Intentional use of carbapenem antibiotics for valproic acid toxicity: A case report.
The interaction between valproic acid (VPA) and carbapenem antibiotics is well described with previous reports suggesting a reduction in VPA half-life between 47% and 90%. As described in this case, this interaction might be beneficial in the setting of toxic VPA ingestion. An intubated, unresponsive patient arrived via emergency medical services after toxic VPA ingestion. Meropenem was prescribed for a suspected pneumonia and to take advantage of the VPA interaction. We observed a 56% decline in half-life with short-term meropenem dosing and an improvement in mental status shortly after administration. Our findings suggest a potential role for short-term carbapenem therapy for VPA overdose.


A collection of 5 steel chairs with a remarkable magnified optical effect. These pieces are light, harmonious and easy to use; they could be read as a result of the distortion given by the lens in Drop Table.

“Chairs that live in harmony, like a family. Whilst they are all similar, each chair has a slightly different shape, and its own personality. There is a wide chair, a tall chair, a short chair, a normal chair and a stool. It’s fun to choose which chair to sit on, depending on the day’s mood. It’s also fun to decide which is your favourite chair. Like a family sitting around a table, so the chairs are arranged around the table.” Junya Ishigami

Technical details
The structure of the chairs is in 12 mm tubular steel, curved using a numeric control machine, and 15 mm tubular steel legs welded together using internal reinforcement. The seat mesh is in 3 mm steel wire with a 40x40 mm diagonal weave, individually rolled for each seat. Finished with epoxy paint in the colours given in the catalogue, or electro-coated in the outdoor version.
Cushions are available cut to shape in seat-only or seat-and-back versions.


How to Make an Elastic Notebook Pen and Pencil Strap

My sister goes nowhere without her organizer, so she asked me to make this handy elastic case strap to hold her pencils and pens. If you never want to lose a pen again, just grab some fabric, a piece of elastic and some velcro tape and you can whip up your own pencil holder in an hour. Really!

1. Make the Fabric Strip

Step 1

Step 2

Fold the fabric in half length-ways. Keep the printed side of the fabric facing inwards. Thread your needle with the red thread.

Start sewing on the lower right corner of your rectangle, where the fabric is folded. You need to sew along the right short side of the rectangle and the long upper side opposite of the fold. Sew the fabric along these two edges leaving a zone of about 1cm (0.5 in.). You can stitch by hand, as it is a fairly small piece of fabric.

Use back-stitch, where the individual stitches are made backward to the general direction of sewing. Bring your needle up on position 1, then back down at position 2 and bring it back up and through at position 3. Repeat for the next stitch.

When you've completed stitching, cut off the upper right corner (be careful not to cut the stitches) without ruining the stitches, allowing the corner to be sharp when you turn the tube inside out.

Step 3

Now, turn the tube of fabric inside out, pulling it through the open end. Iron it to make it neat.

Step 4

Fold inwards the fabric on the open end of the tube. Back stitch the last side of the rectangle to turn the tube into a two-sided fabric strip.

2. Prepare the Elastic

Step 1

Cut a piece of the elastic 35cm (14 in) long. This should work well for a medium-size organizer or notebook around 15cm (6 in) wide. If your notebook is a different size, just add 2-3cm (1 in) to the circumference measurement.

Step 2

Fold inwards the one short edge of your elastic by 0.5 cm (0.2 in). Fold again and stitch to prevent the elastic from fraying. Repeat at the other end of the elastic.

3. Sew the Fabric and Elastic Together

Step 1

Place the the fabric strip on the center of the elastic. Make sure the folding at the ends of the elastic are facing downwards. Now use sewing pins to attach the fabric on to the elastic at both ends.

Don't stretch the fabric on the elastic; keep it a bit loose. Keep pinning, leaving a space of 2.5cm (1 in) between pins. I also created space for a bigger pocket at one end. Make sure the pins are placed perpendicular to the length of the elastic and parallel one to the other. They will serve as guides for stitching.

Step 2

Take two pieces of sew-on velcro straps in black, around 3cm (1.2in) in length. Make sure you have one piece of loop velcro (the soft one) and one piece of hook velcro (the harder, scratchy one), which together will form the closing of the notebook strap.

Now place the velcro hooks at the left end of the elastic, on the inner face directly after the fold. Make sure the hooks are facing outwards and the straight side of the velcro strap is placed directly on the elastic. Pin it in place.

Pin the velcro loops at the other end of the elastic. This time place the velcro strap on the outer face of the elastic. When you pin both velcro straps, check that when you wrap the elastic around the organizer they meet (and stick!).

Step 3

All you need to do now is keep the velcro straps in place with some stitches around their perimeters. Use black thread for this. You might like to use a thimble for this step, as velcro is a bit harder to sew than fabric.

Now thread your needle with some red thread (or a color that matches your fabric), and stitch along the pins attaching the fabric on the elastic.

Step 4

Wrap the pencil case around your organiser, keeping the fabric part on the front. Turn it over and attach the velcro fastener.

Now pop your favorite pens and pencils in the strap.

Keep Your Pens and Pencils in Order!

Well done! Your elastic notebook strap is ready to use (or gift to someone) and you'll always have a pen or pencil handy. My sister loves her notebook strap, so I'm sure you will love yours, too!

In this tutorial I demonstrated how to make a two-faced fabric strip. Then you learned how to sew on a hook and loop fastener, which you can use on several sewing projects, like a strap for holding make up brushes and pencils around a make-up box.

Are you inspired to make this project? Do you have any questions, suggestions or ideas? Please share them in the comments section below!


