text:
Cisco UCS M-Series Modular Servers deliver exceptional value for online content delivery like gaming, web serving, transcoding, and HPC. Built around Cisco's virtual interface card (VIC) technology and the policy-based management of UCS Manager, this new design brings the award-winning architecture and management of Cisco Unified Computing to the world of parallelized workloads.
Tags: Cisco UCS, Modular Servers


text:
Built in 1970s for the Porter family.
- Lexan panels, air chambers, reduce/increase heat, end vent on peaked roof, fans to provide air flow. Swamp coolers, furnace.
- Microclimate totally different from desert – A tool for education.
- Automated control unit opens and closes the vent, turns on/off coolers, heaters or fans to maintain the chosen temperature.
- Interaction with community: Plant donations!
The greenhouse contains an impressive selection of tropical plants such as Orchids, Hibiscus, Sword Ferns, Swiss Cheese Plants, Epiphytes and Bromeliads (air plants).
- Temperatures:
–Butterfly Season: Day/Night 85F/70F
–Summertime: 85-90F/70-80F
- Fish are African Cichlids & Australian Rainbow Fish
Butterfly Magic
Hundreds of tropical butterflies (about 35 species at any one time!) in a beautiful tropical setting.Email this page to a friend

subset: commoncrawl


text:
Third Quarter results
Net sales for the third quarter of fiscal 2008 were a record $503.7
million, an increase of $141.4 million, or 39 percent, compared with $362.3
million last year. Reported net income was $40.0 million, or $0.42 per
share, compared with net income of $17.1 million, or $0.18 per share a year
ago. In the third quarter of fiscal 2008 and fiscal 2007, the Company
recorded several net of tax charges, summarized as follows:
2008 2007
-- Write-off of IPR&D $2.0 $4.8
-- Inventory step-up 2.1 -
-- Restructuring 0.2 0.2
$4.3 $5.0
Excluding the impact of the charges noted above, adjusted net income for the third quarter of fiscal 2008 was $44.3 million, or $0.47 per share. For the third quarter of fiscal year 2007, adjusted net income was $22.1 million, or $0.24 per share.
(Refer to Table II at the end of this press release for additional non- GAAP disclosure information.)
Perrigo's Chairman and CEO Joseph C. Papa commented, "For the second quarter in a row, we delivered record sales and earnings with successful launches of two of the largest products in our 120 year history, Omeprazole and Cetirizine. We also began shipping products from our Galpharm acquisition in the United Kingdom and launched Clobetasol Foam in Rx. The results this quarter clearly show the benefits of our long term commitment to our new product pipeline."
Nine Months Results
Net sales for the nine months ended March 29, 2008 were $1,321.9
million, compared with $1,073.1 million last
|SOURCE Perrigo Company
Copyright©2008 PR Newswire.
All rights reserved


text:
The American Academy of Cosmetic Surgery is a professional medical society whose members are dedicated to patient safety and physician education in cosmetic surgery. Most members of the AACS are dermatologic surgeons, facial plastic surgeons, head and neck surgeons, general surgeons, oral and maxillofacial surgeons, plastic surgeons, or ocular plastic surgeons -- all of whom specialize in cosmetic surgery. AACS is an organization that represents all cosmetic surgeons in the American Medical Association through its seat in the AMA House of Delegates.
|SOURCE American Academy of Cosmetic Surgery
Copyright©2008 PR Newswire.
All rights reserved


text:
Wahl Manscaper
We know you want the goods as soon as possible, which is why orders received by 3pm ET leave our warehouse the very same day. We'll send you an email that day to confirm shipment and the email will include your tracking information.
Our warehouse is closed Saturday & Sunday, so orders received after 3pm ET on Fridays will not ship out until Monday.
Wahl Manscaper is a rechargeable full body hair trimmer perfect for back, shoulders, chest, abs, face, underarms and much more!!
- 12 position rotating head
- Perfect for use all over the body
- Up to 1 hour of usage per charge
- Attachment combs allow for different lengths including: stubble, 1 day growth and 2 day growth
- 3 Interchangeable heads included
- Also includes storage base, cleaning brush and blade oil.
Folica stands behind the authenticity of everything we sell. Since 1998, Folica has made it our mission to provide you with an outstanding selection of hair and beauty products at highly competitive prices.
All the power of a full-size clipper, the Peanut is both small and lightweight at only 4 inches long and 4 ounces in weight. Now available in a new fun peanut shapped design.New Price:


text:
Gerald,Originally Posted by Gerald Koch
I hope didn't come across as condemning auto exposure in general or the EM in particular. I agree completely with what you are saying, however, what I took out of the initial question was that a little more control was desired in order to learn a bit more about photography in general, and eventually go a little more in-depth into it. I think auto-exposure is fine once you learn what it is doing, how its doing it and why - and as such, what to expect from it and how to use it best. That's why I suggested keeping the EM - little financial gain from selling it, and a handy little camera (with emphasis on little) to have when just such a thing is called for.
As to the suggestion of the FG - I would have to say that while it too is a fine camera (I haveone, and love it dearly), it would not be "the" camera I would buy in this case. It is very much a consumer camera which does not have certain features I think someone learning about photography could greatly benefit from (dof preview for one). But if you have the motor drive for the EM already, it does cast a vote in favour of an FG, as it essentially the same chassis with another exposure mode and the ability to go fully manual. The meter is very accurate, at least on my example, when compared with much more "pro" level gear. But I think an FM/FE type camera would serve you a lot better before you find yourself needing anything else in a manual focus 35mm camera (as you might have noticed, many people never do find a need for anything else - that's how fine a camera they are).
Well, I think I deserve to pat myself on the back for my complete lack of any Canon propaganda


