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Unless you've been living in a cave since the start of September, you'll have seen that here at Your Golf Travel we are running quite an exciting promotion….introducing Early Eagle!
The premise of the promotion is simple, if you book a golf holiday for 2018 before the end of October you will be able to make savings of up to £1000 or receive rewards including brand new TaylorMade equipment.
Booking your golf holiday this early brings many benefits beyond clubs and cash. Getting in the door early will secure the best accommodation, the most convenient tee times and will also allow you to bag all the extras you might want or need like club hire or buggies.
However, before you reap the rewards of the Early Eagle, you'll have to take on the small task of choosing the destination for your 2018 golf holiday. If you're struggling for ideas or simply have too much choice in front of you, this article can help. We have asked some of Your Golf Travel's most travelled (you can probably guess one of them) and our fantastic Twitter audience where they want to play in 2018…
Now, when you book through the Early Eagle, the benefits you receive depend on where in the world you book and with what group size. So we decided to ask, independently, where our Twitter faithful want to go in 2018 in the UK, Europe and in the rest of the world.
Before moving on to the more exotic golfing venues around the world, we started with the UK. We sent a tweet out to our followers asking where they want to play in the UK in 2018 and we received a great mix of premier golfing destinations, local favourites as well as some hidden gems. From the responses, we noticed the three top venues that the majority of our followers want to play in 2018…
Wales' top golfing destination was host of the 2010 Ryder Cup and is home to three championship courses. While Roman Roads and the Montgomerie course play much shorter than the Twenty Ten course, they still provide challenges for all golfers due to the undulating fairways, bordering woodland and ultimately a demand for accuracy over length.
However our Twitter followers, like many golfers around the world, are extremely keen to play the renowned Twenty Ten course…and who can blame them?
Built for he 2010 Ryder Cup (hence the name), it is one of modern golf's greatest creations with immaculate fairways, lightning fast greens and tournament standard tests at every turn. From 18 holes ranging across over 7000 yards, the stand out is the par 4 14th where players will have to choose between laying up or going for the long carry with two lakes either side of the fairway.
Par: 71
Yards: 7,493 (championship tees)
Signature Hole: 14th (Par 4)
Despite the many wonders of the Twenty Ten course and the Celtic Manor resort being an obvious attraction, we get the feeling that a certain promise from the club may have been turning heads on Twitter.
The home of golf is one of the most historic, iconic and respected golf clubs in the world with the Old Course a true bucket list destination for golfers everywhere. Considered as golfing paradise, a round in the shadow of the R&A clubhouse with iconic landmarks such as the Swilcan Burn, Swilcan Bridge and the Valley of Sin in play, it is something every golfer should experience at least once in their lives.
The signature hole is one of the most famous in all of golf. The par 4 17th is commonly known as the Road Hole and the tee shot is played in the shadow of the spectacular Old Course Hotel as you hit blind over the left side to the fairway. To play this hole, you must decide your strategy on the tee, a fade to keep right of the fairway gives the best approach shot to the green which has the daunting Road Bunker to the left.
"The reason the Road Hole at St Andrews is the most difficult par 4 in the world is that it was designed as a par 6." – Ben Crenshaw
Par: 72
Yards: 6,933 (championship tees)
Signature Hole: 17th (Par 4)
Ten time host of the Open Championship, two time host of the Ryder Cup and widely regarded as one of the best links courses in the UK, playing golf at Royal Birkdale is a truly unique experience. An extremely rewarding course is typical of links golf with dunes and hills throughout which add character to the holes as well as providing great vantage point for spectators for the many tournaments held there. Despite its prestige, the course is extremely fun to play and will test all aspects of your game so if you can, follow in the footsteps of some of the greatest Open champions ever.
The signature hole is the par 3, 12th which plays at 181 yards but has a green which is scarily well protected by four deep bunkers. To avoid these bunkers, you must reach the green but once you're there it doesn't get any easier as subtle contouring makes for a difficult read.
Par: 72
Yards: 6,817
Signature Hole: 12th (Par 3)
If you were booking a European golf holiday for 2018, which of these destinations would you most like to visit? ⛳???
— Your Golf Travel (@yourgolftravel) August 29, 2017
As we move into European golf holidays, our Twitter following voted in numbers for Portugal as their preferred destination for a 2018 golf holiday. Of course, the Algarve is synonymous with everything golfers want with a golf holiday; fantastic, championship courses, amazing weather and the added extras such as the beautiful beaches, rich culture and bustling nightlife to make it the complete golfing destination. With so many courses to choose from, draw some inspiration from one of our own, Rory has travelled far and wide playing golf and the Algarve is one of his specialities, here are his top 5 courses in the Algarve.
It's not just the Algarve that can boast impressive golfing venues, though. Lisbon is a region which has often sat in the Algarve's shadow but Portugal's capital is also home to some incredible courses and resorts.
The Praia D'El Rey resort is one of Lisbon's finest courses and boasts an exciting layout which makes for fun golf for everyone. Set on the Atlantic coastline, the course overlooks the vast dunes and undulation that run throughout, especially the back nine. With pine trees, sand dunes and bold, deep bunkers, the course is typical of some of the finest links golf in mainland Europe and was recently rated the 7th best in Europe by Golf World magazine, while the International Association of Golf Tour Operators voted Praia D'El Rey the best on the continent.
Par: 73
Yards: 7,036 (championship tees)
Signature Hole: 13th, 14th & 15th
If you were booking a golf holiday for 2018, which of these destinations would you most like to visit? ⛳???
— Your Golf Travel (@yourgolftravel) August 25, 2017
Next up we wanted to know where in the 'rest of the world' our Twitter followers want to play golf in 2018. With South Africa offering an obvious allure (see below) and Mauritius boasting a truly unique and exotic golfing experience, the USA still took the spoils with 46% of the votes.
Golf in America is popular beyond belief and the attraction of diverse, uniquely designed courses, stretches around the world. So it really isn't surprising that our followers chose the US as their preferred destination for a golf holiday outside of the UK and Europe.
America boasts more golf courses than anywhere else in the world and naturally there is a solid selection of truly jaw-dropping golf resorts which are on the bucket list of almost every golfer. From the Amen Corner at Augusta to the 17th at Sawgrass, we run through your top picks for golfing in America.
Sawgrass Golf Resort is home to 5 championship courses but is definitely best known for the incredible TPC Stadium Course which is one of the most famous in the world. Every hole will present all types of hazards and obstacles for golfers but there is one that stands out and is testament to the brilliant and innovative design by Pete Dye & Deane Beman. The 17th at Sawgrass is probably the most famous par 3 in the world; it plays at just 132 yards but the island green surrounded by water strikes fear into the face of the best golfers, even professionals.
Par: 72
Yards: 7,215
Signature Hole: 17th (Par 3)
Augusta National Golf Club is very special for so many reasons. Back in 1933, the legendary Bobby Jones helped design the beautiful course and then went on to co-found the US Masters. Since then Augusta has hosted every single Masters to date and still amazes the hundreds of golfers that play, the thousands of visitors and millions around the world with its natural beauty. Every hole is named after flowers, shrubs or trees to pay homage to the former nursery that used the occupy the property but it still boasts bright and colourful surroundings with vibrant azaleas, especially on Amen Corner.
While playing at Augusta is an extremely exclusive experience, going to the Masters and seeing golf's elite battle it out for the coveted green jacket is the next best thing. Packages to go and see the tournament often include tickets to the Masters, luxury accommodation and the opportunity to play at some of the amazing local courses so you can take note of the pros and put it into practice…
Par: 72
Yards: 7,435
Signature Hole: Amen Corner 11th, 12th & 13th
Ranked as the number one golf course in the world, Pebble Beach is located on the San Francisco coast and is the ultimate once-in-a-lifetime golf course. It has hosted the US Open five times and will make it six in 2019, ready to challenge the best golfers in the world yet again. The championship course really comes to life from the 6th hole where it starts to run parallel to the cliff-side ad the course's design demands strategic shots and rewards jaw-dropping views of the surrounding Pacific Ocean.
It's safe to say that Pebble Beach is a favourite for many…
"This is one of the most special golf courses in the planet." – Graeme McDowell
"A fantastic golf course and venue. It's one of those places that you always look forward to going back to…no matter how many times you have been there before." – Lee Westwood
"I don't think you can get a better venue any place in the world." – Ernie Els
"If St. Andrews is the home of golf, I think Pebble Beach feels like the home of American golf, like the home of championship golf. It has a real sense of history here."- Stewart Cink
"If I had only one more round to play, I would choose to play it at Pebble Beach. I've loved this course from the first time I saw it. It's possibly the best in the world." – Jack Nicklaus
Par: 72
Yards: 7,080 (Championship tees)
Signature Hole: 18th (Par 5)
As you can probably guess, here at Your Golf Travel many of us are in fact, well travelled. Across the departments you can hedge a bet that someone will have played or visited a good chunk of the world's courses. For this reason, we decided to ask the experts in golf travel where they would like to play in 2018.
Billy Morris (YGT Sales) – Woburn
Always hear nothing but positives about the Dukes course. Would probably play there in June – Aug to have half a chance of it being a sunny day.
Nicholas Goldie (YGT Marketing) – Kent Golf Tour
A Golf Tour to Kent to play the prestigious courses of Royal St Georges, Princes and Royal Cinque Ports. Links golf is probably my favourite type of golf and with these 3 great courses within touching distances, it's an ideal trip for myself. I'll probably look to head there next July, feeling inspired after The Open Championship and giving myself the best chance of good weather to enjoy the courses.
Callum Bruce (YGT Events) – St Andrews (Old Course)
Only one place for it, The Home of Golf. I have been lucky to play The Old Course a few times now, and every time I end up playing different shots from different angles and that's what I love about golf!
Oliver Gunning (YGT Marketing) – Ireland
I know I should be looking to do something new but I'm actually looking to repeat a trip I went on a few years ago I went with Andrew Harding (YGT's co-founder) to Dublin, we had an amazing weekend playing The K Club, Carton House, Portmarnock & Portmarnock Links. Things I remember from that trip are…playing golf in Ireland is amazing…even in the wind and rain…and the nightlife, or apres golf if you will, is excellent. We had some great nights out in Malahide and Dublin and when it comes to golf you'd have to travel quite a long way to find something better when it comes to championship parkland and incredible links golf within such a short distance of each other.
Billy Morris – Turkey
Couldn't think of anything better than 7 days in the sun at an all inclusive 5* resort and playing 4 or 5 rounds of golf all in the same week!! Always wanted to have a floodlit round at Carya too.
