決定仔豬F4ac腹瀉易感/抗性的MUC13基因及其應用的制作方法

專利名稱：決定仔豬F4ac腹瀉易感/抗性的MUC13基因及其應用的制作方法
技術領域：
本發明涉及動物遺傳育種領域，具體涉及一個決定仔豬ETEC F4ac腹瀉易感性/ 抗性的MUC13基因及其在種豬遺傳改良中的應用。
背景技術：
仔豬腹瀉是養豬生產中的常見傳染病，給世界養豬業造成了巨大經濟損失。產腸 毒素大腸桿菌F4(Enterotoxigenic Escherichia coli F4，ETEC F4)是引發新生和斷奶前 仔豬腹瀉病的最主要致病菌。以我國和丹麥為例，分別有約35%和40%的仔豬腹瀉病是因 ETEC F4侵染而引起的。在我國，其致死率在10%-30%之間，在個別地方甚至比這個比例 更高，導致養豬出欄率、商品豬質量嚴重下降，經濟效益受損。ETEC F4的致病性取決于其是否與豬小腸上皮細胞表面刷狀緣的受體特異性結 合。這種結合發生后，ETEC F4大量繁殖產生腸毒素，致使腸上皮細胞分泌功能亢進，大量水 和電解質進入腸腔，造成腸腔中大量液體積蓄，超過了腸壁重吸收能力而引起腹瀉。因此， 仔豬小腸上皮細胞有無ETECF4受體是仔豬被ETEC F4感染時是否發病的關鍵。無受體豬 表現為抵抗型，有受體豬則表現為易感型。分離和鑒別豬ETEC F4受體基因及其抗性位點 因而成為豬ETEC F4腹瀉抗病育種的關鍵。已有的研究表明豬ETEC F4受體是單基因控制的，呈顯隱性遺傳模式。攜帶顯性 基因S的豬對ETEC F4的粘附易感，隱性基因s純合子豬則表現為抵抗型。常見西方商業 豬種中，如大白、長白和杜洛克，均有一定比例的易感個體。ETEC易感性可以通過小腸刷狀 緣的體外黏附實驗來判定，黏附的個體表現為易感，不黏附的個體表現為抵抗。ETEC F4根 據免疫學特征分為ab，ac和ad三種亞型，其中F4ac在西方豬群中是最主要的致病菌。因 而F4ac受體基因的定位和鑒別得到了國內外多個研究小組的重視。1995年，瑞典Leif Andersson小組利用歐洲野豬X大白豬資源家系群體為材 料，經過三代系譜的連鎖分析，首次將ETEC F4ac受體定位于13號染色體，與Tf基因緊密 連鎖，并發現F4ab和F4ac受體位點緊密連鎖，認為這兩個受體是由同一受體控制的。2002 年，瑞士 Peter Vogeli小組以大白豬和大白X長白三代家系為材料，利用13號染色體更 多的微衛星標記信息，將ETEC F4ac受體進一步定位于S0068和Swl030之間，與其中的 S0075和Sw225高度連鎖(0 = 0. 00)。這一結果在1年后瑞典LeifAndersson小組和丹麥 Jorgensen小組聯合開展的基因定位研究中得到了驗證，他們還利用fish和輻射雜種細胞 克隆板技術揭示ETEC F4ac受體位于13號染色體q41區(SSC13q41)，分別與人3號染色體 的q28_29和q21區同源。2009年，瑞典LeifAndersson小組、丹麥J0rgensen小組和瑞士 Peter Vogeli小組聯合開展的精細定位將ETEC F4ac受體基因進一步縮小至SSC13q41的 SW207與S0075之間。并提出該受體基因最有可能位于Sw207與MUC4之間的14Mb區域。鑒別SSC13q41區域的位置候選基因是當前ETEC F4ac受體研究的重點。大量的 免疫生化特性研究表明，黏蛋白型唾液糖蛋白(IMTGP)，74kDa的轉鐵蛋白(GV74)和小腸中 性糖鞘H(mLad)符合ETEC F4ac受體的理化特征，其中黏蛋白型唾液糖蛋白與F4ab和F4ac結合，但不與F4ad結合，且該糖蛋白的存在與新生仔豬對ETEC F4ab或F4ac的敏感性高度 相關，因而成為ETEC F4ac受體候選基因選擇的重要依據。國內外研究小組在獲得了 ETEC F4ac受體基因初步的染色體定位信息后，選擇了黏附素糖蛋白編碼基因(MUC基因家族)和 轉鐵蛋白受體基因(TFRC)進行了研究，瑞士 Peter Vogeli小組對TFRC、B3GnT5、B4GALT4 和B4GALT4等4個基因進行了研究，但沒有發現與F4ac黏附表型顯著相關的候選基因和標 記；丹麥J0rgensen小組在歐洲野豬x大白豬資源家系群體中，發現了 Mucin 4基因內含 子7的一個SNP標記與ETEC F4ac的黏附表型共分離，由此申請了一項國際發明專利。申請 人就該位點的影響效應在自身構建的白色杜洛克X 二花臉資源家系中開展了研究，發現 該標記在始祖動物中沒有多態性，而該資源家系的&個體中有著充分分離的ETEC F4ac的 黏附表型，這表明該SNP標記只是與ETEC F4ac受體基因因果突變(causative mutation) 呈一定連鎖不平衡的分子標記，不能解釋所有群體的ETEC F4ac受體遺傳基礎。因此，盡管 國內外其他研究小組在ETEC F4ac受體基因鑒別方面取得了一定的進展，但仍未有重大的 突破，迄今沒有鑒別到真正的ETEC F4ac受體基因及其關鍵變異位點。

發明內容