text:
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I am a new patient of his daughter's and had the pleasure of meeting him today. It's no surprise where she gets her fabulous bedside manner from!
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Great dental Office And Staff. Never a dull moment, professional, friendly, upbeat, smiles and jokes come along with the treatment and this is genuine. I feel at home at Fujiki dental and Bob Fujiki is the best. Both of his children are accomplished dentists practicing along with their father add to the already great experience of Fujiki Dental.
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I came to this practice after a bad experience elsewhere, and I am now a patient of the Fujikis. They run a first-class dental practice! There is never a wait time, and everyone is very professional, friendly and knowledgeable.
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The doctor is awesome!very nice,funny,the best doctor I ever met:-)Outstanding!you guys took care of me like a baby! LOL Thank you so much.
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Dr. Bob is the BEST! I had several very bad experiences before seeing Dr. Bob and I must say, I am not fearful anymore. He is an excellent clinician, great guy and his staff are amazing! I wouldn't go anywhere else. I have been a very satisfied patient for 20 years now. Keep up the good work!
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Dr.Bob, is gentle kind and reassuring, I don't get as nervous as I use to anymore, t he staff is courteous and professional , extremely friendly, they are very accommodating to my physical limits in the chair.
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Been going to Bob since he was a rookie with Dr Starr. This is the best dental practice you could hope to find.
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Excellent service from a caring and competent doctor. Superb, friendly and attractive staff who always seem to love what they do. Two thumbs up, way up!
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After many bad experiences over the years, we finally found the most professional, compitent,gentle, caring, knowlegable dentist who we would recommend to everyone.
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text:
Hungary Sees More Loan Steps as Banks Book $151 Million Loss
Nov. 3 (Bloomberg) -- Hungary's government is working with local lenders on further measures to help foreign-currency borrowers after banks booked more than 33 billion forint ($151 million) in losses on such loans under a Cabinet plan.
"Our aim is to find joint solutions that will take the burden of foreign-currency risk off families," Economy Minister Gyorgy Matolcsy said at a press conference in Budapest today after a meeting with the head of the Banking Association. The government pledged not to implement further measures before a deal with local lenders, Matolcsy said.
Hungarian lawmakers approved legislation on Sept. 19 that allows early repayment of Swiss-franc denominated mortgages, which account for two thirds of such loans, at more than 20 percent below market rates, forcing lenders to swallow losses. Domestic banks turned to the Constitutional Court and the European Union said the plan may breach the bloc's rules.
Hungarian banks may book another 71 billion forint in losses based on indications submitted so far, Karoly Szasz, the chairman of financial regulator PSZAF, said at the same press conference. Households have repaid 133.8 billion forint in such loans and have applied for the early repayment of more than 295 billion forint through Oct. 23, Szasz said.
'Complex Package'
The plan allows borrowers to repay foreign-currency mortgages in a lump sum at a fixed exchange rate of 180 forint per Swiss franc and 250 forint per euro, provided they were taken out at lower exchange rates. Borrowers have until the end of the year to submit their applications for early repayment.
The forint strengthened 0.9 percent to 303.6 per euro by 2:23 p.m. in Budapest, paring losses to 8.8 percent since Sept. 9, when the ruling Fidesz party proposed the early repayment plan. Hungary's currency has depreciated 44 percent against the Swiss franc since July 2008.
Local lenders will present a "complex package" of proposals on foreign-currency loans in two weeks, Mihaly Patai, head of the Banking Association, said at the same press conference.
Households are struggling to repay franc-denominated mortgages after the advance of the Alpine country's currency boosted repayments and triggered defaults.
Repayment Estimates
The central bank expects 20 percent of the 5 trillion forint in foreign-currency mortgages will be repaid under the plan, causing banks losses of 207 billion forint, excluding foreign exchange impact, according to the bank's Report on Financial Stability published yesterday. Applications for early repayment may surge in late November and early December after a leveling out period, Szasz said.
OTP Bank Nyrt., Hungary's largest lender, competes mostly with units of international banks including Erste Group Bank AG, Raiffeisen Bank International AG, UniCredit SpA, Bayerische Landesbank AG, KBC Groep NV, and Intesa Sanpaolo SpA.
Raiffeisen's Hungarian unit may lose as much as 22 billion forint on the early repayment plan, Ferenc Szabo, deputy chief executive, said on Oct. 26. Erste, eastern Europe's second-biggest lender, was the first foreign bank to recapitalize its Hungarian unit by injecting 600 million euros into the subsidiary.
To contact the reporters on this story: Edith Balazs in Budapest at firstname.lastname@example.com; Andras Gergely in Budapest at firstname.lastname@example.com
To contact the editor responsible for this story: James M. Gomez at email@example.com


text:
by the number and final deadness of his imitators. Shakespeare stands without Shirley. Skelton will always remain an example for poets caught up in the coils of a tradition, a decent way of writing, which they feel to be constricting their lives. It is better, always, to be a buffoon than a bore.
From E.M. Forster's 'Two Cheers for Democracy', first published in 1951. I have followed the text of the Abinger Edition (London, 1972), pp. 133-49.
Forster (1879-1970) was one of the most distinguished novelists of the twentieth century. His novels include 'The Longest Journey' (1907), 'Howards End' (1920) and 'A Passage to India' (1924). In addition he wrote volumes of biography and criticism. The lecture printed below was first given at the Aldeburgh Festival in 1950.
John Skelton was an East Anglian; he was a poet, also a clergyman, and he was extremely strange. Partly strange because the age in which he flourished—that of the early Tudors—is remote from us, and difficult to interpret. But he was also a strange creature, personally, and whatever you think of him when we've finished—and you will possibly think badly of him—you will agree that we have been in contact with someone unusual.
Let us begin with solidity—with the church where he was rector. That still stands; that can be seen and touched, though its incumbent left it over four hundred years ago. He was rector of Diss, a market town which


text: More from SI Report: Dodgers release pitcher Freddy Garcia Report: Dodgers release pitcher Freddy Garcia Marshawn Lynch sports gold cleats for Super Bowl Marshawn Lynch sports gold cleats for Super Bowl Louisville vs. FIU: Game time, live stream, TV coverage Louisville vs. FIU: Game time, live stream, TV coverage Pittsburgh-area high school football player dies after... Pittsburgh-area high school football player dies after... Close Tweet0 Share0 Comment0 Share0 Share0 Stumble It0 Print0 More SI Wire Casey Pachall back at TCU after seeking substance-abuse treatment by Brett LoGiurato Email Posted: Mon Jan. 14, 2013 TCU quarterback Casey Pachall has returned to campus and the football team after undergoing treatment for substance abuse. (Cooper Neill/Getty Images)Former TCU quarterback Casey Pachall has returned to the university and is back on the Horned Frogs football team, his father and TCU coach Gary Patterson both told the Forth-Worth Star Telegram.Pachall was suspended from the team after a DUI arrest in October and decided to enter a treatment facility for substance abuse. Patterson said that Pachall participated in team meetings on Sunday and "did everything we wanted him to do" in order to rejoin the team."He looks great," Patterson told the paper. "It's like he looked when he was a freshman. Right now, we feel really good about this. It's never going to be 100 percent perfect but there is a chance we did the right thing and made a difference. You've got to understand, everybody is going to be watching on this."Pachall is expected to be given an opportunity to earn back his starting job. But he has not "lifted weights or thrown a ball in several months," Patterson said. Pachall got off to a roaring start in the first four games of 2012 before his suspension. He completed 66 percent of his passes and threw 10 touchdowns to just one interception. In 2011, Pachall completed 66.5 percent of his passes for nearly 3,000 yards. He threw 25 touchdowns and only seven interceptions that season. Promoted Stories Comments


