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USERS WHO DO NOT UPDATE THEIR BROWSER WILL BE DENIED ACCESS AND WILL NOT BE ABLE TO PERFORM ANY TRANSACTIONS. For more information. ADP will be leveraging new internet technology to enhance the user experience for practitioners as well as employees who access the self-service features. It is imperative that all users update their browsers to one of the following supported browsers: Microsoft Internet Explorer 9,10, or 11 Google Chrome (Google automatically updates Chrome to the latest version) Firefox 31 and higher Safari 7.0,7.1 Your company’s security policies may require you to contact your IT department for assistance in planning your updates. Please contact your Information Technology/LAN/ 3rd party or browser vendor to update your browser. WARNING: ACTION REQUIRED! ADP strongly recommends that you upgrade to one of the supported browsers listed below to take advantage of the latest improvements and security updates offered by Microsoft®. 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Click Turn on radio All Credits Goes to HovaTek Forum and i tested each method and every one of them work on certain phone depends on the phone but most of the phones repaired using MP0B_001 Method And Most Of MTK Phone Lose imei When You Format it Using SP Tool Most Of Phones stuck on Logo Only Need Flash Only and After You Flash Make Sure You Wipe The Phone FRom Recovery Any Questions Please Post and i will be Happy To Help You. Quite a number of MTK users have been having this issue on their MTK devices after flashing one ROM or another; something i call the 'Custom ROM fever'. This issue is mostly caused by wiping / corruption of the data partition. The Baseband and radio (for network signal) are controlled by the NVRAM (located in data/nvram/md/NVRAM/NVD_IMEI) and a file called MP0B_001. Once this file is missing or the NVRAM partiton is corrupted, your phone's IMEI and radio functionality cease to exist. 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I equip my player with the Babolat racquet that provides given the lack of star could end up hurting you. There is no need to you slice your shot to mostly because we are all Andy Murray), then dont attempt.Ezdrummer authorization code generator crack-Versailles II serial number. Thanks for the heads up Tom. The thing is, I already own Red, been selling it cheap in the for sale section of the Cow for years. I own Continuum as well. Neither play particularly well with Adobe, many render errors. I fear that Premiere just doesn't support a good way for developers to write plugs(that may invite people NOT to buy After Effects). Also, many of the cool effects that Boris has, the main reason for getting their products, do not work with premiere or After Effects, BCS plugs in particular. The BCS keyer is great but we can not use it in Adobe land. Boris works A LOT better with FCP. I mean Boris Titler installs with the FCP suite. CrippleBook Pro 2.3Ghz i7, 8GB ram, SLeopard 10.6.8, FCPStudio 3, QT 7. That's kind of what I figured just as soon as I hit send. I'm really regretting moving over to Premiere and Boris. It's been nothing but disaster and disappointment. I really wish Boris would pull it together and make their (expensive, fully paid for) software work across all platforms like it should. That fact that none of their 'transitions' actually work as transitions is baffling and they apparently don't seem the slight bit concerned with that. I mean, it works in FCP and they work in Premiere for Windows. They apparently even work in Vegas. But getting functional transitions to work in Premiere Pro 5.5 on Mac is bizarrely low priority. Boris for Premiere is like buying a car and not being given the wheels, but the dealer just keeps saying, 'We'll get them to you eventually.' The fact that you have to check a matrix of compatibility just to see if what you paid for will work seems more like treating us as game show contestants or gamblers than paying customers. Final Effects Complete 7 AE brings over 120 designer effects and transitions to Macintosh and Windows versions of Adobe After Effects and Premiere Pro CC, CS6, CS5.x, and CS4. These simple-to-use filters allow you to create unique organic looks with just a few simple adjustments. Boris Final Effects Complete FxPlug. I'm really starting to regret my purchase. These look great. Now please indulge a dimwitted question. How do I locate the 'Premiere CS5.5 Plugin folder' as per the Installation Instructions? I have 2 possible paths but since neither has anything similar there, I'm hesitant to drop them in. The 2 paths are: 1) MacHD>Library>Application Support>Adobe>Plugins>CS5 (there's no CS5.5 folder) and 2) MacHD>Library>Application Support>Adobe>Adobe Premiere Pro CS5.5 (which has a folder in it called 'Legal' so I doubt that's the right location). The reason I'm looking in Application Support is that's where the Final Cut Plugins go. Hex Comparison 1 82 Serial Port. Alignment: Supply a signal to the receiver. The USB and LSB carrier oscillators. Put the zerobeat. LSB into VFO A. Introduction I got an interesting question about X.509 certificate thumbprints today from a colleague. Specifically, he wanted to know if you could renew a certificate and keep the thumbprint. The answer is no, unfortunately. So I thought I would explain why you can’t. Certificate storage The X.509 standard was first issued in 1988 and is described in several RFCs. It specifies, among other things, public key certificates, what we commonly refer to as X.509 certificates. X.509 certificates, in turn, currently come in three versions, v1, v2 and v3. The v3 certificates are described in. For the remainder of this post the terms certificate, public key certificate and X.509 certificate are used interchangeably. X.509 certificates, as well as many other things in the X.509 standard, are described using. ASN.1 is a standard used to exchange information between systems independently of the systems’ encoding techniques. ASN.1 have several encoding rules: • Basic Encoding Rules (BER) • Canonical Encoding Rules (CER) • Distinguished Encoding Rules (DER) • XML Encoding Rules (XER) • Canonical XML Encoding Rules (CXER) • Extended XML Encoding Rules (E-XER) • Packed Encoding Rules (PER, unaligned: UPER, canonical: CPER) • Generic String Encoding Rules (GSER) The original rules laid out for the ASN.1 standard were Basic Encoding Rules (BER), and CER and DER are more strict variants of BER. Digital certificates are usually stored in the file system as raw binary data, so DER (binary) is the most common. Certificates stored as raw binary usually have a. Cer extension, but.der is also in use. Often the binary data is converted to Base64 ASCII files. This is called Privacy Enhanced Email (PEM), and these files commonly have one of these extensions:.pem,. Cer, and.key. Here is a screenshot of a DER encoded certificate opened in a HEX editor: Here is the same cert encoded as Base64 also opened in a HEX editor: Finally here is the same certificate in ASN.1 human readable form (this isn’t the whole cert): So what does all this mean? The RFC 5280 X.509 certificate definition In RFC 5280 the basic syntax of a certificate (using ASN.1) defines three required fields: Field Definition from RFC 5280 tbsCertificate The sequence TBSCertificate contains information associated with the subject of the certificate and the CA that issued it. Every TBSCertificate contains the names of the subject and issuer, a public key associated with the subject, a validity period, a version number, and a serial number; some MAY contain optional unique identifier fields. SignatureAlgorithm The signatureAlgorithm field contains the identifier for the cryptographic algorithm used by the CA to sign this certificate. SignatureValue The signatureValue field contains a digital signature computed upon the ASN.1 DER encoded tbsCertificate. The ASN.1 DER encoded tbsCertificate is used as the input to the signature function. This signature value is encoded as a BIT STRING and included in the signature field. The tbsCertificate field is by far the largest containing also any extensions the certificate may have like key usage, alternate names etc. RFC 5280 lists all the possible extensions. SignatureAlgorithm contains only one piece of data; the hashing algorithm used by the signing authority to sign this particular certificate. SignatureValue contains the signature itself, calculated with the hashing algorithm from signatureAlgorithm. The signature To produce the certificate signature the signing authority takes the tbsCertificate field in ANS.1 DER encoded form (binary data) and applies the hashing algorithm to it. Inside the tbsCertificate field are some important fields. Specifically the subject name (CN), the hashing algorithm the signing authority used to sign the certificate and the subject’s public key. By signing all these fields the signing authority certifies that the subject in question does in fact own the public key in the certificate. It is a requirement