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Kermanshah IAU Library
As a sub-section of the deputy of research and technology, and in order to provide appropriate grounds for teachers and student to carry out research, the library was first established in 1367 on the establishment of the university which was then located in a rental building in Jomhuri-e-Eslami square. The library held only 500 books with only one staff in an area of 12 square meters. Little by little as the university expanded both quantitatively and qualitatively, the library expanded. With the construction of phase one of the university which is located in Bagh-e-Ney, the library was moved to a 24-square-mere area. Later on when the deputy of research and technology was allocated an separate building in 1378, the library was given an area of 720 square meters of which 420 square meters was allocated to office work and book bank and 300 square meters to study area. In 1383, when the faculty of literature 2 was established in Imam Khomeini (RA) Complex located in Motakhassesin-e-Faza'ei town, an area of 550 square meters was added to the library. With the construction of the central library building in 1387, the library enjoys an area of 4000 square meters in a three-story building, and is considered the largest academic library in the west of Iran. It gives service to its users and consists of the following sections.
Management, cataloging and preparation, reference section, lending and study area for boys (second floor), lending and study area for girls (third floor), journals, theses and dissertations section, digital library and profiles of articles on the net, reparations and binding section, book store and postal services office.
Management section
The university library management like any other management is in charge of planning, organizing, guiding, controlling, leading the library. All the decisions concerning the macro-plans of the library, providence to further the aims of the library in accordance with the advances made in technology, issuing the license to makes purchase of all the requirements of the library, including research grant, current budget, and following up and implementing all the office work of the library are made in this section of the library.
Since its establishment, the following people have been in charge of managing the library.
Ms. Shahin Karami from 1368 to 1373, Ms. Turan Bagheri from 1373 to 1375, Ms. Elham Ayandi from 1375 to 1376, Ms. Hayedeh Rasuli from 1376 to 1381, Mr. Farid Masudi from 1381 to 1382, Mr. Seyyed Jalal Hosseinpur from 1382 to 1392, and Mr. Aliakbar Kordi from 1392 to 1393.
Right now Mr. Abbas Qomashi is the manager of the library at IAUKSH branch. He has finished his primary school, guidance school and high school studies in Langrud and get into Sistan and Baluchestan University 1375 to do his B.Sc. in Applied Mathematics and graduated in 1379. He continued his post-graduate studies majoring in Applied Mathematics, research in Operations and graduated in 1383 and finally in 1389 he managed to get into Islamic Azad University, Science and Research Branch to do his PhD in Applied Mathematics, research in Operations. He joined the IAUKSH branch as faculty member in 1386.
Cataloging and preparation
The technical and cataloging section which is located on the ground floor of the library building, prepares the books, registers them in the library registry books, and prepare the Bibliographic information such as their descriptive and analytic contents. This section is divided into two sections: a section in charge of selecting and ordering printed and non-printed materials, and a section in charge of organizing the materials (cataloging section). The responsibilities of these sections are as follows:
Selection and ordering printed and non-printed materials section
Collecting requests for purchasing references, processing them to avoid duplicates, prioritizing them and finally registering the references is done in this section
Organizing the material (cataloging section)
Organizing the materials including preparing the descriptive and analytic contents f the materials is done in this section. Cataloging is done by following the Anglo-American rules of cataloging and using the library of congress. Cataloging is done using computers and running the Simorgh Software online.
This section is connected to the Net and uses the library of congress and the national library to catalogue the books. Appropriate technical and cataloging instruments are available at this section.
The Repository section
The repository of the library is located on the ground floor of the library and with a capacity of more tha 400 000 book volumes, it has an area of more than 600 square meters. All the books here are non-reference and provides closed-system services. The books are arranged based on the library of congress system.
Receiving orders from Boys and girls lending area are directed here. After checking the orders and if the books is not lent out, the staff will send the books up through and electric lift to its intended area so that the user can borrow it out. The returned books are also sent here form the lending area so that they could be put away back in their appropriate places on the shelves. Books which are directed here from the technical and cataloging section will be placed on their appropriate places on the shelves based of the library of congress system.
Reference section
The reference section which is located on the ground floor has duties such as placing an order for and taking care of references, dictionaries, encyclopedias, manuscripts, atlases, maps, and valuable and precious books. The materials here are organized following the library of congress code and users have direct access to the shelves. All the books have barcode labels and all checking and shelf-reading are done through barcode readers.
Lending and study area
Lending and study area is divided into two areas: lending and study area for boys with a capacity of 400 students on the first floor, and lending and study area for girls with a capacity of 300 students i=on the second floor. This sections is completely mechanized and there are barcode readers which record all library user membership information and books information on the computers. The users can search for and take out the books using The Simorgh software. Taking out or returning a books can be done in two ways.
1. In person: the user searches for a book using one of the computers and places an order for the book. The order is sent to the book repository and if it's available it will be sent up through an electric lift. The books will be registered on the users name in the system.
2. Off-campus: the user visits the library website www.lib.iauksh.ac.ir and goes to the search for and renting a book. After searching for the book and placing an order for the book, the user enter her/his account using a password which was given to him or her when the user had applied for membership. The book will be booked for the user and sent to him/her through post.
Journals, theses and dissertations section
This section located on the second floor of the library building is in charge of placing an order for and maintaining foreign and domestic journals, theses and dissertations, and newspapers. The service in this section is open-shelf. All the theses and dissertations are cataloged and their abstracts are available on the library website. Moreover, the bibliographic information for the journals is both descriptively and analytically available on the computer and can be accessed through the Simorgh software. This section is connected to the Web and some of the journal subscriptions are made online.
Reparation and binding
This section is responsible for repairing and binding all the books which are torn. It is also in charge of binding all the journals, theses, dissertations, filed student scores, and newspapers. Previously all the binding was done manually, but currently advanced instruments with electric cutting and press, digital display and professional inlay are used to do the binding.
Address : Imam Khomeini Campus, Islamic Azad University, Farhikhtegan Bld., Shahid J'afari St., Kermanshah, Iran


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The Sting of Summer by Katherine Over
Genres: Young Adult, Contemporary Romance
[Goodreads | Amazon | Barnes & Noble | Kobo]
Summer has arrived. For seventeen-year-old Becca,
it marks the start of the wedding season. Her family
operates an event venue in the form of a renovated barn, and nearly every weekend a wedding takes place in their backyard. Becca lends a helping hand for the accelerated schedule, but work is not the only task on her mind. Seth, her longtime crush, has nabbed a photography internship that requires wedding coverage, putting the two of them in close proximity. Becca intends to make Seth something more than a far-reaching fantasy, but it's hard to build a dream when a growing tension in her family life cannot be avoided.
Will Becca succeed in her quest for love?
Or will summer collide with her plans and leave nothing but a sting in her heart?
Becca was just such a good main character. She fit the story and setting so well that I felt like I was truly there with her. She is a very relatable character, and I can definitely see readers sympathizing with her and becoming attached to her. I know I did. The love story is also really well done. I loved the chemistry between Becca and Nolan.
Such a cute book! Seriously, if you're wanting to experience summer you have to read The Sting of Summer. It's a great addition to any summer reading list. It's a quick read and one that readers of all ages can identify with and enjoy!
**I received this book for free in exchange for an honest review with no compensation.
2. If you could choose any TV show to promote your book on, which would you choose?
On the Netflix show Orange Is the New Black (I'm obsessed!), sometimes you'll catch one of the characters reading a novel. There isn't necessarily a verbal acknowledgment of the book. The advertisement is quite subtle. It would be so cool for The Sting of Summer to be promoted that way but maybe on a more age-appropriate show, like MTV's Awkward.
3. What's your dream summer vacation?
Absolutely no resorts! I'm terrible at lounging around a pool and relaxing. I'm happiest when I'm exploring and taking in new experiences. Spanish history/culture is fascinating to me. I would love to visit the country at some point, specifically the city of Seville.
4. How similar are your main character, Becca, and you?
Becca and I grew up with a very different family dynamic. Her specific encounters throughout the book are separate from my life, but at the core I think we share some similarities. I certainly know what it's like to become so involved in a fantasy that you forget to appreciate real life. In high school, I was a head in the clouds kind-of-girl. OK, I'm still that person.
5. Choose one character from The Sting of Summer that you get to go on a road trip with. Who, where, and why?
Without a doubt, I'd choose Nolan. He's traveled all over the United States so he'd make the perfect guide. Some of our stops would include the inspirations for his tattoos, such as the Ferris wheel at the Santa Monica pier.
6. Do you have any quirky writing habits or rituals that you stick to when writing?
When I feel like I've had a particularly good writing day, I'll try to recreate that exact situation the next time I write. The room will be the same, what I'm drinking will be the same, and what I'm listening to will be the same. The result is never, ever the same! The best ideas come from spontaneity. I've gotten better at breaking this habit of believing otherwise.
7. What's your favorite comfort food?
I love food. For all occasions. For all moods. Haha. However my go-to comfort food is usually some kind of baked good that I've made myself. I love to bake. The process is almost as comforting as the eating part. Almost. :)
8. Lastly, what's up next for you? Any projects we should be looking out for?
Currently I'm working on a New Adult Contemporary and a Young Adult Horror novel. I like to wait for a story's completion before discussing any details. It gives me the freedom to scrap the project if necessary. However, my fingers are crossed for happy outcomes, and I hope to have another book released before the end of the year. All updates for future works can be found on my website (www.katherineover.com).
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subset: commoncrawl


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I have to wake up early and get ready for school. And I pick up rubbish. I get dressed for school and me and my sisters tidy up the play room and I clean the room.
The horse has four long legs. And it is a brown horse. It has one white leg. It has long ears. It is so so cute. It is so soft.