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Recent reviews in Earth City
Bhann thai
"Tiny restaurant in a strip mall with great food"
"Thanks to Yelp, I found this Thai restaurant near my hotel on the other side of the highway. It's a tiny place in a strip mall, but has great food, cheap prices, and fast service. Seems to be more geared to a lunch crowd and carry out (due to it being near several large office complexes), but I ha
d a great meal of freshly prepared pad thai. "
More >
Bhann thai
"Perfect little Thai place"
"overall, excellent Food was delicious, smelled great, looks great. It's simple, low priced for great food. Sit down and carry out options. Daily specials. Owners are the chefs and waiters, so they're very motivated and nice. Atmosphere is basic, nothing fancy, just a small store front or half-stor
e front restaurant; but that's what we wanted. "
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Bhann thai
"Very Good Food at reasonable prices"
"I am a big fan of Thai food (been to Thailand several times) and happened to be in Earth City a couple of evenings. I found Bhann Thai online and decided (with the wife) to check it out. It is located right at the corner of the Earth City freeway (exit off of I-70) and Rider Trail South. It is in
a small plaza next to the Mcdonalds and the gas station. My wife and I went for dinner (twice) so we were able to sample a few of the menu items. The place was deserted at dinner time (usually not a good sign), but the area is full of office buildings and hotels, so I imagine the majority of their business is lunchtime. The woman working there welcomed us and offered us our choice of tables. The menu is a mix of traditional Thai and some Chinese (american version) as well. We tried the following: Spicy Noodles (very good), Chicken Satay (very good), Spicy Beef Salad (excellent), Nam-Tak Beef (excellent) and Pad Thai (good). The Pad Thai was tasty but the sauce was a little wat... "
More >
"The wait time was short, the food was brought to us fast, hot & delicious! This is one of my favorite breakfast destinations!"
Bhann thai
"Decent grub - modest price"
"Spicy curry choices are spot on - can't speak for other menu items. I frequently stay at a local hotel and this is my go-to place."
Jack in the Box
"Decent fast food breakfast"
"Had breakfast here twice during the week when we stayed at the Holiday Inn next door. The sausage biscuit sandwiches are tasty and a good deal in the value meal."
Malone's Neighborhood Grill and Pub
"Above average food for a hotel"
"The ribs are good if you like them sweet which I do. The hamburgers are 1/2 lb and very good. The new york strip is really good for the price. Tell Amber behind the bar hi for me."
Malone's Neighborhood Grill and Pub
"Best deal going if you're staying at the hotel"
"Great menu, great service, reasonable prices, good bar. Don't know why you'd drive someplace when this place is on-premises. Would definitely go back!"
Malone's Neighborhood Grill and Pub
"Irish Pub"
"Delicious nachos. Great fish and chips. Top notch Reuben. Service, slow to get going but got better. Stella served in a beautiful, gold lettered, frosty mug. "
Malone's Neighborhood Grill and Pub
"Food a litter better then I expected. Good Bar."
"Food was OK. Nothing special but acceptable. Beer was cold and good draft beer choices. Very nice Bartenders. OK breakfast choices. Priced average too. "
Malone's Neighborhood Grill and Pub
"Good restaurant in the Holiday Inn"
"I was told this was an "Irish Bar", as the name with the shamrock would suggest..... But, other than Guinness on tap, which was poured perfectly, there is nothing "Irish" about this bar. That being said. This is a great place for drinks and bar food. They have an OK tap, would actually like to see
local craft beers, but their bottle selection looked great. I had the rack of Baby Back Ribs, which was very good. And, talking to patrons around me, their food was also great.... I heard a lot of great remarks about the 1/2 burgers. The service was very good, there was a server covering everything but the bar & a bartender. They seemed to work well together getting drinks and food out quickly. Over all, a great place to eat and drink, inside the Holiday Inn. "
More >
Malone's Neighborhood Grill and Pub
"Holiday Food"
"The food is good but the menu is limited. Closing time is 11 pm which is early for the only restaurant in the hotel. Average cost of a meal is less than $15. The soups are quite good. The environment is pleasant and has a small bar with large screen TVs."
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Malone's Neighborhood Grill and Pub
#1 of 6 Restaurants in Earth City
8 reviews
Holiday Food
Good restaurant in the Holiday Inn
Visitor photos (2)
Jack in the Box
#2 of 6 Restaurants in Earth City
3 reviews
Part of an old, unkempt gas statio...
Bhann thai
#3 of 6 Restaurants in Earth City
10 reviews
Nice little place with Great owner...
Wasn't awful but seems to be a tou...
Break Away Cafe
#4 of 6 Restaurants in Earth City
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OverviewGAME Studios was the entertainment division of The Learning Company Inc. and its licensors.
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Bobby Ghajar and Nina Hernandez look at the issues surrounding Kim Kardashian's new fragrance bottle. Read More »
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text: http://androidforums.com – Hi, I am new here, hopefully I have posted this in an acceptable place. Does anyone have a NOTE 3/or android and have problems viewing videos in HD on Facebook? If it's a video that I've taken it looks fine, but the second I upload it to Facebook it looks HORRIBLE. Also, I uploaded an HD video from my computer to Facebook and it looks amazing on my computer. It looks horrible on the NOTE 3. (General)


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Welcome to Bulldog Bio
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|Nardly Stoad's Climb
|24m. Easy climbing direct to a vegetated ledge at 10 metres leads to the first worthwhile gear in the obvious crack which divides the two slabs.Follow this until it peters out 5 metres from the top.Climb broken rock up and right and top out with care.
Photo: Nardy Stoad's © Jelly Mould Surfer


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Attention Everyone please we have only two spots left for the morning The SoMi Fitness JNL FUSION CHALLENGE that is starting on Wednesday January 9th and 10 spots left for the evening class. It will run for two months. Please try to come in before the 9th so we can get started with the measurements and eating plan before the 9th. New year to a NEW YOU !!!!!!
For more information please feel free to contact Unni Greene at SoMi Fitness (305) 775-4128.


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Movie star Mary Marvell consults with Hercule Poirot after receiving threatening letters that warn her to return her diamond, the famous "Western Star," to its rightful owner. But who does own the diamond, and is it even the genuine article?
Editeur: William Morrow Paperbacks ( 8 mai 2012)
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Nombre de pages: 100 pages
Taille du fichier: 803 ko
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Langue: Anglais


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Daniel gave a tremendous prophecy of a rock that struck a large statue and broke it to pieces. The rock grew and became a huge mountain that filled the whole earth. Later, Daniel interpreted this as the kingdom of God destroying the kingdoms of this world and replacing them. The kingdom of God would crush all other kingdoms and would last forever. It starts out as a rock but grows into a huge mountain and fills the whole earth. The rock was cut out of a mountain without human hands. This kingdom would have its origin in God and be a spiritual kingdom. (See Daniel 3) This prophecy began its fulfillment when Jesus came into this world and announced, "The time is fulfilled, and the kingdom of
God is at hand: repent ye, and believe the gospel." (Mark 1:15 KJV) This then was the beginning of a kingdom that would grow into a huge mountain and fill the whole earth.
In March of this year, Alex Dodson, President of Watchmen Radio Ministries International, met with Pastor Samson Roberts in Guntur, India to discuss our radio outreach to India. This picture shows them in front of the Kingdom Gospel Church where Pastor Roberts and his wife Raji have served for many years. They founded the church and have sent out many missionaries to India from there. (See videos of their ministry at
http://www.watchmenradio.org/India ) Please prayerfully consider helping to reach the people of India with the gospel.


