn't want to move backward and waste that kind of time."
Rumor had it that some things needed to be edited to bring an R rating down to a PG-13. Singer shared a bit about what those slight changes were, "Originally we got an R rating, and then just a few minor things of intensity that were trimmed. But, very little. I was very pleased. There's very little blood in the movie. There's some intensity in it, but it's not a gory picture by any means. It's somewhat heroic. Like in the first picture, you have Rogue get stabbed through the chest by Wolverine, but we kind of solved those problems in the story, and it's kind of fun and the audience realizes it is not a terrifying threat, and this is not a massacre. This is just heroes having to defeat villains."
As for if there is any additional pressure with doing a sequel, Singer stated, "No, not really. I think some of the pressure was alleviated by the fact that fans, the core audience have supported the X-Men universe for 40 years, really embraced the first picture. There was some more time and money this time around, and I had a better sense of what I was doing, because I'd already established these characters and already cut my teeth in the genre. So, I felt a little less pressure and actually a little more freedom."
One of the new characters to the X-Men movies is Nightcrawler, played by Alan Cumming. Singer was asked if Nightcrawler's appearance in the film is similar to his appearance in the comic books. "It is very similar, that's the wonderful dichotomy of Nightcrawler. He's a character who's deeply religious, a sympathetic character, but at the same time has the physical appearance of a demon. The one thing we added to it were the scarification on his face, they give an interesting sense of history."
Singer enjoyed the challenges of making an ensemble film with so many diverse and changing characters. "One of the fun parts about an ensemble picture, especially with so many characters where the lines of good and evil are blurring, constantly, there's a lot more room for unpredictability and surprise. When you're following one character on a journey, it's fun to find yourself all of a sudden disrupted by another character, and then another character introduces another mystery, and then that's solved just as more characters are introduced. Unholy alliances are formed, but not quite ... ever since I've made Usual Suspects I've enjoyed the challenge of ensemble pictures. So, this time with X2, I was like, 'Okay, I've got all these characters &#Array; bring on a couple more.' I enjoy that challenge of balancing."
When asked if he will stick around to put out a couple more X-Men movies, Singer replied, " I'm not sure. I have ideas and things have been established in this film that would lend themselves to future pictures, but at the same time, circumstances have to be appropriate. I'd probably like to do something in between. I don't know if it's to get back to my roots &#Array; smaller films or go and make another event picture, I'm not sure yet. I'm developing things."
With the movie coming out in the aftermath of the military action in Iraq, Singer was asked if the troubled times made him nervous about people's reactions to the war theme in this film. "No, not at all, because the soldiers working in this film, they're not even working in the United States. They're working up in Alberta, in a secret base. They're working for a person who's completely rogue from the government. The President of the United States is very on the fence and very concerned, justifiably about issues. There are mutants who posses incredible power and who are terribly violent and dangerous to the human society and mutant society. I view these as henchmen, and in terms of fighter airplanes getting dogged and police getting dogged, no one in that sphere is really harmed. It's not about bullying the authority. I personally have tremendous faith and support of our authorities and military ... Having shown it to friends of mine in the military, they get a kick out of the fact that these soldiers are a bunch of rogue, dirt bags that get what's coming to them. And we see that. We definitely see that. This guy, Stryker, he's operating in his own universe. He's tricking the President, he's conning the President into his operation, so it's quite the opposite if anything. He's more of a terrorist."
As for how big the Mutant war could become and how closely it really does seem to relate to current events, Singer said, "I think a lot of those are very coincidental. The story line was conceived prior to September 11. Remember, the X-Men universe was created in the early '60s in the height of the American Civil Rights movement. So, these ideas of bigotry, tolerance, fear, war ... I think are perpetual ideas. We've had them for thousands of years, ever since man recognized his fellow man and that two people had two different color hair. So, I think it is oddly relevant. I desperately tried to not let current events as they unfolded, impact the process of making this film. This film is for me, primarily a wonderful piece of fantasy entertainment. I think, if it didn't have some relevance to some social issues or some personal issues that it wouldn't be truly entertaining. I think truly entertaining movies affect you. I think Star Wars talked about growing up and religion and myth, and many kind of serious things, but in a spectacular way. I think good science fiction ultimately tells stories of the human condition from an extraordinary perspective. So, in that way, it's ironic, but there's nothing wrong with it. But it is eerie, in certain aspects."


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Omri Altman, the 19 year-old forward and product of the Maccabi Tel Aviv youth system, will join the rest of the Maccabi squad at their training camp in Austria
Maccabi Tel Aviv are in the advanced stages of contacts with the English Premier League side Fulham FC for the season-long loan of Under-21 Israeli forward Omri Altman. Altman, who departed to London two seasons ago, is expected to train for the first time with Maccabi today and join them at their training camp in Austria. The young striker rose through the ranks of the Maccabi youth system and after impressing as an Under-17 was given the opportunity to play for the Maccabi Under-19s even before his 17th birthday. After scoring in all four of his first matches, his talents caught the eye of the Fulham scouts.
Altman's first move in that direction was on loan but afterwards the London club decided to make his move permanent. He started at the Cottagers with the Under-21s but last season moved on to the reserve team where he made 24 appearances, scoring two goals. The 19 year-old was also a member of the Israeli national Under-21 side that competed in the Euro Under-21 championships held in Israel this past month. He made one appearance as a substitute in the 1-0 victory against England.


text:
Chapel - May 4, 2012 - 12:00am
Dr. Dan Bruss, President, Bethany Lutheran College
Other service participants
Student Organist: Catharine Webber
Order of Service
* Prelude
* Hymn, 374, vv. 1, 3 & 5
* Scripture Reading: Ephesians 6:13-17
* Prayer
* Hymn, 55, vv. 1, 4 & 5
* Benediction
Ephesians 6:13-17 Therefore put on the full armor of God, so that when the day of evil comes, you may be able to stand your ground, and after you have done everything, to stand. Stand firm then, with the belt of truth buckled around your waist, with the breastplate of righteousness in place, and with your feet fitted with the readiness that comes from the gospel of peace. In addition to all this, take up the shield of faith, with which you can extinguish all the flaming arrows of the evil one. Take the helmet of salvation and the sword of the Spirit, which is the word of God.