Standardization of the W-CDMA (UMTS) system, which is one of the third generation mobile communication system, is now under development by the 3GPP (3rd Generation Partnership Project). As a theme of standardization, the HSDPA (High Speed Downlink Packet Access) which can provide a maximum transmission velocity of about 14 Mbps for the downlink is specified.
The HSDPA employs an adaptive modulation and coding (AMC) system which includes, for example, a QPSK modulation method and a 16-level QAM method that are switched adaptively in accordance with the radio communication environment between the base station and mobile station.
Moreover, the HSDPA also adopts the H-ARQ (Hybrid Automatic Repeat request) system. When a mobile station has detected an error in the receiving data received from the base station, the data is re-transmitted from the base station responding to the request from the mobile station, while the mobile station executes the error correction decoding process using both received data and the data received from the re-transmission. In the H-ARQ, as described above, if an error is detected, the gain of the error correction decoding is raised and the number of times of re-transmission is controlled by effectively utilizing the received data.
The major radio channels used for the HSDPA include the HS-SCCH (High Speed-Shared Control Channel), HS-PDSCH (High Speed-Physical Downlink Shared Channel), and HS-DPCCH (High Speed-Dedicated Physical Control Channel).
The HS-SCCH and the HS-PDSCH are shared channels in the downlink direction (i.e., the direction toward a mobile station from a base station), and the HS-SCCH is a control channel for sending various parameters of data transmitted by the HS-PDSCH. The parameters, such as the modulation information which indicates the modulation method used for transmission by the HS-PDSCH, number of spreading codes assigned (number of codes), and information such as the pattern of the rate matching for the transmitting data may all be considered.
Meanwhile, the HS-DPCCH is an dedicated control channel in the uplink direction (i.e., the direction toward a base station from a mobile station) and is used to transmit the ACK signal and NACK signal to the base station from the mobile station in accordance with the acknowledgment or non-acknowledgment of reception of the data received via the HS-PDSCH. If a mobile station has failed to receive the data (the CRC error is detected in the receiving data or the like), the base station executes the re-transmission control because the NACK signal is transmitted from the mobile station or neither the ACK signal nor the NACK signal is received by the base station.
Moreover, the HS-DPCCH may also be used by the mobile station which measured the receiving quality of the signal received from the base station to transmit the result of the measurement to the base station as the CQI (Channel Quality Indicator). The base station determines the environment of the radio communication on the basis of the received CQI. When the communication environment is good, the modulation method is switched to the method for transmitting the data at the higher transmission rate. If the environment is not as good, on the contrary, the modulation method is switched to the method for transmitting the data at a lower transmission rate (namely, adaptive modulation is executed).
“Channel Format”
Next, a channel format in the HSDPA will be described below.
FIG. 1 is a diagram illustrating a channel format in the HSDPA. The W-CDMA introduces the code dividing multiplex system and each channel is therefore separated with the spreading code.
The channels not yet described will be described briefly first.
CPICH (Common Pilot Channel) and P-CCPCH (Primary Common Control Physical Channel) are respectively common channels in the downlink direction.
The CPICH is a channel used by a mobile station for estimation of channel condition, searching of cells, the timing reference of the other downlink physical channels in the same cell, and the channel used for transmitting the pilot signal. The P-CCPCH is the channel for transmitting the broadcasting information.
Next, the timing relationship of channels will be described with reference to FIG. 1.
As illustrated, one frame (10 ms) is formed based on 15 slots in each channel. As described previously, since the CPICH is used as a reference channel, the top of frames of the P-CCPCH and HS-SCCH channels are matched with the top of frame of the CPICH channel. Here, the top of frame of the HS-PDSCH channel is delayed by two slots from the HS-SCCH channel. This may be done to realize demodulation of the HS-PDSCH channel using the demodulating method that corresponds to the received modulation type after the mobile station has received the modulation information via the HS-SCCH channel. Moreover, the HS-SCCH and HS-PDSCH channels may form one sub-frame with three slots.
The HS-DPCCH channel is not synchronized with the CPICH channel but this channel is provided for the uplink direction and is based on the timing generated in the mobile station.
The channel format of the HSDPA channel has been briefly described above. Next, the processes up to transmission of the transmitting data via the HS-PDSCH channel will be described with reference to the block diagram.
“Structure of Base Station”
FIG. 2 illustrates a structure of a base station supporting the HSDPA channel.
In FIG. 2, the reference numeral 1 designates a CRC attachment unit; 2, a code block segmentation unit; 3, a channel coding unit; 4, a bit separating unit; 5, a rate matching unit; 6, a bit collecting unit; 7, a modulating unit.
Next, operations of each block will be described.
The transmitted data transmitted via the HS-PDSCH channel (data accommodated within one sub-frame of the HS-PDSCH channel in FIG. 1) is first subjected to the CRC arithmetic process in the CRC attachment unit 1 and the result of the arithmetic operation is added to the last part of the transmitting data. The transmitting data to which the result of CRC arithmetic operation is added may then be inputted to the code block segmentation unit 2 and is then segmented into a plurality of blocks. This process is required to shorten the data length in unit of the error correction coding, considering the load of decoding process in the receiving side. When the data length exceeds the predetermined length, the code block is equally segmented into a plurality of blocks. An integer 2 or larger may be selected as the number of segmentations but the number of segmentations 2 may be selected to simplify the description. If the data length is rather short, segmentation of the block may be unnecessary.
The segmented and transmitted data are respectively processed as the object data of the individual error correction encoding process in the channel coding unit 3. In other words, the error correction encoding process is executed for the segmented first block and second block. As an example of the channel encoding process, a turbo encoding process may be used.
Here, the turbo encoding process will be described briefly. In an exemplary turbo encoding process, when the data is defined as U, the data U′ obtained by the convolutional encoding of the data U, and the data U″ obtained by the convolutional encoding of the data U after the interleave (re-arrangement) process of the data U may be outputted. Here, the data may be referred to as the systematic bits and may be understood, in the turbo decoding process, as the data used in two element decoders and the data having a higher degree of importance because the application frequency is high. On the other hand, the data U′ and U″ are parity bits. These bits are data used in one of the two element decoders and have a degree of importance that is lower than that of the data U because the application frequency is low.
Namely, it can be said that since the systematic bits have a higher degree of importance than that of the parity bits, and the systematic bits are received with greater accuracy, the more accurate decoding result can be obtained with the turbo decoder.
The systematic bits and parity bits that are generated as described above may be inputted as the serial data to the bit separating unit 4. The bit separating unit 4 separates the input serial data into the data U, U′, and U″ of three systems, and then outputs this data as parallel data.
The rate matching unit 5 preferably performs the puncture process for deleting the bits with the predetermined algorithm and also executes the repetition process to repeat the bits in order to store the data within the sub-frame formed of three slots of the HS-PDSCH channel.
As described above, the bits having completed the bit adaptation process to the sub-frame are then inputted in parallel to the bit collecting unit 6.
The bit collecting unit 6 preferably generates bit sequences of four bits indicating each signal point, for example, of 16-level QAM modulation based on the input data and then outputs these bit sequences. At the time of generation of bit sequences, the systematic bits are preferably arranged, for the first transmission, in the side of upper bits in which an error is not easily generated.
The modulating unit 7 outputs the signal of the 16-level QAM modulation to provide the amplitude and phase corresponding to the signal points indicated with the input bit sequences and then provides the signal to the side of the antenna (not illustrated) after conversion to the radio frequency through the frequency conversion.
“Detailed Description of the Rate Matching Unit 5”
FIG. 3 illustrates one embodiment of the rate matching unit 5. The rate matching unit 5 comprises, as illustrated, a first rate matching unit 51, a virtual buffer 52, and a second rate matching unit 53.
The first rate matching unit 51 executes the rate matching process (puncture process) to the first parity bits (U′) and second parity bits (U″) separated in the bit separating unit 4. This process may be executed, considering the capability of the mobile station in the receiving side described below (capacity of memory or the like to store the data obtained by combining the received data and re-transmitted data), in order to keep the maximum amount of data which can be transmitted including the re-transmission under the capability of the mobile station. Accordingly, bits are deleted on the basis of a predetermined rule.
The virtual buffer unit 52 may be provided to store the systematic bits. The first and second parity bits may have completed the puncture process (deletion of bits) in accordance with the capability of the mobile station.
The second rate matching unit 53 performs the puncture process and the repetition process. The puncture process deletes the bits based on the predetermined rule, and the repetition process repeats the bits for the data read from the virtual buffer in order to store the data within the sub-frame formed from the three slots of the HS-PDSCH channel.
The structure and operations of the rate matching unit 5 are described above and one embodiment of data due to the process performed by the rate matching unit 5 will be described with reference to FIG. 4.
The block A in FIG. 4 illustrates the systematic bits (U), first parity bits (U′), and second parity bits (U″) inputted to the first rate matching unit 51.
The first rate matching process unit 51 executes the puncture process to the block A to attain the predetermined amount of data determined in accordance with the capability of the mobile station and then provides an output. Namely, the bits are deleted to result in the amount of data indicated in the block B considering the capability of the mobile station.
Various methods may be assumed for deletion of bits. For example, the block B illustrated in FIG. 4 may be formed by not applying the puncture process to the systematic bits. These systematic bits are important, and the puncture process is applied to the first and second parity bits (indicated as U′ (r) and U″ (r) in order to show execution of the puncture process as the rate matching process). In FIG. 4, the left half bits are deleted but it is preferable to delete the bits at the positions which a dispersed to a certain degree. For example, the even number bits or odd number bits are deleted.
The second rate matching unit 53 executes the rate matching process to store the data within the sub-frame on the basis of the data block B puncture-processed in the first rate matching unit 51 and then outputs the transmitting data.
For example, with the first transmission, the systematic bits U are outputted as the block C after execution of the puncture process. With the second transmission (first re-transmission), the first parity bits U′ (r) and the second parity bit U″ (r) are outputted as block D after execution of the puncture process. The number of times that the re-transmission may occur may be a predetermined number equal to 1 or larger. However, when the third transmission is assumed to be the last re-transmission which includes the first transmission, the block C obtained by the puncture process is transmitted again, for example, to the block B in the third transmission (second re-transmission).
The items regarding the HSDPA channel are disclosed, for example, in the non-patent document “3G TS 25. 212 (3rd Generation Partnership Project: Technical Specification Group Radio Access Network; Multiplexing and channel coding (FDD))”.
According to the background technology described above, the amount of data transmitted to a mobile station is previously limited (limited to the block B), with the (first) rate matching, to the value below the capability of the receiving apparatus (capacity of memory or the like to store the data obtained by combining the received data and re-transmitted data). Thus, the receiving apparatus does not receive data exceeding the capability (capacity of memory) and is capable of executing the error correction decoding process by completely utilizing the first receiving data and the re-transmitted receiving data.
However, such limitation will close the way of utilization of the data part exceeding the capacity of memory of the receiving apparatus. This will result in the disadvantage that the sufficient capability of the error correction decoding process cannot be ensured.
Elimination of such limitation may be achieved by increasing the capacity of the memory of the receiving apparatus. However, when reduction in the size of the mobile station is alienated due to the increase in capacity of memory and particularly when soft-determination data is used for the error correction decoding process, the amount of data increases drastically. Therefore, elimination of the limitation cannot be employed directly.


Q:

Why the overhead when allocating objects/arrays in Java?

How many bytes an array occupies in Java? Assume It's a 64bit machine and also assume there are N elements in an array, so all these elements would take up 2*N, 4*N or 8*N bytes for different types of array.
And a lecture in Coursera says that it would occupy 2*N+24, 4*N+24 or 8*N+24 bytes for a N element array and the 24 bytes is called overhead, but didn't explain why the overhead is needed.
Also objects have overheads, which is 16 bytes.
What exactly are these overheads? What are these 24/16 bytes composed of?
Also, do these overheads only exist in Java? How about C, C++ and Python?

A:

Each Java object has a header that contains information important for the JVM. The most important is a reference to the object's class (one machine word), and there are some flags used by the garbage collector and to manage synchronization (since every object can be synchronized on) which takes up another machine word (using partial words would be bad for performance). So that's 2 words, which is 8 bytes on 32 bit systems, and 16 bytes on 64 bit. Arrays additionally need an int field for the array length, which is another 4 bytes, possibly 8 on 64 bit systems.
As for other languages:

C doesn't have objects, so of course it doesn't have object headers - but may have a header on each separately allocated piece of memory.
In C++, you don't have garbage collection and cannot use arbitrary objects for synchronization, but if you have classes with overridden methods, each object has a pointer to its vtable, just like the Java object's reference to its class. If you use smart pointers that do garbage collection, they need housekeeping data.
I don't know about Python, but I'm pretty sure it also needs a reference to the class, and housekeeping information for the garbage collector.


After Brief Prayer From David Platt, Trump Announces He's Selling All His Possessions And Going On A Missions Trip To Somalia

WASHINGTON, D.C.—In a surprise announcement, President Trump has declared he is selling everything that he has except the clothes on his back, giving the proceeds to the poor, and traveling to Somalia to preach the gospel after Pastor David Platt briefly prayed over him Sunday.

The president picked up a copy of Radical on the way out of the church, read it on the plane ride, and began to rethink his entire life.

"It's really sad how many Christians think the American Dream is synonymous with the gospel," he said as he looked out the window of Air Force One pensively. "Why do we settle for this cheap, empty gospel that says that God loves you and has a wonderful plan for your life, when the real, life-changing gospel is so much more---indeed, when the gospel says we will lose everything but gain the only thing of true value: Christ Himself?"

"Wake up, America. Jesus is the prize of the Christian life, and that is enough."

Trump added that the gospel is "maybe the best deal in the history of deals" as he embarked on his missions trip to reach the dangerous closed countries of the world.

