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People | 5 |
Bedrooms | 1 |
Bathrooms | 1 |
Property type | cottage |
Minimum Stay | 2 nights |
This budget rental listing is a great get-a-way, semi-secluded cottage style property. It has been recently up dated. There is a large open living space and completely furnished kitchen. Nature lovers will love this wooded location next to a private pond.
Sleeps 5 adults. Second floor sleeping area, Master Bedroom has a queen size, Serta Vera Wang mattress set. Hallway sleeping area; one full size bed and one single.
Enjoy the wonders of the Northern Adirondacks and or the Champlain Valley by day and return for a relaxing soak in the Hot Tub while listening to the loons.
Plattsbugh's Hidden A-way, a destination that has to be seen to be believed. Ideal for a family vacation, or a romantic weekend get-a-way with that special someone. This property requires a minimum stay of two nights. Responsible individual renting/staying at the house must be at least 25 years of age.
Please note that a three day advance booking is required, to rent this property.
Keith, we enjoyed our week at your place so much. The write up and pictures were exactly how it was. Very clean and comfortable. Found the location to be perfect, far enough off the road for privacy and close enough to the City/Town of Plattsburgh to be convenient for everywhere you had to go. Mainely Lobster was our favorite restaurant for seafood. The Naked Turtle Bar/Restaurant on the water was very a relaxing place for drinks at the outside bar overlooking the marina. We will stay next time we're in Plattsburgh, beats any motel! Thanks again Keith for opening your home to us. Bridget and Steve
Clean, quiet and a very relaxed setting. We could have spent hours just sitting on the deck reading. The only sounds were the occasional cries of the geese. Would definitely recommend this property for a place to recharge and energize.
We had an absolutely fantastic time at this property. A romantic getaway that we will never forget. So much privacy and yet close enough to get a bite to eat, grocery run or even catch a movie on a rainy day. We will be back soon.
Our family had a terrific stay. The house is in a beautiful spot overlooking a small lake (maybe a reservoir?) We walked trails from our backyard, checked out the goats and horses next door, and the kids spent hours in the hot tub. From the moment we arrived we felt instantly relaxed and comfortable. Keith was easy to contact and helpful at every turn. We hope to return next summer!
Amazing property in the beautiful Adirondack area. Charming cabin with super comfortable bed, amazing hot tub and a great view of the wildlife. Internet was the only thing to not count on but if you are looking to get away from it all, this is a great place to do it. Only other suggestion would be a portable speaker for playing music :) Thanks Keith!
Private, easy to get to, plenty of good restaurants, and a mall. Relax in the hot tub while gazing at the stars. Have your morning coffee with the ducks and the loons. Your closest neighbors are two horses and a goat.
Thanks Keith
My son and his family stayed here. It was a great place for family to get together and spend quality time. Very private, well maintained and close to shopping. Would definitely book it again. The owner, Keith was very nice and accommodating.
Rental Period | Minimum Price | Additional Nights | Weekend Night | Weekly | Monthly | Event Price | |||
Off season rate
6 Sep 2016 -
4 May 2017
2 night minimum stay
for 2 nights
In Season Rate
5 May 2017 -
5 Sep 2017
2 night minimum stay
for 2 nights
My Standard Rate
2 night minimum stay
for 2 nights
Cleaning Fee | £52 |
Refundable Damage Deposit | £200 |
NO Pets
NO Smoking
Discharging of firearms on property is prohibited.


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An Original Full Page coloured Engraving from THE GRAPHIC NEWSPAPER of December 1880. Page size is 16" x 11 3/4". Minor age spots in border area. From a painting by J.CHARLTON.
At Molloy's MEGA ANTIQUES CENTRE, Takapuna, Auckland, New Zealand
Specialists in Pacific Islands Items, Victoriana, And a Fascinating Range of General Collectibles
Shipping & Handling: | To Be Determined |
Insurance: | To Be Determined |
Shipping to: |
USA change |

subset: commoncrawl


text:
College Football Things That Bug Me Part 3
Its been a while since I added to the list of things that bug me. So far there are two articles actually titled that and another that was a little less direct. Anyway just a few more things that have come up that I have to put out there. Polls A poll would mean that multiple people were asked and their answers were put together to calculate the standings. Please stop with the this is my poll if it is just your opinion and no one else was involved in making it. These would be your rankings but not your poll. Recruiting Rankings It isn't the rankings themselves that I have issue with. No it is the high ranking for a class with average talent but like 30 or more recruits. That or a recruiting class that brings in a whole bunch of one positions where the school is already loaded at that same position. Does either situation really deserve high praise? Maybe. But I am not sold on it. It is also the stalk people put in to them. Yes the top schools in the rankings generally have the top recruiting classes too. But that isn't true for all of them and it doesn't mean success is going to come. Divisions With all the talk of conference expansion and creating divisions I have read many articles set forth in speculation for how conferences would divide. Many people seem to think naming the Big 10 divisions for Woody and Bo would make sense. I just have to say that's stupid. As respected as these men were, I find it hard to believe that the other 10 schools would ever agree to it. North/South, East/West even some sort of regional like Mountain and Coastal make more sense. Big Ten Well the conference itself doesn't bug me. None of the teams bug me. Sometimes, some of the fans do, but that's not where I am going with this. No my problem here is when people write something about them and refer to them as the Big Eleven or the Big 10 + 1 or the Big 10 (11) or any other such notions. We all know there are eleven teams in the conference. The official name of the conference is the Big Ten, just go with it. Putting 11 in instead makes it seem as pretentious as THE Ohio State. Expansion Everyone has an idea or two about expansion and realignment. But this is about conferences as a whole. People tend to give their opinion on what conferences should do with only regard to football and forget that conferences play and participate in many other sports. As well as the athletics, there is also academics. Fitting the profile of the conference in academics is just as important as fitting in in athletics. Being in or out of a region is less important if the name of the conference isn't specific, and even then it still happens. Idaho used to be part of the Sun Belt, but no one would think to look for them on a map in that region. Louisiana Tech is in the Western Athletic Conference, I don't think I need to explain why that doesn't really fit. So if the Big Ten or Big XII go after some teams that seem outside their region, don't forget their names don't limit them.
Like the new article format? Send us feedback!


text:
Published in
Velocity Micro and Overdrive PC get Razerised
Systems will come with Razer peripherals
According to a press release issued by Razer and Velocity Micro and Overdrive PC, systems from both companies will now be available with the entire line-up of Razer's peripheral.
In addition to the choice of Razer-praised gaming peripherals, Velocity Micro's Raptor series PCs will exclusively include Razer's mouse and keyboard with each purchase. The mouse in question is Razer's DeathAdder and those that decide to go for Velocity Micro's Raptor series system will also enjoy Razer's Lycosa gaming keyboard.
It is nice to see that Velocity Micro and Overdrive PC have decided to go for Razer, as they are taking a serious leap to be formidable gaming PC system builders.


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Thursday, 8 November 2012
PokerStars heads west for the latest leg of the UK and Ireland Poker Tour with Bristol's Gala Casino the venue for the £700+£70 Main Event which gets under way today.
The first of two opening flights starts at noon with Day 1B following at the same time tomorrow with the winner set to be crowned on Monday.There's plenty of great side action scheduled over the next few days including a £2,000 High Roller as well as PLO, Turbo and Deepstack tournaments.The complete line up can be seen below:Thu 8 Nov12pm £700+£70 UKIPT Bristol Main Event Day 1a 8pm £100+£10 NLHE Turbo Fri 9 Nov12pm £700+£70 UKIPT Bristol Main Event Day 1b 9.30pm £100+£50+£10 NLHE Turbo Bounty (£50 bounty on each player) Sat 10 Nov12pm UKIPT Bristol Main Event Day 2 6pm £2,000+£150 NLHE High Roller Day 1 (8-handed) 8pm £300+£30 NLHE Day 1 Sun 11 Nov12pm UKIPT Bristol Main Event Day 3 1pm NLHE High Roller Day 2 2pm NLHE Day 2 Sun 11 Nov14:00£200+£20 Pot Limit Omaha 3pm £100+£10 NLHE Women's Event 5pm £150+£15 NLHE Turbo Deepstack Mon 12 Nov12pm UKIPT Bristol Main Event Final TableJoin us over the weekend for updates from what promises to be an exciting few days in Bristol.
Juicy Stakes has just launched a range of new online poker tournaments and is celebrating by offering a limited-time 50% re-load deposit bonus worth up to $100.
Intertops Poker and Juicy Stakes are offering online poker players the chance to take part in the main event at the upcoming St Maarten stop of the TK Poker Tour for as little as $1.10.
The Winter Poker Championship Seefeld 2017 is set to kick off from February 16 and one lucky online poker player will be taking part after winning a prize package worth $4,000 into the Austrian extravaganza's main event at Intertops Poker on Sunday.
The Winter Poker Championship Seefeld 2017 is set to run from February 16 to February 26 and online poker players are being offered the chance to win a prize package into the Austrian tournament series' main event for as little as $3.30.
Sunday Six Max and Sundowner.
Intertops Poker and Juicy Stakes.
Amateur wins at Intertops Poker.
Juicy Stakes and Intertops Poker.
Poker's unassuming killer steps back into the limelight.
Meet one of UK poker's rising stars.
We talk to WSOP bracelet winner.
By Paul Oresteen.