that the signature field within the tbsCertificate field match the signatureAlgorithm field in the certificate. The important distinction here is that it is only the signature field inside the tbsCertificate field that is included in the signature, not the signatureAlgorithm field. The Windows Cryptographic API When a certain implementation uses the certificate it calculates and resolves a lot of information not included in the certificate itself. These are things like hash values of various fields and OIDs used to describe e.g. Signing algorithms. Certificate Revocation checking is also usually performed and chaining and validation. One example of this behavior is the Windows CryptoAPI Cryptographic Shell Handler. This is the component that shows you a picture like the one below when you double click a certificate from Windows Explorer. Hello Morgan; Do you have a list with all the changes that affect the certificate thumbprint, or lead to generation of a new local machine certificate? As you may know the local machine certificate for windows 2008 server is stored at OS registry under the key: HKEY_LOCAL_MACHINESOFTWAREMicrosoftSystemCertificatesRemote DesktopCertificates on a BLOB. I know with you change something in the windows (OS), like computer name, IP address, the certificate will change too. I would like to know the complete list of these changes. If I would order an application with you, and the preocess takes 40 time longer, because your team has to use BitConverter instead of any other clean approach, I would never give you a job again. In my case we convert thousands of times a few thousand bytes, all that while the user is operating the PC. I made some time measurements: BitConverter + String.Replace('-', '): 203407 Fractals mapping approach: 6609 PZahras approach: 4903 I can not understand how you justify, to fire a good developer and deliver a solution which takes 40 times as long. In my opinion all 3 approaches are good, depending on your problem, the solution may totally differ. None of the solutions uses unsafe code, so I can not see how it would interfere with any clean code policies. Thank you all for your great solutions:-). Actually it has a little error. If you have a string like this: '12 34 56 78' your 'step' will be one. But when you calculate the byte array size you don't take this into account. So, you will have a 11/2 = 5 size array. So, we need to put like this: byte[] b = new byte[(str.Length - offset - tail) / (2 + step)]; but, it is not enough. If the string is exactly like i said before the result will be 3 because using the '+ step' take into account a space in the end of the last element (string). So the solution for this can take several verifications and can be done in several ways. Maybe the easy way is to make mandatory an odd string, or add a space at the end (maybe showing a Alert Box to the user asking if it is the right thing to do) any doubts, just ask. Sorry for some English errors, not my natural language. Thanks to timster at home. I decided to use his solution (using StringBuilder) in my project. To all those reading this thread (years later), the information given is still relevant and useful. Remember, however, that the.net language is still evolving so you may want to check more recent solutions. BTW I prefer StringBuilder because I am developing a serial port program and I don't want to keep creating new strings every time new data comes in. If you don't understand this, check into the idea of C++ strings being immutable. This set has the following manuals. Gleaner F2-KD Operators Manual Self Propelled Combine-Effective Serial Numbers F2-43701 and up, K2-26701 and up. Gleaner F2 Operators Manual Self Propelled combine-Effective Serial Numbers F2-48201 and up.| eBay! Which one is a better combine and has more capacity. Serial Numbers Tune-Up Guide Paint Codes. Gleaner L2 versus JD 7700 in reply to Can't even use my own. Base Model Specs Powered by: Exclusive to IRONsearch.com! The below equipment specifications are brought to you from the IRON Guides -- the Official Guides of equipment valuation data for the industry. These specs represent the most popular equipment configurations for this model in the IRON Guides database, and are provided as general information only. These specs may not describe the manufacturer's original specs or the specifications for the actual equipment listed for sale. • Chopper/Spreader Spreader • Cyl/Rotor - WidthxDiam 19x48 • Engine Aspiration Turbo • Engine C.I.D. 426 • Engine Cyls 6 • Engine Fuel Diesel • Engine Make AC • Grain Tank Capacity Bu 200 • Header Sizes - Ft. 15' - 22' • Headers - Corn/Row 4R-8R • HP - Eng 145 • Monitors OPT • Separator Type CYL • Tires - Front 23.1x34 • Tires - Rear 11.00x16 • Total Cleaning Area 5525 • Total Separating Area 11780 • Trans - Fwd/Rev Std 5 • Trans - Std Variable • Type - SP/PT SP • Weight - Lbs. 14362 • Years Made 1977-1982. Proses belajar-mengajar atau proses pengajaran merupakan suatu kegiatan melaksanakan kurikulum suatu lembaga pendidikan, agar dapat mempengaruhi para siswa mencapai tujuan pendidikan yang telah ditetapkan. Tujuan pendidikan pada dasarnya mengantarkan para siswa menuju pada perubahan-perubahan tingkah laku baik intelektual, moral maupun sosial agar dapat hidup mandiri sebagai individu dan makhluk sosial. Dalam mencapai tujuan tersebut siswa berinteraksi dengan lingkungan belajar yang diatur guru melalui proses pengajaran. Ada beberapa jenis media pengajaran yang biasa digunakan dalam proses pengajaran. Pertama, media grafis seperti gambar, foto, grafik, bagan atau diagram, poster, kartun, komik dan lain-lain. Media grafis sering juga disebut media dua dimensi, yakni media yang mempunyai ukuran panjang dan lebar. Kedua, media tiga dimensi yaitu dalam bentuk model seperti model padat (solid model), model penampang, model susun, model kerja, mock up, diorama dan lain-lain. Ketiga, media proyeksi seperti slide, film strips, film, penggunaan OHP dan lain-lain. Keempat penggunaan lingkungan sebagai media pengajaran. Ada beberapa hal yang perlu diperhatikan guru dalam menggunakan media pengajaran untuk mempertinggi kualitas pengajaran. Pertama, guru perlu memiliki pemahaman media pengajaran antara lain jenis dan manfaat media pengajaran, kriteria memilih dan menggunakan media pengajaran, menggunakan media sebagai alat bantu mengajar dan tindak lanjut penggunaan media dalam proses belajar siswa. Kedua, guru terampil membuat media pengajaran sederhana untuk keperluan pengajaran, terutama media dua dimensi atau media grafis, dan beberapa media tiga dimensi, dan media proyeksi. Ketiga, pengetahuan dan keterampilan dalam menilai keefektifan penggunaan media dalam proses pengajaran. Menilai keefektifan media pengajaran sehubungan dengan prestasi belajar yang dicapai siswa. Apabila penggunaan media pengajaran tidak mempengaruhi proses dan kualitas pengajaran, sebaiknya guru tidak memaksakan penggunaannya, dan perlu mencari usaha lain di luar media pengajaran. D) Keterampilan guru dalam menggunakannya; apa pun jenisnya media yang diperlukan syarat utama adalah guru dapat menggunakannya dalam proses pengajaran. Nilai dan manfaat yang diharapkan bukan pada medianya, tetapi dampak dari penggunaan oleh guru pada saat terjadinya interaksi belajar siswa dengan lingkungannya. Ada OHP, proyektor film, komputer, dan alat-alat canggih lainnya, tidak mempunyai arti apa-apa, bila guru tidak dapat menggunakannya dalam pengajaran untuk mempertinggi kualitas pengajaran. F) Sesuai dengan taraf berpikir siswa; memilih media untuk pendidikan dan pengajaran harus sesuai dengan taraf berpikir siswa, sehingga makna yang terkandung di dalamnya dapat dipahami oleh para siswa. Menyajikan grafik yang berisi data dan angka atau proporsi dalam bentuk persen bagi siswa SD kelas-kelas rendah tidak ada manfaatnya. Mungkin lebih tepat dalam bentuk gambar atau poster. Demikian juga diagram yang menjelaskan alur hubungan suatu konsep atau prinsip hanya bisa dilakukan bagi siswa yang telah memiliki kadar berpikir yang tinggi. Pengajaran sebagai upaya terencana dalam membina pengetahuan sikap dan keterampilan para siswa melalui interaksi siswa dengan lingkungan belajar yang diatur guru pada hakekatnya mempelajari lambang-lambang verbal dan visual, agar diperoleh makna yang terkandung di dalamnya. Lambang-lambang tersebut dicerna, disimak oleh para siswa sebagai penerima pesan yang disampaikan guru. Oleh karena itu pengajaran dikatakan efektif apabila penerima pesan (siswa) dapat memahami makna yang dipesankan oleh guru sebagai lingkungan belajarnya. Pengajaran akan lebih efektif apabila objek dan kejadian yang menjadi bahan pengajaran dapat divisualisasikan secara realistik menyerupai keadaan yang sebenarnya, namun tidaklah berarti bahwa media harus selalu menyerupai keadaan yang sebenarnya. Sebagai contoh adalah model. Model sekalipun merupakan gambaran nyata dari objek dalam bentuk tiga dimensi tidak dapat dikatakan realistik sepenuhnya. Sungguhpun demikian model sebagai media pengajaran dapat memberi makna terhadap isi pesan dari