Get Free Access To Our Brand New Site: Not the Bee After creating The Babylon Bee in six literal days, Adam Ford rested. But he rests no longer. Introducing Not the Bee — a brand new humor-based news site run by Adam himself. It's loaded with funny content and all the best features of a social network. And the best part? Everyone with a subscription to The Bee gets full access at no extra cost. Get FREE Access *with premium subscription to The Babylon Bee


Bipolar anastomosis technique (BAT) enables "fast-to-do", high-quality venous end-to-end anastomosis in a new vascular model.
The interrupted suture technique is most commonly used for microsurgical venous anastomosis. Needle-stitch trauma and intraluminal suture, however, potentially cause vascular wall damage, thrombosis, intimal hyperplasia or even stenosis. Therefore, the present study aimed to show the feasibility and reliability of a modified cuff technique (bipolar anastomosis technique (BAT)) for venous end-to-end anastomosis in a new chicken throat vascular model. In ex vivo experiments, freshly resected chicken jugular veins (N = 96) were used to find ideal BAT time to current settings for venous end-to-end anastomosis. Thereafter, the left jugular vein of chickens (N = 40) was dissected in vivo and subsequently anastomosed using BAT. The quality of anastomosis was evaluated by Doppler sonography immediately, at two hours and at two, six, 12, 16, and 29 weeks after surgery. Additional histological examination took place at two hours (N = 8) and at two (N = 6), six (N = 6), 12 (N = 6), 16 (N = 6) and 29 (N = 6) weeks after surgery. Immediately after surgery (N = 40) and at two hours (N = 38) venous anastomoses were found to be patent in Doppler sonography. Anastomotic rupture caused death in two animals within one hour after surgery. Thrombotic occlusion was found in one animal at six weeks after surgery. In the remaining animals (N = 37) only minimal stenosis which decreased to almost normal levels was sonographically found. The average time needed for anastomosis using BAT was less than two minutes. BAT allows fast venous end-to-end anastomosis in a chicken throat vascular model.


“Se comunica que al caso reportado de coronavirus oriundo de Río Negro que se informó como descartado el martes 17 de marzo por la Administración Nacional de Laboratorios e Institutos de Salud (ANLIS), dada su evolución clínica y la persistencia de sospecha se le tomó ese mismo día una segunda muestra (aspirado traqueal). Esta segunda muestra (distinta a la primera que se le tomó) fue procesada en la provincia del Neuquén por el Laboratorio Central y luego enviada al Instituto Malbrán. El análisis inicial en nuestra provincia arrojó un resultado positivo para COVID-19. Ahora el Comité de Emergencia se encuentra a la espera del Malbrán, para validar el resultado”, informaron desde el ente provincial.


Upgrade Your Makeup Storage

Mason jars have plenty of uses, and they make great displays for things like your nail polish, lip gloss collection, makeup brushes, and a ton more. One nifty DIY takes your regular mason jar and turns it into a chic and useful storage display, using only hot glue and spray paint.


Behavioural interventions to reduce sexual risk for HIV in heterosexual couples: a meta-analysis.
The current study was conducted to synthesise evaluations of couple-based HIV prevention interventions, assess the efficacy of these interventions in reducing sexual risk, and identify moderators of intervention efficacy. A comprehensive literature search identified 29 interventions (22 reports; N=5168 couples) that met the inclusion criteria, including enrolment of both members of a heterosexual couple, measurement of condom use at baseline and follow-up, and sufficient statistical information to calculate effect sizes. Effect sizes were analysed using fixed-effects and random-effects assumptions; factors related to intervention efficacy were identified using metaregression. Overall, there were significant increases in condom use from baseline to follow-up (d+=0.78, 95% CI 0.48 to 1.09) and significant decreases in partner concurrency (d+=0.37, 95% CI 0.13 to 0.60). Condom use increased to a greater extent when studies were conducted toward the beginning of the epidemic, were located in countries scoring lower on the Human Development Index, enrolled serodiscordant couples, and delivered intervention content in multiple contexts. Couples who had been together longer, reported higher incidence of sexually transmitted infection, were provided voluntary counselling and testing, and provided outcome measures during face-to-face interviews also reported larger increases in condom use. Couple-based interventions are efficacious in reducing unprotected sex within the context of romantic relationships. Future research should continue to improve risk reduction for couples.


Feb142018

Today, more than 75 million people around the world are starving and 800 million lack enough food to live a healthy life.

Every year, poor nutrition causes more than three million children to die from hunger, nearly half of whom are under the age of 5.

Making matters worse, there are an unprecedented four countries – Yemen, Somalia, South Sudan, and Nigeria – experiencing near-famine conditions at the same time, and warring parties are, in many cases, deliberately blocking access to aid.

For decades, American farmers have played a critical role in reducing hunger overseas. Since the Food for Peace program was established by Congress in 1954, more than four billion people and more than 150 countries have benefited from U.S.-supported food security programs.

This relief is thanks in large part to the innovation, expertise, and unmatched capacity of American farmers to produce high-quality foods that are critical to meeting growing global needs.

Other developed countries have also spent billions of dollars in food aid to confront this global state of emergency, but no nation comes close to the United States.

Unfortunately, despite the American people’s generosity, we are not currently able to meet the tremendous demand as conflict hinders access to those most vulnerable to famine.

What you may not know is that we have an opportunity to feed nine million more starving people each year without spending a single additional taxpayer dollar if we modernize the Food for Peace program when Congress reauthorizes the farm bill later this year.

But modernizing Food for Peace does not mean removing the mission-critical role of American farmers.

According to research from the U.S. Agency for International Development, the Government Accountability Office, and research institutions such as Cornell University and Montana State University, merely allowing the program to use a more flexible mix of American, local, and regionally-sourced foods would save up to $300 million, which could then be used to feed up to nine million more people and do so much faster.

Currently, Food for Peace requires almost all food aid to be sourced from the United States. In contrast, other governments provide much of their food aid from local and regional markets closer to starving populations.

In many cases, locally-sourced food can more efficiently reach those in need months sooner than food from the United States while still providing the opportunity for U.S.-sourced foods to help meet longer term needs when and where appropriate.

Furthermore, there are millions of starving people of all ages in war-torn regions where it is nearly impossible for American food products to reach.

Some have raised concerns that reducing the amount of food aid sourced from the United States might hurt American farmers.

The reality is that, while our country provides the lion’s share of global food aid, it represents only 0.2 percent of total U.S. agricultural output. Modernizing Food for Peace will save millions of lives without undermining our farmers, who will continue to be a key component of the Food for Peace program.

Efforts currently underway in Congress that we support would do just that. American farmers have a long, proud history of feeding the world. You can join them in continuing to be leaders in the fight against hunger by encouraging your representatives and senators in Washington to make modernizing the Food for Peace program a priority in this year’s farm bill.

Bob Corker, of Tennessee, is chairman of the U.S. Senate Foreign Relations Committee. Chris Coons, of Delaware, is a member of the U.S. Senate Foreign Relations Committee. Zippy Duvall is president of the American Farm Bureau Federation.


Introduction
============

The chloroplast genome, also known as chloroplast DNA, is often abbreviated as cpDNA. It shows the typical quadripartite structure generally consisting of four parts with a large single copy (LSC), a small single copy (SSC), and two inverted repeats (IRs) ([@ref-22]; [@ref-46]). The two IR regions are in the same sequence, but in the opposite directions. In general, 400--1,600 chloroplast genome copies are contained in plant cells (leaves) ([@ref-76]). The variation of chloroplast genome size is mainly affected by the length of the IR regions. For example, in the evolutionary process of the *Geranium* chloroplast genome, IR regions increased by 76 kb ([@ref-45]). The IR regions of gymnosperms, such as Japanese black pine, which was only 495 bp in length ([@ref-65]), whereas the IR regions of legumes, such as *Medicago*, disappeared completely ([@ref-53]). This polymorphism of chloroplast genome has important research significance in phylogenetic and population genetics.

The chloroplast genome sequence has been successfully applied to the taxonomic and phylogenetic studies of many species. And the molecular evolution rate of the coding and non-coding regions are significantly different ([@ref-43]). However, cpDNA is highly conserved ([@ref-68]), shows maternal inheritance ([@ref-10]), and does not undergo genetic recombination. The control of chloroplast gene expression is affected by environmental factors and its developmental program, operating at several steps, including transcription, post-transcription, translation, and post-translation. The chloroplast gene expression is largely controlled at the post-transcriptional level ([@ref-63]). Therefore, the plant chloroplast genome has significant advantages in revealing the relationship of species, and can provide a large amount of important data on chloroplast genetic transformation.

*Camellia oleifera* belongs to the genus *Camellia* of the family Theaceae. It is mainly distributed in the subtropical mountains of China ([@ref-82]; [@ref-83]). A forest of *C. oleifera* has a good ecological value in maintaining soil and water and in regulating climate. In addition, its tea oil has a high medicinal value ([@ref-19]). In this study, we chose the *Camellia* species with a unique living environment (on Hainan Island), which is different from the other *Camellia* distribution provinces in China. The unique island climatic conditions have produced excellent and unique *C. oleifera* resources ([@ref-81]). However, so far, the background in the genetics and genomics research of *Camellia* is rather weak. Due to frequent interspecific hybridization and polyploidization, the classification and phylogenetic studies of *C. oleifera* are quite difficult ([@ref-20]). Moreover, the *C. oleifera* on Hainan Island is an independent population, has a certain geographic isolation, with a relatively large genetic variation of its population. In the long run, this will gradually lead to the loss of the resources of germ-plasm. Therefore, this study will analyze the chloroplast genomic sequence of the Hainan *C. oleifera* to understand its similarities and differences with the other *Camellia* species in the process of natural selection, and identify its taxonomic groups, thereby providing valuable genomic resources for molecular breeding and phylogenetic research.