Nicholas Goldie – Vale Do Lobo
Vale Do Lobo is next on my list to play in Europe. Having already been to the Algarve and played the Quinta courses, the Dom Pedro courses, as well as few others such as San Lorenzo & Monte Rei, I feel Vale Do Labo is the next big resort to explore in the Algarve this year, and considering how well known it is, I feel I've been missing out by not having played it yet.
Callum Bruce – Le Paris National
I would love to give Le Paris National a visit, due to hold the Ryder Cup next year. I always like to see how I play compared to the professionals, usually a lot worse than them but still a great experience!
Oliver Gunning – Italy
I have already booked my annual trip to The Algarve so I can't really go with that. One destination that I've really enjoyed getting to know more about this year is Italy so am hoping to tick off one or more trips to Italy in 2018. I'd really like to experience a bit of luxury at somewhere like Verdura or Argentario and would like to head to Turin for a city break to play at Royal Park Roveri Golf Club. Italy is sure to become a big player in the golf travel market courtesy of The Italian Open, which is part of The Rolex Series, and The 2022 Ryder Cup which will take place at Marco Simone Golf Club, so it'll be great to experience this as the country's golfing scene grows.
Billy Morris – California
Although I'm not keen on Links golf, to play Pebble Beach in my lifetime would be an absolute dream (even if I was to shoot a 100+ round).
Nicholas Goldie – Fancourt, South Africa
Fancourt in South Africa would be my dream golf holiday, as not only is it a Top 100 Worldwide course, the off the course activities are second to none. From playing golf one day to exploring Safari's and going Shark Diving the next would make my ideal trip. The time I'd be looking to travel here would be around April/May time as I understand this is the start of the dry period and a quieter season so the weather is nice for golf and also it means animals congregated around watering holes so it is the best time to see wildlife, therefore making the most out of a safari visit.
Callum Bruce – Barbados
Easy, Green Monkey Course at Sandy Lane, Barbados. It's always appealed to me, so lets aim for that in 2017 with Your Golf Travel!
Oliver Gunning – Pebble Beach
Whilst I haven't booked it yet, I am hoping to go to Pebble Beach next year. I've actually been there twice and not played golf which seems like a crime given what an incredible course it is. With the likes of Monterey Peninsula Country Club and Spyglass Hill within easy reach as well as the likes of Pasatiempo and Cordevalle under an hour away this is must visit golfing destination that doesn't require an introduction or any selling, it is simply something that every golfer, including me, has to experience at some time in their life.
Rory Anderson – UK, Europe & ROW
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Part Number: ODVILLA-36T
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This model runs under the Villawood fireplace series, offering you a very easy and affordable way to expand your space even in an outdoor environment. It comes with a compact and clean feature which is very easy to install. This highly exceptional fireplace which serves as the perfect addition to other features in your home such as your backyard deck, patios, and even outdoor kitchen. One of its amazing key features is its traditional full refractory firebox which offers a realistic and durable look. It brings a classic style which is perfect for any space outdoors in the privacy of your home.
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Welcome to our new blog! I have no idea what I am doing, sadly, so please be patient. I am very excited to get our pages up and running- hopefully by the end of summer! Any comments, questions, offers to help, etc are welcome!
Good Morning Artists!
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Show your Gutsy with Gutsy Gals Merch
In the age of the internet, we forget how touching a handwritten note can be. Use these beautiful cards to inspire, encourage and share your thoughts with your family and friends.
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a new way to live out green
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text: Roddrick \r(o)-ddri-ck, rod(d)-rick\ as a boy's name is a variant of Roderick (Old German), and the meaning of Roddrick is "famous power". The baby name Roddrick sounds like Rodrick, Roddric and Rodderick. Other similar baby names are Eddrick, Goodrick, Godrick, Goderick, Keddrick, Rorick, Rodryck, Rodrik, Rodric, Roderyck, Roderik, Roderich, Roderic and Rhoderick.


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"Johnston masterfully draws the story to a height, tension and speed rising as the reader comes to see the whole of things... brilliantly and subtly done"
The AV Club
This is the graphic novel that became the movie ATOMIC BLONDE, starring Charlize Theron as MI6 officer Lorraine Broughton on a mission to retrieve missing intelligence in Cold War Berlin — just as the Berlin Wall is about to come crashing down. Originally published as THE COLDEST CITY.
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In the sequel to the critically-acclaimed EXPHORIA CODE, MI6 hacker Brigitte Sharp battles a series of attacks by a hacker known only as 'Tempus', who threatens to destroy the balance of global power. Bridge is drawn into the dark-web world of cryptocurrencies, Russian hackers, and African rebel militias in a desperate race against time.
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The classic game that redefined a genre, recreating survival horror for a new generation. DEAD SPACE takes players aboard the cursed 'planetcracker' ship Ishimura to fight, and try to survive, an extra-terrestrial infection that transforms the crew into physics-defying nightmare creatures.


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On April 5, 1998, I weighed a meager 125 lbs. I was 9 months pregnant. My due date had come and gone (4/3) and I was big and tired. Most of the 125 lbs was made up of baby, fluid and boobs. My arms and legs remained as tiny as always, which made walking around with the 7.5 lb baby even harder. I had spent most days since my due date, walking back in forth in our house, reading a novel. Someone had told me that walking would help labor come one. And I was oh so ready for labor pain. At around 9:30p, I sat on our sofa in the living room and watched some TV. I had been having some braxton hicks contractions and was keeping my eye on them. They weren't painful at all, but I was hoping for a miracle. At around 10pm a friend of mine called. She was 7 months pregnant. We chatted on the phone for a bit. In mid sentence I dropped the phone and clutched my abdomen. The braxton hicks contraction that I was feeling was quite powerful…and painful I might add. I picked the phone back up and told my friend that I was in a bit of pain and I'd talk to her later. I started writing the times of my contractions on the back of an envelope. The times were all over the place. Nothing about them was regular. But they sure hurt like hell, so I called the nurse at the hospital. She told me to try drinking some water and lying down for a bit to see if they would go away and if not, give her a call back. I didn't want to drink no dang water! But I did. I sipped a bit here and there, but I was feeling too well.
I finally decided to go to bed. It was around 11pm and Ray was already sleep. I climbed in the bed and dozed a bit. A sharp (excruciating) pain. I grabbed my abdomen (again) and let out an involuntary moan. A couple minutes later, I felt it again, and it was worse. This was my first child and I had no idea what the pain of labor would be like. I think those first few real contractions were the worst pain I ever felt in my life. I learned to deal with them better after I got used to them, but those first ones scared me so much.
I pushed on Ray and told him I was hurting. He was half asleep and was looking around crazy, until I yelled even louder. He woke up and asked me what I wanted him to do? I told him to call the nurse and to let her know I had called earlier and that the water wasn't working damnit! He got on the phone and in the middle of explaining everything to the nurse, I screamed again in the background. The nurse told him to bring me in right away.
Ray made all the necessary phone calls. My parents, his mom, and a babysitter for Qua (his daughter). I think I was never more helpless. I didn't want to get out of the fetal position, but I had to put on some pants and a jacket. Ray got some clothes for me and asked if I could put them on while he got his daughter ready. (She was asleep in her bedroom). I nodded, but didn't know if I was telling the truth. He got her up and put her in the car and I still had not made it into those awful gray pants that lay at my feet. Ray rushed around and got my bag and helped me get into my clothes. I tried to think of anything else I may have needed, but my brain wasn't working. Now came the hard part…walking. I swear, looking back on this, I think I was in shock more than anything. But I could barely walk. When a contraction hit me, I went down to the floor. It seemed to take forever to get to the car. Ray half carried me (and the bag). I snapped on my seatbelt and took a big breath. This was it…and I was scared out of my mind.
On the ride their, my stepdaughter mocked me and I wanted to smack her, but Ray made her be quiet. I dreaded every bump on the road. I wanted him to speed to the hospital, but I also wanted him to go slow over the bumps. Ray was going the speed limit and I yelled for him to come on. Forget the cops! Usually, he's flying and I'm yelling to slow down.
The ride to the hospital was about 15 minutes. Ray's cousin met us at the door and took Qua with him. He asked me if I was alright and I just looked at him. I finally got out of the stuffy car and the orderly (or whatever you call them) wheeled me up to the labor and delivery floor. I was starting to shiver and shake, which I learned happens when you're in labor. Well no one ever told me so I thought something was wrong. I couldn't even answer simple questions like, "When's your birthday?" It took me so long to answer that question, I'll never forget. I fumbled for my insurance card and wondered why they couldn't do this later…I wanted some drugs!
They said they couldn't put me in a room just yet. *sigh* They needed to check me in…make sure I was in labor and such. *insert eye roll* If I wasn't in labor…I was dying…cause something was definitely going on. They discovered that I was really in labor and dialated to 4cm. Did I want drugs they asked me? I guess they didn't hear me asking for the drug man over and over again. First, they needed to tranfer me to a real room they said. Sure…I've got time. I was so upset at this point. I wanted DRUGS! *lol* Ray watched the contraction monitor and was letting me know when a contraction was coming, and when it was peaking and going down. I mean…he didn't need the monitor…he could have just looked at my face. *smirk*
By the time I got into my room, my mom and dad had arrived. Then I cried. I was in so much pain and I felt like a big baby. I'm sure it wasn't easy for her to see me in such pain. It seemed like an eternity, but the drug man (who was a woman) finally came. And she was the best anesthesiologist I ever had. Everyone (except the nurse) had to leave the room while I got the epidural. The nurse leaned me over a pillow on the side of the bed and held my hand. She said if I felt pain to squeeze her hand, but not to move. Hell, I didn't want to be paralyzed for the rest of my life. No way I was moving. *lol* The thing I was most nervous about was the shot….I felt nothing. A teeny-tiny pinch. Then the area of my back was numb and finished it off. It was NOTHING. (stayed tuned for my other birth stories, where I include the epidural horror stories) I was anxious for it to start working…and it did. Within about 5 minutes (maybe 3 contractions) I noticed that the contractions were less intense. The monitor was still picking them up, but they weren't as painful for me. The drug lady stayed around and made sure my whole tummy got numb. She had me move around a bit to make sure the drug flowed where it was needed. I started feeling good. My toes and legs started getting a bit numb, but I could still move them. They were just heavy. Sitting up in the bed became hard. As my butt got numb, I would slide down. *lol* The nurse or Ray would help me re-adjust. I was still shivering a bit, so the nurse got me an extra blanket and some footies (that I still have).
Then we played the waiting game. Ray got me ice chips to munch on. I called some of my night owl family (it was about 1am). And someone finally turned on the TV. Ray and I discussed names because we still didn't have one. I was thinking of SM, but didn't say it…then all of a sudden he says, how about SM. I was so freaked out! *shivers* Of course, I said okay! Ray decided it should be spelled in a different way. I gave her the middle name that one of my paternal aunts has…Marie.