text:
This Saturday Toki and I geared up for Hamilton Comic Con. I knew it was a busy place so I made sure to keep Toki's safety in mind as well. We steered clear of crowds as best as we could and I had Cory with me to pick her up for me when I was nervous of situations where there were larger crowds (especially in high traffic areas like enterances). I was a little uncomfortable at first with people pointing at me or saying things to each other about myself or Toki. I eventually learned to ignore it and just have fun with my day as best I could. The one thing that really upset me though was the number of parents who just let their kids run up to my dog and pet her. I had one kid in particular who when I was stopped looking at something, decided it was a good time to sit on the floor and start petting Toki. His mother was completely oblivious to the situation and was over looking at something else. I tried to explain to the child that my dog was working and to please leave her alone. The kid decided to just look at me and then continue to pet Toki. I really was on the verge of screaming at the kids mother but instead just left the situation. It really annoyed me with the amount of parents who seemed like my dog was there for their kids entertainment. I will say though, there were a lot of parents there who made up for the others. I could hear them explaining Toki as a working dog and I even heard a few kids sound super excited to have seen a working dog. It definitely made me feel a lot better knowing that most people understood her role. We stayed for a few hours and even bought a few things. It was a great time and I look forward to our next adventure together. For now we are enjoying a relaxing Sunday!
Here's a photo from today:


text:
Published on
These research details are the result of a market research study organized by InSites Consulting in January 2010. This market research mapped the usage of and attitude towards social networks. A total of 2.884 consumers participated in this online survey, spread over 14 countries, i.e. Belgium, the Netherlands, the United Kingdom, France, Germany, Spain, Italy, Portugal, Brazil, the United States, Australia, Romania, Russia & China. The results are representative of each country's Internet population, spread on age (18-55) and gender.


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- Research article
- Open Access
Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrumL.)
© Hedley et al; licensee BioMed Central Ltd. 2010
- Received: 12 March 2010
- Accepted: 14 September 2010
- Published: 14 September 2010
The detrimental effects of mild winter temperatures on the consistency of cropping of blackcurrant (Ribes nigrum L.) in parts of Europe have led to increasing interest in the genetic control of dormancy release in this species. This study examined patterns of gene expression in leaf buds of blackcurrant to identify key differential changes in these profiles around the time of budbreak.
Using leaf bud tissue of blackcurrant, a cDNA library was generated as a source of blackcurrant ESTs for construction of a custom microarray, which was used to identify differential gene expression during dormancy release. Gene activity was lowest in early stages of dormancy, increasing to reach a maximum around the time of budbreak. Genes with significantly changing expression profiles were clustered and evidence is provided for the transient activity of genes previously associated with dormancy processes in other species. Expression profiling identified candidate genes which were mapped onto a blackcurrant genetic linkage map containing budbreak-related QTL. Three genes, which putatively encode calmodulin-binding protein, beta tubulin and acetyl CoA carboxylase respectively, were found to co-localise with budbreak QTL.
This study provides insight into the genetic control of dormancy transition in blackcurrant, identifying key changes in gene expression around budbreak. Genetic mapping of ESTs enabled the identification of genes which co-localise with previously-characterised blackcurrant QTL, and it is concluded that these genes have probable roles in release of dormancy and can therefore provide a basis for the development of genetic markers for future breeding deployment.
- Quantitative Trait Locus
- Gene Ontology
- Gene Expression Profile
- Dormancy Release
- Leafy Spurge
Blackcurrant (Ribes nigrum L.) is grown widely throughout temperate areas of Europe and New Zealand, with an annual production of 160,000 tonnes in Europe and 185,000 tonnes globally [1]. The centres of genetic diversity for blackcurrant are situated in northern Scandinavia and Russia, although species are also found in North and South America, Asia and northwest Africa [2]. The fruit has very high nutritional value, in terms of the content of ascorbic acid and other antioxidants [3], and its primary commercial use is processing for juice. In the UK, varieties are chosen to give a spread of cropping seasons. Timing of leaf budbreak is an important physiological factor in the developmental processes leading to cropping of blackcurrant, as uniformity of development is essential for both successful harvesting and for optimum fruit quality and yield.
The control of budbreak in many woody perennial plants, including berry fruits such as Ribes and Rubus, is dependent on exposure to chilling temperatures during the winter for a sufficient duration to release dormancy, followed by appropriate warmer temperatures in the spring to induce growth [4]. An insufficient amount of chilling during the dormant period can seriously impact key phenological traits, notably time of budbreak, time and duration of flowering and fruit quality at harvest [5]. The chilling requirement, in terms of both duration and range of suitable temperatures, varies between species, between cultivars within species [6, 7], and even between different buds of the same plant [8, 9]. Blackcurrant has a relatively high chilling requirement for dormancy break, from ca. 1,300 h in the case of New Zealand-selected germplasm [10] to over 2,000 h for some late-flowering types such as 'Ben Lomond' from Scotland and most Nordic cultivars [7]. As warmer winters are likely to become increasingly prevalent [11], there is a risk of insufficient chilling becoming more widespread in cultivars requiring high levels of winter chilling, so breeding strategies to address this using low-chill parental material are now required [7].
However, these must be balanced against the risk of spring frost damage at flowering in cultivars that break bud too early after minimal chilling and whilst the incidence of spring frosts is declining across Europe [5], there can still be occasional damage in some areas. Also, the risk of spring frost damage at flowering may depend on temperature variance as well as on specific mean temperatures, and climate projections are predicting a future rise in such variance [12].
Dormancy in woody perennials is an adaptive mechanism for the survival of winter conditions [9] and was classified by Lang [13] into three major types: (i) ecodormancy, where growth inactivity is caused by unfavourable environmental conditions, with a resumption of growth when conditions improve; (ii) endodormancy, caused by endogenous factors within the dormant buds that cannot be overcome even by favourable environmental conditions; and (iii) paradormancy, or correlative inhibition [14], where the dormancy status of buds is influenced by physiological factors in other parts of the plant (eg. apical dominance). The effects of winter chilling on dormancy break at bud-burst are in endodormant tissues, and further investigation of winter chilling in blackcurrant is structured around improved phenotyping of diverse germplasm [7] coupled with genetic analysis of Quantitative Trait Loci (QTL) and genes associated with dormancy-related processes. Putative QTL linked to key developmental traits, including time of budbreak, have recently been identified by Brennan et al. [15].