Materials and Methods
=====================

DNA extraction, sequencing, and annotation
------------------------------------------

Fresh leaves of *C. oleifera* were obtained by germinating the seeds that were collected from Chengmai, Hainan Province (110.00°E, 19.75°N) in China and the seeds were collected in areas that were not privately owned or protected in any way and no specific permits were required for this study. The treatment of plants and DNA extraction refer to the method of [@ref-80] and more experiment details could be got from our announcement.

The genome can be assembled directly by using overlap between sequenced reads; a total of 2.83G raw data was obtained, including 2.09G of clean data. The quality of the sequencing data of the samples was visually evaluated by the software Fastqc v 0.10.0 (<http://www.bioinformatics.babraham.ac.uk/projects/fastqc/>). After the Illumina PCR adapter reads and low-quality reads were filtered from the paired-end and mate-pair library in the quality control step, all good-quality paired reads were assembled to contigs by using SOAPdenovo2 ([@ref-37]). The assembled contigs were joined into multiple scaffolds using SSPACE ([@ref-5]) to obtain the whole-genome sequence.

The programs DOGMA ([@ref-70]) and cpGAVAS ([@ref-34]) were used for the functional annotation and gene prediction of the whole chloroplast genome, respectively. Subsequently, the start and stop codons were adjusted manually by means of the Sequin tool to get the final open reading frame (ORF). The GeneMarkS (version 4.17) (<http://topaz.gatech.edu/>) program was used to retrieve the related coding genes. The predicted protein sequences were aligned using the National Center for Biotechnology Information (NCBI) Conserved Domain Database CDD ([@ref-38]) to predict the Clusters of Orthologous Group of Proteins function of the genes and make the classification statistics ([@ref-77]). The circular chloroplast genome map of Hainan *C. oleifera* was generated with the OGDRAW ([@ref-36]) software. The complete chloroplast genome sequence of Hainan *C. oleifera* has been submitted to the NCBI with GenBank accession number [MF541730](MF541730).

Comparative analysis of chloroplast genomes
-------------------------------------------

The number of protein-coding genes, the GC content and the length of each region in the Hainan *C. oleifera* chloroplast genome and other nine Theaceae plants were compered by manually. A multiple sequence alignment of the 10 Theaceae chloroplast genomes was performed using mVISTA ([@ref-12]; [@ref-39]), with the annotated Hainan *C. oleifera* chloroplast genome sequence as a reference. The following nine species (with their GenBank accession numbers given in parentheses) are members of the genus *Camellia*: *C. oleifera* ([JQ975031](JQ975031)), *C. luteoflora* ([KY626042](KY626042)), *C. grandibracteata* ([NC_024659](NC_024659)), *C. sinensis* ([KC143082](KC143082)), *C. leptophylla* ([NC_024660](NC_024660)), *C. pubicosta* ([NC_024662](NC_024662)), *C. crapnelliana* ([KF753632](KF753632)), *C. huana* ([KY626040](KY626040)), and *C. danzaiensis* ([NC_022460](NC_022460)), which were downloaded from the GenBank database of NCBI (<https://www.ncbi.nlm.nih.gov/pubmed>). The visual comparisons of the IR/LSC and IR/SSC border regions of chloroplast genome from nine different species closely related to Hainan *C. oleifera* were conducted using the drawing software Visio 2013.

DNA barcode compared of Hainan *C. oleifera* and Mainland species
-----------------------------------------------------------------

We selected the important DNA barcode sequences *matK* and *trnH-psbA* to compare Hainan *C. oleifera* and Mainland *C. oleifera*. The following objects: *C. oleifera-matK* ([KP094074](KP094074), [KP094075](KP094075), [KR530501](KR530501), [KX216462](KX216462)) and *C. oleifera-trnH-psbA* ([KX121760](KX121760), [KR533766](KR533766), [KP095297](KP095297), [GQ435325](GQ435325), [GQ435326](GQ435326), [GQ487355](GQ487355), [KP095298](KP095298)) were involved. All registration numbers can be found in NCBI. The nucleotide sequence information of some *C. oleifera* was searched by NCBI database blast, and compared with the same nucleotide position of Hainan *C. oleifera* by the software MEGA 7.0.

SSRs characterization and long repeat sequences
-----------------------------------------------

The simple sequence repeats (SSRs) are also known as the short tandem repeats or microsatellites. Because of the presence of different nucleotides in the repeat units and different number of repeats, a high degree of variability in SSR length was caused. The microsatellite (di-, tri-, tetra-, penta-, and hexa-nucleotide repeats) assays were performed by using the software SSRHunter 1.3 ([@ref-32]) with a minimum number of four repeat units for di- and tri-nucleotides and three for tetra-, penta-, and hexa-nucleotides.

The REPuter ([@ref-29]) software was used to predict the chloroplast genome long repeat sequences, which were divided into four categories, forward match, reverse match, palindromic match and complementary match, according to their comparison. In this paper, we examined the long repeat sequences with a minimum repeat length of 30 bp and a maximum of 60 bp.

Indices of codon usage
----------------------

The relative synonymous codon usage (RSCU) ([@ref-56]) of Hainan *C. oleifera* chloroplast genome was calculated using DAMBE6 ([@ref-71]) based on the sequence of protein-coding genes in the chloroplast genome. The obtained RSCU values were statistically analyzed by SigmaPlot 10.0 and plotted into a histogram. The distribution of codon usage for the 10 Theaceae species is shown in the form of a heatmap, which was constructed using HemI 1.0 ([@ref-11]), according to the RSCU value. As an intuitive representation, it can graphically display the matrix data by describing each value in different color.

Sequence phylogenetic analysis
------------------------------

Evolutionary trees that indicate the evolutionary relationships among species which are thought to have a common ancestor are referred to as phylogenetic trees. The maximum likelihood method was used to determine the phylogenetic relationships of the Hainan *C. oleifera* clade with the clades of 21 other Theaceae species using MEGA 7.0 ([@ref-28]) and an online software Interactive Tree of Life (iTOL) ([@ref-30]). The GenBank databases was used to download the amino acid sequences 21 *Camellia* species, including *C. oleifera* ([JQ975031](JQ975031)), *C. huana* ([KY626040](KY626040)), *C. impressinervis* ([KF156835](KF156835)), *C. pitardii* ([KF156837](KF156837)), *C. danzaiensis* ([KF156834](KF156834)), *C. grandibracteata* ([KJ806274](KJ806274)), *C. leptophylla* ([KJ806275](KJ806275)), *C. sinensis var. sinensis* ([KJ806281](KJ806281)), *C. sinensis var. dehungensis* ([KJ806279](KJ806279)), *C. crapnelliana* ([KF753632](KF753632)), *C. taliensis* voucher HKAS:S.X.Yang3157 ([KF156839](KF156839)), *C. taliensis* voucher HKAS:S.X.Yang3158 ([KF156836](KF156836)), *C. reticulata* ([KJ806278](KJ806278)), *C. sinensis var. pubilimba* ([KJ806280](KJ806280)), *C. pubicosta* ([KJ806277](KJ806277)), *C. sinensis* ([KC143082](KC143082)), *C. sinensis* cultivar Longjing 43 ([KF562708](KF562708)), *C. cuspidata* voucher HKAS: S.X.Yang3159 ([KF156833](KF156833)), *C. yunnanensis* voucher HKAS:S.X.Yang1090 ([KF156838](KF156838)), *C. petelotii* ([KJ806276](KJ806276)), *C. luteoflora* voucher CLUTE20161220 ([KY626042](KY626042)). The model for phylogenetic assessment was based on the Newick tree file generated by MEGA 7.0. The tree was then imported to the iTOL online system. The bootstrap was quickly calculated with the iTOL's in-built system, followed by the construction of the final circular phylogenetic tree. The same color-coded species in the picture represented similar phylogenetic relationships. We constructed phylogenetic trees using the following datasets due to the different molecular evolution rates of different cp genomic regions: (1) Complete chloroplast genome sequence; (2) Protein coding sequence; (3) LSC region; (4) SSC region. On the other hand, barcode labeling has been successfully applied to the study of phylogenetic relationships, and we have also compared the base sequences of DNA barcode (*rbcL*, *matK*, and *trnH-psbA*) of Hainan *C. oleifera* chloroplast genome and other 21 Theaceae plants. These three sites are commonly used research objects in DNA barcodes and are important molecular tools for identifying organisms ([@ref-17]; [@ref-74]).

Results and Analysis
====================

Chloroplast genome characteristic of Hainan *C. oleifera*
---------------------------------------------------------

Like the majority of land plants, the whole chloroplast genome of Hainan *C. oleifera* showed a typical quadripartite genome organization with a size of 156,995 bp, including a LSC region of 86,648 bp and a SSC region of 18,297 bp, which were separated by two IR (IRa and IRb) regions of 26,025 bp ([Fig. 1](#fig-1){ref-type="fig"}).