本發明的第一個目的是提供一個決定新生和斷奶前仔豬ETEC F4ac腹瀉易感性 /抗性的MUC13基因及其關鍵標記位點；通過現代分子生物學技術檢測該基因關鍵標記位 點，利用標記輔助選擇(MAS)選擇有利的等位基因型個體留種，可以顯著提高種群新生和 斷奶前仔豬的抗腹瀉病能力，大大減少仔豬死亡率。本發明的第二個目的是決定仔豬F4ac腹瀉易感性/抗性的MUC13基因關鍵標記 在種豬抗腹瀉病性狀遺傳改良中的應用。本發明的第一個目的是這樣實現的1、實驗動物與易感/抗性表型判定1.1實驗動物本發明用于目的基因定位的家系動物均來自申請人所構建的白色杜洛克X 二花 臉豬資源家系群體。該資源家系以2頭白色杜洛克公豬和17頭二花臉母豬為祖代繁殖& 代，選59頭&母豬與9頭&公豬自交產生F2代，在此基礎上再選擇62頭F2公豬149頭F2 母豬自交產生&代個體。每個資源家系F2/F3群體個體均在240士3日齡屠宰。本發明所使用的用于目的基因精細定位的遠緣群體為292頭純種仔豬，其中代表 我國6個生態類型(包括華南、華中、華北、江海、西南、高原型等)的12個地方豬種(含二 花臉、金華豬、姜曲海豬、沙子嶺豬、玉山黑豬、杭豬、通城豬、藍塘豬、巴馬香豬、萊蕪豬、榮 昌豬、藏豬)148頭，每個豬種選取至少3個父系家系，每個家系選擇3-4個6-8周齡個體； 另外從5個不同省份大型商業種豬場購買144頭杜洛克、長白、大白豬種，每個豬種同樣選 取至少3個父系家系，每個家系選擇3-4個6-8周齡個體。1.2易感/抗性表型判定豬只屠宰后，取小腸空腸部分，提取小腸上皮細胞刷狀緣，利用E. coliF4ac菌株 黏附豬小腸上皮細胞刷狀緣，通過相差顯微鏡檢測細菌的黏附結果。每個樣品檢測20個以 上的刷狀緣，若有10%以上的刷狀緣上至少有2個細菌黏附，判該樣品為黏附型，若至少有 2個細菌黏附的刷狀緣不到10%，但有1-2個細菌黏附的刷狀緣多于10%，判該樣品為弱黏附型，否則判為不黏附型。黏附效果圖如附圖1所示。2、全基因組掃描初步定位ETEC F4ac受體基因本發明首先選用覆蓋19條染色體的來自美國肉畜中心(MARC)網站公開的151個 微衛星標記，在申請人所構建的白色杜洛克X 二花臉資源家系200個F2代個體以及全部 的Ftl與F1個體中進行全基因組掃描。具體如下2. 1微衛星標記PCR擴增條件 微衛星標記聚合酶鏈式反應(PCR)反應體系為15μ 1，其中包含40ng基因組 DNA (脫氧核糖核酸)，0. 2mM dNTPs (合成DNA的四種脫氧核苷酸底物)，0. 2 μ M引物，1. 5mM Mg2+，IXPCR buffer (緩沖液)和 IU DNA 聚合酶(Taq 酶)。PCR 的擴增程序為 94. 0°C 4min ； 40 循環(94. O0C 30sec, 55-65°C 20sec, 72. 0°C 30sec) ；72. 0°C 5min。2. 2微衛星標記擴增產物電泳及分析PCR條件優化好之后，使用資源家系F1代個體基因組DNA進行微衛星多態性檢測， 并根據微衛星引物熒光標記顏色以及片段大小等因素對微衛星進行優化組合以提高毛細 管電泳效率。PCR擴增后產物首先用2%的瓊脂糖凝膠進行電泳，然后用凝膠成像系統進行 凝膠成像檢測PCR產物的質量并確定PCR產物上樣稀釋倍數，然后對PCR產物進行適量稀 釋，取1μ 1組合稀釋產物，加入10 μ 1緩沖液(由購自美國ABI公司的Hi_Di 去離子甲酰 胺與GeneScan _400HD ROX以100 1比例混合而成)，進行混合、振蕩和離心后，上樣于 ABI3130XL自動遺傳分析儀(3130XL Genetic Analyzer，ABI公司，美國)進行毛細管電泳。 經美國ABI公司提供的公用的DataCollection Software V. 30收集PCR產物電泳檢測后 的原始熒光信號，采用美國ABI公司提供的公用的Genemapperf. 7讀取微衛星片段的大小， 并校正錯誤數據后，將Excel格式的數據轉換成CRIMAP (2. 4)軟件要求的數據格式，再次檢 驗所有個體的基因型。根據家系記載，確保所有個體的數據符合家系分離規律。然后利用 公用的CRIMAP軟件進行連鎖分析，結果如圖2所示。從圖中可知，ETEC F4ac受體基因被 初步定位于豬13號染色體(SSC13)的SW207與S0075之間，這個區域大約17cM。3、目的區域精細定位ETEC F4ac受體基因申請人:在ETEC F4ac受體位點初步定位的區域(SW207-S0075)及其兩翼附近區域 新增分子標記至51個(含33個微衛星和18個單核苷酸多態性標記)，各標記引物信息見 表1。將這些標記在白色杜洛克X 二花臉資源家系群體755個F2代個體及全部Ftl與F1個 體中進行基因分型。33個微衛星標記的檢測方法與上述全基因組掃描中的微衛星標記檢測 方法相同。18個單核苷酸多態性標記(SNP)的檢測方法采用SNaPshot方法進行，具體如 下3. ISNP標記的PCR擴增條件在25 μ 1 的 PCR 反應體系中，包括 25ng 豬基因組 DNA，1. OmM MgCl2, IOOmM dNTP, 正反向引物各IOpmol，2單位Taq酶及IXPCR buffer (上海生物工程公司，中國)。擴增條 件為94°C 4min ；94°C 30s, 56-65°C 30s, 72°C 30s，共 35 個循環；最后在 72°C延伸 lOmin。 PCR產物用2%瓊脂糖凝膠電泳檢測并拍照。取3 μ 1 PCR產物，加入1 μ 1 ExoSAP-IT酶 (上海國藥集團公司，中國)37°C孵育15min，然后80°C處理15min滅活酶。PCR產物純化后 稀釋4倍備用。3. 2SNaPshot反應及產物純化
SNaPshot 反應體系為SNaPshot 反應混合物(Multiplex ReadyReaction Mix)2u 1 ；SNaPshot延伸引物0. 5iil(引物終濃度為0. 2iimol/L)；上述純化稀釋PCR產物 1. 5 ill ；去離子水 1 ill。SNaPshot 循環參數為 96°C 10s，50°C 5s，60°C 30s，共 25 循環。取 5 u 1 SNaPshot 反應產物加入 0. 57 u 110 X buffer 和 0. 1 ii 1 CIP (SNaPshot 反應產物純化 酶)在37°C下孵育lh，然后75°C處理15min滅活酶達到產物純化效果，_20°C保存備用。3. 3遺傳分析儀電泳及判型電泳混合體系包括9. 25 u 1 Hi-Di formamide (甲酰胺變性劑)；0. 5 yl上述 經純化后的 SNaPshot 反應產物；0. 