Dormancy break and subsequent physiological events are controlled through the coordinated action of large numbers of genes in woody plants [9] and whilst there have been many studies of the molecular genetics of dormancy and budbreak, many aspects remain unclear. Microarray studies to examine global gene expression during dormancy and budbreak have been used in a range of woody genera, including Vitis, [16–18], Quercus [19] and Rubus [20]. Most of these studies attempted functional classification of the differentially expressed genes from the transcriptome related to budbreak or, in the case of Mathiason et al. [18], to the fulfilment of chilling requirement. Some studies, eg. Mazzitelli et al. [20] and Derory et al. [19], have identified candidate genes for dormancy-related metabolism, and several of these genes were previously implicated in these processes in other species.
The aim of this study was to examine patterns of gene expression in leaf buds of blackcurrant and to identify the key differential changes in these profiles around the time of budbreak. A custom cDNA microarray was utilised which incorporated blackcurrant Expressed Sequence Tags (ESTs) derived from buds at various stages of dormancy. Expression profiling identified candidate genes associated with budbreak which were mapped onto a blackcurrant genetic linkage map. Initial steps have been taken to develop associated markers that can be deployed in the characterisation of diverse germplasm for inclusion in downstream breeding programmes.
Blackcurrant expressed sequence tags
RNA was isolated from blackcurrant leaf bud tissue sampled at five time-points prior to budbreak and, following pooling, was used to construct a targeted cDNA library to maximise collation of relevant ESTs. Single-pass sequencing of approximately 9,000 clones generated 7,400 high-quality ESTs. Following identification of a low-redundancy set of blackcurrant clones, single ESTs (3,633 in total) representing each contiguous sequence were selected and whole cDNA inserts used to fabricate microarrays as described.
Gene expression profiling during dormancy transition
Leaf buds were sampled from blackcurrant plants throughout the different dormancy phases, from mid-winter following onset of dormancy through to spring when bud-burst was prevalent. RNA was isolated from bud samples from winter 2005 to spring 2006: week 0 (early December), week 2, week 4, week 8, week 12, week 14 and week 16 (end of March). For the microarray experimental design, RNAs from consecutive time-points were hybridised on the same arrays in a two-channel loop-design (design and complete dataset available at ArrayExpress [21] accession E-TABM-807). Data were extracted from each array and quality filtering was applied to leave 2,320 reliable replicated expressed probes.
In order to identify and characterise variation within the entire reliable dataset Principal Component Analysis (PCA) was used (Figure 1a). Principal component 1 accounted for 66% of the variance, whereas principal component 2 comprised 12% of the variance, based upon the filtered genelist of 2,320 probes. Principal component 1 separates the samples of the earlier weeks (0, 2, 4 and 8) from those of the later weeks (12, 14 and 16). Principal component 2 in particular separates the week 8 data from the other time points. To analyse this further, an overview of gene activity in blackcurrant leaf bud tissue was obtained using volcano plots to identify numbers of differentially expressed genes between consecutive sample time points (Figure 1b). Early winter (week 2 to week 4) shows the lowest levels of differential gene expression, and in the first two weeks of sampling the majority of genes are down-regulated. Differential gene expression reaches a peak between week 8 and week 12, with similar numbers of up-and down-regulated genes detected. Subsequently, the majority of genes from week 12 to week 14 are reduced in expression levels.
Significant differentially expressed genes were identified over the entire period of sampling using ANalysis Of VAriance (ANOVA). False Discovery Rate (FDR) was applied at high stringency multiple testing correction; identifying 1,040 significantly changing probes (Additional File 1), of which 842 unigenes have significant homology to known Arabidopsis gene loci. The high proportion of significantly changing genes reflects our generation and use of a microarray targeted to dormancy processes in Ribes. Unigenes considered to have significantly changing gene expression profiles following ANOVA analysis were subjected to K-means clustering to group similarly regulated genes together (Figure 2). For simplicity, average gene expression profiles are shown for each group to indicate general trends. The four groups obtained (Set 1-Set 4, Figure 2) consist of 260 (227 annotated), 181 (160), 124 (107) and 475 (348) unigenes respectively.
Validation of microarray gene expression profiling
Genetic mapping and association of candidate genes with dormancy traits
Quantitative trait loci interval mapping previously identified a QTL for budbreak in 2002 on linkage group 3 [15], a region which also contains SERB09L03 (GenBank accession GT022654) and SERB03C05 (GT027101). The recombination frequency between these SNP-markers was zero. The maximum LOD (logarithm (base 10) of odds) score in this region was 3.4. In 2003, the LOD score for budbreak in this region was less than the usual threshold of 3.0, but the QTL effect was the same sign [15]. A more powerful QTL by environment (year) analysis of the combined 2003 and 2004 data [23] showed that SERB09L03 (GT022654) and SERB03C05 (GT027101) were significantly associated with budbreak (p-value < 0.001) and that there was no significant QTL by year interaction. The mean budbreak for the heterozygous genotype at these SNPs was 4.0 days (se 2.35) earlier than the homozygote in 2002 and 1.6 (se 0.84) earlier in 2003. These two ESTs encode proteins with homology to an acetyl-CoA carboxylase (blastx 2e-47) and a calmodulin-binding protein (blastx 2e-17), respectively. Another EST, SERB02K12 (GT026930) on linkage group 4, was found using the QTL by environment analysis to be associated with budbreak (p-value < 0.001). The mean budbreak for the heterozygous genotype at this SNP was 2.5 days (se 1.38) later than the homozygote in 2002 and 2.1 (se 1.44) later in 2003. This has derived amino acid homology to a beta-tubulin (blastx 1e-94). Specific gene expression profiles, including quantitative RT-PCR validation, is presented in Figure 3 and indicates that the genes encoding acetyl-CoA carboxylase and beta-tubulin have similar patterns, with relatively constant expression to week 8, followed by rapid accumulation of transcripts later in the time course. The gene encoding the calmodulin-binding protein however, initially dips in activity at week 2, recovers and gradually declines through the remainder of the sampling period.