![Gene map of the Hainan *C. oleifera* chloroplast genome.\
The outermost colored blocks represent the physical location of different genes on the chloroplast genome, the inner circle is the physical location of the LSC, SSC, and IR regions on the genome and the different colors represent genes of different functional classes. Genes distributed outside the circle are transcribed counterclockwise, whereas genes inside are transcribed clockwise. The dark gray in the inner circle represents GC content, and the light gray represents AT content.](peerj-07-7210-g001){#fig-1}

The chloroplast genome of Hainan *C. oleifera* was found to contain 141 predicted functional genes, including 88 protein-coding genes, 45 tRNA genes and eight rRNA genes, which were classified according to their function. Among the observed genes, 81 protein-coding genes, 30 tRNA genes, and four rRNA genes were unique. Among the protein-coding genes, 74 were single-copy genes and seven (*ndhB*, *rps12*, *rps7*, *rpl2*, *rpl23*, *ycf2*, and *orf42*) were duplicates. Among the tRNA genes, 19 were unique, and 11 (*trnA-UGC, trnI-CAU*, *trnI-GAU*, *trnL-CAA*, *trnL-UAA*, *trnM-CAU*, *trnN-GUU*, *trnR-ACG*, *ttrnT-GGU*, *trnV-GAC*, and *trnV-UAC*) were duplicates. The four rRNA genes were completely duplicated ([Table 1](#table-1){ref-type="table"}). In addition, four overlapping genes (*psbD*-*psbC*, *ndhK*-*ndhC*, *trnM-CAU*-*trnT-GGU*, and *trnP-GGG*-*trnP-UGG*) were detected. Among these genes, nine genes (*atpF*, *rpoC1*, *rpl2*, *ndhA*, *ndhB*, *ycf1*, and *trnI-GAU*) contained a single intron, of which *rpl2* and *ndhB* were identified as repetitive genes. Two genes (*ycf3* and *clpP*) contained two introns. These two genes were similar to those in the Orpheus flower *Haberlea rhodopensis* and *Broussonetia papyrifera* ([@ref-21]; [@ref-73]). Unlike most angiosperm chloroplast genomes ([@ref-23]; [@ref-25]; [@ref-67]), among the 11 genes, four (*atpF*, *rpoC1*, *ycf3*, *clpP*) of which were located in the LSC region, two (*ndhA* and *trnI-GAU*) in the SSC region, other four (*rpl2*×2, *ndhB*×2) in the IR region, and one gene (*ycf1*) was a trans-spliced gene with its start codon located in the SSC region, stop codon in the IRb region, and possibly missing 3′ end. Additionally, the *ndhA* gene contained the largest intron (1,093 bp), and the *ycf1* gene had the smallest one (27 bp) ([Table S1](#supp-1){ref-type="supplementary-material"}).

10.7717/peerj.7210/table-1

###### Genes present in the Hainan *C. oleifera* chloroplast genome.

![](peerj-07-7210-g008)

  Group of genes                 Gene names
  ------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Photosystem I                  *psaA*, *psaB*, *psaC*, *psaI*, *psaJ*
  Photosystem II                 *psbA*, *psbB*, *psbC*, *psbD*, *psbE*, *psbF*, *psbH*, *psbI*, *psbJ*, *psbK*, *psbL*, *psbM*, *psbN*, *psbT*, *psbZ*
  Cytochrome b/f complex         *petA*, *petB*, *petD*, *petG*, *petL*, *petN*
  ATP synthase                   *atpA*, *atpB*, *atpE*, *atpF*[\*](#table-1fn1){ref-type="fn"}, *atpH*, *atpI*
  NADH dehydrogenase             *ndhA*[\*](#table-1fn1){ref-type="fn"}, *ndhB*(×2)[\*](#table-1fn1){ref-type="fn"}, *ndhC*, *ndhD*, *ndhE*, *ndhF*, *ndhG*, *ndhH*, *ndhI*, *ndhJ*, *ndhK*
  RubisCO large subunit          *rbcL*
  RNA polymerase                 *rpoA*, *rpoB*, *rpoC1*[\*](#table-1fn1){ref-type="fn"}, *rpoC2*
  Ribosomal proteins (SSU)       *rps11*, *rps12*(×2), *rps14*, *rps15*, *rps16*, *rps18*, *rps19*, *rps2*, *rps3*, *rps4*, *rps7*(×2), *rps8*
  Ribosomal proteins (LSU)       *rpl14*, *rpl16*, *rpl2*(×2)[\*](#table-1fn1){ref-type="fn"}, *rpl20*, *rpl22*, *rpl23*(×2), *rpl32*, *rpl33*, *rpl36*
  Proteins of unknown function   *ycf1*[\*](#table-1fn1){ref-type="fn"}, *ycf15*, *ycf2*(×2), *ycf3*[\*\*](#table-1fn1){ref-type="fn"}, *ycf4*
  Transfer RNAs                  *trnA-UGC*(×4)*, trnC-GCA*, *trnD-GUC*, *trnE-UUC*, *trnF-GAA*, *trnfM-CAU*, *trnG-UCC*, *trnH-GUG*, *trnI-CAU*(×2), *trnI-GAU*(×4), *trnK-UUU*, *trnL-CAA*(×2), *trnL-UAA*(×2), *trnL-UAG*, *trnM-CAU*(×2), *trnN-GUU*(×2), *trnP-GGG*, *trnP-UGG*, *trnQ-UUG*, *trnR-ACG*(×2), *trnR-UCU*, *trnS-GCU*, *trnS-GGA*, *trnS-UGA*, *trnT-GGU*(×2), *trnT-UGU*, *trnV-GAC*(×2), *trnV-UAC*(×2), *trnW-CCA*, *trnY-GUA*
  Ribosomal RNAs                 *rrn16*(×2), *rrn23*(×2), *rrn4.5*(×2), *rrn5*(×2)
  Other genes                    *infA*, *matK*, *clpP*[\*\*](#table-1fn1){ref-type="fn"}, *accD*, *ccsA*, *cemA*, *orf42*(×2)

**Notes:**

One or two asterisks after genes indicate that gene contains one or two introns, respectively.

The numbers in parentheses indicate the copy number of the gene.

Comparison with other Theaceae chloroplast genomes
--------------------------------------------------

The chloroplast genomic characteristics of Hainan *C. oleifera* were compared with nine other Theaceae species ([Table 2](#table-2){ref-type="table"}). Similar to the reported results of the Theaceae chloroplast genome ([@ref-59]; [@ref-75]), the whole-genome length of Hainan *C. oleifera* was not much different from that of the other species. This change in sequence length depends mainly on the difference in the length of the LSC region ([@ref-33]). The GC contents of the 10 species of *Camellia* were 37.29--37.34%, which were unevenly distributed in the whole genomes, with IRs exhibiting the highest GC content (42.94--43.01%), followed by the LSC (35.29--35.36%) and SSC regions (30.52--30.63%). Among them, the GC content in each area of Hainan *C. oleifera* and *C. oleifera* was the closest. As an important marker of molecular evolution, the IR region has a high GC content, which makes the sequence stable and highly conserved. In general, the protein-coding genes of two *C. oleifera* were the closest, although the total number of genes in Hainan *C. oleifera* was much larger than that of mainland *C. oleifera*, which has the largest number of 141 genes. This phenomenon may be caused by gene recombination and may affect its evolution.

10.7717/peerj.7210/table-2

###### The basic composition of the Hainan *C. oleifera* chloroplast genome and other nine Theaceae plants.

![](peerj-07-7210-g009)

                         Total sequence length (bp)/GC content   LSC length/GC content   SSC length/GC content   IR length/GC content   Number of genes   Protein-coding genes
  ---------------------- --------------------------------------- ----------------------- ----------------------- ---------------------- ----------------- ----------------------
  *C. oleifera* Hainan   156,995/37.29%                          86,648/35.29%           18,297/30.55%           26,025/42.98%          141               88
  *C. oleifera*          156,971/37.31%                          86,515/35.30%           18,288/30.54%           26,084/42.98%          133               87
  *C. luteoflora*        157,166/37.30%                          86,719/35.32%           18,293/30.59%           26,077/42.96%          133               91
  *C. grandibracteata*   157,127/37.29%                          86,656/35.32%           18,285/30.52%           26,093/42.94%          127               91
  *C. sinensis*          157,103/37.31%                          86,645/35.34%           18,276/30.54%           26,091/42.95%          135               91
  *C. leptophylla*       157,102/37.30%                          86,647/35.32%           18,275/30.58%           26,090/42.95%          126               91
  *C. pubicosta*         157,076/37.30%                          86,649/35.33%           18,279/30.54%           26,074/42.95%          127               91
  *C. crapnelliana*      156,997/37.30%                          86,655/35.30%           18,406/30.60%           25,968/43.01%          136               91
  *C. huana*             156,903/37.32%                          86,568/35.34%           18,203/30.63%           26,066/42.96%          133               92
  *C. danzaiensis*       156,576/37.34%                          86,204/35.36%           18,259/30.59%           26,056/42.98%          135               92

In the present paper, the IR/LSC and IR/SSC regions of the Hainan *C. oleifera* chloroplast genome were compared with the corresponding regions of the nine closely related Theaceae species ([Fig. 2](#fig-2){ref-type="fig"}). As can be seen from the other *Camellia* chloroplast genome studies ([@ref-20]), the length of the chloroplast genome was variable, mainly due to the expansions and contractions of the border regions. Consistently, in all of the comparative chloroplast genomes, the IRb/SSC regions were located in the coding sequences of the *ycf1* gene. The *rpl2* (1,494 bp) and *trnH* (74 bp) genes were located on both sides of the IRb/LSC regions and were 106 and 2 bp away from the borders, respectively. However, the *rps19* gene was located in the LSC region because of the expansion of the Hainan *C. oleifera* LSC region border, which was eight bp apart from the IRa/LSC region. Similarly, the *rps19* gene from the mainland *C. oleifera* was also more biased towards the LSC region, whereas in the other eight *Camellia* species, with 232 bp occupying the LSC region and 46 bp occupying the IRa region. Like most plants, the *ndhF* gene involved in photosynthesis was located in the SSC region ([@ref-58]) and was 2,246 bp in length in all *Camellia* species, except in *C. huana* and *C. oleifera*. It should be emphasized that the *ycf1* gene in the IRa regions was not observed in Hainan *C. oleifera*, *C. crapnelliana*, and *C. danzaiensis* because of the contraction of the IRa region border, which was replaced by the *trnN* gene. By comparing the border regions of the chloroplast genomes of 10 Theaceae species, it can be summarized as two different types and main feathered by gene structure of IRa/SSC. The *trnN*-*ndhF* genes located in above region of Hainan *C. oleifera*, *C. crapnelliana* and *C. danzaniensis*, while other species were *ycf1*-*ndhF*.