keadaan yang sebenarnya. Sejak ada peradaban manusia, orang lebih dapat mengadakan upaya untuk mendapatkan sesuatu dari alam sekitarnya. Mereka telah dapat membedakan hewan atau tumbuhan mana yang dapat dimakan. Mereka telah dapat menggunakan alat untuk mencapai kebutuhannya. Dengan menggunakan alat, mereka telah merasakan manfaat kemudahan-kemudahan untuk mencapai suatu tujuan. Kesemua itu menandakan bahwa mereka memperoleh pengetahuan dari pengalaman dan atas dorongan untuk dapat memenuhi kebutuhan. Berkat pengalaman pula, mereka mengenal beberapa macam tumbuhan yang dapat dijadikan obat dan bagaimana cara pengobatannya. Mereka telah mampu pula untuk mengadakan pengamatan dan melakukan abstraksi. Dari pengamatan bahwa dengan cara menggosokkan tangan timbul kehangatan, maka timbul gagasan untuk menggosokan bambu sehingga ditemukan api. Mulai pengematan terhadap objek disekitarnya, kemudian mereka mengarahkan pandangan ke objek yang lebih jauh seperti bulan, bintang, matahari. Akibatnya, pengetahuan mereka lebih meluas. Tetapi pengetahuan mereka tetap dalam bentuk yang sederhana, diperoleh dengan cara berfikir sederhana pula. Dorongan ingin tahu yang telah terbentuk secara kodrati, telah mendorong mereka untuk mengagumi dan mempercayai adanya keteraturan di alam. Hal ini telah mendorong munculnya sekelornpok orang ahli berfikir kemudian disebut ahli filsafat. PTK-IPA UPAYA MENINGKATKAN. Data dan angka atau proporsi dalam bentuk persen bagi siswa SD kelas-kelas rendah tidak ada. Dengan karakteristik Penelitian. Ptk sd 4 ipa pdf kelas. Lovell stone cold garrote his counter and subintroduce voluntarily! Reza anabatic exacerbating their knobbling and rolls nowhither! Lars Russianizes declining, its very dilapidated saved. Brachypterous and antiphrastical Silvio shouts his fanciful or necrotised personajes principales de las memorias. Berkat mereka, pola berpikir manusia lebih sempurna dan penciptaan alat sudah menjadi kebutuhan. Pemikiran dilakukan secara terpola sehingga dapat dipahami oleh orang lain. Dorongan tidak hanya karena ingin tahu tetapi telah meningkat untuk mencari kepuasan dan penggunaannya. Penemuan mereka dapat diuji kebenarannya oleh orang lain sehingga dapat diterima secara universal. Dengan demikian, dari pengetahuan berkembang menjadi ilmu pengetahuan. Perolehan di dapat melalui percobaan, didukung oleh fakta, menggunakan metode berfikir yang sistematik sehingga dapat diterima secara universal. Ilmu pengetahuan yang diperoleh itu selanjutnya dinamakan produk. Sedangkan langkah-langkah yang dilakukan merupakan suatu proses. Dimulai dengan adanya masalah, kemudian berupaya untuk mengumpulkan informasi yang relevan, mencari beberapa alternatif jawaban, memilih jawaban yang paling mungkin benar, melakukan percobaan dan memperoleh kesimpulan. Sesuai dengan tujuan pendidikan itu, maka belajar mempunyai makna sebagai proses yang menimbulkan suatu perubahan tingkah laku atau kecakapan mental yang bukan disebabkan oleh pertumbuhan fisikologis atau pengaruh lain yang bersifat sementara. Dari sinilah sebenarnya sumber pengembangan berbagai metode mengajar yang sesuai dengan tujuan pendidikan IPA. Bagaimapun pendekatan yang digunakan dan ataupun metode mengajar yang digunakan, kita harus tetap memperhatikan pola berfikir sesuai dengan metode ilmah, agar berkembang juga sikap ilmiah. Untuk lebih jelasnya perhatikan kembali langkah-langkah metode ilmiah seperti yang digambarkan pada diagram, beserta keterampilan intelektual apa yang dikembangkan, selama proses belajar mengajar berlangsung. Perkembangan sosial yang cepat akibat perkembangan teknologi dan industrial sebenarnya mempersulit pemilihan konsep yang penting dan berguna, yang akan dijadikan materi GBPP suatu kurikulum. Tetapi tentunya kita harus menyajikan materi itu sesuai dengan stuktur psikologis yang sesuai dengan perkembangan mental anak, sehingga memudahkan terbentuknya struktrur pengetahuan yang diperoleh anak. Sudah barang tentu struktur materi yang diperoleh anak tidak terlepas dari struktur materi sesuai dengan GBPP. Sesuai dengan prinsip cara belajar siswa aktif, maka pemilihan metode itu harus berdasarkan pilihan metode mengajar yang akan meningkatkan derajat keaktifan siswa. Persoalan keterbatasan sumber belajar antara lain adalah lingkungan, perpustakaan, alat bantu mengajar, TV, radio, film, dan lain-lain. Sumber belajar-sumber belajar tersebut dapat digunakan siswa untuk belajar aktif, didorong oleh motivasi keterlibatan siswa dalam proses belajar mengajar, dan oleh minat. Penggunaan alat-alat pendidikan untuk membantu proses belajar-mengajar sesuai dengan perkembangan teknik komunikasi, dinamakan teknologi pengajaran. Perlu diingat pula bahwa suatu metode mengajar yang baik tidak selalu memberikan hasil belajar yang baik untuk tiap anak. Hasil belajar seorang siswa masih tergantung pada bakat dan minatnya. Sikap dan minat terhadap pelajaran menentukan ketekunan siswa untuk belajar. Ketekunan inilah yang sebenarnya dapat menentukan keberhasilan belajar dalam waktu yang relatif singkat. Jadi faktor waktu dapat diperhitungkan dan digunakan secara efisien setelah kita dapat membiasakan belajar secara tekun. Sedangkan faktor minat dan sikap ini dapat dikembangkan kalau siswa diberi kesempatan untuk belajar secara aktif, disertai rasa gembira, dan tidak membosankan. Kebosanan ini dapat dihindari dengan cara menggunakan berbagai sumber belajar yang bervariasi, dan digunakan rnetode yang cocok, atau bervariasi pula. Hasil belajar yang kurang baik, tentu saja akan mengakibatkan nilai yang diperoleh siswa tidak memuaskan. Perolehan nilai kurang ini akan menimbulkan perasaan bahwa pelajaran itu sulit. Ketidakpuasan yang berlebihan menimbulkan rada frustrasi yang pada akhirnya menimbukan kebencian terhadap mata pelajaran tersebut. Tetapi di lain pihak timbul anggapan bahwa pelajaran yang sulit itu adalah lebih berharga. Siswa yang berhasil dalam pelajaran tersebut dianggap mempunyai kelebihan dari lainnya. Sebaliknya bagi siswa yang tidak berhasil akan menimbulkan rendah diri dari perasaan bodoh. Demikianlah usaha para pendidik untuk menyempurnakan proses belajar mengajar IPA, menyesuaikan diri dengan tuntutan perkembangan ilmu dan teknologi, serta mempertimbangkan kebutuhan masyarakat. Kebermaknaan hasil belajar akhirnya tidak hanya ditentukan oleh sejumlah pengetahuan yang banyak, tetapi hasil belajar yang lebih bermakna, dilihat dari perkembangan struktur, kognitif, struktur efektif, dan nilai-nilai ilmiah. Nilai-nilai ilmiah menjadi sangat berperan dalam perkembangan kebudayaan bangsa, dalam zaman moderenisasi sebagai akibat dari perkembangan ilmu dan teknologi yang maju dengan sangat cepat. Langkah lain yang tak kalah penting adalah, mengusahakan agar penemuan siswa lebih bermakna. Biasanya siswa cukup puas kalau semua tugasnya telah selesai dikerjakan. Kepada siswa harus diberikan pengertian, untuk apa jawaban yang diperoleh, dan apa sebenarnya yang diperoleh itu. Alangkah baiknya kalau kepada siswa diberi informasi untuk memberikan penekanan terhadap penemuan siswa. Penemuan ini akan lebih bermakna lagi kalau siswa dapat mengkomunikasikan pada orang lain, temasuk temannya dan gurunya, dapat dalam bentuk diskusi. Mendiskusikan hasil merupakan langkah untuk membuat penemuan siswa lebih bermakna. Kebermaknaan penemuan siswa dapat juga dinyatakan dalam bentuk aplikasi. Siswa dapat menggunakan hasil penemuannya untuk memecahkan masalah lain yang relevan. Kegiatan ini secara sederhana dapat dilakukan dalam bentuk latihan soal. Kemampuan siswa untuk menghubungkan penemuannya dengan pengetahuan lain yang diperolehnya, merupakan suatu pertanda adanya kebermaknaan atas penemuannya. Sebagai hasil kegiatan ini dapat berbentuk struktur konsep, bagan konsep, atau peta konsep. Jadi kebermaknaan pengetahuan yang diperoleh siswa dapat membentuk suatu struktur kognitif yang dapat dipergunakan untuk belajar lebih lanjut, dan dapat menimbulkan motivasi intrinsik untuk perkembangannya lebih lanjut. Dengan demikian anak SD harus sudah dapat mengklasfikasikan sesuai dengan bagian, struktur, dan fungsinya. Dia harus mampu berpikir kebalikan. Misal, Nuri termasuk kelas burung dan burung itu bertelur. Maka anak harus dapat menyimpulkan bahwa nuri dapat bertelur. Meskipun pada tingkatan ini anak belum dapat berfikir abstrak, seperti berhipotesa secara deduktif, tetapi dia sudah dapat membuat hipotesis sederhana, hanya meliputi satu variabel. Dia akan dapat memecahkan masalah dengan baik kalau konkret melakukannya. Sesuai dengan uraian terdahulu, proses belajar-mengajar berkembang sesuai dengan perkembangan ilmu dan teknologi. Perkembangan pengajaran sebagai ilmu ditandai dengan penerapan hasilhasil penelitian, hasil penalaran para ahli psikologi, khususnya psikologi pendidikan. Dengan demikian maka pengajaran termasuk pengembangan teknologi karena didalamnya tejadi proses penerapan teori-teori ilmu pengetahuan, beserta penjabarannya. Di samping itu proses belajarmengajar tetap merupakan seni dan kiat karena dalam pelaksanaannya tetap mempertimbangkan hakikat dari guru dan hakikat dari