Just keep smiling...
By Adam Slutsky
Pushing the envelope for a new poker generation.
Jeff KImber on fish.
So says Dara O'Kearney.
Dara O'Kearney looks at game thoery.
Dara O'K on bounty tournaments.
Rob Yong calls for competition.
A critical eye on the poker world.
Nick Wealthall on unconscious decision making.
Structure changes don't suit recreationals.


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Wisconsin Aspergers Empowerment Message Board › The SE Wisconsin Aspergers Meetup Group Discussion Forum › Thursday Meeting Canceled
Carole B. |
The meeting this Thursday was supposed to feature the architect but he was called out of town for another meeting and has asked to reschedule for next month. I was going to contact everyone and let them know of this change and ask for suggestions on a new topic. However, today my 3-year old niece passed away. It wasn't unexpected, but we were all hoping for a miracle.
In light of these two events I am canceling the meeting. The room will still be available and you are welcome to meet without me. Just inform everyone through this meetup of your intention so that no one shows up and is the only one there. Thank you for understanding. Carole |
Meetup members, Log in
By clicking "Sign up" or "Sign up using Facebook", you confirm that you accept our Terms of Service & Privacy Policy


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Find the best surf spots |
Sunday February 21 2010 09:30:00 AM
Surf spot: Grand Crohot, France
Session's Rate:
Wind Strength: Strong
Nb waves ridden: 12
Length of the surf session: 90 min
Surfboard ridden: 6'0
Weather and water temp: Air: 7°C, Water: 10°C
Waves height: 1.0m-1.5m / 3ft-5ft
Waves Quality:
- Fun
- Bumpy
Waves Shape:
- Multipeaks
The swell was here but the wind was a little too strong. I got funny waves but nothing incredible.
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Modified: Thursday March 4 2010 11:12:24 AM
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Find a copy in the library
Finding libraries that hold this item...
Additional Physical Format: | Online version: Allen, Terry D. Writing to create ourselves. Washington, D.C. : Office of the Assistant Commissioner for Eduacation, U.S. Bureau of Indian Affairs, 1969 (OCoLC)681460957 |
Material Type: | Government publication, National government publication |
Document Type: | Book |
All Authors / Contributors: |
Terry D Allen; John Povey; United States. Bureau of Indian Affairs. Division of Education. |
OCLC Number: | 6585912 |
Notes: | "November, 1969"--Cover. |
Description: | xvi, 165 pages ; 28 cm. |
Contents: | Why write -- Five doors -- Here & now -- What shall I write? -- Now that you have a manuscript -- The raw material of the writing business -- The teaching of poetry -- The writing of poetry -- Building blocks for prose writing -- Before you start to write a story -- Teacher exchange -- Capsule lessons in writing. |
Series Title: | Curriculum bulletin (Washington, D.C.), no. 2. |
Responsibility: | by T.D. Allen ; with foreword & chapters on poetry by John Povey. |
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Roma vs Lazio Live StreamingThroughout the years in Italy and on the European stage, SS Lazio have continued to enthrall fans with free playing flowing football, and their successes of the past century are a testament to the passion that runs through the veins of the club from Rome. Known as Societa Sportiva Lazio, and nicknamed "Biancocelesti", the club are a mainstay in the Campionato, the top division in Italian football and have seen a resurgence in form over the last few years as they look to return to the top stages of European competitions such as the Champions League. With Lazio fans across the world, fans in their thousands look for the latest tv and internet coverage for matches from the Scudetto from the Italian club. The Roma vs Lazio live stream is our next scheduled match and fans can see below for the latest free streams to watch the game online. If your one of those fans unable to attend the fantastic 72,000 seater Stadio Olimpico or unable to watch on TV, read on for more on how to catch the game on the internet. Our Next Live Stream: Roma vs Lazio, latest links posted on 30-03-17 . Live SS Lazio Streaming:Sky Sports and their Calcio TV package is the top source for watching Lazio matches on television in Italy. Across Europe, other satellite providers are available giving fans a chance to follow the club at home. If you are one of those unable to watch SS Lazio games on your tv, why not watch the matches online? Our streams below allow fans to watch Roma vs Lazio live on their pc, mac or iPad! Look for high quality streams and full screen coverage with English and Italian commentary, giving a fantastic user experience for football fans of Lazio.
Roma vs Lazio live stream Links will be posted 1 hour before kick-off
History of Lazio of Rome. Success of Lazio Watch Lazio Live Online © LiveFootballStreams.co.uk - Watch Lazio Live Online |


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Eee! I ended up winning for the month of March! o.o
Time to necro this blog a little with a bit of joyful and fresh news - consulting this page and Avedral's deviantArt journal, the winner in March's contest is...Avedral!Not just honorable mention, but winner now. Looks like Blizzard will be thinking twice when dealing with Wowhead-ers ;)
Man that's awesome. I love how small time artists can win such great honor. :)Congratulations Nancy!
Many congratulations Avedral... may many more winning comics follow! :D


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Identification of novel microRNAs in Hevea brasiliensisand computational prediction of their targets
© Gébelin et al; licensee BioMed Central Ltd. 2012
Received: 5 October 2011
Accepted: 13 February 2012
Published: 13 February 2012
Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis.
Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented.
Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea.
Gene expression miRNA MIR gene Next-generation sequencing Rubber tree Transcription Transcriptome Abiotic stress miRNA editingBackground
Hevea brasiliensis, the sole commercial source of natural rubber, is a tropical perennial species native to the Amazon basin. South-East Asian countries supplied 92% of the 10 million tons of natural rubber produced in 2010, but ever-growing worldwide demand calls for enhanced tree productivity. This goal is increasingly becoming a crucial challenge for the corresponding research activities in a context of global climate change and redistribution of land for food crops. Natural rubber is a cis-1,4 polyisoprene polymer biosynthesized in rubber particles located in specialized latex cells. Latex cells are periodically emitted from the cambium and then anastomosed to form latificer mantels [1]. Natural rubber is harvested by tapping. The cytoplasm of laticifers, containing 30-50% of rubber particles, is then expelled by high turgor pressure maintained in the soft bark tissues. An ethylene generator, 2-chloroethylphosphonic acid (ethephon), is applied to the tapping panel to stimulate latex production. Ethephon increases the duration of latex flow after tapping and its regeneration between two tappings [2]. Tapping Panel Dryness (TPD) is a physiological disease that causes 10-40% annual rubber production losses over the 30 years of a rubber cultivation cycle. This physiological disorder, triggered by oxidative stress, is partially induced by excessive environmental and harvesting stresses [3]. The generation of reactive oxygen species (ROS) in latex cells leads to in situ coagulation of rubber particles [4, 5]. Research on ROS in plants initially focused on their cytotoxicity only, but nowadays ROS are also considered as signalling molecules [6, 7]. A ROS signalling network emerges and is conserved in all aerobic organisms [8]. The ROS-scavenging system acts both to maintain redox homeostasis and to protect cell components from oxidative damage. Biotic and abiotic stress, such as drought, salinity, strong light, temperature, heavy metals, UV radiation, atmospheric contamination, mechanical wounding, nutrient starvation and pathogen attacks are major sources of ROS in plants [9].
MicroRNAs (miRNAs) particularly play a key function in responses to abiotic stress (for a review see [10–13]). Indeed, small RNAs are involved in fine-tuning gene expression in response to physiological, external and developmental stimuli [14, 15]. Of these, miRNAs have been shown to be a large group of small endogenous RNAs that exist in animals, plants and viruses. Since their discovery in Caenorrhabditis elegans [16] and in Arabidopsis thaliana in 2002 [17], miRNAs have been described as negatively regulating gene expression by targeting specific messenger RNAs (mRNAs) for cleavage [18, 19] or translational inhibition [20]. The biogenesis of miRNAs has been amply described in plants and animals [21–24]. Briefly, a MIR gene is first transcribed to primary miRNAs (pri-miRNA) by RNA polymerase II, the 70-500 bp long pri-miRNA forms an imperfectly paired hairpin which is processed by RNAse III-like DICER-like I (DCL1) to generate miR precursors (pre-miRNAs) [25, 26]. Further cleavage of the pre-miRNA by DCLI releases a miRNA/miRNA* duplex. The duplex is then translocated into the cytoplasm by HASTY (plant ortholog to exportin 5 [27]), the canonical mature miRNA of 20-22 nucleotides is selectively incorporated into the RNA-induced silencing complex (RISC) associated with Argonaute 1 (AGO1). In the RISC complex, miRNAs bind to mRNA and inhibit gene expression through perfect or near-perfect complementarity between the miRNA and the mRNA [18]. In plants, the recent discovery of MIR genes generating both canonical miRNA (20-22 nucleotides) and long miRNA (lmiRNA, 23-27 nucleotides) suggests their bifunctionality [28]. The lmiRNAs derived from MIR genes are selectively processed by DCL3, can associate with AGO4 and guide DNA methylation at some of their target loci in trans in rice and moss [28, 29]. The recent and major discovery of the role played by post-transcriptional modifications of miRNA precursors highlighted the complexity of miRNA biogenesis and the way miRNAs act [30, 31]. Indeed, adding an oligo-uridine tail to the 3' end of the miRNA precursor is a mechanism that controls miRNA production in mice [31]. In plants, it has been reported that either a combination of 5' deletion and 3' uridylation of miRNA, or RNA editing events, alter the targeting and preference of Argonaute association [30]. Several miRNAs have been described as being up-regulated or down-regulated by high salinity, drought and low temperatures [32, 33]. Furthermore, the miRNA-targeted gene copper zinc superoxide dismutase (CuZnSOD), which plays a major role in maintaining redox homeostasis, is cleaved by miR398 under stress-free conditions in Arabidopsis [34, 35].