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European Shorthair cat breed
The history of European shorthair cat breeds
European Shorthair breed was developed by the Swedish, Danish inorvezhskimi breeders in the first half of the XX century. The very first European Shorthair cat named Vastl von der Kolyung, he successfully performed at the Berlin feline show in 1932, after which the breed was officially prizanana. The cat was a silver-merle, and stressed its owner, great catch mice and rats. Generally breeders conclusions of the European short-haired cats and cats as a prominent Mouser and rat-catchers. The last recognized the breed of the United Kingdom, because of its similarity to the British Shorthair.
The Persian cat breed
The origin of the breed Persian cats
Homeland Persian cat breed, day-probability, Iran (formerly Persia). Italian traveler Pietro Della Valle has described these cats in his book "Journey to Turkey, Persia and India in the 54's letters", it was in 1620. From Persian cats were brought to Italy, from where they were first to France and then to Britain, where the breeding of this breed I do seriously. In 1887, the Persian cat breed was officially registered in the UK. Among the owners of Persian cats were even Queen Victoria and the Prince of Wales!
Siamese cat breed
History of Siamese cats
Siamese cat breed appeared vdrevnem state of Siam (now Thailand). Their ancestors - the wild Bengal cat. It is supposed that the Siamese cats were first introduced in the capital of Siam, city Ayuda. They lived in temples and royal houses, the people worshiped them and cared about them.
Siberian cat breed
The origin of Siberian cats
There are several theories about the origin of Siberian cats, nokakaya of them true history is silent. Perhaps during the colonization of Siberia, the first settlers brought cats to control mice and rats. In the future, brought cats interbred with wild forest cats, and as a result there was this rock.
Angora cat or a Turkish Angora
The origin of the Angora cats
Angora cats are known sXVI century AD. The name they received in honor of the Turkish town of Angora (modern Ankara). In the XVIII century Angora cat became popular in Europe, they can be found in many wealthy homes. In Russia they were after the Russian-Turkish wars and too popular with the nobility.
History of breed munchkin
Korotkolapye Munchkin cat-emerged by chance vrezultate spontaneous mutation. These mutations sometimes occur - for example, by worldwide periodically cats are born in one way or another nude. Same thing with korotkolapymi Mike Farley - the first news coverage that somewhere a normal cat kitten born with short legs were still in the 30's of last century.
History of breed cats Egyptian Mau
Egyptian mau- is the oldest breed of cats, its history goes back nearly 3,000 years. Age artwork, which depicts a cat that just about three millennia. There was this breed in ancient Egypt and since then has not changed much. The Egyptians worshiped the cat, they built temples in her honor, in a sign of mourning for the dead cat to its owner shaved eyebrows, the who accidentally He knocked to death a cat waiting for a terrible death (his stoned), the first cat was carried out of a burning house ...
The history of Canadian Sphynx
The first mention olysyh cats appeared in ancient times, the Aztecs. However, according to the description of hairless cats that time were very different from all modern sphinxes. Periodically hairless kittens were born in different parts of the world in the usual household and outdoor cats. Attempts to bring bald breed cats were several. In 1966, the domestic cat was born naked kitten, named Prune. Prune almost became the founder of a new breed, it even recognized, but in 1971 the status of breed It was withdrawn. She was too small in number to the same bald kittens are born weak, require special care and often died.
Bengal cat breed
The history of the Bengal breed cats
History of domestic Bengal cat began in 1961 in the United States. She created this breed American biologist, geneticist Jean Mill (Sugden her first husband). While on a business trip on the South-East Asia, Jean was in a natural habitat species Felis Bengalensis wild cats. Unfortunately, these beautiful cats were then on the verge of extinction (and they entered into the Red Book of the present day). The market in Bangkok Jin saw Bengal kittens. Of course Jin could not bear to look at the small unfortunate kittens in a basket from a dealer. She bought one and brought home to the United States. Kitten, who was a small cat, Gene called Malaysia. Since it began domestic breeds Bengal cats.
History of breed Don Sphynx
Don sfinks- is one of the few cat species, bred in our country. History of the breed began in the city of Rostov-on-Don. In February 1986, a native of the city of Krasnoyarsk Elena Kovaleva returns home and saw the boys mock kitten. Elena took away their kitten and took it home. Little cat named Varvara. Once the hair on the back of Barbara's beginning to come out, the treatment did not help - back cat remained bald. In 1988, the cat showed felinology, but it did not make any impression on them. In 1990, Barbara was brought to the European Shorthair cat Basil. It had three kittens, including a cat named Chita, who became the ancestor new breed - Don Sphynx. Excretion breed known felinologist engaged Irina Nemykina.


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Bangladesh Bank eases rice import rules to ensure supply
Bangladesh Bank has instructed banks to allow private traders to import rice without any deposit against letters of credit or LC margin.
The decision is meant to ensure rice supply to local markets in the aftermath of crop losses in recent flash-floods, the central bank said in a notice on Monday, reports bdnews24.com.
The new rules will remain in force until Dec 31, which will allow the traders to import rice first and make the payments afterwards. The LC margin varies from bank to bank depending on relations between bankers and customers.
In a letter to the chief executives of all banks, Bangladesh Bank said the loss of crops caused by recent floods and heavy rains in parts of the country have created some instability in the market.
Rice imports by private traders slumped 58 per cent year-on-year to 103,200 tonnes between July 1 and May 25 of the outgoing fiscal year.
In December 2015, the government increased customs duty on rice imports from 10 per cent to 25 per cent to discourage imports. But the total duty amounts to 28 per cent when 3.0 per cent regulatory duty is added.
As a result, private traders have been avoiding rice imports for the past one and a half years.
However, Bangladesh's stock of rice has also shrunk as 750,000 tonnes of rice were distributed following the loss of harvest in haor areas and as part of a project aiming to provide rice to the poor at Tk 10.
The government had some 193,190 tonnes of rice in stock until June 12, compared to 593,020 tonnes at the same time last year.
These have led the prices of rice to spiral out of control as wholesalers and millers continued the blame game.
The government initially planned to import 600,000 tonnes of rice to keep the market stable.
It has floated three tenders to buy 150,000 tonnes of rice and plans to import 250,000 tonnes of the grain from Vietnam under government-to-government deals.
The government hopes rice prices will ease once imports arrive in the market.
Government data, often lower than market prices, shows a 47 per cent rise in the price of coarse rice while a fine variety saw around 20 per cent rise in a year.
The fine variety is currently on sale at no less than Tk 60 per kg. Last three months were the worst period when people saw a steep rise in prices of rice of almost all varieties.
According to figures by the food ministry, the flash floods damaged Boro crops, which would have yielded around 600,000 tonnes of rice. Unofficial estimates put the number at 2.2 million tonnes.
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Former two-time Tory Party chairman Lord Parkinson has made a bad start to the year. First Flora Keays, his daughter from an 11-year affair with Sara Keays, appeared on TV with her mother to complain about his neglect of her. Now a long-time former business benefactor is in trouble. Ten years ago, Lord Parkinson agreed to become chairman of the offshore-based Dolphin Holdings, master company of a business empire built up by Kenyan Asian businessman Ketan Somaia. Dolphin was set up in the tax haven of Bermuda, but run from offices in Dubai. This arrangement meant there was no public disclosure of financial details, such as Dolphin's profits or Parkinson's pay. Somaia jetted between offices in Dubai and London, where he and Parkinson were to be found in an imposing Mayfair building round the corner from Grosvenor Square.
No longer. The key Dolphin company in Britain collapsed last November. The building is still occupied by the Delphis Bank of Mauritius, but Somaia is no longer chairman of that bank. Meanwhile, another Somaia-linked Delphis bank in Kenya has been closed by the regulators. This followed a run by nervous depositors prompted by news that Somaia had been made bankrupt in London by Indian entertainment tycoon Subash Chandra of Asia TV. Chandra bought Somaia's stake in the London-based satellite TV station in 1995. Somaia's bankruptcy was later annulled, but the Nairobi bank stays closed pending the injection of new capital. Dolphin companies have also been pursued in the courts in London and Kenya by creditors. The giant U.S. Starwood hotel chain is seeking to enforce a multimillion pound judgment in Kenya. And rival Kenyan Asian businessman Kamlesh Pattni, has been seeking to obtain control of Dolphin's Kenyan assets through the local courts, claiming he bought shares in the companies in 1992 and 1993. Somaia denies this.
Luckily for Parkinson, he is no longer on the Dolphin payroll. After eight years he parted company with Somaia in 2000—just before the storm began to break. But his longstanding role with Somaia and the way in which the former Cabinet minister gave him credibility will once again raise questions about his judgment.
Some 10 years ago, Somaia went to a Tory Party fund raiser—the Winter Ball. He was at a table with Pattni—the man now trying to wrest control of his Kenyan assets. At that time, the Tories were keen to stress their attractions to Britain's hard-working and often Conservative Asian community. Somaia knew at least one well-known Tory. He had met Mark Thatcher in Kenya. Thatcher was interested in motor-racing, and Somaia owned a number of motor dealerships.
By the end of 1992 Parkinson had agreed to become the Dolphin chairman. Somaia, a former timber merchant, also knew a lot about political influence. The rapid rise and rise of his Dolphin Group from the mid-'80s had been helped more than a little by his friends in the circle around Kenya's long-serving president, Daniel arap Moi. By the mid 1990s, with Parkinson as chairman, Somaia's empire stretched from India to the UK. It included Kenya's premier hotel chain as well as TV, radio, newspapers, and magazines aimed at the global Indian population. He snapped up the Kenya and Mauritius branches of the collapsed Bank of Credit & Commerce International. Parkinson visited Moi during a subsequent trip to Kenya.
Somaia had recording studios in London and a leisure project in Milton Keynes. Dolphin published the newspaper Asian Age in Britain. Parkinson hosted a party for the newspaper at the Café Royal in London in 1997. Somaia expanded into South Africa, acquiring a 25-year monopoly to develop three flagship game parks in the northern province of Mpumalanga in a secret deal which has spawned controversy and investigations. Parkinson visited South Africa in 1996 to promote the project.
By the late 1990s Somaia was no longer so welcome in Kenya. On four occasions he declined to appear before a Parliamentary committee investigating why money had been paid up front for security equipment which had not been supplied to the Kenya police. Somaia has always denied any wrongdoing over the Kenya contract or that he had to testify before the committee, and his lawyers have been busy contesting the claims by Pattni and others. But pressure on the Dolphin empire appears to be mounting.