murid. Penerapan pilihan serta implementasi tindakan guru maupun murid tetap berdasarkan pertimbangan pribadi maupun instusi, serta sesuai dengan wawasan kependidikan. Untuk lebih jelasnya perhatikan diagram berikut. Kegiatan perencanaan tindakan meliputi tahapan sebagai berikut: (a) membuat Rencana Pembelajaran (Renpel) berdasarkan prioritas masalah yaitu penggunaan metoda demontrasi pada pembelajaran IPA tentang konsep perubahan benda, (b) mempersiapkan alat atau media pembelajaran yang akan digunakan yaitu metoda demontrasi untuk setiap kelompok, (c) membicarakan prosedur pelaksanaan pengajaran IPA tentang konsep perubahan benda menggunakan metoda demontrasi dan (d) menyusun instrumen-instrumen yang akan digunakan. • Children age 5 and under must be directly supervised by a responsible person age 16 or older at all times. • Youth ages 6-8 may participate during scheduled program hours until 8:00 pm as long as there is a person age 16 or older in the building who is responsible for them. • After 8:00 pm, children under the age of 12 must be directly supervised by a responsible person age 16 or older. • Children age 6 and up must use their respective locker room. • We offer many programs for youth and follow the Virginia Beach City Public School calendar for school-age youth programming. Specific program information is available at each recreation center. • To ensure their safety and to prevent anxiety, please do not leave children at the recreation center for long periods of time. Youth are advised to remain in a supervised program area. •. MP3 Players & iPods. Select Kitchen Appliances by Hamilton Beach. MP3 Players & iPods. Turn up the tunes with an MP3 player from. • Membership card or day pass must be presented and scanned in order to use the recreation center and/or participate in programs. • Proper footwear, shirts, and appropriate dry clothing are required in the facility. • Patrons are expected to dress appropriately. Clothing should not depict lewd graphics or obscene language. Undergarments should not be visible. • Please eat and drink in designated areas, such as the snack bar. All Virginia Beach municipal facilities are smoke-free. • Radios, CD/MP3 players and other personal electronic devices require headphones. • Animals and bicycles are not allowed in the facility. Service animals are permitted. • Skateboards and rollerblades may not be used in the recreation center, or near the recreation center front entrance and exits. Skateboards and rollerblades may be brought into the recreation center and secured in a locker. • Only City materials may be posted on City property. Other items may not be displayed or solicited. • Only Virginia Beach Parks and Recreation employees are permitted to provide instruction in the recreation center. • Courteous conduct is expected from all those using our facility or on the facility grounds. 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To ensure your child's safety and sense of security, please pick up your child at least 15 minutes prior to the building closing or, for children under age 12, at the end of the program time. Please verify operating hours and the center schedule for age-appropriate activities prior to leaving your child at the facility. Parents are advised that staff cannot take responsibility for children who are left unattended. Virginia Beach Parks and Recreation staff may contact the Virginia Beach Police Department for assistance if children are left at the facility 30 minutes after closing. •. Abstract Agassiz’s desert tortoise ( Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. Agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. Agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. Agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex. Citation: Tollis M, DeNardo DF, Cornelius JA, Dolby GA, Edwards T, Henen BT, et al. (2017) The Agassiz’s desert tortoise genome provides a resource for the conservation of a threatened species. PLoS ONE 12(5): e0177708. Editor: Pankaj Jaiswal, Oregon State University Department of Botany and Plant Pathology, UNITED STATES Received: November 18, 2016; Accepted: May 2, 2017; Published: May 31, 2017 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the public domain dedication. Data Availability: All genomic and transcriptomic sequence files are available from the NIH-NCBI BioProject database (accession numbers PRJNA352725, PRJNA352726, and PRJNA281763). All genome assembly, transcriptome assembly, predicted protein, transcript, genome annotation, repeatmasker, phylogenetic trees,.vcf and GO enrichment files are available on Harvard Dataverse (doi:). Funding: Author AEK is employed by Alice E. Rm 604 V10 82 Stube Flash mediafire links free download, download RM 604 v10 82 www mobifiles net, RM 604 10 82 Vietnamese, RM 604 V10 81 - rm 604 v10 82 stube flash mediafire files. Free Search Engine for Mediafire. Detect firmware version by flash file; detect existing languages by flash file; detect keyboard codes by flash file ('Keyboard codes detector'); batch flash file. Files from support area) and a lot of disk space on HDD for file storage counter for unlock/flash/other operations easy to use brand/model selection via 'Shell' easy. Karl and Associates. This company provided support in the form of salary for author AEK but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: AEK is commercially affiliated with Alice E. Karl and Associates and this affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction Species at risk of extinction can benefit from human interventions, and effective management of threatened populations depends on an understanding of their genetic variation, including the effects of inbreeding, disease, and adaptation to local environments []. One of six species of desert tortoise estimated to have arisen in North America ~35 million years ago (Ma) [], Agassiz’s desert tortoise ( Gopherus agassizii) has been heavily impacted by habitat loss, a respiratory tract disease [,], and other anthropogenic factors []. For instance, in one area of the species’ range density declined from about 225 individuals/km² in 1979 to about 75 individuals/km² in 1992 []. Such declines prompted populations of G. Agassizii north and west of the Colorado River to be listed as threatened under the U.S. Endangered Species Act []. Despite this protection, desert tortoise numbers continued to decline ~50% between 2004 and 2013 [], warranting further conservation efforts. Effective management of G. Agassizii populations would benefit from genomic resources, which have only recently been developed for the species. Previous population genetic studies based on microsatellites, nucleotide sequences from mitochondrial and nuclear DNA, and RNA sequencing (RNA-Seq) data have laid the groundwork for assessing the distribution of genetic diversity across the range of G. Agassizii [–]. Agassiz’s desert tortoise is a long-lived species that is slow to mature, with a maximum lifespan of over 50 years in the wild [], making the long-term impacts on adult populations and conservation priorities difficult to assess. Genomic analyses of G. Agassizii may help to understand key aspects necessary to its survival, such as immune system responses to diseases, including upper respiratory tract disease []. Functional trait analysis combined with genomics can advance knowledge of physiological [], morphological [], and life history variation among the six species of Gopherus []. Genomic information can also inform management decisions about habitat corridors and reproduction especially in the light of climate change []. A whole blood transcriptome assembly has been published for G. Agassizii []. However, this represents only a portion of the total genetic information, and a complete genome assembly would better facilitate future analyses of polymorphism and the geographic distribution of variation for the species and its congeners. In addition to providing resources that can save threatened or endangered species from extinction, comparative genomics may reveal information about many traits relevant to human illnesses [] and longevity []. Gopherus agassizii possesses many traits common to turtles, or chelonians, including longevity, as well as those specific to desert tortoises such as robustness to harsh environments. Genome assemblies exist for several chelonians: the Chinese softshell turtle, Pelodiscus sinensis [], the green sea turtle, Chelonia mydas [], and the western painted turtle, Chrysemys picta bellii []. Embryonic transcriptomes have been published for the red-eared slider turtle, Trachemys scripta [], a blood transcriptome for the giant Galapagos tortoise, Chelonoidis nigra [], and liver transcriptomes for the common musk turtle ( Sternotherus odoratus), common snapping turtle ( Chelydra serpentina), and African sideneck turtle ( Pelusios castaneus) []. Recent studies have confirmed a sister-taxon relationship between chelonians (including turtles and tortoises, order Testudines) and archosaurs (including crocodilians and