Currently, 15,172 mature miRNAs have been discovered and deposited in the public database, miRBase (Release 16, 2010, http://www.mirbase.org) [36, 37], and 9,277 mature plant miRNAs in the Plant MicroRNA Database, PMRD http://bioinformatics.cau.edu.cn/PMRD[38]. Most miRNAs have been identified in model species such as Arabidopsis, Oryza [39, 40], Populus [12, 41], Physcomitrella [42], Vitis [43], whose genomes have been sequenced. Some miRNAs have also been identified in Glycine max [44], Arachis hypogaea [45], Solanum lycopersicum [46], Brassica napus [47], Phaseolus vulgaris [48] and in the euphorbiaceous species Ricinus communis [49]. Twenty-three miRNA families predicted in Ricinus communis were experimentally validated in four euphorbiaceous plants (Ricinus communis, Jatropha curcas, Manihot esculenta and Hevea brasiliensis) [49]. However, the strategy used did not allow exhaustive identification of miRNA families in Hevea.
The availability of large collections of Hevea Expressed Sequence Tags (ESTs; http://bassigny/cgi-bin/esttik_dev/quick_search.cgi) and next-generation sequencing techniques offers prospects for understanding the post-transcriptional regulation of key functions involved in responses to stress and in latex production in Hevea brasiliensis. This study involved the deep sequencing of small RNAs from Hevea plants grown under various conditions. We report here on the identification of 48 conserved miRNA families, sharing very high homology with that already known in other species. MiRNA precursor sequences were identified for nine families. In addition, ten putative novel miRNA families were also identified with their precursors. A scan of the Hevea EST databases with miRNA sequences revealed their putative targets. Of them, the predicted targets involved in responses to stimuli, the ROS-scavenging systems and transcription regulation are presented, and new insights into the control of redox status in Hevea are proposed.
Classification of small RNAs
Statistics of small RNA sequences from the Hevea brasiliensis small RNA library
Stress small RNA library | Sequence number | Analysis |
read sequences | 4,223,792 | |
clean sequences | 2,378,135 | Solexa sequencing |
unique clean sequences of small RNA | 670,645 | |
no hit | 629,795 | |
hit, unknown | 26,721 | |
coding mRNA | 6,618 | |
transposons/transposable element | 6,800 | |
rRNA | 399 | Blast against Arabidopsis |
pseudo mRNA | 138 | genome |
ncRNA | 73 | |
miRNA | 57 | |
tRNA | 39 | |
sn/snoRNA | 5 | |
no hit | 652,215 | |
hit | 18,430 | Blast against PMRD |
conserved miRNA family | 48 | |
putative novel miRNA family | 10 | LeARN pipeline |
List of the treatments applied to Hevea plants before small RNA isolation
Treatments | Plant material | Tissue | Condition |
Ethylene | in vitro plantlet (2 years old) | leaf, bark, root | 5 ppm for 4 hours |
budded plant | leaf, bark | ||
Methyl-jasmonate | in vitro plantlet (2 years old) | leaf, bark, root | 0.3 μM for 4 hours |
budded plant | leaf, bark | ||
Wounding | in vitro plantlet (2 years old) | leaf, bark, root | 2 hours |
budded plant | leaf, bark | ||
Drought | in vitro plantlet (2 years old) | leaf, bark, root | 10 days |
budded plant | leaf, bark | 4 weeks | |
Flooding | in vitro plantlet (2 years old) | leaf, bark, root | 4 days |
budded plant | leaf, bark | ||
Cold | in vitro plantlet (2 years old) | leaf, bark, root | 4°C for 8 hours |
budded plant | leaf, bark | ||
Light | in vitro plantlet (2 years old) | leaf, bark, root | PAR: 1000-1500 μmol/m2/s for |
budded plant | leaf, bark | 4 hours | |
NaCl | in vitro plantlet (2 years old) | leaf, bark, root | 300 mM for 4 days |
budded plant | leaf, bark | ||
in vitro plantlet (2 years old) | leaf, bark, root | ||
Control | budded plant | leaf, bark | |
in vitro plantlet (1 month old) | leaf, bark, root | ||
callus | callus |
Annotation of the Hevea small RNAs was attempted by BLASTN on the Arabidopsis genome. Most of the sequences did not map onto the Arabidopsis genome sequence. Of the mapped sequences, 26,721 were not annotated. Annotated sequences predicted 6,618 products of mRNA degradation, 6,800 transposable elements, 399 rRNAs, 138 products of pseudogene degradation, 73 ncRNAs, 39 tRNAs, 5 sn/snoRNAs and 57 miRNAs corresponding to 14 families.
Characteristics of conserved and putative novel miRNA families in Hevea brasiliensis
Detection of single-nucleotide mutations in miRNA sequences
Frequency of single-nucleotide mutation and of a combination of 5' deletion and 3' uridylation (-1 + UU) for four miRNA families from Hevea
Family name in Arabidopsis | Single-nucleotide modification | -1 + UU modification | ||
Arabidopsis | Hevea | Arabidopsis | Hevea | |
AtmiR156 | G-A: 14.091% | A-U: 49.382% A-C: 3.571% | yes | no |
AtmiR159a | G-A: 8.597% | G-A: 4.166% | no | no |
AtmiR164a | G-A: 3.967% | G-A: 7.692% | no | no |
AtmiR172a | G-U: 0.599% | G-U: 6.153% A-U: 7.692% C-U: 7.692% | no | no |
Identification of precursor transcripts for conserved and putative novel miRNA families from the hevea brasiliensistranscriptome
List of precursors for conserved microRNAs identified in the Hevea clone PB260 transcriptome sequences
New miR name | Accession Name | Mature sequence | Mature length | miRNA* (Accession No.) | Precursor length | EST |
HbmiRn1 | acc_584359 | CAGGAACUGGUAUCAACCCAGC | 22 | nd | 130 | CL1Contig1853_r1 |
HbmiRn2 | acc_495109 | UAUUGUAGAAAUUUUCAGGAUC | 22 | nd | 116 | CL1Contig490_r1 |
HbmiRn3 | acc_185377 | UAAUGGGCUCUGCAUAGAUGG | 21 | Yes (3) | 108 | CL27Contig1_r1 |
HbmiRn4 | acc_108425 | UUGCAUAUCUCAGGAGCUUCA | 21 | Yes (4) | 214 | CL19922Contig1_r1 |
HbmiRn5 | acc_644211 | AAACGGCUACCACAUCCAA | 19 | nd | 87 | FYGXE6I01B755W_r1 |
HbmiRn6 | acc_501419 | UAGGAUGUAGAAGAGCAUAA | 20 | nd | 90 | F5VNCTM02H9XL3_r1 |
HbmiRn7 | acc_35715 | UAGUUUGUUUGAUGGUAUC | 19 | nd | 113 | FYGXE6I01EKFYA_r1 |
HbmiRn8 | acc_246127 | GAUUGACAGACUGAGAGCUC | 20 | Yes (16) | 93 | FYGXE6I01B7GPL_r1 |
HbmiRn9 | acc_103082 | UUUAUGAAAGACGAACAACUG | 21 | Yes (6) | 87 | CL1Contig2798_r1 |
List of putatively new miRNA precursors identified in the Hevea clone PB260 transcriptome sequences
MiRNA name | Accession Name | Mature sequence | Mature length | miRNA* (Accession No.) | Precursor length | EST |
Hbmir156 | acc_462135 | UUGACAGAAGAUAGAGAGC | 19 | Yes (10) | 119 | FYGXE6I01DJH4X_r1 |
Hbmir159 | acc_17403 | UUUGGAUUGAAGGGAGCUCUA | 21 | Yes (5) | 221 | GETTCZF01AWUSL_r1 |
Hbmir166 | acc_28021 | UCGGACCAGGCUUCAUUCC | 19 | Yes (14) | 127 | CL2671Contig1_r1 |
acc_150486 | UCGGACCAGGCUUCAUUCCCCC | 22 | Yes (14) | 120 | CL1Contig15359_r1 | |
Hbmir319 | acc_19786 | UUGGACUGAAGGGAGCUCCCU | 21 | Yes (7) | 221 | FRZQES201D34W3_r1 |
Hbmir396 | acc_112787 | UUCCACAGCUUUCUUGAACUG | 21 | Yes (15) | 154 | CL1Contig13811_r1 |
Hbmir408 | acc_393516 | AAGACUGGGAACAGGCAGAGCA | 22 | Yes (294) | 128 | FYGXE6I01CVDV9_r1 |
acc_39581 | ACUGGGAACAGGCAGAGCAUGA | 22 | Yes (294) | 120 | CL3908contig1_r1 | |
Hbmir476 | acc_508145 | UAAUCCUUCUUUGCAAAGUC | 20 | Yes (1) | 126 | CL1Contig11471_r1 |
Hbmir2910 | acc_251816 | GAGCGAUUUGUCUGGUUAAUC | 21 | Yes (5) | 126 | FYGXE6I01EDXGX_r1 |
Comparison of the gene ontology classification of predicted targets for conserved and putative novel miRNA families
Comparison of predicted target genes for conserved and putative novel miRNA families involved in "response to stimulus", and in "antioxidant" and "transcription regulation" activities
List of predicted targets for conserved miRNAs involved in response to stimulus, and in antioxidant and transcription regulator activities according to GO terms
MiRNA family | GO term | Predicted target function | mfe kcal/mol | Target name |
Transcription regulation activity | Squamosa promoter-binding protein | -45.50 | CL2120Contig2 | |
HbmiR156/157 | APETALA2-like protein | -24.41 | hevea_454_rep_c24306 | |
Antioxidant activity | flavonoid 3',5'-hydroxylase | -26.53 | CL2495Contig2 | |
AKR (aldo keto reductase) | -32.90 | hevea_454_rep_c15059 | ||
Transcription regulation activity | APETALA2-like protein | -27.80 | hevea_454_rep_c17780 | |
sarcosine oxidase | -29.70 | CL11607Contig1 | ||
serine/threonine protein kinase | -21.39 | CL11Contig15 | ||
cinnamoyl-CoA reductase | -32.88 | CL1Contig7212 | ||
CuZnSOD peroxysomal | -32.14 | CL1Contig3818 | ||
HbmiR159 | Antioxidant activity | ABC transporter C family | -28.02 | CL1Contig3612 |