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Written By: Terrence A. Merkerson
"The voice of intelligence is drowned out by the roar of fear. It is ignored by the voice of desire. It is contradicted by the voice of shame. It is biased by hate and extinguished by anger. Most of all it is silenced by ignorance."
-Karl A. Menninger
I was having a conversation with a colleague of mine (white guy) a few days ago and the topic of Colin Kaepernick came up. I wrote a piece about Kaepernick and the "Black American Paradox" and had this kind of dialogue before, but this was a little different. Now that yet another team in need of a quarterback has seemingly passed on signing Kaepernick (to sign what most consider to be an obvious inferior option), I was interested to hear what defense my colleague would take. Usually, when any conversation with a non-person of color takes a racially or culturally sensitive turn, my immediate impulses are to guard my blackness and prepare to police their whiteness, but because I know this guy (and probably more importantly, he knows I go in for this kinda shit) I decided to soften my guard a little so I can could more acutely feel the impact of his words. He began by clarifying that he agrees that Kaepernick is irrefutably better than 2/3s of the quarterbacks currently signed to NFL rosters (the setup before the sucker punch), but he does not believe Kaepernick is being blackballed. So I explained to him how the 'disruptive Black voice" has been and continues to be positioned in society and how this serves as another example of how institutional structures punish "disruptive" voices, particularly when that voice is one of a Black man or Black woman in America. While he understood and mostly agreed with my assertions, he refused to move on his position that Kaepernick is being deliberately and systematically blackballed. To quote this colleague, "It's just football, man. It's a game people watch for entertainment and I just don't want to hear that shit, bro. Sports is one of the few spaces in society that I don't have to make any critical considerations beyond the scope of the game. I just want to drink brews and watch the game." I replied by saying, "Well, that was a mighty White man thing to say there, bud." (This statement was accompanied by a standing ovation of slow claps and sided-eyes).
My first thought was to dive into my writing focused on the "shut-up and play" attitude directed toward Black athletes, by White folks and/or institutions controlled by White folks whenever a Black athlete expresses a consciousness, concern or thought beyond the catching, throwing, shooting, and EVEN KICKING of a ball if it in some way affects, affronts or disrupts the institutional model. But because we know that this is much deeper than dollars and cents, I knew there was something else here. So I thought back to the conversation that I had with my colleague and a few of his words still resonated with me…
"I just don't want to hear that shit…"
That is exactly what he said. As innocent of a statement as it may seem, even while shrouded in personal preference and subjectivity, it is just as dangerous as it is obviously dismissive. The question is WHY did he (and many other people) "just don't want to hear all that?"
It's because White folks are beginning to feel as though they are running out of "safe spaces". By "safe spaces", I am referring to the physical and non-physical spaces of refuge within American society that White people are not being constantly reminded of their Whiteness. This conception is built around the themes of cultural and racial blindness, invisibility and oblivion (example: I have a white friend that had no idea that Negro League Baseball was ever a thing… Like I had to prove it. Mind blown. SMH). It is no secret that a vast majority of White Americans do not think about race or their Whiteness unless they are directly confronted with it and for most of them, this makes them deathly uncomfortable. People of color don't mind discussing race because we are constantly confronted with it, but there are exceptions. Both Ray Lewis and Michael Vick spoke out "in support" of Kaepernick by suggesting that he, in one way or another, tone down the "Blackness". As two sports figures who have faced their own unique (and felonious) challenges, I'm sure that they meant well by offering their "Apologetically-Black" suggestions. But nobody is here for that "let's not make the White people uncomfortable" shit anymore. There is no growth in comfort and there is no progression in complacency.
So here we are with Kaepernick, in all his Blackness, making White Folks uncomfortable. It takes a certain level of "sociocultural maturity" (I think I may have just coined that phrase… I think that just happened. If I didn't, somebody let me know, but until then… yeah, that's all me, big dog.) to have the necessary discourse to expel that discomfort. Unfortunately, those who have the actual power and direct influence to move these kinds of needles are usually the most socioculturally inept. So they do what children and immature people do when they don't want to deal with something… they ignore it, in hopes that it will go away. The only difference is that institutional structures can MAKE things go away. Hence, the "blackball". So, we gotta do we always gotta should do… we can't let that shit slide. We can not compromise our equity and equality for the sake of White comfort. When they "just don't want to hear that shit", make them hear it. If they try to ignore you, don't let them. Remember, continuums create change. Comfort breeds complacency.
Miss me with that apologetic Blackness. I don't want to hear that shit.
Terrence A. Merkerson is the Founder & Creator of Avenue Fifteen. Terrence earned Bachelor's Degrees in both Political Science and Gender/Race Studies from the University of Alabama. Terrence also completed Graduate School at the University of Alabama, earning a Master's Degree in Communication and is currently pursuing a Master's Degree in Business Administration at Louisiana State University.
He currently resides in Charlotte, NC.
Social: Facebook @tamerkerson | Instagram: @tazmk


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Find Us
Directions to St Mary The Boltons
The Church is located in the middle of The Boltons, and can be reached by car, bus and underground.
By Car
If driving down Redcliffe Gardens, which is one way,turn into Tregunter Road and drive to the junction with The Boltons. The church will be on your left as you turn into the Boltons one way system. Otherwise drive down the Old Brompton Road until you reach the traffic lights at The Boltons. You should drive three quarters of the way round the one way system to park close to the front door and main entrance. On Sundays there is free/unrestricted parking in The Boltons and nearby streets. On most other days there is residents' parking and a few metered bays, mostly at the Old Brompton Road end of The Boltons, or at the junction with Tregunter Road. Be aware that in the football season when Chelsea FC have a home game the volume of traffic is greatly increased and some access may be restricted (usually on the Fulham Road if driving from Fulham Broadway). Parking is also more difficult on match days. The nearest covered (paying) car park is at the Chelsea and Westminster Hospital off the Fulham Road.
By Bus
The church is three minutes walk from bus stops on the Old Brompton Road,which are at Bolton Gardens/Brompton Library. (Buses C1 and430). Walk along the Old Brompton Road to the junction with The Boltons and walk down the right hand side, past the school, and you will see the church spire in the middle of the gardens.
It is about five minutes walk from bus stops on the Fulham Road, which are at Fulham Road/Beaufort Street (14, 414, 211, 345). Walk along the Fulham Road to Gilston Road and walk up Gilston Road to The Boltons. You will be able to see the church spire from the junction with the Fulham Road.
By Tube
The two nearest stations, both about 10 minutes walk to the church are at Earl's Court or Gloucester Road. Both are on the District and Piccadilly Lines, and Gloucester Road is also on the Circle Line.
From Earl's Court walk South down the Earl's Court Road to the Old Brompton Road Junction, turn left along the Old Brompton Road until you reach The Boltons, and turn right into The Boltons. The church will be ahead of you in the middle of the gardens.
From Gloucester Road, turn right down Gloucester Road, leaving the Cromwell Road behind you, and walk down to the junction with the Old Brompton Road. Turn right into the Old Brompton Road and walk about 300 yards to the junction with The Boltons. Turn left into The Boltons and the church will be straight ahead of you.


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On Dec. 10, Duke Divinity School held a community worship service of "Litany, Lament and Liberation" focusing on the recent tragedies in Ferguson, Mo., New York City, and others across the nation.
Eboni Marshall Turman, assistant research professor of black church studies and director of the Office of Black Church Studies at the school, presided, and Dean Richard Hays made brief remarks. Fatimah Salleh, M.Div. '16, preached at the service, which also included a recitation of the names of black people who were killed by police violence. A video of the service is available below.
After the service, the Divinity School hosted a panel discussion, "I Can't Breathe: Theological Responses to Anti-black Violence in 21st Century America," by Divinity School faculty members with responses from the community. Panelists were: Willie Jennings, associate professor of theology and black church studies; Valerie Cooper, associate professor of black church studies; J. Kameron Carter, associate professor of systematic theology and black church studies; and Marshall Turman. Mary Fulkerson, professor of theology, was unable to attend and sent remarks.


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Item Number: 10009420
Poster Title: Flower Festival: Feast of Santa Anita, 1931
Description: Diego Rivera art print of three small girls on their knees, with a woman bending over above them, carrying calla lily flowers on her back. There is a crowd of women in the background. This print is titled "Flower Festival: Feast of Santa Anita, 1931."
Size: 29" x 42" Artist: Diego Rivera
Price: $19.99 (subject to change)
Topic: Flowers Plants and Trees
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|The Eternal Fire lights our way.
| - Hierarch Hemmelfart, Gwent: The Witcher Card Game
Audio version:
Cyrus Engelkind Hemmelfart was the hierarch of Novigrad, a grizzled old man, noted racist and religious leader of the North. He was one of the negotiators of the peace treaty that concluded the second Nilfgaardian War.
The Witcher 3: Wild Hunt Edit
As a leader of the Church of the Eternal Fire, he is mentioned several times by secondary characters and Geralt can see him as one of spectators in the Whoreson Junior's arena but he does not offer a conversation.
Another opportunity to see him is during the meeting of witchers at Kaer Morhen keep, Geralt, Lambert and Eskel can get drunk and then they mistakenly contact the hierarch via Yennefer's megascope.
There is also a possibility to acquire a painting of him and give it to Dandelion or display it in Corvo Bianco.
Associated quests Edit
Trivia Edit
- His middle name Engelkind, means Angelchild in Dutch and German. Fitting for his position.
- His last name, Hemmelfart, is an allusion to the German word Himmelfahrt and the Dutch word Hemelvaart, meaning Ascension in the Christian sense.
- When he is contacted via megascope (described above), it is implied that he is using the toilet at the time. Why he would be in possession of a megascope is never addressed.
- His portrait bears a striking resemblance to John Paul II, a pope and saint of Roman Catholic Church.