The need for blackcurrant varieties requiring lower levels of chilling during the dormant period is likely to increase in the future [7], although current unpredictability of weather events in many of the growing regions means that utilising a range of varieties with varying chilling requirements is probably advisable for most growers. For example, varieties that break bud too early may be vulnerable to damage by spring frosts during the flowering period in some northern areas, notably Scandinavia [24]. However, the selection of varieties based on budbreak characteristics currently depends largely on phenotypic assessments in the field using prevailing weather conditions. To move beyond this into a more focused selection procedure requires detailed knowledge about the genetic control of budbreak and subsequent developmental events in blackcurrant, which will enable development of molecular markers directly linked to traits of interest.
General observations of gene activity in blackcurrant bud tissue
This study provides insight into the genetic components associated with dormancy transition in blackcurrant. A targeted blackcurrant cDNA microarray was constructed and utilised to identify sets of genes with significant differential expression profiles during dormancy release and budbreak. Utilising PCA and identifying differentially expressed probes between sampling stages clearly indicated maximum gene activity between week 8 and week 12 (Figure 1), during the month of February. This was also demonstrated in all four of the primary gene expression profiles determined by K-means clustering (Figure 2), with rapid induction or repression after week 8. Up to this point, the cumulative chilling (temperatures below 7°C) was 1,723 h (see Methods), similar levels to which have been demonstrated under controlled environments in Rubus to develop 50-100% budburst in whole canes or isolated nodes respectively [20]. This was slightly lower than that estimated by Fraser [25] using cuttings, rather than whole plants, which indicated the chilling requirements of commercial UK cultivars of blackcurrant, including 'Ben Gairn' and 'Ben Hope', to be > 2000 h. In Vitis riparia, almost 100% budburst was achieved in canes after 1,500 h of chilling [18]. Accumulated hours of chilling in our study would indicate that our material received sufficient chilling for budbreak between week 8 and week 12 and, combined with the general observations in gene expression profiles, suggest that the blackcurrant samples include bud material in transition from the endodormant to ecodormant phases [25].
Putative roles of the significantly changing genes during dormancy release from this study remain speculative and care must be taken in interpreting the data, however a number of gene homologues identified have been previously described in several related studies. Some of these common genes of interest are briefly discussed in the context of dormancy transition and budbreak.
Association of dormancy transition with signalling, transcription factors and secondary metabolism
Almost half of the significant differentially expressed blackcurrant genes clustered into expression profile Set 4 (Figure 2), whereby gene activity in general was highest in early winter, subsequently declining throughout the remaining sampling period into spring-time. This set was overrepresented by GO terms associated with transcription factors (TFs; Additional File 1). Of note within this group are genes encoding TFs (see Additional File 4), of which there are several zinc finger family members (B-box, CCCH, C2H2, and C3HC4/RING types) which are known to play important roles in signal transduction pathways of plants [26]. A zinc finger protein from Quercus [19] has been implicated in budburst, which is related to a TF involved in seed germination from Arabidopsis [27]. Another class of TF which shows gradual down-regulation through the time course in Ribes encodes a Short Vegetative Phase (SVP) type MADS box transcription factor (Additional File 4). Homologues of SVP have been identified in Japanese apricot [28] and raspberry [20], where the gene has similar expression patterns to that observed in blackcurrant under controlled conditions leading to dormancy release. SVP has been shown in Arabidopsis to act as a direct repressor of flowering [29] and appears to have elevated expression under short-day environments [30]. A TF family highly related to SVP, termed Dormancy Associated MADS-box (DAM), has been described in several other perennial species, including poplar [31], leafy spurge [32, 30] and peach [33]. Expression of two leafy spurge genes encoding DAM proteins reflect the dormancy status within the leaf meristem, with both genes induced by cold temperatures and differentially expressed according to day length. MADS-box TFs are clearly important components in signalling and dormancy transition in several species.
Oxidative stress has been reported to be an important factor in controlling budbreak, and induction of dormancy release by artificial stimuli, such as heat stress and hydrogen cyanamide, leads to the differential expression of several genes associated with this process in grape [34]. From the blackcurrant array data, there is evidence of transient expression of several genes whose products are coupled with this mechanism (Additional File 4), including ascorbate peroxidase (Set 1), calcium-dependent protein kinases (Set 1 & Set 4), glutathione S-transferases (GST; Set 1 & Set 4), catalase (Set 4) and sucrose synthase (Set 4). Similar mechanisms appear to also be involved in dormancy release of raspberry leaf buds, with transient induction of genes encoding ascorbate peroxidase, catalase and GST [20]. Other signalling systems, demonstrated by artificial dormancy control in grape, involve ethylene and abscisic acid (ABA), leading to the hypothesis that there is complex interaction between these mechanisms to control dormancy release [35]. Within the blackcurrant data, there is evidence of ethylene signalling during transition with transient expression of several ethylene response factors (ERFs; Set 4), which have also been observed in leafy spurge [36]. Many of the blackcurrant genes strongly induced later in the time-course (Set 3, Figure 2) are associated with secondary metabolism, specifically those leading to flavonoid biosynthesis. These include genes encoding chalcone isomerase, chalcone synthase, dihydroflavonol reductase and flavonol synthase (Additional File 4), some of which have also been reported to be significantly regulated during dormancy release in raspberry [20] and leafy spurge [14, 37]. Flavonoids provide protection of tissues against UV damage (reviewed in [38]) and are known to control auxin transport in plants, subsequently altering growth of tissues such as axillary buds [39].
Candidate genes associated with dormancy traits
Genetic mapping of significantly expressed ESTs enabled identification of three genes which co-localise with previously characterised blackcurrant budbreak QTL [15], two of which map with high significance to a single locus on linkage group 3 and another which maps to linkage group 4 (Figure 4). These associations provide independent evidence that these genes may be implicated in dormancy processes, however it should be noted that they may also be simply co-located with the causal genes. Each of these ESTs is discussed on the basis of their homologies to known genes and gene products, and in the context of previous work on dormancy in other species.
Acetyl CoA carboxylase (ACCase, encoded by GT022654) functions to provide malonyl-CoA as a substrate for biosynthesis of fatty acids [40] and secondary metabolites, such as flavonoids and anthocyanins [41] which are essential for plant development and protection. It is known that the homomeric form is induced by UV-B radiation for flavonoid production [42]. The gene expression profile derived from this EST (Figure 3) is a member of Set 3 of the clustered groups (Figure 2), which has overrepresentation of terms associated with secondary metabolism and phenylpropanoid biosynthesis. ACCase is associated with rapidly dividing tissues and early stages of cell growth and development and this gene is clearly induced in expression during early spring. In addition, an ACCase has been shown to be induced in floral buds of common bean [42].