cytochrome b6f complex | -36.18 | CL1Contig475 | ||
TATA box binding protein | -29.38 | CL1Contig760 | ||
oxidoreductase family protein | -32.88 | CL781Contig2 | ||
ATP Synthase | -27.31 | CL1613Contig1 | ||
HMG-CoA_reductase (HMGR) | -36.39 | CL8048Contig1 | ||
6-phosphogluconate dehydrogenase family protein | -33.44 | CL1Contig1588 | ||
HbmiR319 | Antioxidant activity | ferritin putative | -35.86 | CL1Contig17258 |
HbmiR160 | Transcription regulation activity | ARF | -52.20 | CL6582Contig1 |
Antioxidant activity | FAD/NAD(P)-binding oxidoreductase-like protein | -23.10 | hevea_454_rep_c15774 | |
HbmiR162 | ABC transporter C family | -26.47 | hevea_454_rep_c28843 | |
Response to stimulus | zinc finger family protein | -31.40 | hevea_454_rep_c20758 | |
HbmiR164 | Transcription regulation activity | NAC-domain protein | -44.12 | hevea_454_rep_c10543 |
amine oxidase | -32.67 | CL21795Contig1 | ||
Transcription regulation activity | NAC-domain protein | -32.43 | CL7990Contig1 | |
PHAVOLUTA-like HD-ZIPIII protein | -46.68 | hevea_454_rep_c12680 | ||
HbmiR165/166 | Antioxidant activity | LRR protein | -30.08 | hevea_454_c28164 |
Antioxidant activity/Response to stimulus | malate deshydrogenase | -38.95 | hevea_454_rep_c94776 | |
Response to stimulus | abscisic acid insensitive protein | -36.33 | hevea_454_rep_c69184 | |
Hbmir167 | Response to stimulus | cap binding protein | -33.60 | hevea_454_c94262 |
Transcription regulation activity | CCAAT-binding transcription factor | -32.79 | CL435Contig5 | |
HbmiR169 | Laccase | -26.51 | CL1Contig13876 | |
Antioxidant activity | aldehyde dehydrogenase | -26.56 | CL1Contig16398 | |
annexin-like protein | -32.71 | CL1Contig2060 | ||
HbmiR170 | Transcription regulation activity | GRAS domain-containing protein | -37.90 | hevea_454_rep_c11063 |
Transcription regulation activity | GRAS domain-containing protein | -39.50 | CL3142Contig4 | |
HbmiR171 | ribonucleoside-diphosphate reductase | -26.99 | CL1031Contig2 | |
Antioxidant activity | oxophytodienoate reductase (OPR) | -27.23 | CL3790Contig2 | |
glyceraldehyde-3-phosphate dehydrogenase | -24.88 | CL150Contig1 | ||
Transcription regulation activity | APETALA2-like protein | -24.25 | hevea_454_rep_c45080 | |
HD-ZIP protein | -33.63 | hevea_454_rep_c13497 | ||
HbmiR172 | Antioxidant activity | NAD(P)H-quinone oxidoreductase | -24.54 | hevea_454_rep_c13256 |
CLAVATA1 putative | -31.73 | hevea_454_rep_c2026 | ||
Squalene monooxygenase | -29.04 | hevea_454_rep_c32943 | ||
Response to stimulus | syntaxin | -31.00 | hevea_454_c68550 | |
aspartate aminotransferase | -33.44 | hevea_454_rep_c4007 | ||
HbmiR2111 | Antioxidant activity | laccase | -26.57 | hevea_454_rep_c5912 |
HbmiR2910 | Antioxidant activity | 2OG-Fe(II) -dependent-oxygenase-like protein | -30.83 | hevea_454_rep_c1596 |
ketol-acid reductoisomerase | -31.31 | hevea_454_rep_c5152 | ||
HbmiR2914 | Antioxidant activity | FAD Binding domain-containing protein | -58.64 | hevea_454_rep_c15793 |
Rboh | -31.88 | hevea_454_rep_c23550 | ||
Transcription regulation activity | Heat shock protein | -30.89 | CL11591Contig1 | |
Hbmir390 | WRKY transcription factor | -33.09 | CL338Contig4 | |
Antioxidant activity | Phosphoenolpyruvate carboxylase | -19.81 | CL362Contig2 | |
hydroxy-acid oxidase | -23.58 | CL8468Contig1 | ||
HbmiR393 | Transcription regulation activity | APETALA2-like protein | -22.79 | hevea_454_c60993 |
Transcription regulation activity | APETALA2-like protein | -24.47 | hevea_454_c37716 | |
Hbmir395 | Antioxidant activity | 2OG-Fe(II) -dependent-oxygenase-like protein | -30.00 | hevea_454_rep_c21129 |
Transcription regulation activity | APETALA2-like protein | -22.40 | hevea_454_rep_c13430 | |
Transparent TESTA protein | -34.16 | hevea_454_rep_c14652 | ||
GRAS domain-containing protein | -27.01 | hevea_454_rep_c8477 | ||
HbmiR396 | Antioxidant activity | zinc finger family protein | -31.84 | hevea_454_c64598 |
Lignin-forming anionic peroxidase | -26.08 | hevea_454_rep_c20896 | ||
Antioxidant activity/Response to stimulus | Glutamyl-tRNA reductase | -35.76 | hevea_454_rep_c14169 | |
fatty acyl-coenzyme A reductases (FAR) | -25.41 | hevea_454_rep_c16435 | ||
cysteine protease | -33.18 | CL1Contig374 | ||
HbmiR397 | Antioxidant activity | laccase | -40.97 | hevea_454_c73766 |
Antioxidant activity | 4-coumarate-coa ligase | -37.60 | hevea_454_rep_c6270 | |
HbmiR398 | CuZnSOD chloroplastique | -37.30 | CL4308Contig2 | |
Response to stimulus | HVA22-like protein | -36.03 | hevea_454_rep_c27557 | |
Transcription regulation activity | APETALA2-like protein | -28.43 | hevea_454_rep_c64305 | |
serine/threonine protein kinase | -32.93 | CL23251Contig1 | ||
Heat shock protein | -26.98 | CL530Contig7 | ||
cytochrome P450 | -37.63 | CL1Contig8196 | ||
HbmiR408 | enolase | -42.49 | CL5843Contig1 | |
Antioxidant activity | ribonucleoside-diphosphate reductase | -37.34 | CL1031Contig6 | |
thioredoxin reductase | -32.80 | CL1401Contig3 | ||
gibberellin 20-oxidase | -37.63 | CL1Contig12612 | ||
4-hydroxy-3-methylbut-2-enyl diphosphate reductase | -30.00 | CL1Contig6885 | ||
HbmiR444 | Transcription regulation activity | WRKY | -36.82 | hevea_454_rep_c7550 |
Heat shock protein | -29.45 | CL2547Contig3 | ||
HbmiR476 | Antioxidant activity | Rboh | -27.76 | CL1Contig1292 |
cationic peroxidase 2 precursor | -31.63 | CL257Contig2 | ||
HbmiR535 | Antioxidant activity | LRR protein | -29.18 | hevea_454_c18080 |
HbmiR827 | Response to stimulus | fructose-bisphosphate aldolase | -26.90 | hevea_454_rep_c59114 |
HbmiR828 | Transcription regulation activity | MYB class transcription factor | -26.76 | hevea_454_rep_c48615 |
List of predicted targets for putative novel miRNAs involved in response to stimulus, and in antioxidant and transcription regulator activities according to GO terms
miRNA family | Accession number | GO term | Predicted target function | mfe kcal/mol | Target name |
HbmiRn1 | Acc_584359 | Response to stimulus | Aquaporin like protein | -47.70 | CL1Contig2288 |
Transcription regulation activity | Helicase like protein | -31.10 | CL18570Contig1 | ||
HbmiRn2 | Acc_495109 | Response to stimulus | glycogen synthase kinase | -21.10 | CL1Contig2247 |
Transcription regulation activity | TAZ zinc finger | -22.40 | CL5132Contig2 | ||
HbmiRn3 | Acc_185377 | Transcription regulation activity | MYC | -27.14 | CL1Contig2024 |
HbmiRn4 | Acc_108425 | Transcription regulation activity | bZIP domain trnscription factor | -29.10 | CL11387Contig1 |
Peroxidase | -28.30 | CL1Contig15444 | |||
Response to stimulus | gamma-glutamylcysteine synthetase | -27.80 | CL786Contig2 | ||
HbmiRn5 | Acc_644211 | CTR1 | -29.18 | CL3295Contig2 | |
Transcription regulation activity | Auxin-responsive protein | -29.20 | CL16079Contig1 | ||
GRAS family protein | -33.17 | CL2606Contig1 | |||
HbmiRn6 | Acc_501419 | Response to stimulus | ubiquitin fusion protein | -37.74 | CL1Contig4048 |
Response to stimulus | heat shock protein | -27.50 | CL4213Contig3 | ||
heat shock protein | -23.79 | CL1Contig6482 | |||
glycerol kinase | -26.20 | CL6130Contig1 | |||
HbmiRn7 | Acc_35715 | glycine-rich RNA-binding protein | -22.90 | CL2515Contig2 | |
ubiquitin-protein ligase | -21.10 | CL7863Contig1 | |||
Transcription regulation activity | NAC domain protein | -27.06 | CL678Contig1 | ||
LRR receptor-like protein kinase | -26.16 | CL6991Contig1 | |||
HbmiRn8 | Acc_246127 | Response to stimulus | SAUR family protein | -27.40 | CL3428Contig1 |
HbmiRn9 | Acc_103082 | Response to stimulus | Peroxidase | -35.30 | CL1Contig7525 |
HbmiRn10 | Acc_482742 | Response to stimulus | calcium-dependent protein kinase | -23.30 | CL1Contig9241 |
longevity assurance factor | -22.15 | CL8027Contig1 |
Twelve members of the AP2/ERF domain-containing transcription factor family were putatively targeted by HbmiR156, HbmiR159, HbmiR172, HbmiR393, HbmiR395, HbmiR396, and HbmiR408. Two HD-ZIP III domain-containing protein members (HbmiR165/166, HbmiR172), a transcription factor with a bZIP domain (HbmiRn4), three NAC domain-containing proteins (HbmiR164, HbmiR165 and HbmiRn7), three Squamosa promoter binding proteins (HbmiR156/157), four WRKY transcription factors (HbmiR156/157, HbmiR390, HbmiR444), four Auxin Response Factor (ARF); HbmiR160 and HbmiRn5) and five GRAS family transcription factors (HbmiR170, HbmiR171, HbmiR396 and HbmiRn5) were also potentially targeted.