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The Daily Guide of last Tuesday published a most intriguing news item, concerning students of a secondary school going on a demonstration about their headmaster. Intriguing because it was a different kind of protest.
This time, unlike what we usually read or hear about, these students were NOT demonstrating for the removal of their headmaster, or against staffing problems, or even against the poor quality of their school meals.
Wonder of wonders, the students were demonstrating IN SUPPORT of their headmaster! They were in effect lobbying on his behalf! The demo was to back their plea for the withdrawal of a transfer directive to the headmaster, Mr Christian Addai-Poku! A rare occurrence!
And for once, there was no unruly conduct, no damage to school property.
According to the report by Guide Kumasi reporter, Ernest Kofi Adu, under the headline, "Students Protest Headmaster's Transfer":
Ghana News Headlines
For latest news in Ghana, visit Graphic Online news headlines page Ghana news page
Students of Oppon Memorial Senior High School (OMESS) at Kokofu in the Bekwai Municipality of the Ashanti Region yesterday held a peaceful demonstration over the transfer of the headmaster, Christian Addai-Poku.
The agitated students requested the regional directorate of the Ghana Education Service (GES) to reverse the decision as they poured on the streets holding placards waving and chanting slogans against the move.
The students, who (marched) some kilometres in the Kokofu town before presenting a petition to the paramount chief, said they would boycott studies until Mr. Addai-Poku's transfer was revoked.
Addai-Poku, former President of the National Association of Graduate Teachers (NAGRAT), was said to have been moved to another school barely six months after assuming duty at OMESS.
According to the student body led (by) Panford Doris Afriyie, the transfer of the headmaster has led to confusion, bad blood and tension in the school with agitations from various quarters against the move.
She stated that the students could not afford to let Mr. Addai-Poku (go) as they had observed, under his leadership and guidance, tremendous improvement in the school.
"Within six months, he has provided biogas and siren for the school; he has brought discipline and water closet toilet facility. Besides, he has the intention of opening up the dining hall and making sure we have a bus," senior school prefect, Hosea Amankwah, said.
He revealed that academic work of the school had also seen improvement… As a result, the school was able to take the second spot in the science quiz for the first time in the history of OMESS.
Amankwa said they would resist the transfer of Mr. Christian Addai-Poku. And he wanted (the) authorities to reconsider the decision because they would suffer its consequences."
Ms Afriyie, who is the Students Representative Council President, added: "We understand that our headmaster went to report to his new station. We have been waiting for the GES officers to address this issue."(Daily Guide, June 26, 2018).
I find it gratifying that even though Mr. Addai-Poku had been at OMESS for only a short time, he had reportedly chalked up the listed impressive accomplishments.
In my view, of particular interest are the provision of water closet washrooms, as indicated, and the high performance of the school in a national science competition. All that within six months?! No wonder the OMESS students are fighting to have his alleged transfer cancelled!
But if it is true that Mr. Addai-Poku has been transferred after only six months of heading that school, what could the reason be? Was it that he himself had asked for the transfer? Or was it that he was assigned to the OMESS as a 'temporary head' – assuming there is such a designation?
Why would a headmaster who is reportedly doing so well, even by the usually extremely critical assessment of students, be moved?
One wonders what the compelling circumstances were which would necessitate a school head being moved to another institution after only six months. The experience in this country is that even in cases where an incumbent has been found grossly incompetent and there is agitation for their removal, it takes a very, long time before the transfer process even commences.
If Mr. Addai-Poku himself did not ask to leave OMESS, conceivably apart from the likely disruption to the school's programme, what about the effect of such a development on the Addai-Poku family? Presumably, six months is just about the period when his family would have settled down at OMESS and now they have to relocate again.
I don't know what the GES rules say about transferring heads of schools, but a six-month appointment doesn't sound right or fair to all concerned.
Anyway, there seems to be a huge communication gap between the GES, the school management and the students. No matter the circumstances, the students deserved to have been given a full explanation before the transfer. SHS students are certainly not kindergarten pupils to be treated in this way.
Whatever the rules are, if they have no stipulations about the importance of school managements COMMUNICATING WITH THE STUDENTS, it is a serious omission.
The OMESS student leaders seem to be extremely level-headed, and the students have behaved in a very mature manner with their grievance, protesting in a non-violent manner, and presenting a petition to their Paramount Chief.
It is hoped that the Regional Directorate of the GES will meet with the OMESS students as soon as possible to have a dialogue with them, if they haven't done so already. If the students are given the impression that their concerns don't matter to the GES, their protest may take another, undesirable form – as they have subtly hinted.
The objective of such a meeting should be: (a) either the GES is able to convince the OMESS students that it was necessary for their headmaster to be transferred after only six months, or, (b) the students convince the GES Directorate to accept their position and Mr. Addai-Poku stays at OMESS.
And, in my opinion, the OMESS students need to be commended by Education Minister Mr. Matthew Opoku Prempeh and the GES leadership for their exemplary conduct.
I also think that students in other institutions need to hear about it. They need to know also that confrontation and destruction of property is not the way to solve problems; that dialogue and peaceful measures can work perfectly well.


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Digital Power List
IN celebration of Online Fashion Week, VOGUE.COM has teamed up with the Online Fashion 100 to exclusively unveil the 2012 list - which documents the most influential names and faces from the digital fashion world and compiled by Leon Bailey Green
"Welcome to the third edition of Online Fashion 100," says its creator Leon Bailey-Green. "Next year is set to be another year of greatness in online fashion. This edition of the list is a narrative of connections, both strong and loose, covering important industry developments over the past few years. Through a string of ties, bonds, associations and things in common, you'll meet the headline-makers as well as the powers behind-the-scenes."
As well as the annual 100, for the first time this year the public were given their chance to vote on who they thought should be honoured - from categories such as Top Directors to Top Bloggers to Best Dressed to Outstanding Contribution. The full list of results is as follows:
Top director:
1. Stephanie Phair, THEOUTNET.COM(42 per cent) 2. Hash Ladha, Aurora (32 per cent) 3. David Worby, HARRODS.COM (10 per cent)
Top blogger:
1. Cillian O'Connor, Male Mode (44 per cent) 2. Queen Michelle, Kingdom of Style (25 per cent) 3. Mrs D, Fashionable Maven (15 per cent)
Top entrepreneur:
1. Jose Neves, FARFETCH.COM (55 per cent) 2. Julia Fowler and Geoff Watts, EDITD (18 per cent) 3. Warren Bennett and David Hathiramani, A Suit That Fits (15 per cent)
Top international store:
1. Oskar Hartmann, KupiVip (93 per cent) 2. Jeff Bezos, Amazon (3 per cent) 3. Robert Gold, Beyond The Rack (2 per cent)
Top family unit: 1. Baukjen de Swaan Arons and Geoff von Sonsbeeck, Isabella Oliver (47 per cent) 2. Joe and Monica Wahla, Monica and Joe (23 per cent) 3. The Harriman Smiths, CHILDRENSSALON.COM (18 per cent)
Best dressed female: Malika Dalamal, DailyCandy London (34 per cent)
Best dressed male: Daniel Richmond, Tic Watches (36 per cent)
Outstanding contribution: Nick Robertson, ASOS (30 per cent)
To request an e-book copy of the list click HERE.
YOU SHOULD SEE: The latest OFW action...
YOU SHOULD SEE: All the highlights from OFW...


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Have you ever found with particular books that fate has decided you either have to read it or own it? There's been a couple of books like this for me where I have maybe spotted it in a book store, or online and thought it sounded interested but then brushed it aside. It seems then that these particular books will pop up everywhere. I'll walk into other book stores and it will be there on the shelf beconing to me, or it will continuously pop up Amazon as a recommended book (this was the case with Emma Donoghue's book 'Room' which was fantastic). This happened to me with a cookbook about a year or so ago. Whenever I walked into a bookshop there it was - the beautiful cover of What to Cook and How to Cook it by Jane Hornby. So eventually I had to have it! It's been on display on my bookshelf since then - after the initial flicking through it - I haven't made anything from it.
I recently opened it up, and noticed a recipe for Butterscotch Banana Bread and I just so happened to have 3 bananas sitting in my kitchen that needed to be used. I usually don't cook a lot with bananas because of the gross factor, but I thought I could get through it due to the appealing adddition of butterscotch in the recipe name. Unfortunately though, at the time I didn't have any cream cheese so I couldn't make the caramel icing/frosting that usually goes with this bread. Still, I'm glad that I gave it a go - it tasted wonderful.
Butterscotch Banana Bread:
3 medium, very ripe bananas
175g softened unsalted butter, plus extra for greasing
175g brown sugar
1/2 tsp sea salt
3 medium eggs
1 tsp vanilla extract
100g plain flour
120g plain wholemeal flour
2 tsp baking powder
50g chopped walnuts, plus extra to decorate if you like
100g cream cheese
1. Rub a little butter over the inside of a 900g loaf tin, then line it with a strip of baking paper, leaving some overhang at either end. Preheat oven to 160 C
2. Peel the bananas and put them in a bowl. Use a fork to mash the fruit to a lumpy pulp.
3. Put the bananas, 120g of the butter, 120g of the sugar, the salt, eggs, vanilla, flours and baking powder into a large bowl, then use electric mixer to beat the ingredients until smooth.
4. Stir in the walnuts, then turn the mixture into the prepared tin.
5. Bake the loaf for 1 hour and 10 mins, or until risen, golden and springy to the touch. Test if the cake is done by inserting a skewer into the middle of the thickest part. If it comes out clean, the cake is ready. If there are traces of batter on the skewer, bake for 10 minutes longer, then check again. Cool in the tin for 10 minutes, then transfer the loaf to a cooling rack and leave to cool completely.
6. While it cooks, make the icing. Put the remaining batter and sugar into a pan with 1 tbsp of water. Put it over a very gentle heat, then wait until the sugar has dissolved. Let the mixture simmer for 3 minutes, until it looks like a silky caramel. Remove from the heat and leave to cool.
7. Put the cream cheese into a bowl, then add the caramel. Beat or whisk the two together to make a smooth, coffee-coloured icing.
8. Spread the icing over the cake, then scatter with a few more nuts to decorate if you like.
Recipe Source:
What to Cook and how to Cook it.
Jane Hornby
Recipe Source:
What to Cook and how to Cook it.
Jane Hornby