The doctor came in periodically and checked my uterus to see if I was progressing. The whole time I'm in no pain, but I was worrying like crazy. I wanted to know what it was going to feel like? How would I know it was time? Would it be painful? I was extremely scared, but I played it off well, I think. *lol* When I was at about 8cm, my water still had not broken on it's own, so the doc broke it for me. I didn't feel a thing.
More waiting and talking. We were so tired. At around 6am Ray went and got some breakfast and I was mad because I couldn't eat anything. So, my mom and Ray ate in my face. I actually have a picture of him with a mouth full of food. *lol* So at around 7:30am, they discover I am at 10cm. They asked me if I felt pressure….no. I didn't feel anything. The doc came in and got ready. They brought out the baby bed. I got more nervous. The nurse explained how I should push…I said okay. When it was time to push, I didn't know what I was doing. I was scared to push…I didn't want to push. So when they said push with all my might, I said, ok…but I didn't. I gave a little push, but faked it like a big push. *lol* I am such a wuss. No matter…after 2 practice pushes and 2 real ones, SM was out! 7:56am, Monday, April 6, 1998. Ray cut the cord, and I was a bit upset because they cleaned her up before they gave her to me. She was 21 inches long and 7.7lbs. Rather large for little ol' me. I needed no stitches at all. The doc delivered the placenta, which I had no idea we were gonna do. *lol* I had to get a catheter because my blood pressure was really high. They figured it was because I needed to "go." I didn't feel it because I was still numb, but it was a ton!! We were moved to the recovery area. We stayed there where we had many visitors and went home on April 7 @ 10pm.


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Hiring Summer Help
Unfortunately, the demand for summer jobs consistently outpaces the supply of open positions—and it's been getting worse over time. Only 34% of U.S. teens had jobs last summer, according to JPMorgan Chase's recent study entitled "Expanding Economic Opportunity for Youth through Summer Jobs." Moreover, the summer youth employment rate has dropped 20 percentage points over the last two decades.
Beware of ACA Issues When Hiring Interns and Part-Timers
Most companies don't offer medical benefits to interns and other temporary employees. Beginning in 2016, this practice may conflict with the Affordable Care Act (ACA) which requires that companies offer affordable medical benefits to 95% of their employees (an increase from 70% in 2015). If you hire a significant number of interns and temporary employees, you may need to change your hiring practices in order to remain compliant with the ACA. Contact your tax or legal adviser for more information.
Tax Break for Summer Youth Workers
In addition to making you a good corporate citizen, hiring summer help may qualify your business for the Work Opportunity credit, if you meet certain requirements. This tax break applies to qualified wages paid to new employees from certain targeted groups, including summer youth workers. The most recent extension of this credit—under the Protecting Americans from Tax Hikes Act of 2015—retroactively renews the credit for 2015 and extends it through 2019.
Special rules apply to summer youth employees: For them, the credit equals 40% of up to $3,000 of first-year wages (or up to $1,200 per qualified worker). It's available only for individuals age 16 or 17 who work for your business between May 1 and September 15. The youth also must reside in an Empowerment Zone. Contact your tax adviser for information about meeting the certification requirements.
There's no limit on the number of eligible summer youths your business can hire and claim the Work Opportunity credit for. In addition, Work Opportunity credits generated by pass-through entities, such as S corporations, partnerships and limited liability companies, pass through to the owners' personal tax returns. If this credit exceeds your tax liability, it may be carried back or forward. You can't claim it for employing your own dependents or relatives, however.
Employee vs. Intern
Young people applying for summer positions can be an inexpensive source of eager, strong and tech-savvy workers, especially if your business peaks in the summer months. But be careful when determining how much you'll pay them. The DOL has strict rules for offering student internships that pay less than minimum wage.
Before bringing unpaid (or underpaid) interns into your workplace, make sure the work is mostly about on-the-job training. Under federal labor laws, employers must pay interns at least the minimum wage unless the experience passes six rules.
- The work performed (the DOL uses the word "training") is an extension of a trade studied by the student or similar to the intern's school training.
- The work (or training) is for the benefit of the student intern.
- The intern doesn't replace regular employees, but works under their close observation.
- The employer derives no immediate advantage from the student intern's activities. The intern's activity is primarily an educational experience and doesn't significantly benefit the employer.
- The intern isn't necessarily entitled to a job at the conclusion of the internship. The employer holds out no promise of future employment.
- The employer and the intern understand that the student isn't entitled to wages for the time spent in the internship.
Beware that interns and other trainees who pass this six-factor test may still be considered employees for other legal purposes. For example, interns are protected by discrimination and harassment laws. And they may be protected by workers' compensation regulations, depending on state laws and specific circumstances.
Reap What You Sow
One way to avoid the DOL's strict rules for internships is to pay your summer youth workers at least minimum wage. Most employers find that summer workers are grateful for the opportunity to earn money while gaining experience. In turn, summer hires who feel well paid and appreciated are not only more productive today, but they're also more likely to work for you again in the future.
For more information about hiring, paying and claiming tax breaks for your summer workers, contact us.


text: Story I was writing and might start again: We Stand Divided Jan 19, 2014 13:29:27 GMT -6 Quote Select PostDeselect PostLink to PostMemberGive GiftBack to Top Post by Commander Of Games on Jan 19, 2014 13:29:27 GMT -6 I was writing this. Haven't updated it in like a month, probably won't for a little while longer.Thought I'd post it. It has a lot of problems, especially with the first chapter.


text:
- © 2012 American Society of Plant Biologists. All Rights Reserved.
To identify the genetic factors underlying the regulation of fruit vitamin C (l-ascorbic acid [AsA]) concentrations, quantitative trait loci (QTL) studies were carried out in an F1 progeny derived from a cross between the apple (Malus × domestica) cultivars Telamon and Braeburn over three years. QTL were identified for AsA, glutathione, total antioxidant activity in both flesh and skin tissues, and various quality traits, including flesh browning. Four regions on chromosomes 10, 11, 16, and 17 contained stable fruit AsA-QTL clusters. Mapping of AsA metabolic genes identified colocations between orthologs of GDP-l-galactose phosphorylase (GGP), dehydroascorbate reductase (DHAR), and nucleobase-ascorbate transporter within these QTL clusters. Of particular interest are the three paralogs of MdGGP, which all colocated within AsA-QTL clusters. Allelic variants of MdGGP1 and MdGGP3 derived from the cultivar Braeburn parent were also consistently associated with higher fruit total AsA concentrations both within the mapping population (up to 10-fold) and across a range of commercial apple germplasm (up to 6-fold). Striking differences in the expression of the cv Braeburn MdGGP1 allele between fruit from high- and low-AsA genotypes clearly indicate a key role for MdGGP1 in the regulation of fruit AsA concentrations, and this MdGGP allele-specific single-nucleotide polymorphism marker represents an excellent candidate for directed breeding for enhanced fruit AsA concentrations. Interestingly, colocations were also found between MdDHAR3-3 and a stable QTL for browning in the cv Telamon parent, highlighting links between the redox status of the AsA pool and susceptibility to flesh browning.
In plants, l-ascorbic acid (AsA; vitamin C) is essential for the detoxification of reactive oxygen species produced under stress or following exposure to pathogens. In addition to these antioxidant functions, AsA has been shown to be involved in a range of important cellular processes, including plant development and hormone signaling, cell cycle, cell expansion, senescence, and as a cofactor for a number of important enzymes (for review, see Davey et al., 2000; Smirnoff et al., 2001; Noctor, 2006). Fruit AsA concentrations have also been correlated with the maintenance of quality during postharvest storage (Davey and Keulemans, 2004; Davey et al., 2007) and have been linked to susceptibility to internal browning in both apple (Malus × domestica; Davey et al., 2006; Davey and Keulemans, 2009) and pear (Pyrus communis; Veltman et al., 1999; Franck et al., 2003). Finally, AsA is clearly an essential dietary component for humans, with a protective role proposed for many disorders and diseases (Diplock et al., 1998). Given its importance for all metabolically active tissues, there is widespread interest in unraveling the mechanisms underlying the genetic control of AsA concentrations in fruits as well as in how AsA interacts with other plant antioxidant pools.
The concentration of AsA will be determined by the net rates of biosynthesis, recycling, degradation, and/or intercellular and intracellular transport, but the relative contribution of these various processes depends on several factors, including genetics, tissue type (Bulley et al., 2009), developmental stage (Hancock et al., 2007; Bulley et al., 2009; Ioannidi et al., 2009), and light intensity (Yabuta et al., 2007; Gautier et al., 2009). The biosynthesis of AsA proceeds via l-Gal (Wheeler et al., 1998), although conclusive evidence for all steps has only relatively recently become available (Conklin et al., 2006; Laing et al., 2007). Alternative biosynthetic routes involving uronic acids (Davey et al., 1999; Agius et al., 2003), l-gulose (Wolucka and Van Montagu, 2003), or myoinositol (Lorence et al., 2004) have been proposed in several plant species, including apple (Davey et al., 2004; Razavi et al., 2005; Fig. 1), but their physiological relevance and contribution to the AsA pool is still far from clear in most plant species, with the possible exception of strawberry (Fragaria × ananassa; Agius et al., 2003; Cruz-Rus et al., 2011; Zorrilla-Fontanesi et al., 2011).
As an antioxidant, AsA is able to accept electrons from a wide range of radical substrates, and in this process it becomes oxidized first to monodehydroascorbate and then to dehydroascorbate (DHA). These oxidized forms of AsA can be regenerated by the ascorbate-glutathione (GSH) cycle, so that GSH and the activities of GSH reductase, dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) maintain the size and redox status of the AsA pool (Noctor and Foyer, 1998; Fig. 1). Indeed, overexpression of an Arabidopsis (Arabidopsis thaliana) MDHAR (Eltayeb et al., 2007) and a wheat (Triticum aestivum) DHAR (Chen et al., 2003) have both been shown to increase foliar AsA concentrations in tobacco (Nicotiana tabacum). MDHAR activity has also been positively correlated with both AsA and fruit firmness in tomato (Solanum lycopersicum) after chilling stress (Stevens et al., 2008).
Tissue AsA concentrations can also be maintained by intercellular transport, and there is evidence for the long-distance transport of AsA via the phloem from source (leaf) to sink (fruit) tissues (Franceschi and Tarlyn, 2002; Hancock et al., 2003). In apple, fruit AsA concentrations have been suggested to be partly dependent on the translocation of AsA from leaves (Li et al., 2009), but in black currant (Ribes nigrum), others concluded that the contribution of phloem AsA transport to fruit AsA concentrations was negligible (Hancock et al., 2007). While the actual mechanisms of long-distance transport of AsA have not been fully determined, attention has focused on the large family of Nucleobase-Ascorbate Transporters (NATs; de Koning and Diallinas, 2000), and NAT homologs have been found to be highly expressed in vascular tissues (Maurino et al., 2006).