Clone GT027101 encodes a gene product with homology to calmodulin-binding protein. Calmodulin itself binds to calcium, which is common regulator of many different protein targets and a transducer of secondary messenger signals [43]. As previously mentioned, oxidative stress is also known to induce calcium signalling in plant cells [34]. Calmodulin-binding proteins exist in many different forms with a range of associated functions and have been demonstrated to control calcium signalling in woody perennial species such as grape [44] and blueberry [45]. The expression profile of the gene in blackcurrant (Figure 3) indicates repression from week 4 and is present within cluster Set 4 (Figure 2), which contains other genes with GO terms associated with transcription factors. The polypeptide encoded by this gene also shows similarity to a putative pheromone receptor protein AR781 in Arabidopsis, whose EST profiles show high levels of expression in bud and seed tissue (data from dbEST [46]; Id 5922760).
A third blackcurrant gene (GT026930), which maps to a budbreak QTL on linkage group 4, encodes a protein with high homology to beta-tubulin, which is an essential component of microtubules within the cytoskeleton. High levels of beta-tubulin expression is a clear indicator of rapidly dividing and expanding cells and the gene expression profile shows rapid induction after week 8 (Figure 2). This profile is a member of Set 1 (Figure 3) which contains many terms representative of increased protein metabolism and biosynthesis. From previous studies, beta-tubulin is consistently induced on dormancy release in buds of a range of woody perennial species, including Malus and Rosa [47], and is proposed as a genetic marker for dormancy status in such species. It has also been demonstrated that a beta-tubulin gene is induced in expression during bud-burst in poplar [48].
Extending this study to look for association of these genes with dormancy-related traits in a wide blackcurrant germplasm collection is planned and will further substantiate their roles in physiological processes related to budbreak. However, it is clear that they have excellent potential as genetic markers to assist future breeding strategies. Further work to confirm the genetic map locations for both phenological and fruit quality traits is currently in progress with an extended blackcurrant mapping population and appropriate phenotyping.
This study provides insight into the genetic control of dormancy transition in blackcurrant, and the use of microarrays over a time course leading up to budbreak shows key changes in gene expression at budbreak. Further mapping of ESTs with significant differential changes in expression around budbreak enabled the identification of three genes which co-localise with previously-characterised blackcurrant QTL, and it is concluded that these genes have probable roles in the dormancy and budbreak processes, and can therefore provide a basis for the development of genetic markers for future breeding deployment.
Plant material
Blackcurrant (Ribes nigrum L. cv. Ben Hope) plants were grown in the field at SCRI, Dundee, Scotland (56° 27'N, 3° 04'W) to four years of age. Leaf buds were sampled at eight time-points: week 0, 7/12/05; week 2, 21/12/05; week 4, 04/01/06; week 8, 01/02/06; week 12, 01/03/06; week 14, 16/03/06; week 16, 29/03/06; week 43, 16/10/06. Air temperature measurements were taken from October and cumulative hours of chilling (below 4°C) determined: week 0, 610 h; week 2, 837 h; week 4, 1,140 h; week 8, 1,723 h; week 12, 2,262 h; week 14, 2,622 h; week 16, 2,847 h. Buds were taken from three biological replicate pools at random positions within the bush, flash frozen in liquid nitrogen and stored at -80°C. Candidate genes were mapped using the reference blackcurrant population designated 9328, described by Brennan et al [15].
RNA extraction
Total RNA was extracted from frozen blackcurrant bud material (100 mg) using the Plant RNeasy Mini Extraction Kit (Qiagen) according to the manufacturer's recommendations (substituting buffer RLC for RLT and including 10% v/v RNA Isolation Aid (Ambion) and 1% v/v β-mercaptoethanol). RNA quality was checked by spectrophotometry and integrity assessed using a Bioanalyzer (Agilent Technologies).
cDNA library construction and EST sequencing
Only very limited numbers of publicly accessible gene sequences are available for blackcurrant (less than 30 sequences present in NCBI [49], 2009) and closely related woody perennial species. Therefore a cDNA library was generated from blackcurrant leaf bud tissue, which was used as a source of probes for construction of a custom microarray. Utilisation of these expressed gene sequences maximised the chances of identifying putative candidate genes associated with dormancy traits and budbreak in blackcurrant. Total RNA from blackcurrant buds (equal quantities of each) were pooled from five time-points (week 0, week 2, week 4, week 8 and week 12) prior to and potentially including budbreak. Messenger RNA was purified from 100 μg total RNA using Dynabeads mRNA Purification Kit (Invitrogen) as recommended. A directional cDNA library was constructed in pSPORT1 from pooled mRNA using the Superscript Plasmid System (Invitrogen) according to the manufacturer's instructions. Randomly selected clones (~9,000) were picked and stored as glycerol stocks. From these, plasmids were prepared using Multiscreen 96-well Filter Plates (Millipore) as recommended. Sequencing reactions were carried out using M13 reverse primer and BigDye version 3.1 Kit (Applied Biosystems) according to the manufacturer's instructions. Completed sequencing reactions were purified using ethanol precipitation prior to analysis on an ABI PRISM 3730 DNA Sequencer (Applied Biosystems). Sequences were trimmed according to chromatogram quality criteria, using the base-calling program Phred [50] at a score >20, and vector-derived sequences were removed using Crossmatch software [50].
In order to estimate the redundancy of the ESTs, related cDNA sequences were assembled as contiguous sequences using the CAP3 program [51] with default settings of all parameters [52]. ESTs were defined as redundant when they exhibited more than 95% identity over aligned regions and were assembled into a single contiguous sequence.
Similarities to previously identified sequences were obtained by searching public databases using the BLASTn and BLASTx algorithms [53] for nucleotide and deduced amino acid sequences respectively. Local databases containing non-redundant nucleotide and protein sequences obtained from the National Center for Biotechnology Information [21] were searched. Matches were considered non-significant (no match) when e-values were greater than 0.01. All EST sequences used to fabricate microarrays have been submitted to dbEST at GenBank [46].