25 ii 1 GeneScan 120LIZ size standard (電泳分子內 標)。體系混合后于95°C變性5min，然后立即置于冰上處理2min。離心上樣。具體判型方 法如附圖3所示。表1用于ETEC F4ac受體基因精細定位的微衛星和SNP標記引物信息 附注*表示用于資源家系F3代基因分型的標記。3.4多點連鎖分析采用公用的ALLEGRO軟件對51個遺傳標記位點與ETEC F4ac受體基因位點進行多 點連鎖分析。多點連鎖分析結果顯示，ETEC F4ac受體基因位點被精細定位于SSC13UMNP997與S0075之間4. 3cM的區域(圖4)。4、斷點重組分析精細定位ETEC F4ac受體基因為了進一步縮小ETEC F4ac受體基因的染色體區域，申請人以0. 5cM間距開發序 列標簽位點(STS)及其SNP標記(引物詳見表2)，判定白色杜洛克X 二花臉F2 (755頭) 和F3個體(450頭)在這些SNP位點的基因型。基因分型方法使用上述的SNaPshot方法 進行。根據F2和F3抗性個體在這些位點的遺傳重組斷點分析，利用特定重組模式的發生并 導致黏附表型改變來進行受體基因的斷點重組判定以實現精細定位。在耳號為1487的& 代抗性個體中，申請人發現在多點連鎖分析精細定位的UMNP997-S0075區域內S0283標記 處發生了斷點重組，由此將ETEC F4ac受體基因確定在UMNP997至S0283之間。進一步在 耳號為3314的F3抗性個體中發現在UMNP997-S0283區域內的UMNP358標記處發生斷點重 組，由此最終將ETEC F4ac受體基因精細定位于UMNP997和S0283之間2. 3cM的染色體區 域。5、ETEC F4ac受體基因的錨定申請人:在UMNP997-S02832. 3cM的目的區域及其兩翼采用直接測序法開展了高密 度SNP搜尋和鑒別，在此區域內設計了 34對引物(詳見表2)，分別在遠緣群體白色杜洛克 和二花臉中選擇表型黏附和抗性個體各2頭，以其DNA為模板進行PCR擴增。PCR擴增條件 同上所述，PCR產物經QIAquick PCR純化試劑盒(QIAGEN，德國)純化，委托上海華大六一 公司測序。使用公用的DNAStar軟件包中的SeqMan軟件分析鑒別SNP，共鑒別到104個SNP 標記。選擇其中分布均勻和具有代表性的70個SNP，在已有經黏附表型測定的15個中、西 方豬種遠緣群體292個個體中使用飛行時間質譜技術(MassARRAY Analyzer Compact)進 行多態性檢測，飛行時間質譜技術略述如下PCR反應體系為5ii 1，其中包含8ng基因組，0. 05mM dNTPs，0. 1 ii M引物，4. OmM Mg2+，1XPCR緩沖液和0. 5U Hotstar Taq DNA聚合酶(Qiagen，德國)。PCR的擴增程序為 94. 0°C 15sec ；45 循環(94. 0°C 20sec，56°C 30sec，72. 0°C lmin) ；72. 0°C 3min ；4. 0°C保存。 SAP酶(Qiagen，德國)純化處理，降解擴增用的dNTP，以確保延伸反應只延伸一個堿基。 SAP酶反應體系為7 iil，其中包含5 ill PCR產物，SAP酶0. 3iil(l. 7U/iil)，10X緩沖液 0. 17 ill。SAP 純化的程序為 37. 0°C 40min ；85. 0°C 5min ；4. 0°C 保存。單堿基延伸 iPLEX 反 應體系為9 u 1，其中包含7 ill經SAP酶純化處理的PCR產物，iPLEX終止混合液0. 2 yl， 10\丨？1^乂緩沖液0.211 l，iPLEX 延伸引物 0. 94ii 1，iPLEX 酶 0. 041 ii 1。iPLEX 反應程序為 94. 0°C 30sec ；40 循環(94. 0°C 5sec, 5 循環(52°C 5sec，80. 0°C 5sec)) ；72. 0°C 3min ；4. 0°C 保存。樹脂除鹽純化反應和上樣取6mg樹脂在384孔樹脂刮板上，均勻覆蓋，刮去多余的樹 月旨，放置15min。將反應結束的384孔板4000rpm離心30s，每孔加入16 y L去離子水，倒置 在樹脂板上面，然后反置將樹脂板扣在384孔板上，敲擊使樹脂落入384孔板，封膜。以384 孔板的長軸為軸心，翻轉384孔板60min，3200rpm, 5min離心后備用。將檢測樣品從384孔 反應板轉移到表面覆蓋基質的MassARRAY SpectroCHIP芯片。將樣品轉移到SpectroCHIP 芯片后，即可放入質譜儀(SEQUEN0M，美國)進行檢測，每個檢測點只需3-5秒，全自動分析。檢測結果采用公用的SHE軟件進行關聯性分析，分析結果如圖5所示。從分析結 果圖中可看出，與黏附表型關聯顯著性最高的SNP位點，即圖中的M13A(SEQ ID N0:1中 46531bp處的SNP)，位于MUC13基因。此外，已有報道ETEC F4ac受體為黏蛋白型唾液糖蛋
9白，在申請人精細定位的UMNP997-S0283受體基因區域中表達黏液素糖蛋白的僅有MUC13、MUC4和MUC20三個基因。申請人通過基因組織表達實驗顯示MUC4基因在小腸組織中呈極低 豐度表達，MUC20基因呈中度且多種組織廣譜表達，而MUC13基因表現為小腸組織的高度特 異性表達(結果未顯示)。結合上述遠緣群體的SNP標記關聯性分析結果，從而鎖定MUC13 基因為ETECF4ac受體目的基因。表2 UMNP997-S0283目的區域SNP鑒別的引物信息 6、ETEC F4ac受體基因(MUC13基因)關鍵突變位點的鑒別6. IMUC13基因新的全長cDNA序列的分離根據已報道的豬MUC13基因的部分cDNA序列(序列號為EU046996)設計引物Fl 和Rl (見表3)，獲得1148bp的cDNA片段。以此序列片段設計引物F2進行5，RACE，獲得部 分cDNA序列。