Genes involved in several of these mechanisms have been proposed to be key regulators of fruit AsA concentrations, including GDP-l-Gal phosphorylase (GGP) or vitamin c defective2 (VTC2) in kiwifruit (Actinidia deliciosa; Bulley et al., 2009, 2012) as well as GDP-Man-3,5-epimerase (GME; Gilbert et al., 2009), l-Gal-1-P-phosphatase (GPP or VTC4; Ioannidi et al., 2009), and MDHAR (Stevens et al., 2007) in tomato. However, apart from GGP (Bulley et al., 2012), overexpression of these structural genes has to date had limited success in altering the fruit AsA pool (Agius et al., 2003; Bulley et al., 2009; Haroldsen et al., 2011).
In this work, we set out to identify potential genetic determinants of fruit AsA concentrations in apple fruit using a combination of molecular and genomic approaches. Initial quantitative trait loci (QTL) analyses of AsA concentrations (Davey et al., 2006) have been expanded to identify QTL for other antioxidants and fruit quality traits over three years, including results in 1 year comparing the concentrations of AsA in fruit and leaves. Alignments of the apple orthologs of genes involved in AsA biosynthesis, turnover, and transport against the whole genome sequence of cv Golden Delicious (Velasco et al., 2010) allowed us to identify candidate genes (CGs) colocating with stable QTL clusters. Using next-generation sequencing (RNA-Seq) data, polymorphic single-nucleotide polymorphism (SNP)-based markers were developed for these colocating CGs, and their positions on individual linkage groups were confirmed by linkage mapping in our mapping population. Finally, associations between allelic variants of these CGs and their expression levels in cultivars with contrasting AsA concentrations allowed us to develop allele-specific markers associated with high fruit AsA concentrations.
Population Variation in Tissue AsA and GSH Concentrations
Fruit AsA-total ascorbic acid (totAsA) and GSH-total glutathione (totGSH) concentrations showed a high degree of variability in the cv Telamon × Braeburn (TxB) mapping population, with mean values for flesh totAsA and totGSH on average varying 10- and 7-fold across the population over the three years of measurements, respectively (Table I). Total antioxidant activity (TAA) was only measured in 2006, with results indicating that the contribution of totAsA to TAA ranged from 4% to 25% in flesh and from 6% to 50% in skin tissues. Both AsA and totAsA (AsA + DHA) and GSH and totGSH (GSH + oxidized glutathione [GSSG]) concentrations in flesh and skin tissues showed normal distribution across progeny of the cv TxB population over the years, but concentrations of the oxidized forms (DHA and GSSG) displayed a statistically normal distribution in only one of the three years of measurement (2006).
Fruit AsA, totAsA, GSH, and totGSH concentrations in individuals of the TxB progeny were positively correlated (P < 0.0001) between flesh and skin tissues, such that fruit with high skin AsA and GSH concentrations also contained high flesh AsA and GSH concentrations (Supplemental Table S1). AsA and totAsA concentrations were also significantly correlated across the years (Supplemental Table S2; P < 0.0001); however, correlations for DHA, GSH, GSSG, and totGSH concentrations were lower or not significant at all. Comparing totAsA concentrations across the years indicated that in 2009, values were 27% and 42% higher in fruit flesh and 40% and 60% higher in fruit skin than the concentrations determined in 2005 and 2006, respectively. The percentage proportion of DHA in the totAsA pool, which is an indicator of the degree of oxidative stress experienced by the tissue, was found to be significantly higher in 2006 compared with 2005 or 2009. Finally, fruit skin tissues contained approximately 40% more totGSH than flesh tissues, and values in both tissues were around 50% higher in 2009 than in 2006.
These year-to-year differences in fruit AsA and GSH traits are probably related to significant differences in the climatic conditions (Supplemental Fig. S1). For example, the length of the harvest season ranged from 32 d in 2006 to 59 d in 2005, even though the first day of the harvesting period was the same each year. The highest mean population AsA and totAsA concentrations were measured in 2009, when both average daily temperature and hours of sun radiation were higher than those of the other years, suggesting an adaption to the high light and high temperature experienced in this year. Similarly, the lowest AsA and totAsA concentrations were measured in 2006, when hours of sunlight were lower than in either 2005 or 2009. A comparison of leaf and fruit totAsA and totGSH concentrations in 2009 indicated that apple leaves contained approximately 65-fold more totAsA and 12-fold more totGSH than fruit flesh tissues (Supplemental Table S3). No significant correlations were found between fruit and foliar AsA and totAsA concentrations in fruit of progeny from TxB, but there were significant correlations between foliar and both fruit flesh and skin GSH, totGSH, and GSSG concentrations and percentage GSSG (Supplemental Table S4).
Population Variation in Flesh Browning and Fruit Quality Traits
The susceptibility of fruit flesh to browning was assessed at harvest in 2006 and 2009 by measuring the time taken for cut surfaces to brown (Br_time) and the final color of the affected tissue (Br_color). Br_time ranged from 15 to 265 min and was negatively correlated with the percentage DHA values of flesh tissues (Supplemental Table S5) but not with the flesh totAsA values. Br_color was positively correlated with the flesh percentage DHA. A strong correlation (P < 0.0001) was also found between flesh AsA-totAsA concentrations and firmness and to a lesser extent with fruit percentage dry weight and soluble solids content (measured as °Brix [P < 0.01]; Supplemental Table S5).
QTL Mapping
We identified a total of 27 fruit AsA- and GSH-QTL that were stable for at least two of the three measurement years. These were clustered into four main genomic regions on linkage groups (LGs) 10, 11, 16, and 17 (Fig. 2). The total population variability explained by these AsA-QTL (QTL for AsA, totAsA, and DHA) ranged from 10.7% to 59.5% per trait but was much lower for GSH-QTL (QTL for GSH, totGSH, and GSSG), where population variability ranged between 9.5% and 13.1% per trait (Supplemental Table S6).
QTL for Fruit and Leaf AsA Concentrations
Twenty flesh AsA-QTL were identified in the cv Telamon parent and 17 in cv Braeburn. These QTL individually explained from 7.6% to 30% of the total population variance (Supplemental Table S6). Clusters of stable (present in two or more years) and significant (log of the odds [LOD] > 3.5) AsA-QTL were identified at the bottom of LG 11 and in the middle of LG 10 in both parental maps (Table II). Additional AsA-QTL on LGs 3, 6, and 16 were not stable across all years, while parent-specific QTL were located on LGs 3 and 17. In the majority of cases, QTL for totAsA colocated with QTL for flesh AsA concentrations in both parents (Fig. 2; Supplemental Table S6). Of particular interest was the QTL cluster for flesh AsA located on LG 11, as these were stable over all three years and explained a relatively high proportion (up to 27.5%) of the total population variability.
Stable clusters of skin AsA-QTL were detected on LGs 9, 10, and 16, which individually accounted for up to 17.3% of population variability, while additional, non-year-stable QTL were found on LGs 2, 3, 11, and 17 (Fig. 2; Supplemental Table S6). The skin AsA-QTL on LGs 11, 16, and 17 colocated with AsA-QTL of flesh, with only the QTL cluster on LG 9 specific for fruit skin AsA-totAsA concentrations.
QTL analysis of leaf AsA, totAsA, and DHA concentrations (2009 only) identified two QTL clusters on LG 6 and 15 (Table III), but only the QTL on LG 6 colocated with that of a fruit AsA-QTL derived from the Telamon parent.
QTL for Fruit and Leaf GSH Concentrations
From measurements in 2006 and 2009, we identified nine skin and nine flesh GSH-QTL, distributed across nine LGs (Fig. 2; Supplemental Table S6). LOD scores for these QTL ranged from 3.0 to 3.4 and were generally lower than those observed for AsA-QTL. Only one region in the middle of LG 16 contained QTL that were stable across parents and tissues (fruit flesh, fruit skin, and leaf). In 2009, leaf GSH concentrations were measured, leading to the identification of QTL on LGs 6, 12, and 16 (Fig. 2; Table III), with the QTL for leaf GSSG on LG 16 colocating with fruit AsA-QTL in both parents (Fig. 2).
QTL for Fruit TAA
In cv Telamon, two significant TAA-QTL for flesh were detected on LGs 6 and 12, accounting for a total of 23.1% of the population variability (Supplemental Table S6). In cv Braeburn, a major TAA-QTL on LG 11 (LOD = 3.9; population variability of 20.6%), and two minor TAA-QTL on LGs 6 and 16 were identified in flesh tissues, all partially overlapping with QTL for flesh AsA-totAsA.
QTL for Flesh Browning and Fruit Quality Traits
QTL for susceptibility to flesh browning (Br_time and Br_color) were located on LGs 3, 10, 16, and 17 (Fig. 2; Supplemental Table S7). Of these, the QTL for Br_color on LG 16 of cv Braeburn and on LG 17 of cv Telamon were stable over both years of measurements and colocated with AsA-QTL on these LGs (Fig. 2). On LG 10 (both parents) and on LG 17 (cv Telamon), QTL for Br_time mapped to the same position as QTL for AsA, totAsA, and DHA concentrations of flesh tissues. Interestingly, stable QTL for flesh browning (Br_time and Br_color) at the bottom of cv Telamon LG 17 colocated with the stable QTL for flesh DHA concentrations, but no comparable QTL were detected in the "high-fruit-quality" cv Braeburn parent. A large cluster of fruit AsA-QTL in the middle of LG 10 in both parents colocated with a range of previously identified stable QTL for fruit traits, including fresh weight, flesh soluble solids content, and firmness, in this population (Supplemental Table S7; Kenis et al., 2008).
Mapping of Candidate Gene Orthologs and Fruit QTL Clusters
From the literature, we identified 22 structural genes involved in AsA biosynthesis, recycling, degradation, and transport. Using the apple reference consensus coding sequence (CDS) set (http://genomics.research.iasma.it/), 99 apple orthologs of these genes were identified, based on similarity to Arabidopsis sequences and by phylogenetic analysis using published sequences from several additional plant species (Supplemental Table S8). Following an in silico analysis of their positions in the apple genome (Supplemental Table S8), SNP-based markers (Supplemental Table S9) were developed for the subset of CGs that colocated within our QTL using RNA-Seq data of both parents, allowing these CGs to be mapped onto our cv TxB genetic linkage maps (Supplemental Fig. S2). A total of 10 AsA CGs were mapped onto the cv Telamon map and 13 onto the Braeburn map. Generally, these CGs mapped to the expected positions predicted from the Golden Delicious genome assembly. The only exception was for MdDHAR3-1 (MDP0000240690; LG 9), where the marker mapped to the homologous region of LG 17, which contains two other paralogous copies of MdDHAR3 (MdDHAR3-2, MDP0000175246 and MdDHAR3-3, MDP0000156763).