![Comparison of the border positions of LSC, SSC, and IRs regions in chloroplast genome sequences of 10 Theaceae species.](peerj-07-7210-g002){#fig-2}

A multiple sequence alignment of the chloroplast genomes of the 10 Theaceae species was presented in [Fig. 3](#fig-3){ref-type="fig"}. The result showed a high degree of similarity in the chloroplast genome sequences, suggesting that the chloroplast genome in the Theaceae species was evolutionarily conserved, despite of the discovery of several different regions in it, while the conservative of Theaceae plants is relatively rare in all angiosperms, which also makes the study of its evolution a certain pressure. Like other plants, the non-coding regions showed higher sequence diversity than the coding regions, whereas the IRs regions were more conserved than the single-copy regions ([@ref-20]; [@ref-21]; [@ref-77]). Among them, the *rps16*, *clpP*, *ndhA*, *ycf2*, and *ycf1* genes showed high sequence divergence in the coding regions that makes such genes reliable markers for phylogenetic analysis. In addition, the non-coding regions showing a high degree of divergence included *trnK-UUU-rps16*, *ycf3-trnS-GGA*, *trnL-UAA-trnF-GAA*, *accD-psaI*, *ycf4-cemA*, *psbE-petL*, *rps12-trnV-GAC*, *trnL-CAA-ycf2*, and *trnV-GAC-rps7*.

![Visualization alignments of chloroplast genome sequences among 10 Theaceae species with Hainan *C. oleifera* chloroplast genome as a reference.\
The abscissa represents the position coordinates of the chloroplast genome of Hainan *C. oleifera*, and the ordinate represents the sequence similarity of the sample genome to the reference genome. Arrows indicate the annotated gene and its transcriptional direction, blue for the protein coding sequence (exons), green for tRNA or rRNA, and red for the conserved non-coding sequence (CNS).](peerj-07-7210-g003){#fig-3}

Alignment of the *matK* gene and *trnH-psbA* IGR with other *Camellia oleifera*
-------------------------------------------------------------------------------

We compared Hainan *C. oleifera* with other mainland *C. oleifera* published in the NCBI database. The base sequences of the *matK* gene and *trnH-psbA* noncoding spacer were shown in [Fig. 4](#fig-4){ref-type="fig"}. We selected some bases with differences, among which *matK* coding genes were compared. Although the protein base sequence is conservative, the results showed that there was a site change in Hainan *C. oleifera*, that is, at the position of 2,255 bp, all other three mainlands *C. oleifera* were base C, and object species was T. Similarly, in the comparison of *trnH-psbA*, it also changed at 435 bp, but it was the same as [GQ487355](GQ487355) and [KR533766](KR533766), which were base G, and the other plants were A or T. This substitution of base pairs may cause changes in gene structure, leading to genetic mutations, which may be related to the living environment of Hainan *C. oleifera*.

![Base sequence alignment of *matK* gene and *trnH-psbA* intergenic region between different *C. oleifera*.\
(A) Partial nucleotide sequence of *matK* gene; (B) partial nucleotide sequence of *trnH-psbA* intergenic region.](peerj-07-7210-g004){#fig-4}

SSR and long repeat sequences distribution
------------------------------------------

The SSRs are repetitive sequences of up to several dozen nucleotides consisting of one to six nucleotide repeat units, which are short in length and widely distributed in eukaryotic chloroplast genomes ([@ref-9]; [@ref-13]). Owing to the presence of different nucleotides in the repeat units and different number of repeats, of which the most common is the dinucleotide repeat type, a high degree of variability is generated in SSR length. Therefore, we detected the SSRs distribution of the Hainan *C. oleifera* chloroplast genome by selecting the perfectly matched sequences, starting from the dinucleotide repeats. The statistical results were shown in [Table 3](#table-3){ref-type="table"}. We detected a total of 51 SSR loci, all having a base composition biased toward A/T. Of these 51 SSRs, 28 were completely composed of A/T; this was consistent with previous studies ([@ref-8]; [@ref-58]; [@ref-78]). The distribution of SSRs in the chloroplast genome was heterogeneous, with up to 28 SSRs in the LSC region, five in the SSC region, and 18 in the IR region. Among all SSR loci, 23 were located in the 14 protein-coding genes (*matK*, *atpA*, *rpoC2*×2, *rpoC1*, *ycf3*, *cemA*, *petA*, *rpoA*, *rol22*, *rpl2*×2, *ycf2*×6, *ndhB*×2, *ndhD*×2, *ndhA*), and 26 in the intergenic and non-coding regions (*trnS-UGA*, *rrn23*×2). In terms of the SSR types, the most abundant repeat pattern was dinucleotide repeat unit (38 SSRs), with the largest type being AT/TA, accounting for 65.8% (25 SSRs). In addition, one tri- and 12 tetra-nucleotide repeats were found in the Hainan *C. oleifera* chloroplast genome. However, compared with other Theaceae species, Hainan *C. oleifera* and *C. luteoflora* were found to have no hexanucleotide repeats ([Table S2](#supp-2){ref-type="supplementary-material"}). More specifically, all *Camellia* species have no pentanucleotide repeats ([@ref-20]). This is why these sequences were well-tagged in the studies of population phylogeny and taxonomy ([@ref-40]; [@ref-49]).

10.7717/peerj.7210/table-3

###### Simple sequence repeats in Hainan *C. oleifera* chloroplast genome.

![](peerj-07-7210-g010)

![](peerj-07-7210-g011)

  ID   Repeat unit   Repeat number   Length (bp)   Start    End      Region   Annotation
  ---- ------------- --------------- ------------- -------- -------- -------- ------------
  1    TA            4               8             2330     2337     LSC      *matK*
  2    AT            4               8             4652     4659     LSC
  3    AGAT          3               12            6696     6707     LSC
  4    TC            4               8             9184     9191     LSC
  5    GTCT          3               12            11990    12001    LSC      *atpA*
  6    AT            4               8             20077    20084    LSC      *rpoC2*
  7    AT            5               10            20842    20851    LSC      *rpoC2*
  8    AT            4               8             21871    21878    LSC      *rpoC1*
  9    GA            4               8             30227    30234    LSC
  10   AG            4               8             31910    31917    LSC
  11   TCTT          3               109           34002    34110    LSC
  12   GA            4               8             37363    37370    LSC      *trnS-UGA*
  13   AT            4               8             38208    38215    LSC
  14   TTTC          3               12            45247    45258    LSC      *ycf3*
  15   TA            4               8             48399    48406    LSC
  16   AT            4               75            49339    49413    LSC
  17   AT            4               8             56936    56943    LSC
  18   TA            4               8             60906    60913    LSC
  19   AAAT          3               12            62713    62724    LSC
  20   TC            4               8             63432    63439    LSC      *cemA*
  21   AT            4               8             64367    64374    LSC      *petA*
  22   AT            4               8             65980    65987    LSC
  23   TTC           4               12            70097    70108    LSC
  24   TA            4               8             70419    70426    LSC
  25   AT            4               8             79616    79623    LSC
  26   TA            4               8             80572    80579    LSC      *rpoA*
  27   AT            5               10            84341    84350    LSC
  28   AT            4               8             85931    85938    LSC      *rpl22*
  29   TA            5               10            87305    87314    IRa      *rpl2*
  30   GA            4               8             88915    88922    IRa      *ycf2*
  31   GA            4               8             89902    89909    IRa      *ycf2*
  32   TCTA          3               12            94648    94659    IRa      *ycf2*
  33   TA            4               8             95494    95501    IRa      *ycf2*
  34   AG            4               8             97417    97424    IRa      *ndhB*
  35   TA            4               8             99589    99596    IRa
  36   CT            4               8             108654   108661   IRa      *rrn23*
  37   CCCT          3               12            110067   110078   IRa
  38   AT            4               8             116288   116295   SSC
  39   GAAA          3               12            118178   118189   SSC      *ndhD*
  40   AATA          3               12            118328   118339   SSC      *ndhD*
  41   AAAT          3               12            121323   121334   SSC
  42   AT            4               8             123859   123866   SSC      *ndhA*
  43   GAGG          3               12            133565   133576   IRb
  44   AG            4               8             134983   134990   IRb      *rrn23*
  45   TA            4               8             144048   144055   IRb
  46   CT            4               8             146220   146227   IRb      *ndhB*
  47   TA            4               8             148143   148150   IRb
  48   ATAG          3               12            148984   148995   IRb
  49   TC            4               8             153735   153742   IRb      *ycf2*
  50   TC            4               8             154722   154729   IRb      *ycf2*
  51   AT            5               10            156329   156338   IRb      *rpl2*

To further explore the evolutionary characteristics of the *Camellia* species, we compared the long-repeat sequences in the chloroplast genome of Hainan *C. oleifera* with those of the other nine species ([Table 4](#table-4){ref-type="table"}). We identified 28 forward, reverse and palindromic repeats with lengths ranging from 30 to 60 bp in 10 Theaceae species. Of these 28 repeat sequences, the *ycf2* gene showed the presence of the largest number (60 bp in the chloroplast genome of *C. pubicosta*), and several repeats were also found in the intergenic regions. This phenomenon has also been reported in several angiosperms ([@ref-26]; [@ref-67]). Among them, seven long repeats ranging from 30 to 46 bp were found in the Hainan *C. oleifera* chloroplast genome. In total, six long repeats were located in the intergenic regions, and only one forward repeat was located in the protein-coding gene (*ycf2*). In the LSC regions, we observed only two pairs of palindromic repeats with lengths of 30 and 46 bp, respectively, and the remaining were located in the IR regions. Compared with the other nine species, the Hainan *C. oleifera* chloroplast genome had the same number of long-repeat sequences as *C. crapnelliana*, which had the least number of long-repeat sequences, while the most detected in *C. oleifera* (18 repeats) and the reverse match sequence was only detected in *C. oleifera*. Among the remaining seven species, eight long-repeat sequences were present in *C. luteoflora*, 12 in *C. grandibracteata*, 12 in *C. sinensis*, 10 in *C. leptophylla*, nine in *C. pubicosta*, 10 in *C. huana*, and 13 in *C. danzaiensis*.