birds), which split ~250 Ma [,], and have estimated that the basal divergence of most modern chelonian lineages occurred ~100 Ma. Gopherus agassizii and C. Picta bellii last shared a common ancestor ~70 Ma [] (), and comparisons using this more recent divergence may provide a better opportunity to understand the evolution of many chelonian-specific traits, such as longevity and adaptations to aquatic or terrestrial habitats, including hot deserts. To assist both conservation and comparative genomics, we report a whole genome assembly and deep transcriptomic sequence-based annotation for Agassiz’s desert tortoise, and provide insights from comparative and population genomic analyses. Sample collection and sequencing The Institutional Animal Care and Use Committee at Arizona State University reviewed and approved this study, and methods of transport, housing, and euthanasia were carried out in accordance with ethical guidelines in Protocol #13-1319R (to DFD). Specifically, an adult male G. Agassizii collected in urban Las Vegas, Nevada under U.S. Fish and Wildlife Service recovery subpermit #FWSDTRO-1 was transported to Arizona under Nevada Department of Wildlife export permit #S37016, housed for two days and euthanized with an intracoelemic injection of sodium pentobarbital and phenytoin solution. Tissues were harvested under sterile conditions and either flash-frozen in liquid nitrogen or placed in RNAlater (Thermo Fisher Scientific) for DNA and total RNA extraction, respectively. DNA was extracted from liver and skeletal muscle using the DNeasy kit (QIAGEN) and total RNA was isolated from lung, brain and skeletal muscle with the total RNA protocol included in the mirVana miRNA Isolation Kit (Thermo Fisher Scientific). RNA quantity and quality was measured by spectrophotometry (Nanodrop) and Agilent 2100 Bioanalyzer. All sequencing was carried out on the Illumina HiSeq 2000 or 2500 platforms. Paired-end DNA libraries were constructed with targeted fragment sizes of 200 base-pairs (bp), 300 bp, and 1,000 bp using the Kapa Biosystems Library Prep Kit by the Collaborative Sequencing Center at the Translational Genomics Research Institute (Phoenix, AZ; ). Mate-pair libraries were constructed and sequenced for DNA fragment sizes of 2 kbp, 4 kbp, and 10 kbp (). Additionally, RNA-Seq libraries for brain, muscle and lung total RNAs were constructed and sequenced at the University of Arizona Genetics Core (Tucson, AZ; ). To confirm the taxonomic identity of the specimen, and to assure that it was not a hybrid of G. Agassizii and G. Morafkai [], we followed described protocols [] using 25 short tandem repeats and mtDNA sequence data for population assignment against a database of 1,258 specimens of Gopherus. We confirmed the specimen to be G. Agassizii from the northern Mojave genetic unit, which is geographically bounded as east of the Ivanpah, California area and northwest of the Colorado River in Nevada, Arizona and Utah []. Genome assembly DNA sequence reads were trimmed to eliminate nucleotide biases and remove adaptors with Trimmomatic v0.27 []. We retained all reads ≥ 37 bp with a quality score ≥ 28. Illumina sequencing errors were corrected using SOAPec v2.01, and overlapping reads from the 200 bp libraries were joined to form single-end reads using FLASH v1.2.8 []. We compared the outputs of different de Bruijn graph assemblers, including ABySS 1.5.2 [], SOAPdenovo2 [], and Platanus v1.2.1 [] for both contig and scaffold assembly, in addition to SSPACE v3.0 [] for scaffold assembly; we then closed gaps using the GapCloser v1.12 module from SOAP (). The assemblies with the most scaffold contiguity (i.e., N50) and reasonable total length given the expected genome size were selected for further analysis. We evaluated completeness of the assemblies by their estimated gene content, using the Conserved Eukaryotic Genes Mapping Approach (CEGMA v2.5) [], which calculated the proportion of 248 core eukaryotic genes present in the genome assembly, and Benchmarking Universal Single Copy Orthologs (BUSCO v1.22) [], which calculated the proportion of a vertebrate-specific set of 3,023 conserved genes that were either complete, fragmented, or missing. We further improved the raw assembly by RNA scaffolding. In brief, assembled transcripts (described below) were searched for open reading frames (ORFs) and filtered to include only sequences that produced a significant BLAST hit to a protein sequence in the UniProtKB/Swiss-Prot database [] using TransDecoder v2.0 [] (). This filtered gene set was mapped to the genome assembly using BLAT [] and identified scaffolds were merged using L_RNA_scaffolder []. Genome annotation The RNA-Seq fastq files for skeletal muscle, brain and lung were filtered for adapters, quality score ≥ 30 and length ≥ 37 bp using Trimmomatic v0.27, and paired-end and single-end reads were assembled together into transcripts using Trinity v2.0.6 []. Previously published whole blood-based transcriptome data were also used (NCBI BioProject PRJNA281763) []. We used MAKER2 v2.31.8 [] in multiple iterations for gene-finding, which incorporated: (1) direct evidence from the transcriptomes, (2) homology to proteins in the UniProtKB/Swiss-Prot database and the predicted proteome of C. Picta bellii (NCBI BioProject PRJNA210179), and (3) ab initio predictions from SNAP ( release) [] and Augustus v3.0.2 []. The first iteration of MAKER aligned the transcript and protein sequences to the assembly, producing draft gene models. Ab initio gene predictors benefit from the training of their Hidden Markov Models (HMM) to the species-specific genome, and we trained SNAP using a two-pass approach in MAKER. First, we ran MAKER a second time with SNAP using G. Agassizii-specific HMMs generated from the initial MAKER iteration. Using the gene models from this output, we then generated an improved HMM for SNAP in a third MAKER iteration. In parallel, Augustus was trained by sampling the SNAP-improved MAKER gene models followed by optimization. A fourth and final run of MAKER was then performed, which incorporated the optimized HMM for Augustus, the final SNAP HMM, and the aligned protein and transcriptome data, resulting in the final set of gene model predictions. Gene predictions were functionally annotated by collecting the top hits from a BLASTP search of the UniProtKB/Swiss-Prot database. Protein predictions from the final gene models were assigned one-to-one orthology with proteins from the western painted turtle genome through reciprocal BLAST. First, BLASTP was used to search for the best hit for every predicted Agassiz’s desert tortoise protein in the western painted turtle proteome (evalue = 1E-6, max_target_seqs = 1), followed by BLASTP of each western painted turtle protein in the Agassiz’s desert tortoise proteome. We also used reciprocal best hits to identify orthologs of Agassiz’s desert tortoise in Chinese softshell turtle, chicken ( Gallus gallus), green anole ( Anolis carolinensis), and human proteomes. Clusters of homologous genes across Agassiz’s desert tortoise, western painted turtle, Chinese softshell turtle, chicken, American alligator ( Alligator mississippiensis), green anole and human were identified using OrthoFinder v0.4 []. Repeat analysis The Agassiz’s desert tortoise genome assembly was masked for repetitive elements, first with RepeatMasker v4.0.5 [] using the complete library of known repeats available in RepBase []. The remaining unmasked regions of the desert tortoise genome were then searched for repeat families using RepeatModeler v1.0.8 []. This output of de novo repeats from desert tortoise was then combined with the first RepBase library to create a combined library, which was used in a final repeat-masking step to classify all annotated repeats in the genome. We also ran RepeatMasker on the western painted turtle genome using RepBase, which includes repeat libraries specific to Chrysemys picta bellii, in parallel for comparison to Agassiz’s desert tortoise. To estimate the amount of evolutionary divergence within repeat families of Agassiz’s desert tortoise, we generated repeat-family-specific alignments and calculated the average Kimura 2-parameter (K2P) distance from consensus within each family, correcting for high mutation rates at CpG sites with the calcDivergenceFromAlign.pl tool included with RepeatMasker. For comparison, the divergence profile for western painted turtle was obtained from the RepeatMasker server (, accessed August 10, 2015). Comparative genomic analyses To understand patterns of molecular evolution across chelonians and other reptiles, the genome assemblies of Agassiz’s desert tortoise, western painted turtle, Chinese softshell turtle, green sea turtle, American alligator (allMis1), budgerigar ( Melopsittacus undulatus, melUnd1), and green anole lizard (AnoCar2.0) were aligned to the reference chicken genome (galGal4) using LASTZ v1.0.2 []. We used chaining to form gapless blocks and netting to select the highest-ranked chains []. Pairwise alignments for zebra finch ( Taenopygia guttata, taeGut3) and