Microarray fabrication
Blackcurrant cDNA inserts were amplified by PCR using plasmid DNA template and M13 forward and reverse primers that span the multiple cloning site of the vector. Each reaction was performed in 100 μl containing 50 ng of plasmid, M13 primers (0.5 μM each), 0.2 mM dNTPs, 2 mM dNTPs, 2 mM MgCl2 and 0.10 U/μl Taq DNA Polymerase (Promega) in 1 × PCR buffer. PCR conditions were 94°C for 3 min for 1 cycle and then 94°C for 30 s, 54°C for 30 s, 72°C for 2 min for 38 cycles, 72°C for 7 min for 1 cycle. PCR products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen) following the manufacturer's recommendations. Purified PCR products were standardised to 400 μg/μl.
A total of 3,633 cDNA amplicons from blackcurrant library clones were prepared for spotting onto modified glass slides (Nexterion Slide A, Schott Glass). Purified amplified cDNA (7.5 μl at 400 μg/μl) was mixed with 2.5 μl of 99% dimethylsulfoxide (DMSO), and transferred to 384-well print plates (Genetix). Probes were printed in triplicate with a defined random pattern onto each slide using a 24-pin robotic system (Q-Array Mini, Genetix) at constant relative humidity (50%) at 23°C. Arrays were allowed to air-dry for 10 min and the cDNA spots were immobilised following the Nexterion protocol. The quality of the printing process was checked using the SpotCheck Microarray Slide QC kit (Genetix). Details of the blackcurrant dormancy array design can be found at ArrayExpress (accession A-MEXP-1694; [22])
Microarray processing
Target total RNA (2 μg) was labelled using the 3DNA Array 900 Kit (Genisphere) as recommended. RNAs from consecutive time-points were hybridised on the same arrays in a two-colour loop-type design (see ArrayExpress accession E-TABM-807 [22]) incorporating three biological replicates for each time point (8 time points, 24 samples, 12 arrays total).
Arrays were scanned using an ArrayWoRx Auto scanner (Applied Precision) at appropriate exposure settings for Cy3 (595 nm) and Cy5 (685 nm) at 9.75 μm resolution, generating separate TIFF images. Exposure levels were adjusted to compensate for slight variations in labelling efficiencies. Data were acquired from images using GenePix Pro software (Molecular Devices) and median signal and background intensities were determined for the Cy3 and Cy5 channels for each spot on each microarray. Background-subtracted intensity values were imported into GeneSpring (v.7.3; Agilent Technologies), whereby data from replicate spots within each array were averaged.
Microarray data analysis
Data sets for each array were normalised in GeneSpring using the LOWESS (LOcally WEighted polynomial regreSSion) algorithm to minimize differences in dye incorporation efficiency [54], prior to re-importing the data as single colour intensity values. Subsequent analysis was carried out using only the first seven sampling points (week 0, week 2, week 4, week 8, week 12, week 14 and week 16) representing winter-spring seasons. Scaling of the data was performed using default GeneSpring settings. Data were filtered by expression level (>100 in 3/21 samples) to remove unreliable, low intensity data points, leaving 2,320 genes for downstream analysis. Data from biological replicates were combined in GeneSpring for statistical analysis: (i) volcano plots (p-value <0.05, fold-change >2 ×) were used to identify differentially expressed genes between sample time points; (ii) 1-way ANOVA was used to estimate differential response between time points (default parameters: parametric test, variances not assumed equal (Welch ANOVA)) and probes were selected (1,040 genes) that were judged to be statistically significant (p-value <0.01) with Benjamini & Hochberg multiple testing correction [55].
Clustering of gene expression profiles across the developmental time series was performed in GeneSpring using the K-means algorithm. Default parameters (100 iterations, Pearson measure as similarity correlation) were used to generate 4 cluster sets from the selected ANOVA gene-list.
Analysis of Gene Ontology terms
Blackcurrant unigenes used to construct the microarray were annotated with the most significant BLAST homologue to Arabidopsis gene loci. These homologues from selected gene-lists were subsequently analysed for enrichment of Gene Ontology (GO) terms using the Term Enrichment Tool [56] at AmiGO (version 1.7; [24]). The whole microarray gene set was used as the defined background and thresholds of p-value 0.05 and a minimum of 1 gene product were used.
Real time RT-PCR validation
Contiguous EST sequences containing probes selected following microarray analysis were used to design Universal Probe Library (UPL, Roche) assays using default parameters [57]. Primer and probe sequences are detailed in Additional File 3 and were designed to the three candidate genes described and also fifteen additional randomly selected unigenes with contrasting expression patterns. Total RNA (5 μg) was reverse transcribed using oligo d(T)18 and You-Prime RT Beads (GE Healthcare) as recommended. cDNA was diluted to 50 μl and 2 μl of each sample used as template for UPL assays with FastStart TaqMan Probe Master ROX (Roche) as recommended, with standard cycling and data acquisition on 7500 Fast Start (Applied Biosystems) equipment. Assay efficiencies were evaluated with standard curves for reference (eIF4A) and each test gene, which determined if ΔΔCt or Pfaffl [58] calculations were used to estimate relative gene expression profiles.
Candidate gene mapping
Inserts from 112 selected EST clones (Additional File 5) identified by ANOVA filtering were completely sequenced using Sanger technology as above, independently with both M13 forward and reverse priming. Sequences were assembled using Sequencher software (v. 4.9, Genecodes) with default parameters. Oligonucleotide primers (Additional File 5) were designed using the Primer 3 programme [59]. If the lengths of sequenced clones were greater than 1,000 bp, two sets of primers were designed. Genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen) as recommended, from the seed parent (S36-1-100), pollen parent (S10-2-27/28) and six segregating progeny. PCR was used to amplify gene fragments from each of the lines in a 20 μl reaction volume containing 10-50 ng genomic DNA, 1.0 U of Roche Taq Polymerase, 1 × reaction buffer containing 1.5 mM MgCl2 and supplemented with 0.2 mM dNTPs and 1.0 μM each primer. Thermocycling consisted of 5 min at 94°C; 7 cycles of 30 s at 94°C, 30 s at 65°C, and 30 s at 72°C decreasing to 58°C at 1°C per cycle, followed by 25 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C, followed by 7 min at 72°C. Single PCR products were gel purified from each reaction using the Qiagen MinElute Gel Extraction Kit according to the manufacturer's specifications and were sequenced using the forward primer used in PCR amplification as above. SNPs were identified by visual inspection in Sequencher v4.6 (Gene Codes Corporation) and Pyrosequencing assays were designed using the Pyrosequencing Assay Design Software v1 (Qiagen).