通過生物信息學方法，利用人、牛、狗、馬、貓和鼠的MUC13蛋白羧基端序列信 息預測MUC13蛋白羧基端的保守編碼區。然后利用牛該保守同源區cDNA序列設計引物F3 再次進行5，RACE從而獲得新的全長cDNA序列。全長cDNA序列特征如序列表SEQ ID NO 1。MUC13基因cDNA序列所編碼的氨基酸序列如序列表SEQ ID NO 3。6. 2MUC13基因全長基因組序列的獲取 根據所獲得MUC13基因的cDNA序列，采用人-豬比較基因組法，結合生物信息學 法設計引物對 M13-1-F/M13-1-R、M13-2-F/M13-2-R、M13-3-F/M13-3-R、M13-4-F/M13-4-R、 M13-5-F/M13-5-R、M13-6-F/M13_6-R(詳見引物表3)。PCR反應體系包括25ng豬基因 組DNA，1.0mM MgCl2, IOOmM dNTP，正反向引物各lOpmol，2單位DNA聚合酶(Taq酶)及 IXPCRbuffeH 上海博彩公司，中國)。擴增條件為94°C 4min ； 94 °C 30s, 55-65 °C 30s， 72°C 70s，共32個循環；最后在72°C延伸lOmin。擴增片段采用QIAquick PCR產物純化試 劑盒純化，由上海華大六一公司測序。使用公用的DNAStar軟件包中的SeqMan軟件分析測 序結果，并結合已有的cDNA序列從而獲取MUC13基因全長DNA序列，全長DNA序列特征如 序列表SEQ ID NO 2。6. 3 MUC13基因關鍵SNP位點的鑒別使用上述分離的MUC13基因全基因序列的6對引物，擴增二花臉和杜洛克抗性和 易感個體基因組DNA，PCR反應條件同上，PCR產物直接純化測序，使用公用的DNAStar軟件 包中的SeqMan軟件分析測序結果，在MUC13全基因序列上共鑒別到179個單核苷酸多態位 點(SNPs)。選擇MUC13全基因序列上鑒別到的104個SNP位點在已有體外黏附表型測定的 12個代表6種生態類群的中國地方豬種和3個西方引進豬種共計292個個體中采用上述 MassARRAY飛行時間質譜技術進行大規模SNP基因分型。將SNP分型結果與ETEC F4ac體 外黏附表型進行關聯性分析。結果如圖6所示。申請人發現5個SNP即SNP C53019T(圖 6 中的 M131009)、SNP A46531G(圖 6 中的 M13A)、SNP A42784C(圖 6 中的 M13C4A)、SNP A32813G(圖6中的M13C1B)和A37617G(圖6中M13I-395)用于判定易感/抗性個體的準 確性大于92%，關聯性P值小于1. 18X10_19，其中最強相關的標記(C53019T)準確率大于 97% (P = 1.59X10-21)。這5個關鍵性SNP位點是與MUC13基因上的因果突變(causative mutation)呈極度連鎖不平衡狀態的標記或本身即為因果突變。根據它們與黏附表型的強 相關，可以很好的將這5個SNP標記用于豬抗腹瀉病遺傳育種生產實踐中，通過分子標記選 育逐世代選擇抗性的個體，以培育抗ETEC F4ac腹瀉病的種豬新品系。表3 MUC13基因克隆引物信息 本發明的第二個目的是這樣實現的本發明利用MUC13基因的5個關鍵性SNP位 點及側翼DNA序列信息，采用分子生物學技術鑒別個體基因型，以判定個體對ETEC F4ac腹 瀉的易感性/抗性，從而針對抗腹瀉病性狀選育改良種豬。申請人:自2003年以來利用自身構建的大規模白色杜洛克X 二花臉資源家系群 體，在755個具有ETEC F4ac體外黏附表型的F2個體和390個F3個體中，開展了基于183 個微衛星位點的全基因組連鎖分析、目的區域高密度標記的IBD分析及斷點重組分析，將 ETEC F4ac受體基因精細定位于13號染色體q41區(SSC13q41)臨近S0283的約2. 3cM區 域。在精細定位基礎上，構建了包括20個功能基因座位的SSC13q41人-豬高分辨率比較 基因圖譜。進一步通過15個遠緣群體292個個體的連鎖不平衡關聯性分析，最終鑒別到了 目的受體基因及能準確判定易感和抗性個體的關鍵突變位點，由此創建了一項高效、準確 的新生和斷奶前仔豬腹瀉病的抗病育種新技術。本發明鑒別了一個決定新生和斷奶前仔豬腹瀉易感/抗性的MUC13基因及其關鍵 標記位點，通過現代分子生物學技術檢測該基因的關鍵突變位點，利用標記輔助選擇(MAS) 選擇有利的等位基因型個體留種，可以顯著提高種群斷奶仔豬的抗腹瀉病能力，大大減少 仔豬死亡率。本發明為種豬ETEC F4ac腹瀉病的抗病育種提供了一種新方法。


圖1ETEC F4ac與刷狀緣的黏附檢測結果，(a)為不黏附型，(b)為黏附型；圖2ETEC F4ac受體基因的全基因組連鎖分析初步定位結果；圖3為C53019T位點的SNaPshot判型圖，其中a為CC型；b為CT型；c為TT型；圖4為ETEC F4ac受體基因的目的區域多點連鎖分析精細定位結果；圖5為UMNP997-S0283目的區域高密度SNP基因型與ETEC F4ac黏附表型關聯性 分析結果圖6為MUC13基因內高密度SNP基因型與ETEC F4ac黏附表型關聯性分析結果圖。