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Kalki The tenth avatara of Vishnu will be Kalki.
Towards the end of the Kali era, all people will be dastardly. They will oppose the Vedas, become robbers and will be concerned only with wealth. The disbelievers will then become kings and these kings will also be cannibals.
Much later, Kalki will be born on earth as the son of Vishnuyasha. He will take up arms to destroy these disbelievers. Kalki's priest will be the sage Yajnavalkya. The norms of he four classes (varna) and the four stages of life (ashrama) will be established yet again. People will honor the sacred texts and become righteous. It will then be time for the dawn of a new Satya Yuga, a fresh period of righteousness. In every cycle (kalpa) and in every era (manvantara) Vishnu is thus born in various forms. It is a sacred duty to listen to the stories of the ten avataras. The listener attains his desires and goes to heaven.
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- 58SHIVA MAHAPURANA: INTRODUCTION First of all, Sage Shaunak expressed his desire to Suta about knowing the means, which could help a man in this era of Kali to attainment lord Shiva, by cleansing all the impurities of his mind and rectifying his inherent demonic tendencies. Suta then described about Shiva…
- 47To continue with the process of creation, Brahma gave birth to a man and a woman from his own body. The man was named Svayambhuva Manu and the woman was named Shatarupa. Humans are descended from Manu. That is the reason they are known as manava. Manu and Shatarupa had…
- 42Bali had a hundred sons, the eldest among whom was Vana. Vana was devoted to Shiva. He was also extremely powerful. He defeated Indra and conquered the three worlds. Indra and the other gods went to Shiva. "Your devotee, Vana is oppressing us," they told Shiva. "Please take care…
- 40Sage Jaimini, a disciple of Veda Vyasa, effected branches of Samaveda. Sage Jaimini had a son, Sumantu. Sumantu in turn had a son, Sukarma. Both of them studied a branch of Samaveda each. Sukarma divided his branch of Samaveda into sub-branches and taught them to his two disciples- Kaushalya Hiranyanabh…