Pyrosequencing assay
SNPs which were identified by direct sequencing as being heterozygous in one or both parents were subsequently mapped in the segregating mapping population using the Pyrosequencing SNP detection platform. Forward and reverse primers, and internal sequencing primers within a few bases of the identified SNP, were designed using the Pyrosequencing Primer Design Software. One of the external primers was biotinylated and 25 μl standard PCR reactions were performed as recommended, with thermocycling consisting of 5 min at 94°C, 40 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C, followed by 10 min at 72°C. Biotinylated products were immobilised onto Sepharose beads and the samples were analysed using a PSQ 96MA system together with SNP reagent kits and SNP software (Qiagen). Two mapping parents and 93 segregating progeny were scored and mapped using the methodology developed by Brennan et al. [15].
Simple sequence repeat identification and analysis
SSRs were identified using the Sputnik programme and primers were designed using Primer 3 software [59]. Each 10 μl PCR reaction contained 20 ng genomic DNA, 1 μM each forward and reverse primers (one of which was labelled with 6-FAM), 0.2 mM dNTPs, 1.0 U of Roche Taq Polymerase, in 1 × reaction buffer containing 1.5 mM MgCl2. Thermocycling conditions were 5 min at 94°C, 7 cycles of 30 s at 94°C, 30 s at 65°C, and 30 s at 72°C decreasing to 58°C at 1°C per cycle, followed by 25 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C, followed by 7 min at 72°C. Following PCR, fragments (along with 500 ROX-labelled size standard) were separated on an ABI Prism 3730 DNA Analyzer according to the manufacturer's instructions (Applied Biosystems). Genotypes were identified using GeneMapper v3.7 software (Applied Biosystems).
Genetic mapping and QTL analysis
As discussed in Brennan et al. [15], the mapping population S36-1-100 × S10-2-27/28 contains both sibs from these parents and selfs from SCRI S36-1-100. The new markers were added to the existing linkage map using JoinMap 3 [60] to combine data from the selfs and sibs, as described [15]. QTL analysis of the budbreak data was carried out with the new map and the sibs from this cross using MapQTL 5 [61], as described in Brennan et al. [15]. The new markers were also tested using a more powerful mixed model analysis that combines the sib and self phenotypic data in a QTL by environment (year) analysis of the budbreak data from 2002 to 2003, as described [23].
The authors are very grateful to Jim McNicol (BioSS, Dundee) for providing advice on microarray experimental design and GO analysis, and Mark Young (SCRI) for supplying meteorological data. Thanks also to Dr Louise Shepherd & Dr Mary Woodhead for their constructive comments on the manuscript. This work was funded under the Scottish Government RERAD Programme.
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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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Tuesday, May 5, 2015
17 delusion-like beliefs of British adults
I was taught that logos (logical argument) was an important part of public speaking, but now I'm having second thoughts. Recently I read a magazine article by psychologists Rachel Pechey and Peter Halligan titled The Prevalence of Delusion-like Beliefs Relative to Sociocultural Beliefs in the General Population that appeared in 2011 on pages 106 to 115 in Vol. 44 of Psychopathology magazine. You can read the abstract here (and find the full text by a Google search of the title in quotes with the added phrase filetype:pdf).
They developed the Cardiff Beliefs Questionnaire (CBQ), which includes 46 questions. 19 are about political, social and science-related beliefs. 10 are about paranormal and religious beliefs, and 17 are about delusion-like beliefs (10 bizarre). A telephone survey using the CBQ was done on a sample of 1000 British adults (52.1% female and 47.9% male). (The CBQ does not ask if you believe that Cardiff is the capital of Wales).
First, let's look at ten bizarre delusion-like beliefs in British adults. The total percent who strongly, moderately, or weakly believe them is shown first, and the percent who strongly believe them also is shown in [square brackets]. The type of delusion is shown in parentheses.
1. 44.3% - You are not in control of some of your actions [10.8%] (Controlled actions).
2. 38.7% - Certain places are duplicated, i. e. are in 2 different locations at the same time [6.8%] (Reduplicative paramnesia (place)).
3. 33.6% - Your thoughts are not fully under your control [6.2%] (Controlled thoughts).
4. 32.7% - There is another person who looks and acts like you (5.4%) (Subjective doubles).
5. 26.2% - Some people are duplicated, i. e. are in 2 places at the same time [4.1%] (Reduplicative paramnesia (person)).
6. 24.9% - People you know disguise themselves as others to manipulate or influence you [4.4%] (Fregoli).
7. 18.4% - The reflection in the mirror is sometimes not you [2.6%] (Mirrored-self misidentification).
8. 6.1% - Part of your body does not belong to you [1.1%] (Somatoparaphenia).
9. 5.8% - Relatives or close friends are sometimes replaced by identical looking impostors [0.4%] (Capgras).
10. 5.4% - You are dead and/or do not exist [0.9%] (Cotard).
Comedian Flip Wilson used to do a specific version of Number 1 - where his character Geraldine Jones lamented that The Devil made me do it. Numbers 2 and 5 violate the tagline in the Highlander films that There can be only one.
Another seven delusion-like beliefs of British adults are:
11. 46.4% - Your body or part of your body is misshapen or ugly [10.8%] (Body dysmorphia).
12. 40.5% - You are an exceptionally gifted person that others do not recognise [3.8%](Grandeur).
13. 38.5% - People say or do things that contain special messages for you [7.0%](Reference).
14. 33.8% - Certain people are out to harm or discredit you [6.5%] (Persecution).
15. 12.9% - The world is about to end [1.7%] (Nihilism).
16. 12.4% - You are infested by parasites [2.8%] (Parasitosis).
17. 6.9% - Some well known celebrity is secretly in love with you [1.0%] (Erotomania).
I saw the magazine article mentioned in an article by Graham Lawton on pages 28 to 33 of the April 4, 2015 issue of New Scientist titled Beyond Belief that you can read here. It was also slightly delusional - only mentioning Peter Halligan and ignoring the senior author, Rachel Pechey.