具體實施例方式下面結合實施例并對照附圖對本發明作進一步詳細說明。選擇種豬核心群個體，利用SNaPshot方法檢測MUC13基因SNPA37617G、SNP A32813G、SNP A42784C、SNP A46531G和SNP C53019T等5個位點的基因型，選擇有利的等 位基因型個體留種，群體繼代選育后改良種群的抗仔豬腹瀉病性能。實施例利用144頭純種仔豬包括杜洛克、長白、大白豬種作為實驗動物，這些豬只從5個 大型商業種豬場購買，每個豬種選取至少3個父系家系，每個家系選擇3-4個1-2月齡個 體。這144頭純種仔豬屠宰后，開展豬小腸上皮細胞刷狀緣ETEC F4ac體外黏附表型測定 實驗，實驗方法及判型標準如前所述。采用上述SNaPshot 方法對 MUC13 基因 SNPA37617G、SNPA32813G、SNPA42784C、 SNPA46531G和SNP C53019T等5個位點在上述144頭純種仔豬中進行SNP基因分型，擴增 引物見表4。對各SNP位點的基因型與ETEC F4ac體外黏附豬小腸刷狀緣的表型進行相關 性統計分析(見表5)。表4用于判定MUC13基因5個關鍵SNP標記的引物信息 結果表明MUC13基因 SNP A37617G、SNP A32813G、SNP A42784C、SNP A46531G和 SNP C53019T等5個位點的基因型均與黏附表型基本共分離。以其中的SNP位點SNP C53019T 為例，T等位基因為易感基因，C等位基因為抗性基因；基因型TT和CT個體對ETEC F4ac的 侵染易感，CC型個體對ETEC F4ac的侵染具有抵抗性。CC型個體是抗腹瀉病型豬的選育改 良所需的基因型個體。在商業種豬核心群中，繼代選育CC型個體，可以顯著地改良種群的 抗腹瀉病性狀，減少仔豬的死亡率，并最終可望育成抗新生和斷奶仔豬腹瀉病的專門化新 品系。表5五個MUC13關鍵SNP位點在實驗豬群體中的基因型與ETEC F4ac黏附表型的 cgcaggggcaagcaccagcgctccggccgcaggggcaagcaccagcgctccgtctgtggc420
ggcaagcaccagcgctccggccgcaggggcaagcaccagcgctccttccgctgcaagcac480
gagcgcaccggccgcaggggcaagcaccagcgctccagccgctgcggcaagcaccagcgc540
tccagcccccgcggcaagcaccagcgctgccgcggcatcaagcaccagcgctccggccgt600
ggcggcaagcaccagcgctccggcccccgcggcaagcaccagcgctccctccgctgcggc660
aagcaccagtgctccggccgcaggggcaagcaccagcgctccggaccccacggcaagcac720
cagcgctcaggccgcagaggcaagcacaagcgctccgtcggcggcggcaagcaccagcgc780
tccggccgcaggggcaagcaccagcgctccgtctgtggcggcaagcaccagcactccggc840
cgcaggggcaagcaccagcggtccggccgccacggcaagcaccagcgctccggccacagg900
ggcaagcaccagcgctccgtctgtggcggcaagcaccagcactccggccgcaggggcaag960
caccagcggtccggccgccgcggcaagcaccagcgctccggccgcaggggcaagcaccag1020
cgctccgtctgtggcggcaagcaccagcactccggccgcaggggcaagcaccagcggtcc1080
ggccgccgcggcaagcaccagcgctccggccacaggggcaagcaccagcggtccggccgc1140
ggcggcaagcaccagcgctccggctgtaggggcaagcaccagcggtccagccgcgtcagc1200
aagcaccagcgctctgtctgaggcggaaagcaccagctctccagccgcaggggcaagcac1260
cagcgctccgtctgtggcggcaagcaccagcgctccagccgcggcggcaagcaccagcgc1320
tccttccgctgcaagcaccagcagtccggaccccacggcaagcaccagcgctccggccgc1380
aggggcaagcaccagcgctccctccgctgcggcaagcaccagcgctccggccgcaggggc1440
aagcaccagcgctccctccgctgcggcaagcaccagctctccggccgcaggggcaagcac1500
cagcgctccttctgctgcaagcaccagcgctccgtctgtggcggcaagcaccagcgctcc1560
ggcccctgtggcaagcaccagcgctccggccccaggggcaagcaccagcgctccttctgc1620
tgcaagcaccagcgctccggccacaggggcaagcaccagtgctccctccgctgcggcaag1680
caccagcgctccgtccacggcggcaagcaccagcgctccggccgcaggggcaagcaccag1740
cgctccggcccccgcggcaagcaccagcgctccggcccccgcggcaagcaccagcgctcc1800
agcccccgcggcaagcaccagcgctccggctataggggcaagcaccagcgctccgtccgc1860
tgcaagcaccagcgcaccgtctgctgcaagcaccagcgctccggccgcaggggcaagcac1920
cagcgctccgtctgtggcggcaagcaccagcgctccggcccctgcggcaagcaccagcgc1980
tccggccgcaggggcaagcaccagcgctccttccgctgcaagcaccagcgctccagccgc2040
aggggcaagcaccagcgctccggccgcggcggcaagcaccagcactccagaccccacggc2100
aagcaccagcgctccggccgcaggggcaagcaccagcgctccggcccctgcggcaagccc2160