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Primula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China. Studies on population genetics, speciation and phylogeny of Primula have been impeded by a lack of genomic resources. In the present study, we sequenced the transcriptomes of two closely related primrose species, Primula poissonii and Primula wilsonii, using short reads on the Illumina Genome Analyzer platform.
We obtained 55,284 and 55,011 contigs with N50 values of 938 and 1,085 for P. poissonii and P. wilsonii, respectively, and 6,654 pairs of putative orthologs were identified between the two species. Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 877 of the pairs may be under positive selection (Ka/Ks > 0.5), and functional enrichment analysis revealed that significant proportions of the orthologs were in the categories DNA repair, stress resistance, which may provide some hints as to how the two closely related Primula species adapted differentially to extreme environments, such as habitats characterized by aridity, high altitude and high levels of ionizing radiation. It was possible for the first time to estimate the divergence time between the radiated species pair, P. poissonii and P. wilsonii; this was found to be approximately 0.90 ± 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Primers based on 54 pairs of orthologous SSR-containing sequences between the two Primula species were designed and verified. About half of these pairs successfully amplified for both species. Of the 959 single copy nuclear genes shared by four model plants (known as APVO genes), 111 single copy nuclear genes were verified as being present in both Primula species and exon-anchored and intron-spanned primers were designed for use.
We characterized the transcriptomes for the two Primula species, and produced an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two Primula species not only revealed a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. Furthermore, we developed two sets of genetic markers, single copy nuclear genes and nuclear microsatellites (EST-SSR). Both these sets of markers will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.
Keywords:Adaptive radiation; East Himalaya-Hengduan Mountains; EST-SSR; Single copy nuclear gene
Adaptive radiation, 'the rise of a diversity of ecological roles and attendant adaptations in different species within a lineage' is one of the most important processes bridging the gap between ecology and evolution [1]. Usually, the genetic divergence between species within adaptive radiations is very small, and only a handful of genes with large effects are responsible for differences in ecologically significant traits and reproductive isolation between species. Due to the lack of availability of molecular markers for rapidly evolving taxa, especially from nuclear genome, most plant molecular systematic studies on adaptive radiation have hitherto failed to provide resolved phylogenies. The same is true for speciation studies, which rely heavily on there being sufficient intraspecific genetic variation. Moreover, we still have little understanding of how divergent natural selection may have acted on the genomes of such species within the short evolutionary time span since their common ancestor [2].
Transcriptome analysis is not only an effective way to study gene expression in specific tissues at specific time, and it also provides unprecedented opportunities to address comparative genomic-level questions for non-model organisms. RNA-sequencing (RNA-seq) is an efficient new technology for large scale transcriptome investigations. With the rapid development of next-generation sequencing (NGS), RNA-sequencing becomes more efficient and less expensive, and is increasingly being used to study the evolutionary origins and ecology of non-model plants [3,4]. For instance, a large number of microsatellite markers or single-copy nuclear genes in yam (Dioscorea alata) [5], buckwheat (Fagopyrum) [6] and big sagebrush (Artemisia tridentata) [7] have been identified by making use of RNA-sequencing. Since RNA-sequencing is still somewhat expensive at present, few RNA-seq studies to date have included for more than one species at the same time [6,7]. However, comparative RNA-sequencing studies between closely related species can in principle not only provide additional genomic resources such as genus-specific SSR primers or single copy nuclear gene primers, but also give information about the processes of speciation or adaptive evolution, e.g. divergence time estimations, or detection of adaptive loci.
Primula with around 430 species, is one of the three great garden genera [8], and southwestern China, in which ca. 187 species of the genus are distributed, is its diversity centre [9,10]. In this region Primula shows a typical patterns of adaptive evolution and explosive speciation; however, research has been hampered by the fact that few Primula genomic resources are available. Up to now, only a few SSR primers from the three Primula species P. vulgaris, P. obconica, and P. sieboldii have been developed [11-13], and only one large EST collection, consisting of 5,651 ESTs generated from Primula sieboldii were available [13]. Paucity of genetic data such as genome sequences, transcriptome sequences and associated molecular markers has made Primula breeding or evolutionary analysis a challenging task.
Primula section Proliferae Pax, which contains ca. 25–30 species and is centred on southwestern China, is regarded as a taxonomically well-known group circumscribed by possession of numerous whorls of flowers [14]. Within this section, Primula wilsonii and P. poissonii are two closely related species with very similar morphological characters, and the two diagnostic characters used to distinguish them are the corolla structure and the aromatic fragrance of fresh leaves; for P. wilsonii, the fresh leaves are fragrant and corolla limbs are slightly opened, whereas, P. poissonii has no obvious fragrance and widely opened corolla limbs [14]. These closely related species represent a useful resource for addressing two questions: how did Primula species in southwestern China radiate within a short period of time, and what was the driving force underlying the process of rapid adaptive evolution? As the first step towards answering these questions, in this study, we obtained transcriptomes for Primula poissonii and P. wilsonii using the Illumina platform, and carried out a comprehensive analysis of them. Our aims were to 1) characterize the transcriptomes of P. poissonii and P. wilsonii, and increase the genetic resources available for Primula breeding or evolutionary analysis; 2) determine the evolutionary dynamics of the two species, including obtaining a divergence time estimation, signatures of adaptive evolution between the two species; and 3) discover genus-specific SSR markers and single-copy nuclear gene markers from both species.
Results and discussion
De novo assembly and functional annotation of contigs
After cleaning of raw sequences, ca. 55 million 75-bp paired-end reads were obtained for both P. poissonii and P. wilsonii,. We obtained 55,284 contigs with a mean length of 655 and an N50 value of 938 for P. poissonii, and 55,011 contigs with a mean length of 722 and an N50 value of 1,085 for P. wilsonii (Table 1). Contig with lengths between 200 and 500 bp were overrepresented, making up about 56% of the total number of contigs for P. poissonii, and 53% for P. wilsonii, the next most abundant size class was 500–1000 bp, constituting about 24% and 24% of the total, respectively (Figure 1). To evaluate the quality of de novo assembly, we obtained a total of 16,346 peptide sequences from Vitis. For P. poissonii, 34,660 contigs were annotated to 13,800 (84.4%) Vitis proteins, of which, 6,869 (49.8%) proteins were covered for at least 70% of the full length. For P. wilsonii, 34,930 contigs were annotated to 13,955 (85.4%) Vitis proteins, of which, 7693 (55.1%) proteins were covered at least 70% of the full length. The GC content for P. poissonii and P. wilsonii sequences is 41.3% and 41.2%, respectively.
Table 1. Summary of assembly and annotation results for P. poissonii and P. wilsonii using Trinity
Figure 1. Length distributions of contigs for two Primula species. Grey bar, P. poissonii; black bar, P. wilsonii.
In BLASTX homology research with the cutoff E-value set at 1E-6, 36,239 contigs (65.6%) for P. poissonii and 35,857 contigs (65.1%) for P. wilsonii gave hits. For both species, the three top-hit species were Vitis vinfera, Populus trichocarpa and Ricinus communis (Figure 2). A total of 28,435 (51.4%) and 28,302 (51.4%) contigs were assigned at least one GO terms for P. poissonii and P. wilsonii, respectively. For the biological process category, the two mostly highly represented terms among the 23 level-2 categories were cellular process and metabolic process; for the molecular function category, among the 13 level-2 categories, binding and catalytic activity were overrepresented; for the 14 level-2 categories in the cellular component category, cell, cell part and organelle were the most abundant terms (Figure 3). These categories were similarly distributed in both species.
Figure 2. Top-hit species distribution for sequences from two Primula species submitted BLASTX against the NCBI-nr. database. Grey bar, P. poissonii; black bar, P. wilsonii.
Figure 3. Comparison of GO terms distributions between two Primula species. Grey bar, P. poissonii; black bar, P. wilsonii.
Orthologous contigs, substitution rates, and transcriptome divergence between two Primula species
We identified 28,482 pairs of putative orthologous contigs between P. poissonii and P. wilsonii using the reciprocal best hit method with BLASTN algorithm. After incorporating the Vitis peptide sequences [15], 7,006 pairs of putative orthologs were obtained using the RBM triangulation method [16]. This reduction in ortholog numbers was caused mainly by the exclusion of the relatively young orthologs specific to Primula, which were discarded as being low similarity to Vitis. After excluding alignments with unexpected stop codons, lengths less than 150 bp or Ks values above 0.1, 6,654 pairs of orthologs were retained for subsequent analysis.
Using the Vitis proteins as reference, the coding regions of 6,654 pairs of orthologs from P. poissonii and P. wilsonii were extracted, in some cases, 5′-UTR (1,315 pairs of orthologs) or 3′-UTR (2,051 pairs of orthologs) were also determined. The average genetic divergence of coding regions between the two Primula species is 0.011 ± 0.007 according to the K2P model. The genetic divergence between the two species is 0.019 ± 0.017 for 5′-UTR and 0.018 ± 0.013 for 3′-UTR regions. The accelerated substitution rate observed in the 5′UTR and 3′ UTR relative to the coding region, is indicative of relaxed functional constraint on the evolution of the UTR than on the coding region at the genome level, which is consistent with the evidence from other model species-pairs [17].
Among the 6,654 pairs of orthologs between P. poissonii and P. wilsonii, 165 pairs were identical, 1,327 pairs had only either synonymous or nonsynonymous substitutions, and 5,162 pairs had both types of substitutions, for which the Ka/Ks ratio were calculated. The mean values of Ka, Ks, and the Ka/Ks ratio of all orthologous pairs were 0.007 ± 0.005, 0.027 ± 0.017 and 0.322 ± 0.324, respectively. Of the 5,162 pairs of orthologs, 233 pairs with a Ka/Ks value > 1 were found. Taking a more appropriate threshold of 0.5 for the Ka/Ks ratio as an indicator of positive selection [18], 644 pairs with a Ka/Ks value between 0.5 and 1 were also found.
Peaks in the Ks value distribution of orthologs between closely related species often indicates speciation events [19], and this approach has been successfully used in the inference of such events [20]. In this study, a peak of Ks distribution between P. poissonii and P. wilsonii was observed at 0.027 ± 0.017 (Figure 4). The low level of Ks between P. poissonii and P. wilsonii indicated their close relationship and confirmed the previous taxonomic treatment. Based on the data derived from ESTs of Asteraceae and several model plants provided by Kane [21] (personal communication), we found a mean Ks value of 0.03 ~ 0.10 between congeneric species. According to these criteria, the differentiation between P. poissonii and P. wilsonii is obviously very recent.
Figure 4. The Ks distribution of orthologs between P. poissonii and P. wilsonii, their divergence is shown by the peak of Ks at 0.03 ± 0.017.
The peak synonymous rates (Ks) for orthologous transcript pairs can be used to estimate the times of divergence between species. To obtain a rough estimate of the divergence time (T) between P. poissonii and P. wilsonii, we followed the simple formula: T = K/2r [22], where r is the mean rate of synonymous substitution, and is considered to be 1.5E-8 substitutions/synonymous site/year for all dicots [23]; K is genetic divergence expressed in terms of mean number of synonymous substitutions between orthologs. The age of the speciation event between P. poissonii and P. wilsonii is approximately 0.90 ± 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Bearing in mind disputes about the substitution rate [24], this divergence time is only an appropriate estimate based on the coding region of orthologous genes, nonetheless, it is useful because there is as yet no adequate fossil dating the divergence of the two Primula species.
Functions under positive selection and implications for adaptive evolution between two Primula species
In enrichment analyses, we categorized the orthologs into two datasets: a test dataset with Ka/Ks > 0.5, and a reference dataset with Ka/Ks < 0.5. In an analysis of GO terms with at least five hits, 20 GO-terms annotated to 98 pairs of orthologs were found to be over-represented (Fisher's exact test, P-value < 0.05) in the test dataset (Table 2, Additional file 1: Table S1). For the 98 selected genes, we used BLASTX search to find their orthologous genes in Arabidopsis, and the results showed that the genes with function in DNA repair, stress resistance were overrepresented (Tables 2 and 3). Among the candidate genes under positive selection with Ka/Ks > 0.5, almost one-quarter of them were involved in the DNA repair. DNA repair is essential for maintenance of genomic stability in all organisms. For instance, in our study, the ortholog pw11166, and pw24180 were found to be homologous to Ku70 and Ku80, which are involved in the repair of DNA double-strand breaks (DBSs) by non-homologous end joining (NHEJ) [25]; other orthologs, pw42431, pw06163, pw54400 were homologous to SMC5, ETG1, ROR1, respectively, which are all involved in DNA repair by homologous recombination [26-28]. The finding that gene families Ku, and SMC have been under positive selection gives an indication of why P. poissonii adapted to the habitats of higher altitude and ionizing radiation than did P. wilsonii.
Additional file 1. Table S1. Candidate orthologs under positive selection between P. wilsonii and P. poissonii. Table S2. Characteristics of conserved microsatellites primers based on two Primula orthologs. Table S3. Characteristics of primers for Primula-specific single copy nuclear genes homologous to APVO gene sets.
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Table 2. Gene Ontology terms significantly over-represented in the test dataset versus the reference set
Table 3. Partial list of candidate orthologs under positive selection between P. wilsonii and P. poissonii