Sequences encoding protein involved in protein degradation, such as cysteine protease (HbmiR396) and ubiquitin protein ligase (HbmiRn6 and HbmiRn7), were identified for genes classified in the "response to stimulus" category.
Three sequences related to ABA signalling were also identified: an ABA-insensitive protein (HbmiR166, and HbmiR2910), and an HVA22-like protein (HbmiR398). In addition, one sequence encoding CTR1, a negative regulator of the ethylene signalling pathway, was targeted by HbmiRn5. MiRNA biogenesis was also highlighted by the presence of a cap binding protein predicted to be targeted by HbmiR167. Interestingly, two enzymes involved in natural rubber biosynthesis, HMG-CoA reductase and 4-hydroxyl-3-methylbut-2-enyl diphosphate reductase, were predicted to be targeted by HbmiR159 and HbmiR444, respectively. For the "antioxidant activity" category, several ROS-scavenging enzymes were identified. The chloroplastic and peroxisomal isoforms of CuZnSOD were putatively targeted by HbmiR398 and HbmiR159, respectively. Another enzyme involved in glutathione biosynthesis, gamma glutamyl cysteine ligase (GCL), was predicted to be targeted by HbmiRn5. Rboh (or NADPH oxidase) is an enzyme involved in ROS production, which is likely to be targeted by two miRNAs (HbmiR2914 and HbmiR476). In addition, targets that are subjected to or are involved in the redox status of proteins, such as genes encoding proteins involved in lignin synthesis, cynnamoyl CoA reductase, lignin forming anionic peroxidase precursor and 4-coumarate:CoA ligase, were also found to be targeted by HbmiR159, HbmiR408 and HbmiR398, respectively.
List of target genes experimentally cleaved by miRNA
miRNA family | Target name | Heveasequence name | miRNA accession | mfe kcal/mol | Alignment | ||||||
Hbmir156 | Squamosa promoter binding protein (HbSquamosa) | CL2120Contig2 | acc_480780 | -45.5 | miRNA Target | 23 766 | CUACACGAGAGAGAGAAGACAGU : ::::::::::::::::::::: GUUGUGCUCUCUCUCUUCUGUCA ▲ 8/14 | 1 788 | |||
Hbmir160 | Auxin Response Factor (HbARF) | CL6582Contig1 | acc_370 | -52.2 | miRNA Target | 21 524 | ACCGUAUGUCCCUCGGUCCGU ::::::.:::::::::::::: UGGCAUGCAGGGAGCCAGGCA ▲ 27/30 | 1 544 | |||
HbmiR398 | Chloroplastic copper zinc superoxide dismutase (HbCuZnSODchl) | CL4308Contig2 | acc_420 | -37.3 | miRNA Target | 23 456 | UAGU-C-CCCGCUGGACUCU-UGUGU .::: : :::.::::::.:: :::.: GUCAUGCGGGUGACCUGGGAAACAUA ▲ 6/14 | 1 480 |
Size distribution of small RNAs in Heveamay differ from others species
Next-generation high-throughput sequencing techniques are powerful tools for miRNA identification. The sequencing and analysis of the Hevea small RNA library generated more than 2 million sequences. In their vast majority (86%), the sequenced small RNAs were only represented by one or two reads. Similarly, 65% of unique miRNA sequences were found in Arabidopsis by 454 sequencing techniques [52]. Interestingly, we found that the size distribution of Hevea small RNAs differed from other species. The most abundant small RNAs had 17, 19 and 27 nucleotides in Hevea whereas they have 21 and 24 nucleotides in other species. Indeed, in Arabidopsis thaliana [52], Arachis hypogea [45] and Oryza sativa [53], the fraction of 24 nucleotides has proved to be predominant, accounting for 60%, 45% and 36% of unique small RNAs, respectively. The predominant size has been found to be 21 nucleotides in Pinus contorta [53] and Taxus chiniensis [54]. In Hevea, the high percentage of 17- and 19-nucleotide small RNAs resulted from the increased amount of RNA degradation products and transposable elements in response to abiotic treatments according to the mapping results against the Arabidopsis genome (data not shown), as previously observed in Brachypodium [55]. Indeed, a change in small RNA size distribution has been reported in Brachypodium in response to cold treatment. Under normal growth conditions, the 21- and 24-nucleotide classes were the most abundant small RNA families, whereas it was the 19-nucleotide class after cold treatment [55]. Our dataset was generated from pooled small RNAs of various tissues subjected to several types of abiotic stress. The size distribution may have been differentially affected by each type of abiotic stress and may explain the difference in size distribution in Hevea.
Long conserved and novel miRNAs of 23-27 nucleotides consisted of short miRNA sequences with an extension. This lmiRNA structure suggested that they were generated from the same MIR genes. Our observation suggests dual functions for 14 canonical MIR genes producing 20-22 nt and 23-27 nt lmiRNA in Hevea, namely HbMIR169, HbMIR2911, HbMIR482, HbMIR472, HbMIR408 and HbMIRn3-10. Across the plant kingdom, lmiRNA were previously classed as heterochromatic small interfering RNAs (hc-siRNA) [28]. In Arabidopsis, the hc-siRNA derived from the miRNA-generating site is dependent on DCL3, RDR2 and PolIV associated with AGO4 and direct DNA methylation for some target sites [28]. As regards the higher number of reads for the 24 nucleotide compared with the 20-22 nucleotide miRNAs, the 14 miRNA families producing 20-22 nt and 23-27 nt miRNAs might mostly regulate their targets through direct DNA methylation in response to abiotic stress. This may confirm the results of a recent study showing the existence of site-specific abiotic stress-induced DNA methylation for five genes in Hevea [56].
Ancient and recently differentiated Hevea brasiliensismiRNAs provide additional information for evolutionary studies
The 48 Hevea brasiliensis conserved miRNA families were more or less similar to those of other species. In Hevea, five miRNA families (HbmiR319, HbmiR156/157 and HbmiR165/166) have been found in more than forty plant species [57], and nine correspond to ancient miRNA already present in the common ancestor of Embryophytes (HbmiR156, HbmiR159/319, HbmiR160, HbmiR165/166, HbmiR171, HbmiR408 and HbmiR395), two in the common ancestor of Tracheophytes (HbmiR397 and HbmiR398) and nine in the common ancestor of Spermaphytes (HbmiR162, HbmiR164, HbmiR168, HbmiR169, HbmiR172, HbmiR393, HbmiR394, HbmiR399 and HbmiR827) [58]. Interestingly, the so-called monocotyledon-specific HbmiR444 family was identified in this study [58]. This suggests that HbmiR444 appeared before the speciation between monocotyledons and dicotyledons. Three woody species-specific miRNA families were also found in Hevea brasiliensis: HbmiR473 (Citrus sinensis and Populus trichocarpa), HbmiR476 (Populus trichocarpa) and HbmiR479 (Populus trichocarpa, Vitis vinifera, Gossypium hirsutum and Citrus sinensis) and were probably recruited independently by woody species during their evolution. The 10 novel miRNA families identified in this work are likely to be more recent, after Hevea diversification, as they have not been identified up to now in other euphorbiaceous plants. Furthermore, this information complements previous studies on euphorbiaceous species [49] in which only 25 conserved miRNAs were identified.