cagcgctccggccgcagggacaagcaccagcggtccagcccccgcggcaagcaccagcgc2220
tccggccgccgcggcaagcaccagccctctggaccccacggcaagcaccagcactccggc2280
cgcaggggcaagcaccagcgctccatcggcagcggcaagcaccagcgctccttctgctgc2340
aagcaccagcgctccttctgctgcaagcaccagcgctccggccgcaggggcaagcaccag2400
cgctccggccgtggaggcaagcaccagcgctccggcccccgcggcaagcaccagcgctcc2460
ggctgcaggggcaagcaccagcgctccgtccgctgcaagcaccagcgctccggccgcagg2520
ggcaagcaccagcgctccggccgcggcagcaagcaccagcgctccggcccccacggcaag2580
caccagcgctcccgccgcggtggcaagcaccagcgctgcggcccccgcggcaaccaccag2640
cgctctggccgcggtggcaagcaccaaccctccggccgccggggcaagcaccagctctcc2700
ggccgcaggg gcaagcacca gcgctccgtc tgtggcggca agcaccagcg ctccggcccc2760tgtggcaagc accagcgctccggccgcagg ggcaagcacc agcgctcctt ccgctgcaag2820caccagcgct ccggccgcag gggcaagcac cagcgctccg gcccccgggg caagcaccag2880cgctccggcc cccgcggcaa gcaccagcgc tccttccgct gcaagcacca gcgctccggc2940cataggggca agcaccagcg gtccagcccc cgcggcaagc accagcgctc cggccgccgc3000
ggcaagcacc agcgctccgg accccacggc aagcaccagc actccggccg caggggcaag3060caccagcgct ccgtcggcag cggcaagcac cagcgctccg gcccccgcgg caagcaccag3120cgctccttct gctgcaagca ccagcgctct ggaccccacg gcaagcacca gcgctccgtc3180cgctgcaagc accagcgctccggccgcagg ggcaagcacc agcgctccgg cccccgcggc3240aagcaccagc actccggctg caggggcaag caccagcgct ccttctgctg caagcaccag3300cgctccggcc gcaggggcaa gcaccagcgc tccggccccc acggcaagca ccagcgctcc3360cgccgcggtg gcaagcacca gcgctgcggc ccccacggca agcaccagcg ctccggccgc3420ggcagcaagc accagcactctggcccccac ggcaagcacc agcgctccgg ctcccgcggc3480aagcaccagt gctccgtccg cggcaagcac aagcgctccg gccgcggcgg caagcaccag3540cgctccggcc cccgcggcaa gcaccagcgc tccggctggg gcggcaagca ccagcgctcc3600ggctgcaggg gcaagcacca gcgctccatc cgctgcaagc accagcgctc cggccgcggc3660ggcaagcacc agcgctccgg ccgcaggggc aagcaccagc gctccggccg ccgcggcaag3720caccagcgct ccggcccccg ctgcaagcac cagcgctccg gcggcggtgg caagcaccag3780cgctccggcc ccaggggcaa gcaccagcgc tccatccgtg gcaagcacca gcgctccggc3840ccccgctgca agcaccagctctccggccgc aggggcaagc accagcgctc catccgcggc3900aagcaccagc gctccggtccccgctgcaag caccagctct ccggccgcag gggcaagcac3960cagcgctccg gcccccgctg caagcaccag ctctccggct gcaggggcaa gcaccagcgc4020tccggccgcg ggggcaagca ccagcgctcc ggccgcaggg gcaagcacca gcgctccagc4080ccccgctgca agcaccagctctccggccgc aggggcaagc accagcgctc cggcccccgc4140tgcaagcacc agcgctgcgg ccgcgggggc aagcaccagc gctccatccg cggaaagcac4200cagcgctccg gctgcagggg caagcaccag cgctccggct gcaggggcaa gcaccagtgc4260tccggccgcg ggggcaagca ccagcgctcc atccgcggca agcaccagcg ctccggccgc4320cgcagcaagc accactcctg cctctgcagc