From the l-Gal biosynthetic pathway, one ortholog of GGP (MDP0000172222, VTC2, MdGGP1), with 71% protein sequence identity to Arabidopsis At4g26850.1, mapped to LG 11 of cv Telamon within the cluster of stable flesh AsA-QTL in both parents (Fig. 2). A clone of this gene from Malus sylvestris (GB CN915822) has been shown to have GGP activity when expressed in Escherichia coli (Supplemental Table S10). A paralog of MdGGP1, MdGPP2 (MDP0000288088; 69.5% identity to At4g26850.1 and 89.6% identity to MdGPP1), mapped to LG 3 of cv Braeburn within the flesh AsA-QTL cluster in cv Telamon that was only detected in 2006. Again, a homologous gene from apple (GB CN939721) has been demonstrated to be active (Supplemental Table S10). A third more distantly related ortholog of Arabidopsis GPP, MdGPP3 (MDP0000191488; 30.1% identity to At4g26850.1 and 34% identity to MdGGP1), mapped to LG 10 of cv Telamon and also colocated within a stable AsA-QTL. The markers developed for both MdGGP1 (2006 and 2009) and MdGGP3 (2005) were also the best markers describing the respective QTL (Table II). None of the other genes involved in the l-Gal biosynthetic pathway colocated with any of our AsA-QTL, apart from a putative GDP-mannose pyrophosphorylase (GMP or VTC1; MDP0000323050, MdGMP2-1) with 83.5% identity to At1g74910, which colocated with the non-year-stable flesh AsA-QTL on LG 6 (Fig. 2).
From the alternative AsA biosynthetic pathways, a marker for a putative d-GalUA reductase (MdGalUR1, MDP0000309417; 57.5% identity with At2g37790) mapped to LG 12 of cv Braeburn but did not colocate with any QTL (Fig. 2). However, two MdGalUR2 sequences (MDP0000135496 and MDP0000857724) mapped in silico within the flesh AsA-QTL on LG 16 (Fig. 2; Supplemental Table S8). It was not possible to develop markers for these others, however, as expression levels were too low to identify unique SNPs from our RNA-Seq data.
We identified six DHAR and six MDHAR apple orthologs involved in the recycling of oxidized DHA (Supplemental Table S8). Of these, MdDHAR3-3 (65% identity with AtDHAR3) mapped to the bottom of LG 17 in cv Telamon and colocated with a stable QTL for flesh DHA concentration (2005 and 2009 data) and a minor QTL for flesh AsA concentration (2005; Fig. 2). The other paralogs of MdDHAR3 located in silico to LG 9 (MdDHAR3-1, MDP0000240690 and MdDHAR3-4, MDP0000530903), also within the QTL intervals for skin AsA concentrations (2005 data). There were no colocations, however, between any of the MdMDHARs and antioxidant QTL.
Twenty-one apple orthologs of the NAT protein family were identified in the reference CDS set. Four of these are highly similar to AtNAT7 and AtNAT12, which are the two members considered to be specifically involved in AsA transport, and are located on LGs 11, 13, 15, and 16 (Supplemental Table S8). MdNAT7-2 (MDP0000320308; 58% identity with AtNAT7) mapped to LG 16 of cv Braeburn, colocating with the significant QTL for flesh AsA-totAsA concentrations in both parents (Fig. 2).
Finally, in silico alignment of genes of GSH metabolism (glutathione reductase and glutathione synthase; Supplemental Table S8) identified only one potentially interesting colocation on LG 4 between MdGS2-2 (MDP0000188669; 61% identity to At5g27380) and GSH-QTL for both flesh and skin tissues detected in cv Braeburn.
Sequence Variations in Candidate Regulatory Genes for Fruit AsA Concentrations
High-throughput RNA sequencing (RNA-Seq) was used to detect polymorphisms in the transcript sequences of AsA CGs and to identify allelic SNPs linked to enhanced fruit AsA-totAsA concentrations. Aligning the RNA-Seq reads to the cv Golden Delicious reference consensus CDS set (http://genomics.research.iasma.it/) resulted in a mean read coverage for MdGGP1 (LG 11) of 164.6- and 123.9-fold for cv Braeburn and Telamon, respectively, indicating high transcript expression and good read depth. A total of 10 different SNPs along the 1,704-bp MdGGP1 cv Golden Delicious reference CDS were identified (Supplemental Fig. S3; Supplemental Table S11). This corresponds to one SNP every 170 bp, which is consistent with previous reports in apple cDNA sequences (one SNP every 149 bp; Chagné et al., 2008). These alignments were used to correct the MdGPP1 reference gene model, which was found to contain an incorrectly predicted start codon and two extra codons (Supplemental Figs. S3 and S4). As a result, the majority of the SNPs in MdGGP1 shift into the 5′ untranslated region and only two SNPs are found within the CDS, at reference positions 414 and 885 bp. Neither SNP leads to an amino acid change, but SNP885 is unique to cv Telamon and was developed into a diagnostic SNP marker for mapping and genotyping.
For MdGGP2 (LG 3), mean read coverage following alignment to the cv Golden Delicious reference consensus CDS set was 122.5- and 111.2-fold for cv Braeburn and Telamon, respectively, again indicating high expression and good read depth. A total of eight SNPs were identified in the MdGGP2 CDS, of which three were heterozygous in cv Braeburn and six in cv Telamon (Supplemental Fig. S5; Supplemental Table S12). Both cv Telamon and Braeburn possess nonsynonymous SNPs at positions 74 and 169 bp. However, as both SNPs are present in both parents, they are unlikely to be responsible for the parent-specific allelic differences observed. Both also have a homozygous, but different, SNP at reference position 564, which was used for mapping and genotyping.
Mean read coverage of MdGGP3 following alignment to the cv Golden Delicious CDS was 34.8- and 31.3-fold for cv Braeburn and Telamon, respectively. Both the cv Telamon and Braeburn CDS were noticeably more divergent from the reference CDS than MdGGP1 and MdGGP2, and we identified 79 SNPs in cv Braeburn and 75 SNPs in cv Telamon (Supplemental Fig. S6). A unique nonsynonymous SNP in cv Telamon at reference position 211 was developed for mapping. Both parents contain nonsynonymous SNPs that could influence MdGGP3 protein function, and both also possess a homozygous 33-bp deletion at reference position 272 at the beginning of intron 4, leading to the loss of 11 amino acids. In addition, in cv Braeburn, a heterozygous single-nucleotide deletion at reference position 986 leads to a frame shift and a different and truncated N-terminal amino acid sequence (Supplemental Fig. S7). This suggests that cv Braeburn contains an MdGGP3 allele that codes for a truncated and possibly dysfunctional protein. Alignment of cv Braeburn de novo contigs supports this conclusion, with fragments of both cv Braeburn alleles apparently being present.
The mean read coverage for MdDHAR3-1 (LG 9) was 14.0- and 20.0-fold for cv Braeburn and Telamon, respectively; however, these alignments contained a large region of nonspecifically matched reads, so that only a single SNP could be reliably identified. This SNP-based marker mapped to the homologous region on LG 17, which contains both MdDHAR3-2 and MdDHAR3-3 (MDP0000175246 and MDP0000156763). However, the read coverage for these latter two sequences was too low to allow additional MdDHAR3-specific markers to be developed.
For MdNAT7-2 (LG 16), the mean read coverage was 33.1- and 34.9-fold for cv Braeburn and Telamon, respectively. However, coverage was not uniform, and it was not possible to generate a full-length consensus sequence in either case. In addition, the consensus read-mapping sequences from both parents showed a considerable degree of polymorphism from the cv Golden Delicious reference CDS, with 252 variants detected in cv Braeburn and 151 in cv Telamon.
Allele Association Studies
Orthologous CG sequences, which mapped within AsA-QTL, highlighted several genes with a potential role in regulating fruit AsA concentrations. Of particular interest are the paralogous copies of MdGGP, as all three were mapped within significant flesh AsA-QTL, and two of these QTL were further stable for at least 2 years. In addition, MdNAT7-2 colocated with QTL for flesh antioxidant concentrations on LG 16. To further investigate the involvement of these four genes in the control of apple AsA concentrations, correlations between the allele SNP variants were examined in the TxB progeny as well as in a set of commercial apple cultivars with a wide range of totAsA concentrations.
TotAsA concentrations of flesh tissues in progeny of the TxB population were significantly correlated with SNP variants for MdGGP1 (all three years), MdGGP2 (1 year), MdGGP3 (2 years), and MdNAT7-2 (2 years; Table IV). Specifically, genotypes homozygous for the T allele (T/T genotypes) of MdGGP1 at position 885 had statistically significantly higher flesh totAsA concentrations than the T/C genotypes over the three years of measurement. Similarly, G/A genotypes for MdGGP2 (SNP564) or the T/T genotypes for MdGGP3 (SNP211) also had higher flesh totAsA concentration than G/G or T/C, respectively, but only in 1 or 2 years of measurements (Table IV). For MdNAT7-2 (SNP92), the T/G genotypes had also higher fruit totAsA concentrations in 2006 and 2009 compared with G/G genotypes. Comparing the flesh totAsA concentrations of progeny with the "best" and "worst" SNP variant combinations for these genes showed that the best SNP allelic variants resulted in fruit mean totAsA concentrations that were up to 10-fold higher than those carrying the unfavorable allelic variants (data not shown). In all cases, cv Braeburn was the parent that transferred the beneficial allelic variants for high totAsA concentrations.
To validate these associations outside the mapping population, a selection of 22 apple commercial cultivars with known fruit totAsA concentrations (Davey et al., 2004; I. Mellidou and M.W. Davey, unpublished data) were genotyped with the same SNP markers. In the case of MdGPP2 (LG 3) and MdNAT7-2 (LG 16), these markers yielded complex melting curve profiles in the high-resolution melting (HRM) SNP assay and so could not be used with this germplasm. This is presumably the result of additional polymorphisms present in the PCR amplicon region. However, the SNP variants in MdGGP1 and MdGPP3 were successfully determined for 22 and 11 of these cultivars, respectively (Table V). For the remaining 11 cultivars, the complex melting curve profiles for MdGPP3 again precluded us from establishing SNP genotypes. Results show that once again the MdGPP1 T allele and the MdGGP3 T allele were both associated with higher fruit totAsA concentrations in these cultivars. Furthermore, in the 11 cultivars for which we have the SNP genotypes for both MdGGP1 and MdGGP3, the favorable T+T allele combination was always associated with an average 3-fold higher fruit totAsA concentration and a maximum 6-fold higher concentration than those with the C+C combination.