Some orthologs related to abiotic stress were also found to be positively selected. For example, pw23141 is homologous to GPX7, which regulates cellular photooxidative tolerance and immune response [29]; pw12283 is homologus to ARP3, related to light-induced stomatal opening [30]; pw37795 is homologous to LEW1, the product of which catalyzes the biosynthesis of dolichol [31] and confers acclimation to drought stress, which may partially explain why the two Primula species were able to inhabit habitats with different level of aridity; pw10616 is homologous to SBP1 and pw09970 is homologous to FER2, which are involved in the cadmium stress [32] and iron deficiency [33], respectively; these results shed further light on how the two Primula species differentially adapted to extreme environments. In addition, two positively selected genes are worth notice, one gene pw13953, is homologous to AP3, a key component in the ABC mode of flower development [34], and may provide a clue about the origin of the differences in corolla structure between P. poissonii and P. wilsonii; the other gene pw42171, is homologous to MYB12, which functions as a R2R3-MYB transcription factor in phenylpropanoid biosynthesis [35], also may give some hints on the leaf fragrance differentiation between P. poissonii and P. wilsonii.
Overall, in this study, we detected a dozens of gene under positive selection between the Primula species pairs, and these findings will not only shed light on how differentiations between two Primula species occurs, but also open the door to increased understanding of how plants living in plateau environments adapt to different characteristics of high altitude, such as strong radiation, aridity and so on.
Identification of microsatellites and single copy genes
Usually, SSR markers derived from expressed sequence tags (EST-SSRs) are more transferable between species than random genomic SSRs, and they are more advantageous for revealing adaptive differentiations at the population level. Traditional strategies for SSR marker development are labour-intensive and costly. In the case of Primula, up to now, only a few microsatellite primers have been available for Primula obconica, P. sieboldii and P. vulgaris[11-13], and this has impeded genetic analysis of this important garden plant. Based on the two Primula transcriptomes, 7,571 and 8,272 SSRs were found in P. poissonii and P. wilsonii, respectively. The most abundant repeat types were dinucleotides followed by trinucleotides (Table 4). The dominant classes of sequence repeat in the contigs were AG/CT, AT/TA and AC/GT, followed by AAG/CTT repeats (Figure 5), and most SSRs located close to the ends of contigs and were not suitable for primer design (Figure 6). In order to maximize the universal applicability of markers developed in this study and hence reduce their cost, we searched for SSRs in the 6,654 pairs of putative orthologous contigs, and found 1,207 SSRs distributed among 1,073 pairs of contigs (Table 4). Taking only those with a repeat-unit length of at least 16 bp, 421 pairs of SSRs contained in 342 pairs of orthologs were selected for primer design, and 54 pairs of sequences with conserved, sufficiently long flanking sites were used to design primers successfully (Additional file 1: Table S2). To evaluate the reliability of these primers, we tested 36 out of the 54 pairs and 24 pairs produced clear fragments with the expected sizes in both Primula species (Figure 7).
Table 4. Summary of microsatellite loci in Primula poissonii and P. wilsonii
Figure 5. Comparison of the distribution of SSR repeat motifs between two Primula species. Grey bar, P. poissonii; black bar, P. wilsonii.
Figure 6. The location distribution of SSRs for the two Primula species; the location is defined by the ratio of the start site position to the total length of the SSR-containing contigs. Grey bar, P. poissonii; black bar, P. wilsonii.
Figure 7. Validation of a subset of the microsatellite primer pairs for the two Primula species by agarose-gel profiling. Results are shown for one individual from each species with pairs name on top; the band profiles for the 50 bp maker ladder are illustrated; additional bands over 300 bp for some primer pairs are non-specific products.
One noteworthy fact about the SSR primer development based on the Illumina platform is the lower proportion of contigs suitable for primer design compared with Sanger sequencing, in our study, 367 out of the 421 pairs of contigs with SSRs were excluded from primer design because they had insufficient flanking regions caused by NGS assembly algorithms or sequencing [36]. As an alternative, the 454/Roche sequencing platform, which delivers longer reads, has promise as a way of reducing bias.
Using the APVO gene sets [37] to carry out TBLASTN queries against our Primula dataset orthologs, 612 of the APVO genes were found to give hits against orthologous contigs between P. poissonii and P. wilsonii over at least 600 bp; these are most likely to be single copy genes in the two Primula species. When we set a threshold identity of 75% with Arabidopsis thaliana and specified facultative intron sizes not less than 300 bp in Arabidopsis thaliana, we were successful in obtaining primers for 111 of the 612 APVO genes (Additional file 1: Table S3); we randomly selected four primer pairs to test, and of these, three of the pairs amplified successfully and when the products were sequenced directly, they yielded the expected gene model, and only one primer pair failed due to the presence of extremely long intron variants, which made the products unsuitable for Sanger sequencing.. The availability from this study of dozens of single copy nuclear genes with heterogeneous rates of variation, will undoubtedly facilitate phylogeny resolution for the radiative Primula species, and open a doorway to understanding the dynamics of speciation using a population genetics approach.
We developed sets of two types of molecular markers, and these two widely-used marker types, each of which has its own advantages, were applied for characterizing population structure, parentage analysis, genotyping, gene flow inferences and phylogenetic construction. The large number of novel single nuclear gene will greatly increase the resolution of phylogenetic reconstruction for this adaptive taxon. Moreover, these markers with their diverse evolutionary rates will provide unprecedented opportunities to answer the following important questions: What demographic histories underlie the phylogeographic patterns of Primula species? Which evolutionary forces drive the explosive radiation of Primula species in the extreme habitats?
In this study, we characterized the transcriptomes for the two Primula species, and obtained an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two species not only yielded a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. In addition, we developed two sets of genetic markers of popular types, single copy nuclear genes and nuclear microsatellites (EST-SSR). These marker sets will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.
Plant material, RNA extraction and sequencing
Primula poissonii (2n = 22) is widespread in the mountain areas of northwest Yunnan and southwest Sichuan, China. It inhabits alpine meadows at an altitude of 3200–3500 m. Primula wilsonii (2n = 22) is distributed in central Yunnan and grows on open lands in evergreen broad-leaved forest at an altitude of ca. 2500 m [8]. During July 2011, we sampled P. poissonii from Zhongdian plateau in northwest Yunnan (28°06'55.24"N, 99°47'48.49''E, alt. 3314 m), and P. wilsonii from Ailao Mountain in central Yunnan (24°32'34.47''N, 101°01'41.48''E, alt. 2450 m), respectively, and fresh leaves and whole flowers of multiple individual plants for both species were stored in RNAlater solution (Takara Biotechnology Co. Ltd., Dalian, China) to preserve the RNA state for use immediately after harvesting. After mixing an approximately equivalent weight of fresh leaves and flowers, total RNA was extracted using a modified CTAB method and precipitated with 5 M LiCl2 at -20°C overnight, and the resulting RNA pellets were suspended in about 100 μl DEPC-treated water. After assessing RNA quality by means of electrophoresis and an Eppendorf AG 2231 BioPhotometer Plus (Hamburg, Germany), quantified total RNA (concentration ≥ 100 ng/μL; rRNA ratio ≥ 1.5) were delivered to The Beijing Genome Institute (Shenzhen, China) for further treatments. The cDNA library for transcriptome sequencing was prepared using a cDNA Synthesis Kit (Illumina Inc., San Diego, CA, USA) following the manufacturer's recommendations. The cDNA library was then sequenced using a HiSeq2000 (Illumina Inc, San Diego, CA, USA) to obtain short sequences of 90 bp from both ends of each cDNA.
Sequence cleaning, assembly, contig annotation
Raw reads were firstly subjected to cleaning by removal of adaptors, reads with too many Ns, and reads with quality scores lower than 20. The cleaned reads were assembled de novo using Trinity [36] with the default parameters and contigs with length less than 200 bp were discarded due to a low annotation rate [38]. The filtered reads for P. poissonii and P. wilsonii were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR629689 and SRR640158, respectively.
Functional annotation was implemented using the online program Blast2GO v.2.6.0 [39]. All the assembled contigs were firstly subjected to BLASTX against the NCBI's non-redundant protein database with an E-value threshold of 1E-6. The predicted gene name for each contig was assigned according to the best BLASTX hit. Gene Ontology [40] terms were retrieved from BLASTX hits at E-value threshold 1E-6. Finally, the distributions of level-2 GO terms for all contigs were plotted with the program WEGO [41]. In addition, we download the Vitis vinfera proteome [15], and queried against all the assembled contigs using TBLASTN with an E-value threshold of 1E-10 [42].
Identification of orthologous contigs and estimation of substitution rates
The reciprocal best matches (RBM) method [43] is widely used for identifying orthologs, and a modified version program RBM triangulation [16], allows a third species to be incorporated, which can increase reliability and detect large numbers of conserved orthologs, so we used the following approach for this study. First, we used BLASTN with the RBM method to find orthologs between the two Primula species setting the E-value cutoff at 1E-10. Next, to avoid misspecification caused by the absence of a true ortholog from either Primula species, the third species Vitis was added as positive control. All the reciprocal best hit orthologs were subjected to BLASTX against the Vistis peptide sequences at a threshold E-value of 1E-10, and only those pairs of orthologs with the same reciprocal best hit with Vitis were kept.
With the Vitis peptide sequence as reference, 5′UTR or 3′UTR sequences for some Primula contigs were determined. According to the best-match Vitis peptide sequences, the coding region sequences (CDS) of all the contigs were extracted with custom Perl scripts, and subsequently aligned using the MUSCLE algorithm [44] implemented in MEGA5 [45]. Alignments with unexpected stop codons, or less than 150 base pairs in length, were discarded after checking manually. For the remaining orthologs, synonymous substitution rates (Ks) and non-synonymous rates (Ka) were estimated using a maximum-likelihood method [46] implemented by yn00 in the PAML toolkit [47]. For the closely related species pair, P. poissonii and P. wilsonii, orthologs with Ks > 0.1 were excluded to avoid paralogs [48]. Divergence in the CDS sites and UTRs were calculated using the K2P model with a custom Perl script.
On the basis of the Ka/Ks value, setting a threshold at 0.5, the orthologs were sub-categorized into two dataset: a test set with Ka/Ks above 0.5, and a reference dataset with Ka/Ks value less than 0.5. The significance of the difference in GO term abundance between the two datasets was tested using the Fisher's exact test with the GOSSIP package [49] implemented in BLAST2GO V.2.6.0 [39].
Simple sequence repeats (SSRs) identification and mining of single copy nuclear genes
The program MISA (http://pgrc.ipk-gatersleben.de/misa/ webcite) [50] was used to identify and localize microsatellite motifs in the two Primula species, and only those contigs with motifs containing at least five repeats were selected. The alignments of 6,631 pairs of orthologs were extracted as the input file for the MISA program. Using the detailed information on SSR loci obtained from the output of the MISA program, primers for each SSR-containing sequence with a repetitive at least 16 bp in length were designed with Program Primer Premier 5 (PREMIER Biosoft Int., Palo Alto, CA). To validate the SSRs identified in silico identified SSRs, primer pairs shared between the two Primula species were synthesized (Invitrogen Trading Shanghai Co., Ltd, Shanghai, China), and amplified with one individual of each species as templates. PCRs were performed in a 25 μl volume containing 25 ng of template genomic DNA. The PCR reactions were carried out under the following conditions: initial denaturation at 95°C for 2 min, 35 cycles at an annealing temperature ranging from 45 ~ 60°C for 50 s, and a final extension at 72°C for 10 min. The PCR products were checked on 1.5% agarose gel.
Duarte et al. (2010) identified about 959 sets of single copy nuclear genes shared by Arabidopsis, Populus, Vitis and Oryza (known as APVO genes). We extracted the protein sequences encoded by the APVO gene from the TAIR10 database and queried them against the Primula orthologous EST database using TBLASTN [42] with a threshold E-value of 1E-10. All the queries with hits were considered to be single copy nuclear genes in the Primula species, and the consensus contigs of best- matched orthologous pairs of the two Primula species were extracted for degenerate primer design using the SeqMan 5.0 program (DNASTAR Inc, Madison, WI, USA). The consensus sequences were queried against the Arabidopsis thaliana protein database using BLASTX with an identity threshold of above 0.75, then subjected to exon-anchoring and intron-spanning primer design according to the corresponding Arabidopsis thaliana gene models with Program Primer Premier 5 (PREMIER Biosoft Int., Palo Alto, CA, USA). To validate these primers, four of them were randomly chosen to amplify with genomic samples from one individual of each of the two Primula species, and the products were sequenced.
Competing interests
The authors declared that they have no competing interest.
Authors' contributions
XJG and WW conceived and designed the project. WW and HFY performed the experiments. LZ, WW, XJG analysed and interpreted the data. LZ, WW and XJG drafted the manuscript. HFY and HY revised the manuscript. All authors read and approved the final manuscript.
We thanks Dr Nolan Kane (University of British Columbia, Canada) for providing data on substitution rate estimations for Asteraceae, thanks are also given to the members of the Adaptive Evolution Lab in Sun Yet-sen University (Guangzhou, China) for helps with bioinformatics analysis. We'd really like to thank Dr. Joelle Hoggan for helping us polish the manuscript. This work is financially supported by the National Natural Science Foundation of China (Grant Nos. 31170205, 31270009).
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I'm so close to the end, and yet there's still nothing I can do but wait. Oh, I can pre-wash the cloth diapers and drink my uterus-strengthening tea. I can swim the world's slowest 800 meters and blend healthy strawberry-banana smoothies. I can bump into counters. But mostly, I wait.
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Sam Allardyce facing a tall task at Sunderland
The bookmakers and media pundits were not alone in tipping or even rooting for Sam Allardyce as the new manager of Sunderland AFC.
He was also the supporters' choice and Friday's announcement of his appointment was broadly welcomed.
Allardyce famously divides fans. Admirers point out that he has a habit of not allowing his teams to be relegated. He makes his players work and organises them in all areas of the pitch, especially at the back.
Detractors complain that he encourages, even requires,...