text:
A performance super thin and lightweight multi-use waterproof ankle sock. Created for conditions where minimal insulation, maximum waterproof performance, high breathability and moisture wicking properties are required.
- 100% Waterproof, Windproof & Breathable
- 33% Lighter than our "thin" waterproof socks
- Reduced water ingress at cuff with our silicon free Hydrostop band, providing a more secure fit
- Moisture Control, natural cooling & anti-microbial properties
- Natural eco-friendly bamboo lining offering next to skin comfort
- Temperature regulating & breathability–Hollow fibre liner construction and performance membrane
- Additional ankle support–Elasticated ankle and instep
- Blister prevention & moisture wicking–Super soft lining
- Odourless feet–Excellent anti-microbial properties prevent bacteria from thriving
- Hydrostop Usage Guide–SealSkinz Hydrostop technology creates a seal between the sock cuff and skin, helping to reduce the amount of water from running down the leg and into the sock. Due to this close bond between sock cuff and skin, we recommend that Hydrostop socks are only used outdoors and not for a prolonged period of time. Incorrect or prolonged use can result in excessive rubbing against the skin and may in a very small minority of cases cause minor skin irritation. The Hydrostop seal material has been fully tested to EN ISO 10993-5: 2009 and is OEKOTEX compliant.
Sealskinz make a range of waterproof hats, gloves and socks that battle the elements to help keep you dry, warm and active for longer. Designed to perform and protect, every Sealskinz product builds on the 20 years' experience that Sealskinz have battling extreme weather conditions.
In severe weather, our extremities are the first to suffer from the cold. By developing their own waterproof and breathable membrane, Sealskinz have brought you the perfect ally in the fight against the elements. From your fingers to your feet, ears to your toes, Sealskinz will make you more comfortable outdoors.
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Head Shots
Your headshots are the first chance people have to get to know you.
What are your photos saying?
Regardless of your industry, people want to get to know you...
Photographs are the first opportunity people have to get to know you. A professional headshot can communicate a number of key messages to your audience. Are you a confident, friendly realtor, or a tough edgy musician. Having a photographer that understands your brand and can communicate it through image is key! Karma's photography team will work with you to plan out your photo shoot and ensure your headshots send the right message...whatever that may be.
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We help you to make the visitors do what you want them to on your site, our team is ready to help optimize your site and boost the conversion rates, providing a successful a long-term effect CRO strategy for your business.
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Work for your money while making your money work for you! Invest your marketing budget into our acclaimed CRO strategy, developed by our professional team, and get an immediate return on your investment.


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|Android Developer – Flex time
|To be determined
|Norfolk, VA or Danville, VA
|Job Information
This is a flex time position requiring a minimum of 20 hours to a maximum 40 hours per week. In other words, you set your own schedule as long as each sprint is completed in a timely manner. Telecommuting is also available to those qualified candidates.
The current opening for a Android Designer/Developer is specifically for an employee working in or around Norfolk, VA or Danville, VA area. We are open to remote positions and will accept resumes for potential future openings.
Netarus, LLC. is looking for Android designer/developers with knowledge of and experience related to some or all of the following:
The successful candidate will have expertise in some or all of the above, and will be capable of working in a small-company environment with minimal supervision.
|Apply Now
Job: Android Developer – Flex time
All applications must include a resume. Thank you for your interest in Netarus.


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Courtney & Jason travelled to New Zealand to celebrate their 9 year wedding anniversary. At the end of their trip they had a photo session. The weather was less than ideal, but we were all happy to head out, get a little wet. We had some light rain on The Ledge, which added to the drama, before finishing in Queenstown Gardens as the rain came down heavier. Nice work rocking the wet weather!
If you would love to stay up to date with more of our work, like our Facebook Page or follow us on Instagram – @williamsphotography_nz


text:
Post by: Patollo(52903)
2005-05-07 10:29:58
|I am currently a donater to this fantastic game and plan to do so for some time, but in my time in the game have noticed a significant difference to tech wrestlers (Inferno being my first character) and Aerialists (Manic Diver being my second) as a tech, i'm still to win a championship as Inferno and he is border-line SSWO, and my second wreslter, Manic Diver is current SPWA Rage in a Cage champ. As a donater I find it strange that my lower ranked wrestler is able to have much more fame than my main. I don't know if this is same for others or that only pin and submission type matches are favouring tech types while the rest of the classes are favouring the others. I love the game and will stay, but if I become a legend and knowing what I do at the moment, Aerialist is definately the fave for my next class!
Post by: BubbaG(33460)
2005-05-07 12:07:07
|I guess everyone has their own preferences. I had the most success with my Stiff-Neck but my new Tech is kicking some serious Butt. We'll see and I'll still got Trad to go.
Post by: Crosson(37742)
2005-05-07 12:09:35
each type has a strategy of their own. Some types match up well against other types and some bonuses are better than others, although others might say differently.
As to your issues, it could be that you are basically trying to use the same strategy for both wrestlers, which will likely not work.
Post by: The Legend Killer(21778)
2005-05-07 16:28:53
It also depends on the people that are in the tourneys aswell.
LK before retirement only won the SSWO Trad tourney (4 times), after retirement (as an aerialist), he has already held 3 titles and won another tourney.
Same goes with my alts who have won various tourneys that LK hasn't won.