Gene Expression Analysis
To understand the basis for the differences in totAsA contents in fruit of cultivars with different SNP alleles, quantitative PCR expression analysis of CGs was carried out in the flesh of fruit from 10 apple genotypes with high or low AsA concentrations (Fig. 3). In all cases, the expression levels of MdGGP1, MdGGP3, and MdDHAR3-3 were positively and significantly correlated with fruit totAsA concentrations (Fig. 3, B–G), with the mean differences in their relative expression among the high and low AsA groups being 2.0-, 1.5-, and 2.0-fold, respectively. In contrast, there were no statistically significant differences in the mean relative expression of MdGGP2 and MdNAT7-2, although in both cases, cv Braeburn once again had a higher relative expression than cv Telamon tissues.
Analysis of the RNA-Seq data shows that the mean expression levels of MdGGP1 (LG 11) and MdGGP2 (LG 3) are much higher than that of MdGGP3 (LG 10). Reads per kilobase exon model per million (RPKM) values for MdGGP1, MdGGP2, and MdGGP3 in cv Braeburn and Telamon, respectively, were 84.1 and 56.8, 61.4 and 50.4, and 13.9 and 15.3. Although these values are derived from RNA samples isolated during bud and flower development, comparison with the RPKM values of these genes in the low-AsA cv Royal Gala at the mature fruit stage (132 d after full bloom) again shows that MdGGP1 (RPKM 58) and MdGGP2 (RPKM 52) are much more strongly expressed than MdGGP3 (RPKM 5; W.A. Laing, unpublished data). Together with the QTL results, this suggests that MdGGP1 expression is particularly important for the regulation of tissue flesh AsA concentrations.
Antioxidant Concentrations and QTL in the cv TxB Mapping Population: Genetics versus Environment
The absolute concentration of antioxidants varied considerably from year to year, with totAsA and totGSH concentrations being greatest in 2009. This is presumably due to the higher light and temperatures experienced prior to harvest in 2009, factors that are known to influence totAsA concentrations (Davey et al., 2000, 2007; Dumas et al., 2003). However, there was no clear correlation between totAsA concentrations and mean daily temperatures or sunlight hours across the years, underlying the fact that it is difficult to dissociate the individual contributions of temperature, light, development, and genetics from the regulation of antioxidant pools. Nonetheless, the fact that stable (present in at least two measurement years) fruit flesh AsA-QTL (LGs 10, 11, and 17) were found demonstrates a strong genetic component to the control of fruit AsA concentrations in apple.
Environmental factors such as light intensity are expected to have a larger impact on AsA concentrations in the skin compared with flesh tissues and therefore could mask the underlying genetic control mechanisms. Despite this, significant QTL for skin AsA and totAsA concentrations were identified, and most of these colocated with flesh AsA-QTL. Only the QTL cluster on LG 9 (both parents) and on the cv Telamon LG 16 were specific for skin AsA concentrations. These regions on LG 9 and LG 16 have both been shown to contain major QTL for apple fruit polyphenolic contents (Chagné et al., 2007, 2012; Khan et al., 2012). We have previously reported that the sun-exposed (red) side of apples has higher anthocyanin and AsA concentrations and is more resistant to both biotic and abiotic stress than the shaded (green/yellow) side (Davey et al., 2004, 2007), and recently, Bulley et al. (2012) demonstrated that transgenic fruits from tomato and strawberry with elevated AsA contents also had an approximately 50% larger polyphenolic pool. Finally, we report here positive correlations between TAA, AsA, and totAsA concentrations in both flesh and skin tissues and colocations between flesh AsA- and TAA-QTL on LGs 6, 11, and 16. Therefore, the skin-specific AsA-QTL on LGs 9 and 16 may be related to interaction with the polyphenol contents in apple skin tissues. In this regard, the recent report indicating that the accumulation of anthocyanins in Arabidopsis leaves is fine-tuned by the AsA redox state is of particular interest (Page et al., 2012).
For the first time, to our knowledge, we also present QTL for fruit GSH concentrations, but apart from QTL for flesh GSSG concentrations on LG 16 that colocalized with AsA-QTL in both parents, all the other QTL had low LOD scores, and no significant associations between AsA- and GSH-QTL were detected.
The active transport of foliar AsA to developing sink tissues has been demonstrated previously (Franceschi and Tarlyn, 2002; Hancock et al., 2003; Tedone et al., 2004). While both apple fruit and leaf tissues have been shown to be capable of AsA biosynthesis (Davey et al., 2004; Razavi et al., 2005; Li et al., 2010), there were no significant correlations between fruit and leaf totAsA concentrations and only one colocation between leaf and fruit AsA-QTL on LG 6. This would also need to be confirmed, as it is based on only 1 year of measurements. Regardless, these results indicate that screening progeny on the basis of foliar AsA concentrations to identify cultivars rich in fruit vitamin C concentrations will not be highly informative.
Links between Vitamin C Concentrations, Flesh Browning, and Other Fruit Quality Traits
Susceptibility to browning has been linked to flesh DHA concentrations in apple (Davey et al., 2006), and in pears, the development of internal postharvest browning disorder occurs when AsA concentrations decline below a certain threshold value (Franck et al., 2003). Here, the time taken for cut apple flesh to complete browning and the final color intensity following browning were both correlated with DHA concentrations and the percentage DHA of flesh tissues. In addition, stable QTL for browning colocated with stable QTL for flesh DHA concentrations on LG 17. This browning QTL was only detected in cv Telamon, which produces poor-quality fruit and in which the flesh percentage DHA was on average 3-fold higher than in cv Braeburn (data not shown). Taken together, these results suggest that factors that lead to an oxidized AsA pool are associated with susceptibility to flesh browning.
In cherry tomatoes, fruit firmness and AsA concentrations have been reported to be closely correlated (Gilbert et al., 2009), and here, we show that firm apples also generally contain higher flesh AsA concentrations. Flesh AsA-totAsA concentrations and percentage DHA are also significantly correlated with fruit flesh soluble solids content, suggesting a link to the supply of sugar substrates for AsA biosynthesis. However, the significance of this is not clear, as a large cluster of QTL for fruit physiological traits, including fruit firmness, soluble solids content, fresh weight, and flesh browning, all colocated with the fruit flesh AsA-QTL on LG 10; therefore, the correlation with fruit AsA-totAsA concentrations could be due to indirect effects on fruit physiology and quality.
Candidate Genes Regulating AsA Concentrations
Candidate Regulatory Genes from the AsA Biosynthetic Pathways
GMP (VTC1) catalyzes the conversion of d-Man-1-P to GDP-d-Man (Fig. 1), and we observed a colocation between a putative MdGMP (MdGMP2-1) and the flesh AsA-QTL cluster on LG 6. However, these QTL explained only a relatively low proportion of the population variability (10%) and were detected only in 1 year. In tomato (Ioannidi et al., 2009) and kiwifruit (Bulley et al., 2009), fruit AsA concentrations were also not correlated with GMP expression.
GME has been proposed to be critical for the regulation of plant AsA concentrations by several groups (Wolucka and Van Montagu, 2007; Gilbert et al., 2009), and in tomato, SlGME1 colocates with a cluster of stable fruit AsA-QTL (Stevens et al., 2007), while in apple, MdGME transcript levels are highly correlated with AsA concentrations during apple fruit development (Li et al., 2011). However, the data presented here support the results obtained in peach (Prunus persica; Imai et al., 2009) and tomato (Ioannidi et al., 2009), which suggest that this step is not a major point of control in mature apple fruit, at least under our field conditions.
GGP (VTC2; Fig. 1) catalyzes the first committed step of AsA biosynthesis and has also been suggested to be the rate-limiting step for AsA biosynthesis in plants (Linster and Clarke, 2008; Bulley et al., 2009, 2012). Our results here provide strong evidence that MdGGP1 is the only structural gene of AsA metabolism that is tightly linked to flesh AsA concentrations in apple and that this is independent of the environmental conditions, since significant AsA-QTL were detected over all three years of measurement. While we cannot exclude the possibility that these QTL regions contain other regulatory genes, it is interesting that the two other MdGGP paralogs (MdGGP2 on LG 3 and MdGGP3 on LG 10) also mapped within fruit AsA-QTL.
The next step of the l-Gal pathway is catalyzed by GPP (VTC4). Although GPP has been suggested to be important in regulating AsA concentrations in tomato fruit (Ioannidi et al., 2009) and apples (Li et al., 2011), these conclusions are not supported by our results. None of the other genes involved in the main AsA biosynthetic pathway (PMI, PMM, GalDH, or GLDH) mapped to positions located within our AsA-QTL, suggesting that these have no major control over AsA homeostasis in mature apple fruit under Belgian field conditions.
From the alternative (uronic acid) AsA biosynthetic routes (Fig. 1), the reaction catalyzed by GalUR (Enzyme Commission [EC] 184.108.40.206) in strawberry has been the most extensively studied (Agius et al., 2003). d-Galacturonate has been suggested to be an alternative substrate for the synthesis of AsA in fruit skin by precursor feeding experiments in apple fruit tissues (Li et al., 2008), and biosynthesis proceeding via GalUR has been suggested to play a more significant role in the later stages of fruit ripening when breakdown of cell wall pectin takes place (Melino et al., 2009; Cruz-Rus et al., 2011; Badejo et al., 2012). The in silico colocation of two MdGalUR paralogs within flesh AsA-QTL clusters on LG 16 of both parents supports a possible role for GalUR in mature fruits, but there were no other colocations between genes of the alternative AsA biosynthetic pathways and AsA-QTL.
To summarize this section, except for paralogs of the first dedicated step of the l-Gal biosynthetic pathway (MdGGP1-3), our results do not support a key role for any of the other genes of the main l-Gal biosynthetic pathway. However, as reactions before the GGP step also supply substrates for cell wall polysaccharide biosynthesis, mutations in these genes could affect the flux of substrates through to GGP.
CGs from the AsA Recycling Pathway
DHA, the oxidized form of AsA, is unstable and can be readily degraded if not rereduced by the activity of MDHAR (EC 126.96.36.199) and/or DHAR (EC 18.104.22.168). In tomato, an ortholog of MDHAR (SlMDHAR3) has been found to colocate with a stable QTL for fruit AsA contents (Stevens et al., 2007), and MDHAR enzyme activity has been associated with elevated AsA concentrations under chilling stress (Stevens et al., 2008). In apple, we found no colocation between MdMDHARs and any antioxidant QTL. However, MdDHAR3-3 mapped to the bottom of cv Telamon LG 17 within a highly significant, stable QTL for flesh DHA concentrations, and MdDHAR3-1 mapped in silico within QTL for skin AsA concentrations in LG 9, suggesting that orthologs of DHAR could be important in helping to maintain the reduced AsA pool in apple fruit. The colocation of MdDHAR3-3 within a stable QTL for flesh browning suggests that genetic regulation of redox status of the AsA pool via DHAR is important for postharvest fruit quality traits in apple.