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Flood Maps. Pacific trash vortex showing drift of ocean pollution. Flood warnings. Science Daily: News & Articles in Science, Health, Environment & Technology. Warmlist. A complete list of things caused by global warming Apologies for a temporary delay in updating the dead link list Advice of any omissions (with sources) or broken links is welcome at email@example.com Note : All links were live at time of posting.
Inevitably some will disappear, particularly from Yahoo News. Thanks to correspondents for additional entries; especially, as always, Our Man in Puerto Rico. David Suzuki Foundation: Climate Change. Global warming is a serious problem, and we're working to decrease carbon emissions in Canada.
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text: Sheryl Lee | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Sheryl Lee Performer ( b. Apr 22, 1967 Augsburg, GERMANY ) Female Productions ProductionsDate of ProductionsSalome[Play, Drama, Revival] Starring: Sheryl Lee [Salome]Jun 28, 1992 - Jul 29, 1992 Find out where Sheryl Lee and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


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Court Allows Kenyan Transgender Rights Group to Officially Register
July 30, 2014 by HRC staff
Post submitted by Jane Thirikwa, Global Engagement Fellow
Kenyan transgender rights group, Transgender Education and Advocacy (TEA), won a landmark victory after a court ordered the Kenyan Non-Governmental Organizations (NGOs) Coordination Board to formally register the advocacy group.
The NGO Board, which is responsible for registering nonprofit organizations in Kenya, previously denied the registration, citing that the names of the three transgender women listed in the application, did not reflect their gender.
Justice George Odunga said the reasons for the board's refusal to register the group lacked legal basis since the constitution upheld the freedom of assembly regardless of gender orientation. He also criticized the Board's decision as "unfair, unreasonable, unjustified and in breach of rules of natural justice." The judge also ordered the Board to compensate the Audrey Mbugua, Maureen Muia and Annet Jennifer for the cost of the three year old litigation.
Audrey Mbugua welcomed the ruling, stating that "this is a watershed moment for the transgender community in Africa and a show of the capabilities of the transgender community to extricate themselves from State sponsored discrimination and marginalization."
Mbugua has also filed a separate case suing both the Kenya National Examinations Council and the Attorney General for failing to recognize her gender identity.
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LO had diarrhea for a couple days and his poor little butt was super chapped. I was changing him and DH was looking over my shoulder and poor LO was screaming. DH was like there has to be something else we could do for him! I was like I have desitin babe, that's all we can really do. He was like well i'm going to go to the store.
He came back and was like 'I asked the pharmacist and they told me that this clear ointment will help it heal faster than the desitin. And then I also got some tummy drops and pedialyte so he can stay hydrated. And don't worry, I got the clear ones." (LO has a red dye allergy).
It almost made me get a little teary eyed lol :) Not only did he take it upon himself to find stuff for LO, he even remembered about his allergy without me saying anything! I love my DH so much, I'm so lucky to have him and LO is lucky to have such a great dad :)