Our data could be used to study the evolution of MIR genes. We showed that 70% of the putatively new MIR genes were able to produce lmiRNA of 23-27 nucleotides, compared to 29% for conserved MIR genes. This corresponds to what has been observed in Arabidopsis, where it was previously suggested that young MIR genes consistently produced siRNAs, while ancient MIR genes produced predominantly canonical miRNAs, showing that the evolution of MIR genes is associated with changes in the use of DCL, resulting in specific miRNA size classes [59]. In addition, several so-called plant species-specific miRNAs were also found in Hevea, such as HbmiR1310 initially found only in Pinus taeda [53], HbmiR2910, HbmiR2914, HbmiR2915 and HbmiR2916 found in Populus euphratica [60] and HbmiR476 in Populus trichocarpa [61]. Our data suggest that those miRNAs should no longer be considered as species-specific miRNA.
Our study also revealed some conserved and divergent mechanisms at post-transcriptional level. Indeed, four families of miRNA were subjected to RNA editing events at the sites previously observed in Arabidopsis. However, for the thirteen families left, RNA editing was not observed under our experimental conditions. Under our experimental conditions, the -1 + UU modification was not observed in Hevea for the nine families selected from Ebhardt et al. [50]. Once the Hevea genome sequence is available, it will be possible to conduct an exhaustive analysis of the discrepancies between members of a multigenic family and RNA editing events.
Predicted target/miRNA pairs conserved between plant species and putative new ones in Hevea brasiliensis
In comparison with other plants, several regulation mechanisms seem to be conserved in Hevea brasiliensis, such as the regulation of HD-ZIP III protein by HbmiR166 [62], Squamosa promoter binding protein by HbmiR156 [63], NAC domain protein by HbmiR164 [64], APETALA2-like protein by HbmiR172 [65–67], CCAAT-binding transcription factor by HbmiR169 and cysteine proteinase by HbmiR396 [44]. These ancient miRNAs regulate ancestral transcription factors that coordinate highly conserved functions, such as organ polarity and separation, cell division, or hormonal control [68, 69]. We predicted potentially new miRNA/target pairs in Hevea. New target/miRNA pairs were found for transcription factors, such as, for example, the NAC domain protein by HbmiR165 and HbmiRn7, the GRAS domain protein by HbmiR170, HbmiR396 and HbmiRn5, WRKY by HbmiR396 and HbmiR444, and the APETALA2-like transcription factor by HbmiR156, HbmiR159, HbmiR393, HbmiR396 and HbmiR408. (Duan et al., submitted). In addition, we also detected new pairs involved in protein degradation (ubiquitin protein ligase by HbmiRn6 and HbmiRn7), in response to hormones (ABA-insensitive by HbmiR166, ABA responsive element binding protein by HbmiR2910, and HVA22-like protein by HbmiR398, CTR by HbmiRn5) and in the regulation of miRNA (cap binding protein by HbmiR167). Further analyses are needed to validate the inhibition of these targets at transcriptional or translational level and to unravel specific regulation in Hevea brasiliensis regarding the response to stress in latex cells, and latex production.
Conserved and divergent regulation of redox homeostasis in Hevea brasiliensis
Several enzymes involved in ROS production or detoxification were found in our analysis. Two ROS-producing enzymes, Rboh, are likely to be targeted by HbmiR2914 and HbmiR476. They are key regulators in the ROS signalling network as they integrate different signal transduction pathways, such as calcium, protein phosphorylation and lipid signalling with ROS production [6]. Careful attention was also paid to targets classified in the GO term "antioxidant activity". For conserved miRNAs, two CuZnSOD chloroplastic and peroxisomal isoforms were predicted to be targeted by HbmiR398 and HbmiR159 respectively. Surprisingly, the CuZnSOD cytosolic isoform was not predicted by the LeARN pipeline and, even manually, no correct alignment was possible with HbmiR398. So far, validation by 5' RACE PCR has failed under our experimental conditions. By contrast, previous studies in Arabidopsis showed that HbmiR398 targeted both chloroplastic and cytosolic isoforms [34].
Some miRNA families have been reported to be H2O2-responsive in rice, such as OsmiR169, OsmiR397, OsmiR528, OsmiR827, OsmiR319 and OsmiR408 [70]. In rice, OsmiR397 targets laccase genes involved in lignin biosynthesis and those targets were also found for HbmiR397 in Hevea.
A library of small RNAs was deeply sequenced to identify conserved and novel miRNAs in Hevea brasiliensis. Combined with the Hevea transcript database, this led to the identification of 48 miRNA families and 10 novel miRNAs. Our study revealed new insights for evolutionary studies on woody plants and the potentially specific regulation of redox homeostasis in Hevea. All the putative targets of interest for Hevea stress biology and latex production will be experimentally validated and the target genes and their corresponding miRNAs will undergo co-expression analysis.
Production of in vitro plantlets
Embryogenic friable callus from line CI05538 of clone PB 260 was maintained on MM medium in the culture room at 27°C, with 60% humidity in the dark [71]. In vitro plants were regenerated according to the procedure described in [72]. Briefly, somatic embryogenesis was initiated by sub-culturing 1 gram of callus in 250 mL flasks containing 50 mL of a semi-solid Induction Medium. Pro-embryos were then developed in a temporary immersion system (RITA®, CIRAD, France) with 1 min of immersion per day in the liquid development media DEV1 and DEV2. For plant regeneration, only well-shaped mature embryos were collected after 8 weeks of culturing [73]. They were transferred to glass tubes on a semi-solid DEV3 medium for one month under a light intensity of 60 μmol m-2 s-1 and a 12 h light/dark photoperiod for embryo conversion into plants. Plantlets were then acclimatized in the greenhouse at 28°C with 60% relative humidity.
Plant material cultivation and treatments
Budded plants and two-year-old in vitro plants of clone PB260 were grown in the greenhouse at 27°C, with 50% humidity under natural light conditions. Budded plants and two-year-old in vitro plants of clone PB260 were exposed to cold at 4°C for 8 h, light (PAR = 1000-1500 μmol/m2/s for 4 h), drought (4 weeks for young budded plants and 10 days for two-year-old in vitro plants without watering), flooding (covered with water for 4 days), salinity (300 mM NaCl for 4 days) and wounding for 4 h (Table 2). Hormonal treatments were applied in hermetically sealed boxes, either with 5 ppm of ethylene for 4 h or 0.3 μM of methyl jasmonate for 4 h as described in [74, 75].
Isolation of small RNAs
For all treatments, leaves, bark and roots were sampled in liquid nitrogen and then frozen at -80°C pending small RNA isolation. Small RNAs were purified with the mirPremier microRNA Isolation Kit (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer's instructions for plant tissues. Briefly, 100 mg of the plant tissue powder was mixed with a lysis mix that released small RNA and, at the same time, inactivated ribonucleases and interfering secondary metabolites that might exist in plant tissues. Large RNA and genomic DNA remained insoluble and were removed from the lysate along with other cellular debris by brief centrifugation. Small RNA was then captured on a silica binding column in the presence of alcohol. Residual impurities were removed by wash solutions, and purified small RNA was eluted in RNase-free water.
Deep sequencing of small RNAs
Small RNA samples from callus and from control and treated plants were pooled. A Hevea small RNA library was constructed by the SKULTECH Company (France). Briefly, small RNAs were separated on a denaturing 15% polyacrylamide gel. The gel slices containing RNA with a size of about 15-50 nucleotides were excised and the RNA was eluted. Small RNAs were ligated to 5' and 3' adapters using T4 RNA ligase. Products from the second ligation were gel purified then reverse transcribed and amplified using PCR to produce cDNA. The PCR was performed with two primers that annealed to the ends of the adapters. PCR products were separated on 15% polyacrylamide gel with ethidium bromide staining. The gel slices containing DNA with a size of about 92 nucleotides were excised and the DNA was eluted. The resulting cDNA was sequenced using Solexa/Illumina technology (Illumina Inc., San Diego, CA, USA). Raw reads were trimmed using Trim_Adaptor application (non Open source application) with the minimum clip length fixed at 6 nucleotides.
Bioinformatic analyses
This work was supported by the high performance cluster of the SouthGreen Bioinformatics platform (southgreen.cirad.fr - UMR AGAP - CIRAD) comprising 26 compute nodes (208 Nehalem cores) and a 16 to storage capacity connected through a low latency Infiniband network. Sun Grid Engine was used as the workload manager to schedule jobs on the different compute nodes.
Identification of the conserved heveamiRNAs
Adapters and low-quality sequences were removed from the raw reads obtained from Illumina sequencing using cross-match software http://www.phrap.org/phredphrpconsed.html; the non-redundant dataset was produced using WU nrdb software. The unique small RNA sequences were then compared with BlastN [76] against the Arabidopsis genome (Phytozome V6) and the PMRD database [38]http://bioinformatics.cau.edu.cn/PMRD in order to classify the small RNAs and identify mature miRNAs in Hevea already known in several species.