CGs Related to Intercellular AsA Transport
Intercellular AsA transport via the NATs could represent an important mechanism of maintaining sink tissue AsA concentrations (Maurino et al., 2006). MdNAT7-2 mapped to the same position as significant fruit AsA-QTL on LG 16 of the cv Braeburn genetic map, and another ortholog of NAT (MdNAT12-2) colocated in silico with a QTL for leaf DHA concentrations on LG 15 of both maps. As NATs are highly expressed in vascular tissues (Maurino et al., 2006) and they colocated with AsA-QTL in both fruit and leaf tissues, these results indicate that they may be involved in the long-distance transport of AsA in apples.
Molecular Markers for Fruit AsA Concentrations
Here, we show that allelic variations in the nucleotide sequences of MdGGP1, MdGGP3, and MdNAT7-2 are associated with up to 10-fold differences in totAsA concentrations of fruit from individuals of our cv TxB mapping population. The remaining MdGGP paralog, MdGGP2, also mapped within an AsA-QTL cluster on LG 3, but this cluster was only detected in 2006. Importantly, fruit totAsA concentrations of 22 commercial apple cultivars were also associated with the same allelic variations in MdGGP1 and MdGGP3, and the T+T combination of alleles for MdGGP1 and MdGGP3 is consistently associated with an up to 6-fold higher fruit totAsA concentration. Sequence analysis of the (corrected) MdGGP1 CDS showed that there are no nonsynonymous SNPs (Supplemental Table S11), while in MdGGP2, nonsynonymous SNPs result in three amino acid changes (Supplemental Table S12), but none of these are predicted to alter structure or function. By comparison, MdGGP3, MdDHAR3-3, and MdNAT7-2 CDS contain multiple synonymous and nonsynonymous SNPs. Further study of the impact of these sequence differences, however, was limited by the complexity and relatively low coverage of the MdDHAR3-3 and MdNAT7-2 transcript sequences. Since the SNPs in MdGGP1 and MdGGP2 are not associated with altered protein function, it is likely that the observed allelic associations are due to linkage with polymorphisms in the promoter region that alter allele expression. Gene expression analyses in a range of phenotypically diverse cultivars demonstrated that MdGGP1 and MdGGP3 as well as MdDHAR3-3 expression levels are all strongly correlated with fruit totAsA concentrations (Fig. 3). Expression levels of MdGGP2 (LG 3) or MdNAT7-2 (LG 16), in contrast, were not significantly correlated, but this could be because they mapped within a non-year-stable AsA-QTL cluster and the expression analyses were carried out in fruit harvested in a different year. Conceivably, therefore, these CGs might still be important only under certain environmental/developmental conditions. Finally, our RNA-Seq data demonstrate that mean expression levels of the third paralog of MdGGP (MdGGP3) are considerably lower than the expression of the other two gene copies in developing tissues as well as in mature fruit. This, in combination with the high number of SNPs and other polymorphisms, suggests that MdGGP3 is less important for the regulation of tissue AsA concentrations and that the translation product may even be nonfunctional.
High fruit AsA concentration is considered to be a desirable trait for consumers, but in apple, as in other hard fruit species, the long juvenile period of plants means that fruit-specific traits cannot be determined until progeny reach maturity around 3 to 5 years after germination. As such, there is much interest in the development of genetic markers for the early selection and/or screening of progeny for fruit AsA concentrations. Our QTL, CG mapping, and analysis of allelic variations across a selection of germplasm show that MdGGP paralogs are consistently linked to fruit totAsA concentrations, clearly indicating a major role for these genes. Gene expression analyses suggest that the regulation of flesh AsA concentrations in apple is primarily through control of the expression levels of MdGGP1, MdGGP3, as well as MdDHAR3-3. The SNP-based markers developed here are thus excellent candidates for the early screening of progeny for increased fruit AsA concentrations. Despite conflicting reports in the literature, our work does not support a major role for the other biosynthetic genes in apple fruit AsA homeostasis, at least under Belgian climatic conditions. However, we propose that MdDHAR3-3 helps regulate the redox state of the AsA pool in apple fruit and that increased flesh DHA concentrations are associated with susceptibility to flesh browning.
Mapping Population
An F1 mapping population consisting of 257 individuals was created from a cross between the apple (Malus × domestica) cultivars Telamon and Braeburn, using cv Telamon as the female parent (Kenis and Keulemans, 2005). For the 2005 and 2006 work, a population of mature trees more than 4 years old was used. Trees were grown on M.9 rootstock at the Rillaar Experimental Field Station in Aarschot, Belgium. For the 2009 work, a regenerated copy of the same population was propagated on M.27 rootstock, grown on the same field.
Harvest and Meteorological Measurements
Fruit from individual progeny were harvested up to twice per week when considered commercially ripe. Ripeness was assessed by experts based on ripening parameters. A minimum of 10 healthy apples per genotype were sampled for AsA, totAsA, GSH, totGSH, and TAA analysis and processed essentially as described previously (Davey et al., 2006). Fruit were immediately transported to the laboratory for analysis. Leaf tissue from 180 genotypes was collected before fruit harvest and stored at −80°C until further analysis. Mature apple fruit were also collected in 2011, pooled into three biological replications, each one consisting of at least three healthy apples, and processed immediately for AsA measurement or stored at −80°C for gene expression studies. Data for mean daytime air temperature (°C) and sun radiation (W m−2) during a period of 5 weeks before the harvest period, starting on August 1, were obtained from the university meteorological station (Heverlee), located 25 km from the experimental field.
Fruit Physiological Measurements and Flesh Browning
For all years of QTL analysis, fruit fresh and dry weight were measured immediately after harvest. Flesh firmness was determined using a penetrometer fitted with an 11-mm-diameter plug, while at the same time, the juice released by penetration of the probe was used to measure the soluble solids concentration (mainly sugars; °Brix) using a digital refractometer (Pocket PAL-1; Atago), essentially as described by Kenis et al. (2008). In 2006 and 2009, the susceptibility of the apple flesh to browning was assessed as Br_time, and Br_color was also evaluated (Davey and Keulemans, 2009).
AsA and GSH Analysis
Samples for metabolite analysis were obtained from skin and flesh fractions from 10 individual fruit by blending using a metaphosphoric acid/EDTA/polyvinylpolypyrrolidone extraction, essentially as described previously (Davey et al., 2003, 2006). Extracts were filtered and injected into an HPLC system (Waters 2690 separation module) equipped with a C18 rocket column (Grace; 53 mm × 7 mm). AsA and GSH were detected at 243 and 197 nm, respectively (Waters 996 photodiode array detector). AsA analysis usually occurred on the same day within a few hours of harvest, although at peak harvest time it was sometimes necessary to store apples for a maximum of up to 48 h in a cool cell at 2°C before extraction. In total, the fruit from 136, 140, or 163 individual progeny were analyzed for AsA, totAsA, and DHA concentrations in the season of 2005, 2006, and 2009, respectively, whereas GSH, totGSH, and GSSG measurements of the same samples were carried out in fruit only in 2006 and 2009.
The acid-soluble TAA of the fruit tissues harvested in 2006 was determined as the sum of the 2,2-azinobis (3-ethylbenzthiazoline-6-sulfonic acid)-reactive substances present in aliquots of the same extracts used to measure AsA and GSH concentrations (Davey et al., 2007). A734 was measured in three technical replicates of all samples using the Multiskan Spectrum Microplate spectrophotometer (Thermo Labsystems). AsA was used as an external standard, and the TAA of each extract was expressed as AsA equivalents.
Linkage Map Analysis and Development of a Bin Mapping Set
The available genetic maps of cv Telamon and Braeburn (Kenis et al., 2008) were further saturated with 14 additional microsatellite loci and CG-based markers to improve marker density in key chromosome regions. The updated linkage maps of cv Telamon and Braeburn were constructed using the JoinMap 3.0 software (Van Ooijen and Voorrips, 2001), essentially as described previously (Davey et al., 2006; Kenis et al., 2008).
A total of 324 and 339 molecular markers were initially ordered in 17 linkage groups, corresponding to the 17 apple chromosomes. To improve the resolution of QTL analysis, markers that were less informative (scored on less than 50% of genotypes), markers with distorted segregation, or markers with two alleles heterozygous in both parents (hk × hk segregation) were discarded, unless the gap between neighbor markers was greater than 10 centimorgans. A subset of 298 markers was used to construct the cv Telamon genetic map, with 96% of the markers highly informative (ab × aa, ef × eg, ab × cd), whereas 313 markers were employed for the cv Braeburn genetic map, which contained less than 10% less informative markers (hk × hk). A bin set of 14 highly informative genotypes was created essentially as described by Chagné et al. (2008). Successful markers were considered those that were heterozygous in at least one parent, had a normal segregation ratio in the bin set, and mapped onto the expected position on the bin maps.
QTL Analysis
QTL for AsA, GSH, and fruit physiological traits were identified using phenotypic data from 2005, 2006, and 2009 and updated genetic maps of the TxB population. QTL mapping was carried out using the MapQTL 4.0 software (Van Ooijen et al., 2002). QTL were detected using interval mapping in combination with restricted multiple QTL model mapping (rMQM) for normally distributed traits with a step size of 1 centimorgan or using the Kruskal-Wallis nonparametric test for nonnormally distributed ones. In rMQM, the most informative marker with the highest LOD score within the intervals of a QTL was selected as a cofactor, and multiple rMQM runs were performed until the cofactors selected were stable, as described previously (Davey et al., 2006). The LOD threshold for the significance of a QTL was selected based on the genome-wide level, using 1,000 permutations at 90% for each trait (around 3.5 or 3.0 for fruit and leaf tissues, respectively). A QTL was classified as significant/nonsignificant and major/minor based on a LOD threshold of 3.5 and 20% of explained population variability. QTL identified were described by the marker with the highest LOD score in the corresponding QTL region, and QTL regions were defined as the 1.5-LOD support interval. Linkage maps and QTL were drawn using MapChart 3.0 software (Voorrips, 2002).