The image was adapted from one for Acute Dementia found at the Images from the History of Medicine web site.
Posted by Richard I. Garber at 8:42 AM
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There's nothing quite like a glass of fresh, ice-cold water straight from the fridge. Aware of this, Latin American supermarket Exito recently developed a special bottle with seven layers, designed to preserve the taste of the H2O inside.
In a print campaign to promote the new product, advertising agency Sancho BBDO developed this clever series of ads, featuring some of the more pungent things one might keep in the fridge, including salmon and green onions, being poured into glass containers.
The message is simple. If you don't want your water to take on the taste of the contents of your fridge, keep it in an Exito water bottle.
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- Illustrator tutorials: amazing ideas to try today!
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Have you seen an innovative print ad lately? Let us know in the comments box below...


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In San Francisco's Cow Hollow neighborhood, M-Projects and Larson Shores Architecture + Interiors transformed an outdated two-bedroom home into a wood-wrapped modern marvel. Totaling 4 stories, the completely rebuilt property is saturated with natural light thanks to copious skylights, a glazed interior courtyard and six outdoor decks. But 1864 Greenwich St.'s most distinctive feature is its hand-cut cedar facade, which balances the home's clean modern angles with visual texture and a natural touch. We recently had a chance to tour this new project through AIA SF's Architecture and the City Home Tours - hit the jump to take a look inside.
When M-Projects purchased 1864 Greenwich St. in 2012, it was a modest two-bedroom with some quirky features, like a stove in the dining room. M-Projects blew out the interior and built up the property to stand four stories high with over 4,500 square feet. The home's multi-tiered roof is topped with connected decks, the tallest of which offers panoramic views of the neighborhood and the nearby Golden Gate Bridge.
M-Projects and Larson Shores Architecture + Interiors took a "bespoke" approach to the renovation, as evidenced by the unique facade made from strips of hand-cut cedar. This motif is echoed in the interior, where vertical slats of wood frame the stairwell and define some of the interior spaces.
Skylights shower the rooms with natural light, and a sunny courtyard cuts through the second floor living space to illuminate the interior like a lantern. Natural materials and finishes reign throughout, and the designers lowered the home's energy footprint by installing energy-efficient appliances, LED lighting, and a radiant heating system controlled by Nest smart thermostats.
The home is currently on the market, although interested buyers will have to dig deep. Its asking price is $6.99 million – steep even by San Francisco standards.
+ Larson Shores Architecture + Interiors
+ Architecture and the City Home Tours
Photos by Mike Chino for Inhabitat


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Red Pine Studios is moderating our annual filmmaking workshop with the Boulder Adventure Film Festival. Here is our blurb from the Festival catalog…should be a fascinating evening with some outstanding image makers who are all involved in unique productions. Join us for the only local conversation of its kind…shooters and producers talking with each other.
A complete festival schedule is available at: www.adventurefilm.org


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If you are hopping along with us today: You should have come here from The Twinery blog. If you did, you are in the right place! Welcome!
If you aren't already a follower, I'd love to have you follow my blog!
If you are on Facebook, please "like" Ink Blots by TRD.
If you are one of my regular readers: Thanks for coming back! I love having you! Please feel free to join along in the hop - you can start at the The Twinery blog.
The Hop Order:
Tifany DeGough <--- You are here
The Giveaway:
Comments will close at midnight (EST) on Tuesday, November 3rd.
Winners will be posted on The Twinery blog on Thursday, November 5th.
The Storage:
striped and tri-color twines:
The Twinery is allowing each participating designer on the hop to give away a fabulous Striped Twine Sampler! That means there are EIGHT of these beauties being given away:
Comments will close at midnight (EST) on Tuesday, November 3rd.
Winners will be posted on The Twinery blog on Thursday, November 5th.
The Storage:
My craft supplies are stored in every crafter's best friend (an Expedit bookcase from IKEA - no longer available, but the new version is called Kallax). Within that, I store my twine in two wire baskets. (Mine came from a company that is now out of business, but you can find cute wire baskets in a variety of places. My baskets are approximately 11" x 13" x 4 1/2". Here is something similar I found online, but slightly smaller dimensions and slightly taller sides - approximately 10" x 11 1/8" x 5 1/4". Still, it looks like a great size for twine storage.)
I love pulling my twine out and seeing all those beautiful rolls to choose from when I am creating! I also love how it looks on the shelf - cute and contained.
Because I don't have a dedicated craft room (or even craft table), all of my craft supplies need to be easy to get out and easy to put back - so storing them in easily accessible (& portable) containers on my bookcase works well for me.
Because my craft bookcase is in easy view (and not in a dedicated craft room), I wanted to make it both functional and pretty. I have a variety of baskets, boxes, bins, tins, totes, jars...you get the idea...along with a few small decorative items placed throughout the cubbies of my bookshelf.
I stack them in one cubby in my bookcase. Nothing fancy, but it works for me!
How do you store your twine? I'd love to see!
Want to add to your twine stash?
The Twinery is offering a special discount just for this hop!
Use code: STASH25 to get 25% off any order through 11/6/2015.
Shop here: http://shop.thetwinery.com/
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Thanks for stopping by today!
The Next Stop:


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Author: Sydney Salter
Description: Welcome to the summer of passion. For seventeen-year-old Jory Michaels, that means three sun-and-fun filled months of spending time with her best friends, obsessing over her crush, trying to find something she is passionate about, and…saving for a nose job. Jory is determined to lose the big, honking, bumpy monstrosity she calls the Super Schnozz—the one thing standing between her and happiness.
So accident-prone Jory takes a job delivering wedding cakes to save up for surgery; she even keeps a book filled with magazine cutouts of perfect noses to show the doctor. To find her passion, she tries yoga; she tries becoming a foreign film buff; but nothing is quite as interesting as finding a boyfriend. And that can't happen until Super Schnozz disappears…right?
Jory is in for some big surprises when passion finds her—and she discovers the beautiful people she longs to look like are not as perfect as they seem. In her hilarious and heartbreaking debut novel, Sydney Salter delivers a story about broadening your horizons, accepting yourself, and finding love right under your nose.
This is such a cute video! Don't you agree?


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