Identification of HeveamiRNA precursors
Conserved miRNAs and putative novel miRNA precursors were identified in the PB260 transcriptome http://bassigny/cgi-bin/esttik_dev/quick_search.cgi using the LeARN pipeline http://symbiose.toulouse.inra.fr/LeARN/release_1;5.0[50], a platform for detecting, clustering and annotating non-coding RNAs. Novel miRNAs were separated into 4 classes according to their position on the stem-loop structure. Class 1 precursors only produced small RNAs corresponding to the predicted miR and miR* (i.e., small RNAs in a 25-bp region around the miR:miR* predicted region to be considered with two nucleotide overhangs linked to DCL action); class 2 comprised hairpins containing both predicted miR and miR* and additional small RNAs, of lower abundance, outside this region; class 3 involved precursors with reads on the defining small RNA region only (3a, at least two reads; 3b, only one read); class 4 consisted of precursors containing the defining small RNA plus reads of lower abundance outside the predicted miR:miR* duplex. Finally, class 5 contained hairpins that generated small RNAs of greater abundance than the defining RNA outside the predicted miR:miR* region [77]. Each miRNA precursor was checked manually following the rules described in [45]. Specifically, we considered dominant, mature sequences residing in the stem region of the stem-loop structure and ranging between 20-22 nt with a maximum free-folding energy of -25 kcal mol-1. A maximum of six unpaired nucleotides between the miRNA and miRNA* was allowed. The distance between the miRNA and miRNA* was fixed between 5 and 240 nucleotides.
Identification of putative HeveamiRNA targets
Computational identification of miRNA targets was performed with the Miranda toolbox included in the pipeline with the default parameters (gap_value = 2, mm_value = 1, gu_value = 0.5, score_threshold = 3, min_length_alignment = 20 and no_mismatch_positions = 10;11) for conserved miRNA and secondly checked with psRNAtarget http://plantgrn.noble.org/psRNATarget/(length_alignment = 18), [78]. For the putative novel miRNAs, psRNAtarget server (length_alignment = 18) and Miranda (gap_value = 2, mm_value = 1, gu_value = 0.5, score_threshold = 3, min_length_alignment = 18 and no_mismatch_positions = 10; 11) were used to scan the Hevea EST databases (Duan et al., to be submitted, http://bassigny/cgi-bin/esttik_dev/quick_search.cgi). A first assembly set was generated from reads of leaves, bark, latex, embryogenic tissues, and roots separately to create tissue-specific transcript databases with sequence names CLxxcontigxx. Then, reads from all tissues were used to generate one transcript sequence database for the Hevea clone PB260 with the sequence name hevea_454_xxx.
Gene Ontology analyses were performed with the Blast2GO program [79, 80]. In detail, sequence analyses, such as alignments and BLAST, were performed with Geneious software [81].
Total RNA extraction
Total RNAs from leaves, bark and roots subjected to abiotic stress treatments were extracted using the caesium chloride cushion method adapted from Sambrook [82] by Duan et al. [75]. One gram of fresh matter was ground and transferred to a tube containing 30 ml of extraction buffer consisting of 4 M guanidium isothiocyanate, 1% sarcosine, 1% polyvinylpyrrolidone and 1% ß-mercapto-ethanol. After homogenization, the tubes were kept on ice and then centrifuged at 10,000 g at 4°C for 30 minutes. The supernatant was transferred to a new tube containing 8 ml of 5.7 M CsCl. Ultracentrifugation in a swinging bucket was carried out at 89,705 g at 20°C for 20 hours. The supernatant and caesium cushion were discarded, whilst the RNA pellet was washed with 70% ethanol. After 30 minutes of air drying, the pellet was dissolved in 200 μl of sterile water. RNAs were conserved at -80°C.
Experimental validation of miRNA targets
The GeneRacer kit (Invitrogen, Carlsbad, CA, USA) was used to ligate a 5'RNA adapter to pooled RNAs prior to reverse transcription. The resulting cDNAs were used as a template for PCR amplification using one Gene-Specific Primer (GSP) designed downstream of the predicted miRNA:target binding site and one GeneRacer 5' forward primer. The primers used were HbCuZnSODchl R: GGGTAACCAGCAAATGCAAGCAGC, HbARF R: CTTGTGTTGAGTTGCAGCGCG and HbSquamosa R: TGGAGCCTAATTGGCTTCCTTCTGC. The first PCR product was then used for nest amplification performed with a nested GSP primer and a nested GeneRacer 5' forward primer. The nested primers used were HbCuZnSODchl nested R: GCAGGGAACAATGGCTGCC, HbARF nested R: ATGTTCAGCTCAGTGGACACGG, and HbSquamosa nested R: TTGTTTGGCACCACGCTTGTGGAAG. The PCR products were gel purified, cloned into PCR2.1 TOPO TA vector (Invitrogen, Carlsbad, CA, USA) and sequenced.
Complementary DNA (cDNA) synthesis and stem-loop RT-PCR
Primer list for the stem-loop RT-PCR and the real-time PCR and their efficiencies
Precursor name | Forward primer | Reverse primer | Unique melting pick | PCR efficiency |
Hbpre-miR156 | TGGTGATGTTGTTGACAGAAGATAGAGAGC | GCACAAAGGAGTGAGATGCAGAGTCC | Yes | 1,790 |
Hbpre-mir159a | GGTTAAGAAGTGGAGCTCCTTGAAGTC | GCTCCCTTCAATCCAAACAAGGATC | Yes | 1,958 |
Hbpre-mir159b | GTGGAGCTCCTTGAAGTCCAATAGAGG | AGAGCTCCCTTCAATCCAAACAAGG | Yes | 1,881 |
Hbpre-mir166b | GGGGAATGTTGTCTGGTTCGATG | TCAAATCAAACCCTGTTGGGGG | Yes | 1,738 |
Hbpre-mir396 | TGACCCTCTTCGTATTCTTCCACAGC | CCCACAGCTTTATTGAACCGCAAC | Yes | 1,782 |
Hbpre-mir408b | GACATACAAAGACTGGGAACAGGCAG | GCCACAAGCCAGGGAAGAGGC | Yes | 1,792 |
Hbpre-mir476 | GCCTTGTATGTTTCATTTAGTAATCCTTCT | GATAATCCTTCTATGCAAAGTCTTTTATGC | Yes | 1,732 |
Hbpre-mir2910 | CAGGTCCAGACATAGTAAGGATTGACAG | CGTTAACGGATTAACCAGACAAATCGC | Yes | 1,728 |
Hbpre-miRn1 | ACCAGGAACTGGTATCAACCCAGC | TGCTACCAATGAATCGGACCCACC | Yes | 1,837 |
Hbpre-miRn2 | CAGTAAATAGCAGTATCGTGGATAGGG | GTCCAATCATTGATCCTGAAAATTTCTAC | Yes | 1,828 |
Hbpre-miRn3 | TGGATTGGAGCCCAATACTGTGAC | CTGCTCCATTGATTTTACCATCTATGC | Yes | 1,873 |
Hbpre-miRn4 | TGCGTGGGTAGATTGAGCTGC | GCTCCTGAGATATGCAAGCCACAAG | Yes | 1,863 |
Hbpre-miRn6 | ACCTAGGATGTAGAAGAGCATAAC | ACTACATGAGTGGATATATAGGAATCC | Yes | 1,787 |
Hbpre-miRn7 | ATCTGCTACTCGGATAACCGTAG | GCAGCAAATCTTCCATAGCATCC | Yes | 1,925 |
Hbpre-miRn8 | TTTCTTGATTCTAGTGGGTGGTG | GGAATTAACCAGACAAATCGCTC | Yes | 1,905 |
Hbpre-miRn9 | AGTCGGGGGCATTCGTATTTC | GCAAATACTTTCGCAGTTGTTCG | Yes | 1,956 |
Hbpre-miRn10 | TGGATTTATGAAAGACGAACAACTGCG | TTCGAGCCCCCCAATTTCG | Yes | 1,712 |
- miRNAs:
Tapping Panel Dryness
Reactive Oxygen Species
Plant microRNA database
RNAse III-like DICER-like I
plant ortholog to exportin 5
RNA-induced silencing complex
Argonaute 1
Expressed Sequence Tag
This work was supported by the Institut Français du Caoutchouc. The authors thank Gérald Oliver and Maryannick Rio for technical assistance. We are grateful to Dr Kuswanhadi and Tetty Chaidamsari for RNA isolation from the latex and bark of trees grown at the Sembawa Research Centre (Indonesia). The authors thank Peter Biggins for his revision of the English.
Notes: All precursors and mature miRNA sequences will be submitted to the MiRbase registry after acceptance of the manuscript for publication
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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How does the Bible portray its villains?
Eminent scholars explore the textual accounts and archaeological evidence of some not-so-nice notables from the Old and New Testaments: from a ruthless Assyrian king to a confused Judean governor, and from Roman executioners to a betraying friend.