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A paws in the AquaSox action
Friends, and dog owners, Andrea Sanford (left), Mary Schurr (center) and Lisa McCoy play with their pooches at "Bark in the Park," the annual dog-friendly AquaSox game in Everett on Wednesday night at Memorial Stadium.
Four-legged fans were allowed in their stands to enjoy the game along with dog activities.
Four-legged fans were allowed in their stands to enjoy the game along with dog activities.
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Dream shared on social-media prompts EvCC to boost security 6:15 p.m. Arlington thrift shop gives special-needs kids real-world skills Former county ombudsman John Koster files $950K claim Driver wipes out 5 cars at Everett repair shop Tulalip Tribes give nonprofits $5.8M in 'Raising Hands' initiative Cascade senior wants to expand her interests in robotics Lawmakers seek input on education funding in Everett on Tuesday Audit: City of Snohomish over-claimed $125K for roundabout costs
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Poker, Flying in ABC Team's Dreams
NEW YORK George Stephanopoulos will soon find out if he really has a poker face. The 'Good Morning America' anchor will travel to an Atlantic City, N.J., casino. It's part… More
NEW YORK George Stephanopoulos will soon find out if he really has a poker face. The 'Good Morning America' anchor will travel to an Atlantic City, N.J., casino. It's part… More
You have to respect Ashton Kutcher for his honesty. Yesterday his fans were twittin to him asking how he feels about autographs. "Truth… autographs R annoying. I try 2 do… More
Add author to the resume of ex-Hills resident, Lauren Conrad. Her novel hits bookstores June 16th and an advance review came courtesy of Billy Bush Sunday night, "Just finished Lauren… More
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KING OF PRUSSIA, Pa., Nov. 30 /PRNewswire/ -- Cadient Interactive, a Cadient Group company specializing in interactive marketing services and solutions for the healthcare industry, today announced the launch of its HATCH Consulting services group (www.cadientinteractive.com/hatch). Aligning strategic experts across a range of emerging areas of focus in healthcare marketing, the HATCH Consulting team is committed to guiding companies and brands to optimal market performance by applying a keen understanding of today's leading interactive strategies combined with creative, expert insight into what lies on the horizon of interactive healthcare marketing.
"We have found that healthcare and life sciences companies are often dissatisfied with the accessibility, agility and experience of their current marketing partners' strategic leadership teams," said Stephen Wray, president and CEO, Cadient Group. "We created the HATCH program as a way to address this industry challenge. Our clients have direct access to a team of interactive marketing specialists with deep industry expertise in a multitude of areas who offers breakthrough strategic thinking, relevant marketing solutions and peerless creative execution. Our internal teams also benefit from their access to our HATCH subject matter experts when developing campaigns, initiatives and solutions for our clients."
The HATCH team is segmented into distinct interactive healthcare marketing disciplines to best align with Cadient Interactive's clients' most critical marketing needs. These disciplines include: Market Development, Mobile Marketing, Consumer RM, Social/Emerging Media, Program Optimization/Analysis, Customer Ac
|SOURCE Cadient Interactive
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The Descent Of Man And Selection In Relation To Sex
Kessinger Publishing, 2004 - Science - 756 pages
In the discussion on Sexual Selection in my 'Descent of Man, ' no case interested and perplexed me so much as the brightly-coloured hinder ends and adjoining parts of certain monkeys. As these parts are more brightly coloured in one sex than the other, and as they become more brilliant during the season of love, I concluded that the colours had been gained as a sexual attraction.
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