text:
Visit the front page for rants, reviews, and stories about Nacho's Hammer
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Warning: topic is currently/will be locked!Only admins and moderators can reply.
shortcuts: hit alt+s to submit/post or alt+p to preview
Um...shall we organize a movie date guys?
Five years on my brain not being scarred by the knowledge of it's existence.
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Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin‐like phospholipase A. In all surveyed inducer lines, NLD carries a 4‐bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild‐type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild‐type NLD protein. In conclusion, an intact pollen‐specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.
See also: D Jackson (March 2017)
The function of the patatin‐like phospholipase A NOT LIKE DAD (NLD) in the sperm cells of maize pollen is necessary for successful fertilization, whereas its disruption promotes the development of haploid embryos, which represent an important plant breeding tool.
Fine mapping restricted a major QTL (quantitative trait locus) responsible for haploid induction in maize to an interval containing a single gene coding for NLD.
A 4‐bp insertion in NLD leading to a predicted truncated protein is responsible for haploid induction.
Expression of NLD is restricted to sperm cells.
Wild‐type NLD is anchored to the plasma membrane, whereas the truncated protein present in inducer lines loses its membrane attachment.
Mutation of NLD orthologs may allow to establish doubled haploid breeding techniques in crop species lacking such tools.
The mixing of male and female genetic information during sexual reproduction is considered as key to the evolutionary success of higher eukaryotes and is the basis of plant breeding. At the same time, asexual reproduction occurs in nature and is exploited in breeding to fix interesting genetic combinations. One form is gynogenesis, in which an unfertilized egg cell develops into a haploid embryo upon stimulation by a male gamete. After spontaneous or artificial chromosome doubling, diploid plants can be obtained, which are completely homozygous and reflect a fixed state of the mother plant. Such doubled haploids are routinely used in plant breeding to simplify logistics and reduce time to market. Consequently, the elucidation of the molecular mechanisms triggering gynogenesis represents a major scientific and economic interest (Geiger & Gordillo, 2009).
In maize, spontaneous gynogenesis is very rare with frequencies in the 0.1% range (Chase, 1949). Beyond genetics, stress treatments such as strong heat shock (Randolph, 1932), X‐radiation, or UV light (Mathur et al, 1980) have been reported to influence haploid induction rates in maize (Chase, 1949). Much higher frequencies suitable for hybrid breeding are achieved by pollination with the so‐called inducer lines, which all seem to have stock 6 (Coe, 1959) as common ancestor (Weber, 2014; Hu et al, 2016). The haploid induction rate is determined mainly by the genetic constitution of the inducer line but also by that of the female line receiving inducer pollen (Chase, 1969). Eight paternal quantitative trait loci (QTL) were uncovered in inducer lines (Barret et al, 2008; Prigge et al, 2012) and two maternal QTLs in receiving females (Wu et al, 2014). Among the paternal QTLs, the one located on chromosome 1 and called successively gynogenesis inducer1 (ggi1) (Barret et al, 2008) and quantitative haploid induction rate 1 (qhir1) (Prigge et al, 2012) has by far the strongest effect explaining in certain crosses up to 66% of the genotypic variance for haploid induction. The ggi1/qhir1 locus is supposed to be mandatory for haploid induction ability, and has been associated with poor transmission of inducer pollen leading to segregation distortion (Barret et al, 2008; Prigge et al, 2012). The ggi1/qhir1 locus was fine‐mapped to a region of 243 kb on chromosome 1 (Dong et al, 2013). A second important QTL, named qhir8, was found to explain 20% of the genotypic variance and to be important for enhancement of the inducing capacity of ggi1/qhir1. The qhir8 locus was fine‐mapped to a region of 789 kb on chromosome 9 (Liu et al, 2015).
It has been challenging to elucidate the mechanisms underlying gynogenesis induced by stock 6 and its derivatives for two reasons: Firstly, the partial nature of the induction makes it difficult to predict for a given pollen tube/embryo sac interaction, whether it will result in gynogenesis or not. Secondly, comparative studies between inducer and non‐inducer lines are hampered by the heterogeneity of the genetic background, since no near‐isogenic material is available due to the polygenic nature of the induction capacity. Consequently, it is not surprising that some of the published results remain contradictory. In inducer lines, all steps up to the development of the trinucleate pollen are normal, since there are no biologically relevant differences in the number of nuclei between inducer and control lines, neither in mature pollen nor in germinated pollen tubes (Mahendru & Sarkar, 2000). First differences appear during pollen tube germination since pollen from non‐inducer lines germinates faster than pollen from inducer lines and outcompetes pollen from inducer lines in successive pollination experiments (Xu et al, 2013). At the level of the embryo sac, a cytological study detected remnant sperm cell nuclei after pollination with inducer pollen but not with wild‐type pollen (Swapna & Sarkar, 2012) and molecular studies even revealed remnants of paternal genetic material in haploid embryos (Zhao et al, 2013). Finally, the presence of haploid embryos on ears pollinated by an inducer line is clearly correlated with a general decrease in kernel number and an increase in the number of defective kernels (Xu et al, 2013).
Phospholipases (PLs) are enzymes that cleave phospholipids, the major component of all cellular membranes. The hydrolysis site within the phospholipid molecule defines four PL types. Whereas PLAs and PLBs remove hydrophobic fatty acid tails from the glycerol backbone, PLCs and PLDs remove either the hydrophilic phosphate head or side chains attached to it (Wang, 2001; Park et al, 2012; Vines & Bill, 2015). PLs have broad and diverse roles, and it is now clear that they have both structural and signaling functions: PL enzymes take part in lipid metabolism and membrane remodeling but also generate lipid‐derived messengers involved in membrane associated lipid signaling pathways that regulate multiple cellular processes (Wang, 2001; Park et al, 2012). Within the PL family, patatin‐like PLs represent a group of proteins found in diverse organisms (bacteria, yeast, plant, and animal) that share high similarity to the catalytic domain of patatin, an abundant protein of the potato tuber (Scherer et al, 2010). This catalytic center consists of the esterase box GXSXG and the phosphate‐ or anion‐binding element DGGGXXG. Patatin‐like PLs preferentially hydrolyze fatty acids in the sn‐2 position (Banerji & Flieger, 2004; Scherer et al, 2010).
Pollen development is one of the processes for which PL activity is essential. In Arabidopsis, three PLA2 genes were found to be expressed in mature pollen, and knockdown of their expression by RNAi results in pollen lethality (Kim et al, 2011). On the subcellular level, these three PLA2 enzymes located to the endoplasmic reticulum and/or Golgi (Kim et al, 2011). In addition, mutants in the chloroplast‐localized PLA DAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) are impaired in anther dehiscence and show defects in pollen grain maturation (Ishiguro et al, 2001). Earlier aberrations in pollen development as well as defects in the female gametophyte were observed in the plc2 mutant of Arabidopsis (Li et al, 2015).
This work presents the positional cloning of the ggi1/qhir1 QTL and provides evidence that a mutation in the patatin‐like phospholipase A NOT LIKE DAD (NLD) is the key factor triggering gynogenesis in maize after pollination by inducer lines.
Fine mapping of the ggi1/qhir1 locus
The starting points for the fine mapping of the ggi1/qhir1 locus were the creation of 96 highly recombinant inbred lines (F2i3S2) and 18 single‐seed descendants (selected for induction over seven generations) of a cross between the inducer line PK6 and the inbred line DH99 (Barret et al, 2008). Based on haploid induction tests and mapping with 10 markers in the umc1144/bnlg1811 interval (Fig 1A), the two inducer lines with the shortest PK6 genome region were backcrossed to the recurrent DH99 parent. In parallel, the use of additional markers narrowed the confidence interval down to the region between markers SYN24144 and PZE‐101081844 (Fig 1A and Appendix Table S1). After selfing plants derived from the backcrosses heterozygous at the locus, 10,275 plantlets were screened for new recombination with markers GRMZM2G100497_10, GRMZM2G152877_6, and SYN35770 (Fig 1A and Appendix Table S1) located outside of the redefined QTL interval but inside the original breakpoints; 531 recombinant plants were obtained.
The phenotypic characterization of the new recombinants exploited the strong correlation between haploid induction rate and segregation distortion (Barret et al, 2008; Prigge et al, 2012; Xu et al, 2013). For a subset of 48 recombinants with breakpoints in the redefined confidence interval, the segregation distortion was determined by genotyping for each recombinant a small population obtained by self‐pollination with 16 markers. The strongest segregation distortion was observed with marker GRMZM2G471240_1 (Fig 1B and Appendix Table S1).
Among the 48 families, 28 contained plants homozygous for the recombinant allele. This subset was used as pollen parents to assess their haploid induction rate in test crosses (Fig 1D). The results indicated that the locus responsible for haploid induction was located between markers GRMZM2G471240_13 (breakpoint of the recombinant named 71‐19‐29) and 155K10_55 (breakpoint of recombinants named 71‐39‐162 and 71‐29‐69) in a 166‐kb interval on the B73 reference genome (Fig 1D and Appendix Table S1).
Structure of the confidence interval in the PK6 genome
The occurrence of 30 consecutive dominant PAV (Presence–Absence Variant) markers (absence in PK6 genome) between positions 68.24 Mbp and 68.36 Mbp of the B73 reference genome was a strong indication for important structural differences between the two genomes (Appendix Table S1). Consequently, it was necessary to determine the DNA sequence of the corresponding genomic interval in the genotype PK6 to assess the number and type of genes predicted at the gim1/qhir1 locus. Thus, a BAC library was created from PK6 gDNA. The alignment of the consensus sequence of five overlapping BAC clones (GenBank accession KX852318) with the B73 reference genome revealed the replacement of a 156‐kb region of B73 by an unrelated sequence of 70 kb in PK6, explaining the absence of additional recombination between the breakpoints defining the 166‐kb interval in B73, which was reduced to 80 kb in PK6 (Fig 1C). It also revealed the absence of genes GRMZM2G062320 and GRMZM2G062313 in the PK6 sequence. In addition, several other insertions or replacements in the PK6 genome existed in the proximity of the gim1/qhir1 locus (Fig 1C).