10.7717/peerj.7210/table-4

###### Long repeat sequences in the chloroplast genome of 10 Theaceae species.

![](peerj-07-7210-g012)

  Type   Repeat sizes (bp)   Location   Region
  ------ ------------------- ---------- -------- ---- ---- ---- ---- ---- ---- ---- ------------------------------------- ----------
  F                          56                  56   56   56             56   48   *ycf2*; *ycf2*                        IRa
  P                          56                  56   56   56             56   48   *ycf2*; *ycf2*                        IRa, IRb
  P                                              48   48        60                  *ycf2*; *ycf2*                        IRa, IRb
  F                          56                  48   48   56   56        56   48   *ycf2*; *ycf2*                        IRb
  P                          48                                                     IGS (*rpl2-trnH*; *rps19-rpl2*)       LSC, IRa
  F                          48                                                     IGS (*rpl2-trnH*); *rpl2*             LSC, IRb
  F                          47                                                     IGS (*rpl2-trnH*)                     LSC, IRb
  P      46                                                                         IGS (*petB-petD*)                     LSC
  P                          46         46       42   42   42   42   46   46   46   *petD*; *petD*                        LSC
  F      42                  42         41       42   42   42   42   42   42   42   IGS (*rps12-trnV*); Intron (*ndhA*)   IRa, SSC
  P      42                  42         41       42   42   42   42   42   42   42   IGS (*trnV-rps12*); Intron (*ndhA*)   IRb, SSC
  F      38                  38         38       38   38   38   38   38   38   30   *ycf2*; *ycf2*                        IRa
  P                          38                  38   38   38   38   38   38        *ycf2*; *ycf2*                        IRa, IRb
  F                          38         38       30   30   38   38   38   38   30   *ycf2*; *ycf2*                        IRb
  F      38                                                                         IGS (*trnL-ycf2*)                     IRb
  P                                     38                                          *ycf2*; *ycf2*                        IRb
  P      38                                                                         *ycf2*; IGS (*trnL-ycf2*)             IRa, IRb
  F                                                                            34   IGS (*rrn4.5-rrn5*)                   IRa
  P                                                                            34   IGS (*rrn4.5-rrn5*; *rrn5-rrn4.5*)    IRa, IRb
  F                                                                            34   IGS (*rrn5-rrn4.5*)                   IRb
  R                          32                                                     IGS (*atpB-rbcL*)                     LSC
  R                          31                                                     IGS (*atpB-rbcL*)                     LSC
  F                          31                                                     IGS (*atpB-rbcL*)                     LSC
  F                                     34                                          IGS (*petA-psbJ*)                     LSC
  P      30                  30         30       30   30   30   30   30   30   30   IGS (*psbI-trnS*; *trnS-rps4*)        LSC
  R                          30                                                     IGS (*atpB-rbcL*)                     LSC
  F                          30                                                     IGS (*atpB-rbcL*)                     LSC
  P                                              30   30                       30   *ycf2*; *ycf2*                        IRa, IRb

Characteristic of codon usage
-----------------------------

In the Hainan *C. oleifera* chloroplast DNA, the 88 protein-coding genes had 27,270 codons. More than 94% of the protein-coding genes used ATG as the start codon, and less than 6% of the remaining used ATT (*petB* and *rps19*), ATA (*ndhD*), and ATC (*rpl16* and *orf42*) as start codons. Furthermore, ATA, ATC, ATT, and TTG were used as start codons in the *Aquilaria sinensis* chloroplast DNA ([@ref-67]), and GTG and ACT served as start codons in the *rps19* and *rps2* genes in *Morus atropurpurea*, respectively ([@ref-31]). Among the stop codons, TAA was the most common, followed by TAG and TGA, with 73.86% of the stop codons ending in A, thus strongly reflecting the AT bias in codon usage. The RSCU value of the Hainan *C. oleifera* chloroplast genome was shown in [Fig. 5](#fig-5){ref-type="fig"}; its value increased with the number of codons. Leucine accounted for the highest codon usage (10.84%), followed by serine (9.17%) and isoleucine (8.42%). Nearly one third of the total codons were represented by these three amino acids.

![20 amino acid codon and stop codon of the island plant Hainan *C. oleifera* chloroplast genome.\
The color of the histogram corresponds to the color of the codon.](peerj-07-7210-g005){#fig-5}

The distributions of codon usage in the form of heatmaps for 10 Theaceae species were shown in [Fig. 6](#fig-6){ref-type="fig"}. It can be seen that about half of the codons with low RSCU values (shown in blue in the heatmaps) were infrequently used. All codons with RSCU \>1 ended with A/T. A similar phenomenon has been found in other plant lineages, indicating that the A + T bias plays an important role in the plastid genome. In addition, we found that the codon usage of Hainan *C. oleifera* chloroplast genome was most similar to that of *C. oleifera*. As more chloroplast genome data are being continually explored, further validation of these findings will allow us to understand the relationship between different codon usage patterns and hosts, as well as the interactions among nuclear genomes ([@ref-35]).

![The distributions of codon usage in the form of heat maps for 10 Theaceae species.\
Color indication: red represents the larger RSCU values and blue represents the smaller RSCU values.](peerj-07-7210-g006){#fig-6}

Phylogenetic relationship with near source species
--------------------------------------------------

As an important part of plant cell organelles and photosynthetic organs, chloroplasts play an important role in the long history of biological evolution ([@ref-24]). In this study, we not only constructed the phylogenetic tree of complete chloroplast genome of 22 *Camellia* species, but also extracted protein sequence information, LSC region sequences and SSC region sequences, and constructed the phylogenetic tree. The results showed a different evolutionary relationship, in which the whole chloroplast genome showed that Hainan *C. oleifera*, *C. oleifera*, and *C. crapnelliana* were independent branches and have similar evolutionary relationships ([Fig. 7A](#fig-7){ref-type="fig"}). The protein coding sequence showed that Hainan *C. oleifera* was closely related to *C. crapnelliana*, *C. danzaiensis*, *C. pitardii*, *C. huana*, and *C. impressinervis*, which was closest to *C. crapnelliana* ([Fig. 7B](#fig-7){ref-type="fig"}). The results of the LSC region ([Fig. 7C](#fig-7){ref-type="fig"}) and the SSC region ([Fig. 7D](#fig-7){ref-type="fig"}) were similar to those of the complete chloroplast genome sequence, showing that the three Camellia species of Hainan *C. oleifera*, *C. oleifera*, and *C. crapnelliana* were the same family. The evolutionary relationship was clear. There were similar parts in these results, especially *C. oleifera* and *C. crapnelliana*, but the protein coding sequence is more stable and not susceptible to the external environment, so its phylogenetic relationship is more informative.

![Phylogenetic relationships of 22 species of *Camellia* plants inferred from different data partitions.\
(A) Whole chloroplast genome; (B) protein coding region; (C) LSC region; (D) SSC region Species with similar evolutionary relationships are displayed in the same color.](peerj-07-7210-g007){#fig-7}

The base sequence comparison of DNA barcodes (*rbcL*, *matK*, and *trnH-psbA*) revealed a high degree of conservation of the chloroplast genome sequence of the *Camellia* plants ([Table S3](#supp-3){ref-type="supplementary-material"}). There were 10 locus in *rbcL* with differences (marked by red triangles below), but Hainan *C. oleifera* has the same base composition as most species. There were 22 locus differences in *matK*, similar to *rbcL*, the most occurred in a single base region of other species. Unlike the other species, Hainan *C. oleifera* was replaced by T at one base, and the rest were C. In the *psbA-trnH* comparison, there were nine positions in which the base changes slightly, while in the second change point, Hainan *C. oleifera* was C, and other species were A. Therefore, these single site changes can be used as good molecular markers.

Discussion
==========

With the rapid development of molecular biology techniques, new molecular markers and detection methods are emerging in an endless stream. Several DNA analysis techniques suitable for chloroplast genome research on plant phylogeny and evolution will be available. Moreover, the research on population genetic changes will play an important role in the protection of biological resources and biodiversity ([@ref-72]). As an important evolutionary feature of the chloroplast genome, the codon usage bias has become an analytical tool widely used in several organisms ([@ref-41]; [@ref-55]; [@ref-66]). In this study, we systematically analyzed the RSCU value of the Hainan *C. oleifera* chloroplast genome. The results showed that nearly half of the values were higher than 1.00 and nearly half were lower than 1.00, and all high-valued codons ended with A or T, whereas the RSCU value was 1.00 without any codon usage bias ([@ref-18]). Therefore, we hypothesized that the codons in Hainan *C. oleifera* chloroplast genome preferably ended with A/T. This conjecture was also confirmed in this paper, and has also been found in other plant studies ([@ref-67]). It is generally accepted that codon usage bias reflects the balance between the mutational bias and natural selection for translation optimization ([@ref-7]; [@ref-57]; [@ref-60]). In order to further clarify the role of the two in the evolution of the Asteraceae species, some researchers used the neutrality analysis, which reflected that the natural selection pressure played a major role in shaping codon usage ([@ref-41]). However, the *C. oleifera* population on Hainan Island has a certain geographical isolation; the genetic variation of population might occur. Whether the mutation or natural selection plays a leading role needs further confirmation.