medium ground finch ( Geospiza fortis, geoFor1) to chicken were downloaded from the UCSC Genome Browser. A guide tree representing the phylogenetic relationships of the included taxa [,] was used to construct a 10-way whole genome alignment (WGA) from the pairwise alignments with MULTIZ v11.2 []. The WGA was filtered to include alignment blocks containing at least nine out of the 10 species. Divergence at fourfold degenerate (4D) sites, which approximates the neutral mutation rate [], was used to compare variation in DNA substitution rates of chelonians and the other sauropsids. We extracted 4D sites from the WGA based on the Ensembl v82 chicken gene annotations using msa_view in the PHAST v1.3 package []. To reconstruct the phylogeny of the 10 sauropsids included in the WGA using the 4D data, we used RAxML v8.2.3 []. We generated 20 maximum likelihood (ML) trees under the GTRCAT substitution model and conducted 500 bootstrap replicates to assess statistical support for the ML tree with the highest likelihood. We used the topology of the best ML tree from RAxML and obtained branch lengths in terms of substitutions per site by fitting a time-reversible model (REV) to the 4D site data using phyloFit in PHAST. We then estimated lineage-specific substitution rates in sauropsids using the penalized likelihood method implemented in r8s v1.8.1 [] while placing constraints on nodal ages obtained from TimeTree []. The age of the ancestral sauropsids node was fixed at 280 Ma and the ancestral archosaur node at 245 Ma; these were the median age estimates. Estimates of divergence time for most chelonian families differed widely in the literature. Thus, we placed minimum and maximum age constraints on the nodes representing Cryptodira (encompassing all four represented chelonian families, 97.4 Ma to 250 Ma) and Drurocryptodira (excluding P. Sinensis, 36 Ma to 183 Ma). The divergence time of Testudinae ( G. Agassizii) and Emydidae ( C. Picta bellii) was estimated (1) without constraint and (2) with constraints, which fixed the node age as the median estimate from TimeTree (74.2 Ma). To identify protein-coding genes potentially responsible for adaptations in Agassiz’s desert tortoise, we calculated the ratio of non-synonymous to synonymous substitutions (Ka/Ks) in pairwise comparisons separately to western painted turtle (chrPic2), Chinese softshell turtle (pelSin1), green sea turtle (cheMyd1) and chicken. Syntenic genome alignments were generated as described above, and the Stitch Gene Blocks tool in Galaxy [] was used to extract orthologs using the chicken reference annotation (Ensembl v82). We removed gene alignments with less than 50% coverage, ambiguous codons, and internal stop codons using AlignmentProcessor (). Estimated Ka/Ks for the filtered gene alignments were obtained using KaKs_calculator2.0 [], and we collected the RefSeq (western painted turtle, green sea turtle, Chinese softshell turtle) or Ensembl (chicken) IDs of accelerated genes (Ka/Ks > 1). These genes were enriched for non-synonymous and potentially functional substitutions resulting from amino acid changes []. A Bonferroni adjustment for multiple hypothesis testing [] was used to identify genes with highly significant Ka/Ks > 1, which indicates positive selection. Many genes in chelonian genome assemblies lacked clear orthology to an organism in RefSeq. Thus, human orthologs were obtained by the ID Mapping of associated gene names in UniProt. Next, Ensembl BioMart [] was used to for retrieve Gene Ontology (GO) terms. REVIGO [] semantic clustering was used to visualize GO terms for biological processes, cellular components and molecular function. To compare GO terms, analyses used medium similarity (0.7) for semantic overlap, clustering of terms by ‘uniqueness’ and the default whole UniProt database. In 2011, divergence across genetics, ecology and behavior was used to recognize two different species of desert tortoise in the southwestern United States: Agassiz’s desert tortoise ( G. Agassizii), found in the Mojave Desert of California, Nevada, Utah and northwestern Arizona, and Morafka’s desert tortoise ( G. Morafkai), found in the Sonoran Desert of Arizona and Sonora, Mexico []. More recently, genetic data were used to characterize and describe the third desert species, Goode’s Thornscrub tortoise ( G. Evgoodei), which resides in the states of Sonora and Sinaloa, Mexico []. These three species are thought to have diverged nearly simultaneously ~5.5 Ma [] and occupy diverse habitats, including the high desert of the Great Basin shrub steppe in Utah, to the Joshua tree forests of California, through the coastal desertscrub along the east coast of the Sea of Cortez and the lush tropical deciduous forests of Sinaloa, Mexico []. The three species of desert tortoise have likely adapted to different climatic regimes and landscapes. To find genomic regions responsible for species-specific desert tortoise adaptations, we analyzed fixed unique variants within three species of the desert tortoise species complex. We mapped previously published blood RNA (NCBI BioProject PRJNA281763) data from three individuals per species to our new G. Agassizii reference genome using STAR v.2.5.0a [], with default parameters for the two-pass method set to “basic” to find splice junctions. We added read groups and removed PCR duplicates using MarkDuplicates from Picard v1.140 (). We then used the Split’N’Trim tool in the Genome Analysis Tool Kit pipeline (GATK v3.4–46) [] to split reads into exon segments and clip sequences that overlap introns. The split, de-duplicated alignment files from each individual were then merged using Picard. We used the CallableLoci GATK tool to filter the genome for areas that met default coverage criteria for each alignment file, and concatenated, sorted and merged the output BED files using bedtools version 2.24.0 [], with–d set to 20 to join nearby yet non-contiguous callable sites. Using the merged callable loci as a target file, we called variants on all nine alignments with FreeBayes v1.0.1 []. We identified fixed differences between the three species using the Filter command in SnpSift v4.0 [], filtering by quality (≥ 30) and read depth (≥ 20). We filtered for only SNP variants within the unique variants file per taxon, excluding STRs and insertion-deletions. We cross-referenced these unique SNP variant files to orthology tables for human ( Homo sapiens), chicken ( Gallus gallus), and green anole ( Anolis carolinensis) (see Genome Annotation Methods) for each Gopherus species (9 comparisons in total). Each set of cross-referenced orthologous Ensembl IDs were: 1) converted to Gene Ontology (GO) terms using g:Convert (, accessed July 31, 2016) [], to plot and analyze semantically with REViGO, and 2) entered into g:GOSt [] to test for statistical enrichment of GO terms per Gopherus species. For enrichment analysis in g:GOSt we retained only significant results, used hierarchical sorting without filtering, and used the default significance threshold (g:SCS) correction for multiple hypothesis testing. For visualization in REViGO, we allowed medium similarity (0.7) for semantic overlap, clustered terms by ‘uniqueness,’ and used the default whole UniProt database for comparison of GO terms. Gene clusterings and GO-term enrichments were similar across reference taxon (human, chicken, or the green anole), so we only show results using orthology assignments from the green anole. Genome sequencing and assembly For whole genome sequencing, we used multiple insert size genomic libraries (). We generated a total of 2,877,286,716 small-insert library reads with targeted insert sizes of 200 bp, 300 bp, and 1,000 bp (average insert sizes of 189 bp, 265 bp, and 788 bp, respectively). Average read length was 104 bp, and after trimming we retained 85% of those reads for an average read length of 96 bp. Using a k-mer of 27, the k-mer frequency spectrum of the trimmed short insert library data () revealed peaks at 35X k-mer depth for heterozygous regions and 71X k-mer depth for homozygous regions. Using the formula genome size = total k-mers ÷ peak k-mer depth, we estimated the genome size for G. Agasizzi to be 2,350,283,237 bp or ~2.4 gigabase-pairs (Gbp), which is smaller than a previous estimate of 2.9 Gbp based on Feulgan densitometry (FD) of red blood cells []. This 17% difference may reflect either error in experimental quantification or intraspecific variation in genome size. Another potential explanation for this discrepancy could be that the FD quantification of genome size was published in 1965, long before the taxonomic reclassification of the desert tortoise species complex [,], and thus may have been performed using a species other than G. Using our k-mer based estimate of genome size, we further estimate that with the short insert libraries we obtained genome coverage of 128X before trimming and 97X after trimming. We also generated mate pair libraries with targeted insert sizes of 2 kbp, 4 kbp, and 10 kbp (average insert sizes of 911 bp, 2,220 bp, and 4,446 