Ab initio analysis predicted 13 genes in the 80‐kb interval of genotype PK6 (Appendix Table S2). Ten of them carried annotations related to retrotransposon families with multiple copies across the genome. Two others genes, FGENESH_18 and FGENESH_19, had more than 10,000 and 182 copies in the B73 reference genome, respectively. The only single copy gene was FGENESH_8 (GRMZM2G471240) annotated as patatin‐like phospholipase, which was the only functional annotation not related to retransposons among the 13 genes. In comparison with B73 and DH99, the PK6 allele carried a 4‐bp insertion in exon 4 corresponding to marker GRMZM2G471240_14. A survey of this marker in a genetic diversity panel of 92 lines and a collection of 12 haploid inducer lines (including PK6, the widely used RWS and the initial stock 6) detected the PK6 insertion in all inducer lines but in none of the lines of the diversity panel, whereas other polymorphisms of GRMZM2G471240 were not restricted to inducer lines (Appendix Table S3). Thus, the 4‐bp insertion represented not only a discriminant marker for haploid induction but also its possible cause. Reinforcing this idea, lines possessing the same haplotype as PK6 in the corresponding interval and differing only by the 4‐bp insertion (EM1201, Nys302, Mo1W, and Tx303) were not able to induce haploids (Appendix Table S3). This result ruled out a direct role of the two candidates, GRMZM2G062320 and GRMZM2G062313, in haploid induction. The sum of the mapping and sequencing data identified GRMZM2G471240 as the only low copy gene candidate in the PK6 80‐kb confidence interval for haploid induction. This candidate gene was named NOT LIKE DAD (NLD) because the PK6 allele induces maternal embryos without paternal contribution. Parallel independent studies named the same gene MATRILINEAL (MTL) and Phospholipase A1 (ZmPLA1) (Kelliher et al, 2017; Liu et al, 2017).
NLD codes for a patatin‐like phospholipase expressed in pollen
The analysis of the deduced amino acid sequence of NLD was based on transcript variant GRMZM2G471240_T01 (Appendix Fig S1), in which the second of the four exons was larger than in variant GRMZM2G471240_T02. A single RT–PCR band of the size expected for variant T01 indicated that this variant was the main transcript in maize. The deduced NLD amino acid sequence contained a patatin/phospholipase domain (IPR002641, Fig 1E), and phylogenetic analysis of maize and Arabidopsis patatin‐like PLA placed NLD among class II patatin‐like PLA (Appendix Fig S2). The 4‐bp insertion in NLD exon 4 found in haploid inducer lines was also found in cDNA. This 4‐bp insertion causes a frameshift that replaces the last 49 amino acids of the wild‐type protein by an unrelated amino acid sequence of 20 aa followed by a premature STOP codon (Fig 1E and Appendix Fig S3).
NOT LIKE DAD expression in the maize plant was restricted to the mature stage of the male reproductive organ (tassel) as revealed by gene‐specific qRT–PCR experiments (Fig 2A), in agreement with public microarray data (Appendix Fig S1). Compared to the non‐inducer line DH99, the inducer PK6 line showed slight but significant increase of NLD expression in mature tassel (Fig 2B). In light of the high and specific expression of NLD in mature tassel, it seemed interesting to increase the spatial resolution and to distinguish between sporophytic (anther) and gametophytic (pollen) expression. A 2.6‐kb upstream fragment from genotype B73 was transcriptionally fused to the GUS reporter gene (pNLD::GUS) and used for maize transformation. GUS staining was found in pollen grains whereas it was absent in anther tissues (Fig 2C). In a time course experiment during pollen development, the onset of NLD promoter activity coincided with the second pollen division, which leads to tricellular pollen with sperm cells (Fig 2D and E), specifying an expression of NLD in mature pollen. After pollination of wild‐type plants with transgenic pNLD::GUS pollen, observation of germinating pollen, silks, and ovules 17 h after pollination (HAP) showed GUS staining in pollen tube and at the interface of pollen tube and ovule (Fig 2F). Some GUS staining was still observed at 48 HAP, but disappeared at 72 HAP (Fig 2F). Even if the important half‐life time of the GUS protein makes precise statements impossible, these results suggest that the NLD promoter is active during the entire fertilization process.
Wild‐type NLD abolishes the haploid induction phenotype of the inducer line
To test the hypothesis that the 4‐bp insertion in NLD was responsible for haploid induction, complementation of the mutation was carried out in an A188 genetic background. This choice was motivated by the fact that A188 represents one of the rare maize inbred lines that can be readily transformed. In a first step, the PK6 NLD locus was introgressed over four generations into A188 to create a near‐isogenic inducer line (A188NLD–PK6). The haploid induction rate of the successive backcrosses dropped from 3.59% in BC0S1 to 0.50% in BC3S1 (Table 1), confirming the need of other favorable loci for enhanced haploid induction capacity (Prigge et al, 2012; Liu et al, 2015). This rather low induction rate remained significant because A188 was a non‐inducer line with an extremely low spontaneous haploid induction rate (0 haploids out of 4,487 seedlings tested, Table 1). In parallel, A188 was transformed with a genomic version of NLD from the non‐inducer line DH99 under the control of the constitutive rice Actin promoter. The resulting NLDDH99‐OE line, which showed a ninefold increase in NLD transcript accumulation in pollen compared to A188 wild type (Appendix Fig S4), was crossed with A188NLD–PK6. No haploid induction was observed with plants homozygous for both the NLD‐PK6 mutation and the NLDDH99‐OE T‐DNA cassette (Table 1), demonstrating that the presence of wild‐type NLD abolished the A188NLD–PK6 inducer capacity. These data led to the conclusion that the PK6 allele of NLD was responsible for maternal haploid induction at the ggi1/qhir1 locus.
Truncation of NLD protein leads to loss of plasma membrane localization
To investigate the mechanism by which the truncation of NLD interfered with double fertilization, the subcellular localization of both the full‐length wild‐type NLD and the truncated NLD‐PK6 was examined. C‐terminal fusions with CITRINE fluorescent protein were introduced under the control of the constitutive UBI10 promoter in Arabidopsis (Fig 3A–D). Confocal imaging of roots showed a clear plasma membrane localization together with localization in cytoplasm and in endomembrane compartments for NLD::CITRINE (Fig 3A and B). In contrast, NLD‐PK6::CITRINE signal was absent in the plasma membrane and observed exclusively in cytoplasm and to a lesser extent in endomembrane compartments (Fig 3C and D). This result suggested that the truncated NLD‐PK6 was mis‐localized and its absence in the plasma membrane was probably the cause for haploid induction underlying the gim1/qhir1 locus in the PK6 inducer line.
To address the subcellular expression pattern in maize pollen, both wild‐type NLD and truncated NLD‐PK6 were expressed under the NLD promoter. Confocal imaging of mature pollen and germinating pollen tubes showed a clear fluorescence signal in sperm cells for full‐length wild‐type protein, whereas only background auto‐fluorescent signal was observed for the truncated protein (Fig 3E–H and Appendix Fig S5). The rather small size and the high nucleus/cytoplasm ratio of sperm cells hampered subcellular observation with conventional confocal microscopy. The use of structured illumination microscopy (SIM) allowed to localize the wild‐type NLD::citrine signal to what appears to be the plasma membrane of the sperm cells, confirming the results observed in Arabidopsis roots (Fig 3I and Appendix Fig S5D). An in silico analysis of the wild‐type NLD protein sequence predicted the absence of a signal peptide but the presence of an S‐palmitoylation site at C10 and an S‐palmitoylation or S‐farnesylation site at C423 (Xie et al, 2016) (Appendix Fig S6). The presence of two lipid anchor sites was coherent with a localization in the plasma membrane and the loss of the C‐terminal lipid anchor in the truncated NLD‐PK6 protein provided a plausible explanation of its mis‐localization.
Structural rearrangements at the gim1/qhir1 locus between inducer lines and the reference genome
Fine mapping of the gim1/qhir1 locus identified NLD as the only low copy gene with a functional annotation not related to retrotransposons in the 80‐kb confidence interval of the inducer line PK6. The candidate genes GRMZM2G062320 and GRMZM2G062313 present in the B73 reference genome, but absent in PK6 interval, can be ruled out because lines with similar haplotype missing these two genes are not able to induce haploids. This makes NLD a prime candidate to be responsible for both haploid induction and segregation distortion. More precisely, the left border of the confidence interval is determined by two recombinants within NLD, reducing the causal region to the 3′ end of the gene. A 4‐bp insertion in the last exon of the inducer allele is likely the causal polymorphism, since it is found in all surveyed inducer lines and absent in a panel of lines representing the genetic diversity of maize. In particular, lines EM1201 and Nys302, which have the same haplotype at the gim1/qhir1 locus as the inducer line PK6 except for the 4‐bp insertion, do not induce gynogenesis. This diagnostic indel is also absent in the lines Mo1W and TX303 used as non‐inducer controls in a GWAS study of gim1/qhir1 (Hu et al, 2016). This later result highlights the preponderant role of the sub‐region qhir11 (containing NLD) compared to the sub‐region qhir12 (downstream of NLD) in haploid induction phenotype.
The fact that the right border of the interval is at a much larger physical distance from NLD is readily explained by important structural differences of PK6 with DH99 and B73. The presence of 70 kb (PK6) or 156 kb (B73) of unrelated sequence just downstream of NLD prevents genetic recombination in this region, which is reestablished further downstream in the region with synteny between the genomes. Similarly, structural differences upstream of NLD push the next recombination breakpoints at a distance of more than 62 kb in the B73 reference genome. The presence of a NLD‐related pseudogene (GRMZM2G062313 annotated as lncRNA) in the B73 genome, which is absent from the PK6 sequence in this region, may be interpreted either as a possible cause for genome rearrangements in this region or as a consequence of inherent genome instability. The fact that the NLD gene is the only conserved genetic entity in this otherwise heavily rearranged region hints at a vital role in maize reproduction. Very recently, similar conclusions were reached by two independent studies (Kelliher et al, 2017; Liu et al, 2017).
Possible roles of NLD phospholipase in fertilization
Several lines of evidence indicate that NLD is a membrane‐localized phospholipase necessary for successful fertilization: (i) the NLD promoter is strongly active in mature pollen and growing pollen tubes, (ii) the NLD protein is found in sperm cell plasma membranes, (iii) a 4‐bp insertion in the NLD coding sequence promotes the formation of haploid embryos, (iv) the localization of NLD to the plasma membrane is lost by the truncated protein predicted in the inducer lines, and (v) its annotation as a patatin‐like phospholipase A suggests that NLD is implicated in membrane lipid homeostasis of the sperm cells. Either a structural role in the maintenance of membrane integrity or a signaling role in the generation of signaling precursor from the membrane may be hypothesized. Due to the broad functions attributed to patatin‐like phospholipases (Scherer et al, 2010; Murakami et al, 2011), the molecular and cellular mechanisms linking NLD phospholipase activity to haploid induction require further investigations, which are not facilitated by the low penetrance of the induction phenotype (6–10%). Based on the loss of plasma membrane localization of the truncated NLD protein present in haploid inducer genotypes, one may speculate that defects in sperm cell/female gamete fusion could be at the origin of haploid induction. This scenario allows the connection of haploid induction and seed abortion to the same molecular mechanism (Xu et al, 2013). Indeed, haploid induction and seed abortion may reflect fusion failure of a sperm cell/egg cell and sperm cell/central cell, respectively. In Arabidopsis, RNAi knockdown of three pollen expressed PLA2 genes that do not code for patatin‐like PLA leads to developmental defects of pollen and results in reduced pollen fertility. However, the cellular and molecular mechanisms involved remain unrevealed (Kim et al, 2011).