aaatgcttct tctgctattt cgagtacacc4380tattgcctcc acacccacctcaggttccag gaacccttgc cagcaagatt cctgtaaggg4440tggttcttca tgtgttagtctgaataatac gtttttttgc ctgtgttcag aagggtatta4500ctataactct tccacctgca ataaaggaaa gattttccct ggtaaaatta cggtcaaaat4560atcagaaaca actgatctag aacatgaaaa ttctgtgacc tatcaaaatt tataccttca4620agttacaaat ttttttaatg acacatttag caattctgag tacggacaga ccgtaattct4680taaagtaagc acaactccttcagcaagata tgaaatgcgt gccagcaacc agcttgttga4740ggtagaagta gtaaacatttttagagaaac tacaacagaa aatgagagtt ccatatttaa4800tgccattgac caagcaatta aaagcaaccc aagtgacttt acagaatata tccagcaaga4860tcggtgtgat tattatggatgtaagaggca aaatgagcaa gatgagtgcc ccagtggttt4920actgtgtgag tgcaaagagg ggctggagag 3-CC3.3-3.CCCc, cagatccctc tgtgtgttgc4980tttgggtcca gcatgctctg atacctgcaa tgcagagaac aataggcaat gcctcgtgag5040
gagcgctggg aacgccgaat gcttgtgcct gcctggctac aaggaggata aaaacaagat5100ctgccaagcg tgtccatttg gctacagtgg agttggctgt gaaaacccat atcagctgat5160cctcaccatc gtgggcacca tcgctggcat cctcattctc agcctggtga ttgcattcgt5220
cctctcatta agatcaaacagaatgtggaa gaacagaact tgattgagaa5280ygactttcaa aacctgaaac tgcaggagac tggctttagc aacctaggag caggaaacct5340ctttcctaag atcaaggtgg acatttccag aggtggtcag ccacagaatc cctatataac5400ccmgggtagt ataccccgcc ctgactatta gaatggtaaa aatttggaac catccatggc5460cgtcaaccca gcacatgaga tttttgtgtg agggtttgat agactgatgg agaaaagaga5520tctggccttt agtatttttc ctccatccaa catctggtct ctctgaatgg aagctgtaac5580tgtttgcatt gaattcagcg tgtttgaatc taagtaccta tgaagttaaa tgcacacata5640aatgctatta gcacttgtaa gatagttgat ttgctccagt acagtggtta ggtgtttgtt5700tgttttttcc tggtgggaca acaataacca tttccaaact agagggaagc tccccaggat5760tgttcattcc tggaagagtc taagtgttgc tttgagttga tagcatgcag ataagtgacc5820taattctcgg aaattcttac acatcagctg tggaattcca gtagacggga ggagacacca5880tccttaaagg tactcagtgg accctgaaga ttcattctct agcaagttaa aataaggatg5940ctacttctgg tcctcaaatg tgggaatttc attaccctta aactgcttgc gacctggctt6000cacaccaaga ggacagaaaa caagacatgt ctcccagctt tcaggaagga gttcctggga6060atccaggacc tttgagatgt taattaaaag atgtggactt gggagttccc gtcgtggcgc6120agtggttaac gaatccgact aggaaccatg aggttgcggg ttcggtccct gcccttgctc6180agtgggttaa tgatccggcg ttgccgtgag ctgtggtgta ggttgcagac gcggctcgga6240tcctgcgttg ctgtggatct ggcgtaggcc ggaggctaca gctccgattc gacccctagc6300atgggaacct ccatatgccg cgggagtggc ccaagaaata gcaaaaaaaa aaaaaaaaaa6360