bp, respectively), with an average read length of 100 bp after trimming for additional 19X coverage. Genomic sequence summaries are listed in, and raw data is made available through NCBI BioProject PRJNA352726. Assembly statistics for the genome of Gopherus agassizii and comparison with Python bivittatus and Alligator mississippiensis. (A) Two k-mer frequency peaks exist for G. Agassizii: a peak at 35X coverage, representing heterozygous regions and a peak at 71X coverage, representing homozygous regions. (B) Gene-based scaffolding greatly increased scaffold N50 from 169.2 kbp to 251.6 kbp, which places it within the range of recently published genomes of reptiles including the Burmese python [] and the American alligator []. Statistics for candidate assemblies are found in the Supporting Information online ( and Tables). Assembling with SOAPdenovo2 and a user-set k-mer value of 47 results in a contig N50 of 42.7 kbp, a scaffold N50 of 169.2 kbp, with placing 86.9% of contigs on scaffolds, 43.19% GC content, and 1.51% consisting of Ns after gap-closing, which we select for further analysis. RNA-scaffolding vastly improves the contiguity of the initial assembly by increasing the scaffold N50 to 251.6 kbp and with placing 88.7% of contigs on scaffolds, and 1.55% consisting of Ns (). The total length of the draft assembly is very close to our k-mer based estimate of genome size at 2.4 Gbp, which is longer than 1.2 Gbp for the chicken [] but comparable to 2.6 Gbp for C. Picta bellii [], 2.2 Gbp for P. Sinensis, and 2.2 Gbp for C. We detect 235 out of 248 (94.8%) core eukaryotic genes (CEGs) in the final assembly in at least partial status, and 194 (78.2%) CEGs are complete. We identify 92% of the 3,023 vertebrate-specific BUSCOs in the assembly, including 2,324 (77%) complete and 459 (15%) fragmented (). Summarizes the final assembly statistics. The distribution of GC content across the assembly is similar to that of other published turtle genomes, and differs from more phylogenetically distant reptiles (). Genome annotation To maximize the representation of annotated genes, we executed deep transcriptome sequencing of tissues with divergent expression profiles and developmental origins: brain (ectodermal origin), skeletal muscle (mesodermal origin), and lung (endodermal origin). RNA sequencing (NCBI BioProject PRJNA352725) produced 93,284,795 Illumina reads from brain, 107,105,716 reads from lung, and 86,205,948 reads from skeletal muscle, all of which assemble in a transcriptome containing 245,326 contigs including 2,642 (87%) complete and 143 (4.7%) fragmented vertebrate BUSCOs (). Genome annotation also uses published data from blood transcriptomes of G. Agassizii (NCBI BioProject PRJNA281763) [] and publicly available protein sequences. The annotation pipeline identifies 20,172 protein-coding genes (including 85% of vertebrate BUSCOs, ), 17,173 5’ untranslated regions (UTRs), and 20,417 3’UTRs. The number of protein-coding genes in the G. Agassizii assembly (20,172) is comparable to the number of predicted genes in other published reptilian genomes, including 21,796 in the western painted turtle [], 19,327 in the Chinese softshell turtle [], 19,633 in the green sea turtle [], 22,962 in the green anole [], and 15,508 in the chicken []. The number of shared one-to-one gene orthologs between Agassiz’s desert tortoise and other reptiles include 15,220 with western painted turtle (), 12,505 with Chinese softshell turtle (), 11,491 with chicken (), and 11,983 with green anole (). The number of shared one-to-one gene orthologs between Agassiz’s desert tortoise and other reptiles is comparable to the number shared with the human genome (13,284, ). Predicted orthology groups identified by the OrthoFinder analysis shared across human, turtles, archosaurs, and lepidosaurs are made available to the comparative genomics community (). Gene structure parameters for the G. Agassizii genome build are comparable with that of other reptile genomes, with Agassiz’s desert tortoise being more similar to the chicken than to the two aquatic chelonians (). The total coding DNA sequence (CDS) length for Agassiz’s desert tortoise is similar to that of the chicken, but it has fewer genes below the 10 kb CDS length than do the western painted turtle and Chinese softshell turtle (). The frequency distribution of exon length is similar among the four species () but intron length is most similar for Agassiz’s desert tortoise and chicken; both have fewer large intron genes compared to the other chelonians (). The low number of large introns in Agassiz’s desert tortoise and chicken may be the result of a reduction in intron size in the common ancestor of chelonians and archosaurs [,] followed by lineage-specific increases in intron length in western painted and Chinese softshell turtles [], or may result from independent changes in Agassiz's desert tortoise. The future availability of additional chelonian and archosaurian genomes will help to address this question. Repeat analysis Repeat identification of the G. Agassizii genome using known RepBase elements results in 31% of the genome masked (). Additional de novo repeat identification with RepeatModeler improves the annotations of several classes of repeats, resulting in 43% of the genome being assigned to repeats. These include a diverse array of transposable elements, which comprise 33% of the genome, including DNA transposons, long terminal repeat (LTR) elements, long interspersed nuclear elements (LINEs), and short interspersed elements (SINEs). The genomic proportion of repeats in Agassiz’s desert tortoise is greater than that for the western painted turtle (29%). Slight differences in the abundance of repeat classes exist between the analyzed chelonians, although relative abundances are similar except for DNA transposons, which are more abundant in Agassiz’s desert tortoise (12.4% versus 9.8% in the western painted turtle), as are LTR retrotransposons (7.7% versus 5.2%, respectively). The genomes of both Agassiz’s desert tortoise and the western painted turtle possess a large proportion of CR1 subfamilies at ≤ 5% K2P divergence from their consensus sequence (), suggesting they are recent inserts. Therefore, these elements comprise the most active and common retrotransposons in both chelonians and archosaurs [,]. The high percentage (8%) of unclassified interspersed repeats identified by RepeatModeler in the Agassiz’s desert tortoise’s genome, for which we could not find matches in either RepBase or GenBank, suggests that novel elements exist in the genomes of chelonians. Slow DNA substitution rates in chelonians The filtered whole genome alignment (WGA) is just over 217 Mbp in length, including 15% gaps, with an average of 9.23 species represented in each alignment block. The RAxML analysis included full statistical support for the a priori expected relationships, including the monophyly of C. Picta bellii and G. Agassizii, P. Sinensis as the outgroup to all other included chelonians, and a monophyletic archosauria. When analyzing 1,815,350 4D sites, branch lengths in terms of substitutions per site on the sauropsid phylogeny (, ) suggest limited neutral divergence since the most recent common ancestor of chelonians. In contrast, the avian clade has longer internal branches, consistent with faster evolutionary rates in birds compared to other archosaurs [,]. Incorporating different divergence times between Gopherus and Chrysemys () affects the absolute estimated rates but not relative results. These results are similar to previous findings that chelonians have accumulated fewer DNA substitutions over time than other sauropsid lineages [,] (, ). This may be due to the unusually long generation times of many chelonians, which correlate with lower substitution rates in reptiles []. Comparative genomics of turtles The G. Agassizii genome alignments to western painted turtle, green sea turtle, Chinese softshell turtle and chicken are 1.9 Gbp, 1.8 Gbp, 1.3 Gbp, and 0.44 Gbp in length, respectively. There are 223 genes with Ka/Ks > 1 in the tortoise-western painted turtle comparison, and 75, 31, and 23 in the comparisons with green sea turtle, Chinese softshell turtle and chicken, respectively (). This results in 58, 29, 6, and 23 accelerated genes with clear orthology in the respective comparisons (), though none passed the p-value threshold for positive selection after the conservative Bonferroni correction for multiple hypothesis testing. Notwithstanding, we report results of all genes with Ka/Ks > 1, because both positive selection and relaxed functional constraint can affect the evolution of phenotypes []. The western painted turtle is the taxonomically closest genome-enabled species to Agassiz’s desert tortoise, and as a temperate aquatic species its comparison with desert tortoise can shed light on the evolution of many desert tortoise traits. Indeed, the desert tortoise-painted turtle comparison yielded the highest number of genes with Ka/Ks > 1. Many of the 58 accelerated genes may be relevant to tortoise