BART D. EHRMAN Who Killed Jesus? Pontius Pilate & the Jews
CRAIG EVANS The Art and Archaeology of Execution in the Roman World
DAVID USSISHKIN Sennacherib's Attack on Lachish: What We Have Learned from Archaeology
MARVIN MEYER The Gospel of Judas: What It Has Taught Us Thus Far
ITEM: 9H292 Run time: 4 hours ISBN 978-1-935335-76-4


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I kinda like the graphic on this tank. It's channeling Ocean Pacific, circa 1980.
The Run: Energy short sleeve shirt and the Zoom Knee Shorts (I think it's those) made a cute running outfit.
The blog where lululemon fans discuss & feed their addiction. Your resource for photos, reviews, & lulu news since 2008.
Friday, May 21, 2010
4 comments:
I really wish they made prints for the technical tanks like the CRBs. The MRB just doesnt do it for me.
- May 21, 2010 at 4:38 PM
LOVE the modern racer!!
- May 21, 2010 at 4:46 PM
i love the modern racer better than the crb -- love this print too -- and i'm not a fan of gray -- but this is pretty!
- May 22, 2010 at 5:49 AM
do we know the color of the modern racer?
- May 24, 2010 at 10:54 AM
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text: We decided to how to Romantica god our 10th Wedding Anniversary meal after reading reviews on Trip Advisor. We could not have been more disappointed. The good was bland, the service terrible and waitress stood chatting with the table next door for about 20 minutes before coming back to our table after our main course to see if we wanted... More


text: Home > Nation Mar not part of PH delegation to Russia By Willard Cheng, ABS-CBN News Posted at Sep 07 2012 04:07 PM | Updated as of Sep 08 2012 07:58 PM Share Save Facebook Twitter GPlus LinkedIn Pinterest MANILA, Philippines - Newly appointed Department of the Interior and Local Government Secretary Mar Roxas is not joining the President's trip to Russia for the APEC Summit, contrary to an earlier statement of presidential spokesperson Edwin Lacierda. "I think that was a mistake. I asked Secretary Roxas and hindi talaga siya kasama," Presidential Communications Development and Strategic Planning Office (PCDSPO) Secretary Ricky Carandang told reporters. Aquino is attending the APEC summit in Russia's east coast city of Vladivostok this weekend. Among the officials joining the President in the trip are Foreign Affairs Secretary Albert del Rosario, Trade Secretary Gregory Domingo, Energy Secretary Rene Almendras, and Laura del Rosario, Undersecretary for International Economic Relations, APEC National Secretariat. The Palace said the APEC summit is an opportunity to foster "greater cooperation" among the member nations, as well as strengthen relations and economic cooperation. Lacierda said that existing issues between the Philippines and China "should not be a hindrance" to discussions on economic cooperation among the member nations. Share Save Facebook Share on Twitter GPlus LinkedIn Pinterest Read More: Mar Roxas APEC /sports/03/25/17/pba-star-dismantles-globalport-silences-terrence-romeo-in-return/news/03/25/17/joining-earth-hour-shows-commitment-to-fight-climate-change-malacaang/news/03/25/17/deped-orders-probe-into-mercury-leak-at-manila-science-hs/sports/03/25/17/womens-volleyball-ust-bolsters-final-4-bid-with-4-set-win-over-feu/overseas/03/25/17/lights-go-out-around-the-world-for-10th-earth-hour


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Full profile →'">
The author is a Forbes contributor. The opinions expressed are those of the writer.
@giorodriguez why supporting Latino technology entrepreneurs benefits everyone
A couple of months ago, I got an email from Jesse Martinez -- a devoted and tireless leader of Latino entrepreneurship in Silicon Valley -- asking me to speak at a leadership summit. He also asked me what I wanted to speak about.
A great question, because while I have spoken at many events like this, my interest in Latinos in business has evolved. Or should I say: Latinos in business have evolved, and so have my views.
There was a time when I used to speak about the special assets that Latinos bring to startups, particularly in the world of social technology. Now I like to talk about the category itself -- Latino entrepreneurship -- because the category itself has special value.
New markets
But in case you were wondering, make no mistake: lots of folks have questioned the strategy of developing entrepreneurship around ethnicity, gender, and other kinds of identity.
To be clear, lots of folks in Silicon Valley have questioned the strategy.
First it began with a conversation about the absence of African American leaders in tech. Then it became a conversation -- though we've had this conversation before -- about the absence of women leaders in tech. Recently, I found myself in a silly argument with a journalist who had convinced himself that identity-based strategies for empowering people had the ultimate effect of marginalizing them ("ghettoizing them," is the phrase I believe he used). I'm happy to report that I got him to see the situation differently ... but only after examining a number of ways that identity-specific approaches help everyone, not just the group in question.
First -- and this should be obvious -- the group in question may actually have access to new markets that typically elude people who are not in the group. When it comes to Latinos, what we are looking at is the access that comes to markets where there is a language barrier or a cultural barrier. This is, by the way, why more and more businesses -- including large tech companies like and Facebook-- are investing in multicultural marketing. There's a growing recognition that it's no longer about the mainstream but the manystreams that make up the new global marketplace.
New services
Second -- and I am sure this is less obvious -- the group may actually have ideas for services that typically elude other entrepreneurs. A while back, I wrote about a Silicon Valley technology startup -- Interersante.com -- that had a special spin on how to serve Latin American immigrant communities in the US through ecommerce (for example, Argentinians who miss shopping in Buenos Aires). To pull this off required a special knowledge not just of language and culture but the special needs (think products and services) of the increasingly transient Latin American professional community. People less familiar with Latin America think simply about immigration. People more familiar with Latin America think deeply about diasporas and their positive economic impact.
New narrative
But for me, there's a more powerful reason for supporting Latin American entrepreneurship and creating more Latinopreneuers: it changes the narrative of Latin Americans from being a community of have-nots to a community of haves, from a community that is solely focused on getting jobs to a community that creates jobs ... for everyone.
There's a reason why Silicon Valley and other tech hubs are so crazed about entrepreneurship these days. At some point, everyone realized that one of the fastest paths to economic growth is company formation (the other is household formation -- i.e., the creation of new households, which helps to drive other parts of the economy, including retail).
But just as it's no longer about the mainstream in marketing, it's no longer about the mainstream in technology acceleration. There are many new markets in the new multicultural marketplace, and a strategy that puts people on the supply side of the equation -- particularly people who have not seen themselves on the supply side -- is, in my opinion, the right strategy. And it's the one that organizations like Manos Accelerator and Jesse Martinez's org -- the Latino Startup Alliance -- are following.


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Montross Early OriginsThe surname Montross was first found in Forfarshire part of the Tayside region of North Eastern Scotland, and present day Council Area of Angus, where a Henry de Monros, a chaplain, was on record when he witnessed charters by Gregory, bishop of Brechin in circa 1218-22. Mestre Matheu de Monros, a clerk of Perth, rendered homage to King Edward I of England in 1296. Montross Spelling VariationsMontross Spelling Variations Spelling variations of this family name include: Montross, Montrose, Monros and others. Montross Early HistoryMontross Early HistoryThis web page shows only a small excerpt of our Montross research. Another 169 words (12 lines of text) covering the years 1385, 1464, 1505, 1513, 1548, 1608, 1612, and 1650 are included under the topic Early Montross History in all our PDF Extended History products and printed products wherever possible. Montross Early Notables (pre 1700)Montross Early Notables (pre 1700)Notable amongst the Clan from early times was William Graham, 4th Lord Graham (1464-1513), who became the Earl of Montrose in 1505. John Graham (1548-1608), 3rd Earl of Montrose was the Chancellor of the... Another 34 words (2 lines of text) are included under the topic Early Montross Notables in all our PDF Extended History products and printed products wherever possible. Montross In IrelandMontross In IrelandSome of the Montross family moved to Ireland, but this topic is not covered in this excerpt. Another 117 words (8 lines of text) about their life in Ireland is included in all our PDF Extended History products and printed products wherever possible. The Great MigrationThe Great MigrationSome of the first settlers of this family name were: Montross Settlers in Canada in the 18th Century
MottoMottoThe motto was originally a war cry or slogan. Mottoes first began to be shown with arms in the 14th and 15th centuries, but were not in general use until the 17th century. Thus the oldest coats of arms generally do not include a motto. Mottoes seldom form part of the grant of arms: Under most heraldic authorities, a motto is an optional component of the coat of arms, and can be added to or changed at will; many families have chosen not to display a motto. Motto: Ne oublie Motto Translation: Do not forget. Montross Family Crest ProductsMontross Family Crest ProductsSee AlsoSee AlsoCitationsCitations
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Dress it up or dress it down — either way you'll be happy as could be wearing this 3/4 sleeve dress. For a dressier look, style it with black heeled sandals, statement jewelry, and a clutch. For a dressed down look, try it with slide on sneakers and a cro
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$38.00We love a fabulous mini dress — especially one that features some fun pinstripes! Try this gem styled with black heels a statement necklace.
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$38.00Love the midi length? Than this dress is for you! The best part about this dress is that it can be worn to a huge variety of places — like to the office with a blazer and heels, to the beach with a bathing suit underneath, or for a Summer night out with


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Christmas Came Early
My great friend and herbie gal pal, Susan Belsinger must have been catching up on her food reading last night because she sent me a message about the December 5 Food Section of the Washington Post.Seems that venerable newspaper had listed 30 of the Top Cookbooks for 2012.
Guess what? Preserving was number six in the list!!