Previous studies have shown that the chloroplast genome structure is highly conserved ([@ref-27]). In this study, the genomic composition, gene sequence, GC content, and codon preference of the Hainan *C. oleifera* chloroplast genome were not much different from that of the other *Camellia* species, and were similar to the typical angiosperm chloroplast genome ([@ref-61]; [@ref-67]; [@ref-75]), which revealed that the chloroplast genome of Hainan *C. oleifera* is highly conserved in structure and evolution. The conservation of these sequences makes Hainan *C. oleifera* have some pressure on evolutionary research, so that it difficult to determine the differential genes it contains. This also forced research on the evolution of *C. oleifera* on Hainan Island. In addition, we focused on the junctions between the IR/LSC and IR/SSC regions of the chloroplast genome, where more sequence data were available. In the long process of evolution, the structural order of the LSC, SSC, and IR regions remained unchanged. The differences among the chloroplast genomes of different species are mainly reflected in the length and orientation of the IRs regions. Among them, a similar situation was produced in Hainan *C. oleifera*, *C. crapnelliana*, and *C. danzaiensis* due to the change of IRa region, and the similarity at this level indicated that they shared a common ancestor. This can initially determine its source to reduce the pressure of its taxonomic research. As an important indicator of chloroplast genome evolution, the contractions, and expansions of the IR/LSC and IR/SSC boundaries often determine the chloroplast genome length variation ([@ref-14]; [@ref-79]). Moreover, the boundaries of the plastids of various species are often different even between members of the same family ([@ref-51]). Therefore, it would be useful to compare the IR/LSC and IR/SSC junctions of chloroplast genome in different *Camellia* species for alleviating its evolutionary pressure.

It was difficult to study the classification and phylogeny of the genus *Camellia* because of various reasons, such as its frequent interspecific hybridization and polyploidization ([@ref-20]; [@ref-75]). However, the samples of *C. oleifera* used in this study were grown in a unique island environment, and their genetics and evolutionary history are still vague. Even if we used the complete chloroplast genome information for analysis, it was not enough to completely solve all phylogenetic relationships, as can be seen in previous studies ([@ref-47]; [@ref-62]; [@ref-69]). Besides, the comparison object we selected were no plant groups involved in the other genera of the family Theaceae, which might provide useful information for the evolutionary study of Hainan *C. oleifera*. In summary, our research can promote the exchange of information between the nuclear genomes of *Camellia* species and provide valuable genomic resources for phylogenetic studies.

Conclusions
===========

In this study, we sequenced the complete chloroplast genome of *C. oleifera* on Hainan Island, China, using the Illumina high-throughput sequencing technology. The chloroplast genome of Hainan *C. oleifera* was found to have an intact quadripartite structure by genome sequencing and reassembly. The annotation and comparison with other *Camellia* species showed that the genomic composition, gene sequence, GC content, and codon preference of the Hainan *C. oleifera* chloroplast genome were not much different from that of the other *Camellia* species, and were similar to the typical angiosperm chloroplast genome. Therefore, we performed in-depth analysis of the border regions, long repeats, SSRs, and sequence diversity in the chloroplast genomes of 10 *Camellia* plants, indicated that their chloroplast genomes were highly conserved in structure and evolution. Long-sequence repeats, SSRs and DNA barcodes can be used as novel molecular markers. By studying the gene composition of the marginal regions, codon distribution and phylogeny, it was found that Hainan *C. oleifera* has similar evolutionary relationships with *C. crapnelliana*. Therefore, the data obtained in this study will be helpful to further study the evolutionary history of Hainan *C. oleifera* and to protect its excellent germplasm resources, and to provide valuable genome resources for phylogenetic and taxonomic studies and for the exchange of information between nuclear genomes.

Supplemental Information
========================

10.7717/peerj.7210/supp-1

###### The genes with introns in the Hainan *C. oleifera* chloroplast genome and the lengths of introns and exons.

^\*^The *ycf1* is a trans-spliced gene with the start located in the SSC region and the end in the IRb region, and possibly missing 3' end.

######

Click here for additional data file.

10.7717/peerj.7210/supp-2

###### Distribution of simple sequence repeats (SSRs) in 10 Theaceae chloroplast genomes.

######

Click here for additional data file.

10.7717/peerj.7210/supp-3

###### Comparison of base changes in DNA barcodes (rbcL, matK and trnH-psbA).

######

Click here for additional data file.

Additional Information and Declarations
=======================================

The authors declare that they have no competing interests.

[Wan Zhang](#author-1){ref-type="contrib"} analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper.

[Yunlin Zhao](#author-2){ref-type="contrib"} conceived and designed the experiments.

[Guiyan Yang](#author-3){ref-type="contrib"} conceived and designed the experiments, contributed reagents/materials/analysis tools.

[Jiao Peng](#author-4){ref-type="contrib"} performed the experiments, contributed reagents/materials/analysis tools.

[Shuwen Chen](#author-5){ref-type="contrib"} performed the experiments.

[Zhenggang Xu](#author-6){ref-type="contrib"} analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

The following information was supplied regarding the deposition of DNA sequences:

The complete chloroplast genome sequence of *C. oleifera* is available at GenBank: [MF541730](MF541730).

The following information was supplied regarding data availability:

Data access in NCBI: all complete chloroplast genome sequence are available with the following accessions Hainan *C. oleifera* ([MF541730](MF541730)), *C. oleifera* ([JQ975031](JQ975031)), *C. huana* ([KY626040](KY626040)), *C. impressinervis* ([KF156835](KF156835)), *C. pitardii* ([KF156837](KF156837)), *C. danzaiensis* ([KF156834](KF156834)), *C. grandibracteata* ([KJ806274](KJ806274)), *C. leptophylla* ([KJ806275](KJ806275)), *C. sinensis var. sinensis* ([KJ806281](KJ806281)), *C. sinensis var. dehungensis* ([KJ806279](KJ806279)), *C. crapnelliana* ([KF753632](KF753632)), *C. taliensis* voucher HKAS S.X.Yang3157 ([KF156839](KF156839)), *C. taliensis* voucher HKAS S.X.Yang3158 ([KF156836](KF156836)), *C. reticulata* ([KJ806278](KJ806278)), *C. sinensis var. pubilimba* ([KJ806280](KJ806280)), *C. pubicosta* ([KJ806277](KJ806277)), *C. sinensis* ([KY626042](KY626042)), *C. sinensis* cultivar Longjing ([KF562708](KF562708)), *C. cuspidata* ([KF156833](KF156833)), *C. yunnanensis* ([KF156838](KF156838)), *C. petelotii* ([KJ806276](KJ806276)), *C. luteoflora* ([KY626042](KY626042)).

The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.


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It's basically a small loop attached to the center of the bar, making it possible to lock your bar in full power position by hooking the microloop to a small hook on the CL assembly. Here's an example of a microloop and how it works:

and you use it for tricks like the one below, when you need to take both hands off the bar, or want more hang in your trick.

A stopper works as well, but a microloop is better, since you can be quicker in and out, specially at the end of a jump you want the power to be out of the kite for the landing.

A perfect old school bar has both, since with some tricks it is also good to have a stopper, since this way you don't have the bar too close when you are rotating no handed.

Here you can see when I use the microloop and when not. I mainly use the microloop when I don't spin and use my right arm for the boardoff, since the left arm is not strong enough to keep flying longer.

Ive seen u around the KF, but its the firest video I saw. Cool moves bro, thats what Im trying to learn while kiteboarding.

Is a 2010 12m Cab SB good for onld school moves? I just got a second hand one, and im evolving on big airs and one foots. Doig some unhooked moves and kitel,oops, but i cant see myself doing what u r doing, hehehe

thx!once you have too much power to unhook, you can work on the old school moves.Set the kite to a slower setting, that will help, since it makes the kite more controllable while up there.The problem is, that you need a working stopper or a powerlock to do many of those tricks, specially the ones where you need both hands, like the board pass you see above.If not available, make sure to hold the bar down with your stronger arm, and handle the board with the other arm.

Make your straps wider, use a handle. Sit down in a chair and train the board on and off.Lift your legs up, don't bend down.

u c, since I love kiteboarding somethimes I get confused on wich moves I like to learn first. Thats why I'm always trying to learn more. I love waves, buit i also love doing kiteloops, big airs, old school move (that Im still learning) and now Im unhooking. Since we got so many options, its hard to decide wich one to follow.

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Q:

Action support on change event conflicts with apex:inputfield on change

I am calling javascript function from apex:inputfield and also doing rerendering page same block section Item using action support as I am using depended picklist on my Visual Force page.
but after adding this
 <apex:actionSupport event="onchange" reRender="pbsiType" />

it has stopped calling my javascript function on change of value event.
Below is the code I am using fro Page block section item
<apex:pageBlockSectionItem Id="pbsiType">
                     <apex:outputLabel value="Type"/>

                        <apex:inputField value="{!opp.Type__c}" id="OppType" onchange="Type('{!$Component.OppType}');">
                             <apex:actionSupport event="onchange" reRender="pbsiType" />
                        </apex:inputField>

                </apex:pageBlockSectionItem>

and here is the javascript function I am calling from within.
function Type(Receivetype){
        SelectedType = document.getElementById(Receivetype).value;
        openLookup();
             }

A:

Change your ActionSupport to:
<apex:actionSupport event="onchange" onsubmit="Type('{!$Component.OppType}');" reRender="pbsiType" />

and remove the onchange event from the element