adaptations (), such as CD34. This gene codes for an adhesion molecule on circulating cells that contribute to vascular repair following glomerular injury []. Optimal renal function is critical for organisms living in the desert, such as G. CSTA and SDR16C5 may also link to important chelonian adaptations, as they are involved in keratinocyte differentiation (GO:0030216) and proliferation (GO:0043616), respectively. This implies involvement in the formation of reptilian scales and feathers including the chelonian shell []. Other accelerated genes control responses to oxidative stress, including VIMP and SOD1. This is consistent with the initial analysis of the western painted turtle genome, which showed molecular evidence for adaptations for anoxic conditions []. Accelerated genes between the Agassiz’s desert tortoise and western painted turtle involve function in immunity, including SOD1, FFAR4, CXCL11, CXCL8, CD4, IL10, and NFAM1. These genes regulate cytokine activity. The comparison with green sea turtle () also yields amino acid changes in genes involved in immune function such as TNFSF4 and IFNG, both of which regulate CD4 + T cell/antigen presenting cell interactions, and PGLYRP1 which is a peptidoglycan-recognizing protein involved in response to bacterial infection. Agassiz’s desert tortoise and the green sea turtle share several accelerated genes involved in apoptosis and aging, including BCL2A1 and GZMA. Comparative genomics of chelonians. Representation of Gene Ontology terms for biological processes shared by genes with pairwise Ka/Ks > 1 in comparisons of Gopherus agassizii to (A) Chrysemys picta bellii and (B) Chelonia mydas. Treemap boxes are both sized and colored by uniqueness. Abbreviations in (A): RB, receptor biosynthesis, LCFACB, long chain fatty acyl-CoA biosynthesis, MTMD, membrane to membrane docking. Analyses fail to detect many genes with Ka/Ks > 1 in the comparison of Agassiz’s desert tortoise and the Chinese softshell turtle, yet CD4 is also accelerated as in the comparison with the western painted turtle. Also, as in the green sea turtle comparison, the chicken comparison shows accelerated evolution in TNFSF4. Given their phylogenetic relationships, these changes to the adaptive immune system appear unique to Agassiz’s desert tortoise within these taxa. We also find amino acid differences between chicken and desert tortoise in genes such as MPV17, which is involved in mitochondrial homeostasis, suggesting different adaptive pathways controlling metabolism in endotherms (chicken) and ectotherms (tortoise). Finally, AQP10 is a potentially important accelerated gene from the chicken comparison, which functions as a water-selective channel that can permeate neutral solutes such as glycerol and urea []. Altogether, these analyses of protein coding genes suggest that chelonian adaptations relate to shell development and anoxia, and tortoise-specific adaptations involve longevity, osmoregulation and water balance, immune function, and metabolism. Such results provide a foundation for future chelonian studies in conservation and organismal function. Comparative genomics of the desert tortoise species complex After filtering for quality and coverage, G. Agassizii, G. Morafkai and G. Evgoodei have 10,086, 2,593 and 23,261 fixed unique SNPs, respectively. Of these variants, the numbers occurring in genes with assigned orthology to Anolis carolinensis are 910, 360, and 1,340, respectively. Analyses using g:GOSt identify 59 (BP: 27, CC: 28, MF: 4), 23 (BP: 1, CC: 20, MF: 2), and 95 (BP: 42, CC: 39, MF: 14) enriched GO categories, respectively. The conversion of genes to GO terms in g:Convert produces 8,928 GO terms for G. Agassizii, 3,590 for G. Morafkai, and 13,455 for G. Evgoodei, including duplicates by different genes yielding the same GO term (– Tables, ). The GO categories most enriched for G. Gene Ontology categories most enriched for fixed unique SNPs in G. Agassizii compared with G. Morafkai and G. These results are for Biological Processes, for a complete listing see – Tables. Basic cellular processes are necessary for the higher functions G. Agassizii uses to survive the environmentally harsh Mojave Desert, including circadian rhythm, aging, response to UV, regulation of urine volume, and humoral and innate immune responses (). The enriched GO category of macromolecule modification (GO:0043412) includes genes involved in such biological processes that may be important to the adaptation of G. Agassizii to its environment (– Tables). CRY1 (Cryptochrome-1), and CSNK1D (Casein kinase I isoform delta) are also involved with circadian rhythm (GO:0007623). In the northern house mosquito, functional CRY1 is required for overwintering diapause or dormancy [], although the role of circadian genes in regulation of brumation remains to be determined. Five genes overlap with response to UV (GO:0009411): ERCC6 (DNA excision repair protein ERCC-6), HYAL2 (Hyaluronidase-2), PPID (Peptidyl-prolyl cis-trans isomerase D), UBE2B (Ubiquitin-conjugating enzyme E2 B), and USP28 (Ubiquitin carboxyl-terminal hydrolase 28). HYAL2 overlaps with other genes in the category for regulation of urine volume (GO:0035809). With an estimated lifespan of up to 53 years in the wild [], Agassiz’s desert tortoise allows for investigations into the genetic regulation of aging, making it a potentially important species for the understanding of many human diseases [,]. Six genes overlap with aging (GO: 0007568): ABL1 (Tyrosine-protein kinase ABL1), CARM1 (Histone-arginine methyltransferase CARM1), HYAL2, PRKCD (Protein kinase C delta type), RPN2 (Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 2), and TGFB1 (Transforming growth factor beta-1). These genes with functional enrichment across Gopherus spp. And which overlap with relevant desert-related processes warrant further investigation. Indeed the role that such macromolecular modification genes play in desert adaptation is a question with broad relevance, and may be important to understanding the biology of this threatened species. Overlapping genes in the two most enriched GO categories for fixed unique SNPs in G. Agassizii compared with key functional categories. No overlap was identified with GO:0002456 T cell mediated immunity. Immune response is important for G. Agassizii due to the respiratory tract disease that may be driving population declines (). No genes with fixed variants overlap with T cell mediated immunity (GO:0002456), yet three genes overlap with humoral/B cell mediated immunity (GO:0019724): PAXIP1 (PAX-interacting protein 1), PRKC (Protein kinase C delta type), and TGFB1. Nine genes overlap with innate immune response (GO:0045087): ABL1, CDC37 (Hsp90 co-chaperone Cdc37), IFIH1 (Interferon-induced helicase C domain-containing protein 1), NOD1 (Nucleotide-binding oligomerization domain-containing protein 1), PELI1 (E3 ubiquitin-protein ligase pellino homolog 1), PRKCD (Protein kinase C delta type), SRPK1 (Serine and arginine rich splicing factors protein kinase 1), TAB1 (TGF-beta-activated kinase 1 and MAP3K7-binding protein 1), and TGFB1. Only SRPK1 overlaps with the RNA processing group (GO:0006396) and the more functionally focused groups for innate immune response (GO:0045087). Given the susceptibility of G. Agassizii to upper respiratory disease, variants that would influence the immune response are of high interest to understand host factors involved in pathogenesis. Genes with fixed differences between species of the Gopherus desert tortoise species complex, as well as genes found to be under accelerated evolution across chelonians, are summarized in. Conclusions Agassiz’s desert tortoise ( Gopherus agassizii) is listed as a threatened species. Here, we report a de novo genome assembly and annotation for G. Agassizii to serve as a conservation resource for assessing genetic diversity across the species’ range, identifying genes involved in adaptation to warm deserts, and genomic responses to the respiratory disease that is threatening desert tortoise populations. We generated a draft genome assembly and annotation based on deep transcriptome sequencing of four tissues from adult tortoises. The genome assembly represents 2.4 Gbp with a scaffold N50 length of 251.6 kbp, and a 43% genomic proportion of repetitive elements. Genome annotation identifies 20,172 protein-coding genes, including an estimated 95% of core eukaryotic genes, and is comparable to predicted gene numbers in turtles. We demonstrate that this draft genome will be a useful reference for the future mapping of markers in genetic studies of the evolution of G. Agassizii, such as defining the boundaries of the hybrid zone with Morafka’s desert tortoise ( G. Our analyses of gene sequences lay the groundwork for understanding the genetic basis of adaptations to arid environments for this species and its congeners, and potentially other desert-adapted species, including identifying genes involved in response to UV and regulation of urine volume. 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