Candidate Gene Mapping
The sequences of AsA-related CGs from Arabidopsis or other plant species were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), PLAZA 2.0 (http://bioinformatics.psb.ugent.be/plaza/), Phytozome Genome Browser (http://www.phytozome.net), or the SOL Genomics Network (http://solgenomics.net). Orthologous sequences were retrieved from the cv Golden Delicious assembly (Velasco et al., 2010) using BLASTX versus the apple consensus CDS set. Gene positions on the genome assembly and their sequences were extracted from the IASMA genome browser (http://genomics.research.iasma.it). The amino acid sequences for each CG were aligned using the BLOSUM62 matrix, and phylogenetic analysis was conducted using the neighbor-joining method and 1,000 bootstrap replicates in Geneious software version 5.4.6 (Drummond et al., 2011) in order to determine the number of copies present in apple for each Arabidopsis accession. The positions of these homologs were aligned in silico across the 17 linkage groups of the cv TxB map, based on the physical positions of framework simple sequence repeats.
SNP Marker Development
RNA-Seq data from cv Telamon and Braeburn were used to identify SNPs and polymorphisms in CG sequences. In total, 48.1 and 73.6 million 75-bp paired end reads, respectively, were generated from RNA pooled from eight different developmental stages of tree bud development and two tissue types. Reads were trimmed and then mapped against the apple consensus CDS set, downloaded from the GDR Web site (http://www.rosaceae.org/), using default mapping parameters within CLCBio Genomics Workbench version 5.5. CLCBio software was also used to carry out the de novo assembly of reads into contigs for each cultivar separately, again using default alignment parameters and the automatic scaffolding function. SNPs were called relative to the cv Golden Delicious reference CDS on the basis of using a minimum read depth of 6-fold and a percentage proportion coverage of greater than 25%. Finally, to control gene annotation, the consensus sequences from both the mapped reads and the de novo contigs were extracted and aligned against the annotated genomic DNA contigs used to create the apple genome assembly, again downloaded from GDR and using the Geneious version 5.6 software package. Functional descriptions of consensus CDS and translated protein sequences were confirmed using Blast2GO software (http://www.blast2go.com/b2ghome) as well InterPro (Geneious version 5.6). To validate gene function, clones of GGPs were expressed in Escherichia coli, and GGP activity was measured essentially as described previously (Laing et al., 2007).
PCR primers were developed to identify length polymorphisms, or flanking SNPs, to allow progeny genotyping using the HRM technique (Chagné et al., 2008; Studer et al., 2009). Gene-specific SNP primer pairs were designed using Primer3 software version 4.0 (http://frodo.wi.mit.edu/primer3/) employing the following conditions: product size between 75 and 200 bp, with optimum 120 bp spanning the identified SNPs; GC% ranging from 40% to 55%; self-complementarity and 3′ self-complementarity were set at 4 and 1, respectively; and all the other parameters were set at default. All CG-derived markers developed were screened in the cv TxB bin mapping set and both parents.
Genomic DNA from leaves was extracted from approximately 100-mg powdered aliquots using the Genomic DNA Purification kit (MBI Fermentas). HRM conditions were as follows: the PCR for each sample was carried out in 15 μL containing the HRM Master Mix (Type-it HRM PCR Kit; Qiagen), 10 to 20 μm of each primer, and 5 ng of genomic DNA. Cycling conditions were incubation at 95°C for 5 min, followed by 40 cycles of 95°C (10 s) and 55°C (30 s). HRM analysis was followed immediately by denaturation from 65°C to 95°C, rising by 0.1°C in each step. The HRM analysis was carried out in a Rotorgene Q thermocycler software (Qiagen). Markers that mapped to their expected positions and gave an expected segregation rate were further screened over 100 individuals from the cv TxB population to construct new genetic maps, as described previously.
Allelic Typing and Gene Expression Studies
Gene expression studies were performed following Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (Bustin et al., 2009). Total RNA was isolated from flesh fruit tissues using a modified cetyl-trimethyl-ammonium bromide method (Gasic et al., 2004) and the RNeasy Plus Mini Kit following the manufacturer's instructions (Qiagen). The purity of total RNA extracted was determined as the 260/280-nm or 260/230-nm ratio with NanoDrop 2000 (Thermo Scientific), and the integrity was checked by electrophoresis on a 1% agarose gel (Gel Doc EZ Imager; Bio-Rad). One microgram of total RNA was reverse transcribed to cDNA with SuperScript II (Invitrogen) using random oligo(dT) primers. The expression profiles of CGs considered to be key control points for AsA accumulation (MdGGP1, MdGGP2, MdGGP3, MdDHAR3-3, and MdNAT7-2; primer pair sequences are listed in Supplemental Table S9) were validated by real-time quantitative PCR using SYBR Green I technology on a Rotor Gene Q (Qiagen). 18S-rRNA (5′-GTTACTTTTAGGACTCCGCC-3′ and 5′-TTCCTTTAAGTTTCAGCCTTG-3′), Malus spp. eIF-4A (5′-ATCAGGCTCATCCCGTGT-3′ and 5′-AGCAACACCCTTCCTTCC-3′; Zubini et al., 2007), and Arabidopsis Actin11 (5′-GGACCTTGCAGGCCGTGACC-3′ and 5′-AACCTCCGGGCAGCGGAATC-3′) were used as reference genes. The stability of the expression of the references genes was assessed with NormFinder software (http://www.mdl.dk/publicationsnormfinder.htm). All reactions were set up in duplicate, containing a 1-μL cDNA template (50 ng), 7.5 μL of Absolute QPCR SYBR Green Mix, and 1 μL of each primer (3.75 mm) in a final volume of 15 μL. The cycling conditions were as follows: denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 20 s, annealing at 63°C for 20 s, and extension at 72°C for 20 s. A melting curve analysis was performed, ranging from 55°C to 95°C, with temperature increasing in steps of 0.5°C s−1. The criteria of acceptance for reaction efficiency ranged from 0.83 to 1.03 and both r and r2 > 0.97. For each run, a standard curve was included based on cDNA pooled from all samples to be analyzed in a range of different dilutions. The relative quantification of expression levels was performed using the comparative cycle threshold method (Pfaffl, 2001). Two technical replications were performed for each of the three biological replications per sample. All expression data were calculated as an expression ratio relative to the geometric mean of the expression of 18S-rRNA and Actin, as these showed the highest stability in our sample set, with stability values (Vandesompele et al., 2002) of 0.34 and 0.47, respectively.
Descriptive Statistical Analysis
Statistical analysis of all traits was carried out using the SAS 9.2 software package (SAS Institute). Mean values, sd values, ranges, and skewness of the F1 progeny were calculated for each year and trait, while the normality of the distributions was tested using the Shapiro-Wilk test (Shapiro and Wilk, 1965). Pearson correlation coefficients were employed to compare mean values of the traits over the three years. The significance of the mean fold difference in totAsA concentrations of genotypes carrying different alleles/allele combinations, as well as the significance of the mean relative expression of CGs of high/low AsA genotypes, were tested with Student's t test (*P < 0.05, **P < 0.01, and ***P < 0.001).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Climatic conditions prior to fruit harvest.
Supplemental Figure S2. Genetic linkage maps of cv Telamon (TEL) and Braeburn (BR) used for QTL analysis.
Supplemental Figure S3. Alignment of the MDP0000172222 (MdGGP1) consensus CDS from apple cultivars Braeburn (Br), Telamon (Tel), and Golden Delicious (GD) with the full-length EST sequence from cv Royal Gala (gi|225380879).
Supplemental Figure S4. Alignment of the MDP0000172222 (MdGGP1) consensus CDS from apple cultivars Braeburn (Br), Telamon (Tel), and the full-length EST sequence from cv Royal Gala (gi|225380879) with the genomic DNA scaffold MDC003775.57.
Supplemental Figure S5. Alignment of the MDP0000191488 (MdGGP2) consensus CDS from apple cultivars Braeburn (Br), Telamon (Tel), and Golden Delicious (GD).
Supplemental Figure S6. Alignment of the MDP0000191488 (MdGGP3) consensus CDS from apple cultivars Braeburn (Br), Telamon (Tel), and Golden Delicious (GD).
Supplemental Figure S7. Alignment of the MDP0000191488 (MdGGP3) consensus peptide sequences (translation products) from apple cultivars Braeburn (Br), Telamon (Tel), and Golden Delicious (GD).
Supplemental Table S1. Correlations between fruit antioxidant metabolite concentrations.
Supplemental Table S2. Correlations between fruit antioxidant metabolite concentrations over the three years of measurements.
Supplemental Table S3. Overview of population mean values and distribution for foliar AsA, DHA, totAsA, GSH, GSSG, and totGSH concentrations.
Supplemental Table S4. Correlations between GSH traits in foliar and in fruit flesh and skin tissues.
Supplemental Table S5. Correlations between fruit AsA concentrations and physiological traits.
Supplemental Table S6. QTL mapping for fruit antioxidant concentrations.
Supplemental Table S7. QTL mapping of fruit physiological traits.
Supplemental Table S8. Apple orthologs of candidate genes of AsA metabolism.
Supplemental Table S9. List of selected candidate gene orthologs of AsA metabolism mapped on the Telamon × Braeburn genetic linkage maps.
Supplemental Table S10. Activities of the clones of Malus sylvestris (GB CN915822) and apple (CN939721) with 100% identity to MdGGP1 and MdGGP2, respectively, at the transcript level when expressed in E. coli.
Supplemental Table S11. Polymorphisms present in cv Braeburn and Telamon MDP0000172222 (MdGGP1) consensus CDS, and read coverage per allele.
Supplemental Table S12. Polymorphisms present in cv Braeburn and Telamon MDP0000288088 (MdGGP2) consensus CDS, and read coverage per allele.
We acknowledge stimulating discussions with Otto van Poeselaere.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mark W. Davey (mark.davey{at}biw.kuleuven.be).
↵1 This work was supported by Katholieke Universiteit Leuven (FLOF fellowship to I.M.) and by the New Zealand Institute for Plant and Food Research Limited (travel grant to I.M. and Pipfruit Research Investment Project funding to D.C. and W.A.L.).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- quantitative trait loci
- AsA
- l-ascorbic acid
- glutathione
- dehydroascorbate
- dehydroascorbate reductase
- monodehydroascorbate reductase
- candidate gene
- single-nucleotide polymorphism
- totAsA
- total ascorbic acid
- totGSH
- total glutathione
- TxB
- Telamon × Braeburn
- total antioxidant activity
- oxidized glutathione
- Br_time
- time taken for cut surfaces to brown
- Br_color
- final color of the affected tissue
- linkage groups
- log of the odds
- coding sequence
- high-resolution melting
- reads per kilobase exon model per million
- rMQM
- restricted multiple QTL model mapping
- Enzyme Commission
- Received July 17, 2012.
- Accepted September 19, 2012.
- Published September 21, 2012.