Biotechnological applications
Haploid induction represents the initial step in the production of doubled haploid plants characterized by complete genome homozygosity (Geiger & Gordillo, 2009). Doubled haploids are an important breeding tool since they rapidly fix novel genome combinations. In maize, haploid inducers represent an inexpensive in planta system that does not require labor‐intensive in vitro steps (Dwivedi et al, 2015). An alternative in planta strategy based on genetic engineering of CENTROMERIC HISTONE3 (CENH3) has recently been transferred from Arabidopsis (Ravi & Chan, 2010) to maize (Kelliher et al, 2016). It has the advantage to induce both maternal and paternal haploids, but the maternal haploid induction rate (< 1%) is far below the rate obtained using high inducer lines derived from stock 6 (~10%). The identification of NLD as the gene responsible for in planta haploid induction in maize opens new possibilities for (i) improvement of haploid induction rate in maize and (ii) translation of this breeding tool to other species. In maize, new alleles can be obtained by TILLING or by targeted mutagenesis with CRISPR‐Cas9 (Barrangou & Doudna, 2016). Translation to other crops will first require the identification of functional orthologs, which is likely straightforward in monocots but complicated by the presence of numerous co‐orthologs in dicots (Appendix Fig S2). The use of additional criteria such as expression in sperm cell or the presence of lipid anchors may facilitate the selection of candidate genes. Beyond NLD, it needs to be recalled that gim1/qhir1 represents the predominant but not the only QTL (Barret et al, 2008; Prigge et al, 2012). The existence of at least seven other paternal QTL underlines the necessity to discover other molecular components. Putting together the different pieces of the puzzle will be valuable to better understand the double fertilization process, to improve doubled haploid production in maize and to extend the technology to other crops.
Materials and Methods
Plant material
Zea mays (maize) plants were either cultivated in fields located in Lyon (France) and Saint Martin de Hinx (France) or grown in a S2 greenhouse with a 16‐h illumination period (100 Wm−2) at 24/19°C (day/night) and without control of the relative humidity (Javelle et al, 2010). The derivation of highly recombinant inbred lines from a PK6 × DH99 cross has been described earlier (Barret et al, 2008). The public inbred lines of the core diversity panel were a gift from A. Charcosset (Camus‐Kulandaivelu et al, 2006).
Score of haploid induction
The lines to be tested for haploid induction were crossed as male parent with hybrid F564 × DH7 homozygous for the glossy1 mutation and/or hybrid PDH3 × PDH8 homozygous for the liguleless mutation (Melchinger et al, 2013). A minimum of 100 kernels was germinated and scored for the glossy (bright leaf surface, adhering water droplets) or liguleless (absence of ligule at the sheath/blade junction) phenotype indicative of haploid plantlets. Haploid induction rate was determined as the percentage of glossy or liguleless plantlets among germinated plantlets. Haploid state of glossy and liguleless plantlets screened were double‐checked by genotyping polymorphic markers between the hybrid parents. Only glossy and liguleless plantlets with all markers at homozygous state were scored as haploid.
Segregation distortion
Heterozygous plants with a recombinant and a non‐recombinant allele between markers GRMZM2G305400b_2 and SYN20148 (Appendix Table S1) were selfed. For each plant, 48 kernels, or less if the selfing was not successful, were sown and plantlets genotyped with 16 markers as indicated in Appendix Table S1. For each marker, the segregation distortion was expressed as the number of PK6 alleles divided by the number of DH99 alleles.
Two leaf punches (diameter 6 mm) were collected in plates with 96 deep wells and ground with two metallic balls (diameter 4 mm) in a 2010 Geno/Grinder® (SPEX Sample Prep, Stanmore, UK). Genomic DNA was extracted using a CTAB type lysis buffer and a magnetic bead based purification. KASP markers were designed and genotyped in 1,536‐well microtiter plates as described (He et al, 2014) using a Meridian dispenser (LGC Standards, Molsheim, France), a Fusion3™ laser plate sealer (LGC Standards, Molsheim, France), a Hydrocycler™ thermal cycler (LGC Standards, Molsheim, France), and a PHERAstar FS HTS microplate reader (BMG Labtech, Champigny‐sur‐Marne, France).
BAC library screen
The BAC libraries Zma‐B‐PK6 (HindIII) and Zma‐B‐PK6e (EcoRI) were constructed in the pIndigoBAC5 vector in strain DH10B‐T1R and screened as described (Chalhoub et al, 2004). Selected BAC clones were sequenced with a PacBio RS II long read sequencer (Pacific Biosciences). Sequence reads were assembled with the SMRT Analysis software (version 2.3.0) and the resulting consensus sequence was given the GenBank accession number KX852318.
Phylogenetic analysis
A complete list of putative orthologs of Arabidopsis patatin‐like PLs was obtained by BLAST search of the maize RefGen_v3 database (http://www.maizegdb.org/). The predicted amino acid sequences were aligned using MUSCLE in the SEAVIEW program (Gouy et al, 2010). Well‐aligned amino acid blocks were selected for phylogenetic analyses using Gblocks. Maximum‐likelihood phylogenetic analyses were performed in PhyML (Guindon et al, 2009) incorporating 1,000 bootstrap replicates and using LG substitution models for amino acid sequence data.
Prediction of lipid modification sites
For the prediction of lipid modification sites, the function "Search for palmitoylation, N‐myristoylation, farnesylation geranylgeranylation post‐translational modifications" and the threshold setting "High" of the "GPS‐lipid" web site predictor (Xie et al, 2016) were used.
RNA extraction, reverse transcription, and PCR
For RNA extraction from pollen grain, a CTAB method was used (Messias et al, 2014). For others tissues, fresh tissue was quick frozen in liquid nitrogen and ground to powder either with a Qiagen MixerMill 3MM or with a mortar and pestle. Approximately 100 mg was used for RNA extraction with TRI reagent (Molecular Research Center), DNase treatment with RNase‐free DNase (Ambion), and reverse transcription using reverse transcriptase without RNaseH activity (Fermentas) according to the instructions of the suppliers. RT–PCR and qRT–PCR were carried out and relative expression levels calculated by the ΔΔCT method as described (Javelle et al, 2010).
Constructs for plant transformation
The plasmids used for the production of NLDDH99‐OE plants contained the backbone of vector pSB11 (Ishida et al, 1996), a Basta resistance cassette (Oryza sativa (rice) Actin promoter and intron, Bar gene, and Nos terminator) next to the right border, a GFP cassette (CsVMV promoter and FAD2 intron, GFP gene, and Nos terminator), and the NLDDH99 genomic coding sequence (primers PLPK6_HDPK_F1 and PLPK6_HD99_R1; see Appendix Table S4) under the control of the constitutive rice Actin promoter and intron.
A NLD upstream fragment of line B73 (containing 2,657 bp upstream of the ATG start codon) was amplified with the primer pair attB4_prom_PL_2576_B73/attB1r_prom_PL_2576_B73 and recombined into pENTR P4‐P1R (Invitrogen) by BP reaction (pNLD). Transcriptional fusions to the GUS reporter gene were obtained by triple LR reaction between the putative promoter fragment, the GUS gene from pEN‐L1‐SI‐L2, pmock3′, and the destination vector pBb7m34GW (Karimi et al, 2007).
NLD‐CDS (without STOP codon) from DH99 and PK6 was amplified from mature anther cDNA with primer pairs PL‐CDS‐F_Dtopo/PL‐CDS‐HD99‐R2 and PL‐CDS‐F_Dtopo/PL‐CDS‐PK6‐R2, respectively, and cloned into pENTR/D‐topo (Invitrogen). The cassettes pUBQ10::NLD‐CDS::CITRINE were obtained by triple LR reaction with the fragments pUBQ10 and mCITRINE (Jaillais et al, 2011) into the destination vector pK7m34GW (Karimi et al, 2005). The cassettes pNLD::NLD‐CDS::CITRINE were obtained by triple LR reaction with the fragments pNLD and mCITRINE (Jaillais et al, 2011) into the destination vector pBb7m34GW (Karimi et al, 2007).
Plant transformation
Agrobacterium‐mediated transformation of maize inbred line A188 was executed according to a published protocol (Ishida et al, 1996). For each transformation event, the number of T‐DNA insertions was evaluated by qPCR, and the integrity of the transgene was verified by PCR with primers situated in the AtSac66 terminator downstream of the construct of interest next to the left border.
Arabidopsis ecotype Col‐0 was transformed with the floral‐dip method (Clough and Bent, 1998) and a modified procedure for Agrobacterium preparation (Logemann et al, 2006).
Fluorescence of transgenic plants harboring the citrine reporter was detected with a Zeiss LSM 710 Laser Scanning Microscope using 520‐ to 580‐nm bandpass filters after excitation at 510 nm. Image data were analyzed with ImageJ software. For SIM, pollen was imaged with a 63× Apo NA 1.4 oil objective of a Zeiss Elyra PS1 system equipped with 488‐nm laser and 495‐ to 575‐nm band pass filter. Image stacks were taken with 0.125‐μm z‐steps and 15 images per section (five phases and three angles). Images were then processed using Zen software using following parameters: manual processing, baseline‐shifted, auto‐noise filtering, and 1.5 weighting.
Histochemical detection of GUS activity
GUS (beta‐glucuronidase) histochemical staining was performed by dipping tissues into the following solution: 1 mM X‐Gluc (5‐bromo‐4‐chloro‐3‐indolyl‐beta‐d‐glucuronic acid), 0.05% Triton X‐100, 100 mM sodium phosphate (pH 7), and 0.5 mM potassium ferrocyanure and 0.5 mM potassium ferricyanure. The enzymatic reaction was performed overnight at 37°C after vacuum infiltration.
Author contributions
PMR and TW were involved in conceptualization; JPM, JC, HB, PMR, and TW contributed to methodology; LMG, AK, JBL, SC, GG, JL, GB, VB, PMR, and TW performed investigation; HB, PB, JC, and JPM provided resources; LMG, PMR, and TW were involved in writing the original draft as well as review and editing; PMR, JPM, HB, and TW were involved in supervision; JPM, PMR and TW were involved in project administration and obtained funding acquisition.
Conflict of interest
LMG, JBL, JC and JPM are employees of LIMAGRAIN Europe. Pending patent applications PCT/EP2016/060202 (published as WO2016177887), EP3091076.
Expanded View
We thank Alexis Lacroix, Yannick Rasmus, Priscilla Angelot, and Justin Berger for maize culture; Hervé Leyral and Isabelle Desbouchages for media preparation; and Frédérique Rozier, Annick Dedieu, Véronique Boltz, Pierre Chambrier, Wiebke Bretting, and Erika Consoli for technical assistance. We are grateful to Gwyneth Ingram for thoughtful discussions. We also acknowledge the contribution of the INRA field station in Saint Martin de Hinx (Carine Palaffre), the greenhouse technical platform of FR3728 BioEnviS at Université Lyon 1, and the AG platform (Bariza Blanquier) as well as the PLATIM imaging facility of the SFR Biosciences Gerland‐Lyon Sud (UMS344/US8). Funding for this work has been refused twice by the French granting agency ANR. ASK was supported by a fellowship of the Egyptian Government and LMG by a CIFRE fellowship of the ANRT (grant N° 2015/0777).
FundingEgyptian Government
- © 2017 The Authors


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