aaagatgtgg acttgagatg tcttccatga ggatacattc tagagtcttt6420gaaatgctgg tgggtgagac caagagtagg agcaacagag gcagaaggaa gctgagctgg6480gaggaagaag gtacccaaga cccaccttca tcctcagatt cccctcccca ataaacaggt6540gggcactttc tctaatgcat gccctaccta tgactcaaga cctatgaaag gatgtgaaga6600taaaaaccct ggagcaagag tttggagtca ctttgaaatt gtagacattc tgtctggctg6660atggtggaga agtgatgcag attccagagg tacttcacca aagtctcagc cttccctggg6720gacagaggct tagcagtggc caccaacaca cacacgcaca tacacagcca agaatggggc6780agccacctgc cgcacaagct t6801SEQ ID Ν02<210>2<211>56728<212>DNA<213>Jiangxi Agricultural University〈220〉<221>exon<222>(20240). . (20373)〈220〉
<221>exon<222> (22970) · . (27238)<220><221>exon<222>(32212). . (32334)<220> <221>mutation = A/G<222>(32813). . (32813)<220><221>exon<222>(33499). . (33605)<220><221>mutation = A/G<222>(37617). . (37617)<223>Y = C/T<220><221>exon<222>(39137). . (39192)<220><221>exon<222> (42484) · . (42647)<220><221>mutation = A/C<222>(42784). . (42784)<220><221>exon<222>(46287). . (46412)<220><221>mutation = A/G<222>(46531). . (46531)<220><221>exon<222>(46763). . (46892)<220><221>exon<222>(47846). . (47883)<220><221>exon<222>(50196). . (50280)
〈220〉<221>exon<222>(52971). . (53169)〈220〉 <221>mutation = C/T<222>(53019). . (53019)<223>Y = C/T〈220〉<221>5/ UTR<222>(55361). . (56728)<400>2accgcggcca tagtcgatcg ttctgctgtg catgatggga attcccataatggttgtaat60tttgatgtgt ctgtgggagg aggtgaattc agagttcttc tattccaccatcttggccac120acccaatagc agacattata tgagccatag gaaatgaatt ctatgtcccatttctagtct180ctgaggaaat gccaatgctt caacattttc tttcttaaat caccaatgcatatgcatcat240gacaaaaaaa aaaagaaggt aaaaatggac ttcaagtgcc attcttttaaatcccagtat300ggattatttt agactttgaa gtatggatta tttttttatt caacttgatggcaaagcatt360atctatcatg cagtgacact gttggtgggc taaaataata cattatgttaatttaccaga420ctaagcacgt atcacagtat ttccaactca cagaaaagca accatcagggaaaatagaaa480gttttaagcg gaatatctca ttgtagcaga atttccccgt aaaattaaataatgaaaatg540aggctgcaat caaagcaagt tttcaaatgg ctcatttgtt agccaagccaggaaagccat600ttagggatga gttaattaaa ctatttgatt gcagtagtca aagaaatgtgtctagaggaa660agaaacttgc ttaagactgt ttcagcaaga acagttactc agaatgttgagaacagtagg720aacagaatca atggtcaatt aataaacaag gtacataatt ttgaatggtttccttggctc780atgatgagtt gataggtgtt accaatagca ctcaattttt gtttattccaggattcaatg840acaagtttga agtgactgaa taattatcct ctgtgaacag attctgtggaacaactatag900gaaagaatat tttcaaaata aaattgtata ttttaaatgg ataaattataagtgaattaa960atctcaataa ggctcttttg taaaaaagaa tgatttcaaa gaagttgagaaaacactatt1020ttcagtataa actgaagtag aatctgccaa gatacaactg aagatagtagaaatatgtgg1080ggagtagaaa aagttatagt tggattcatt tatagtgtgt gaaaatgttagacatttaaa1140tcctctggtt attcattaca ttatttatga ataagtattt tgtgggaaatatttaaatac1200gtcatatgct atcgagccag cactgtaagg gtgaacttca ttcattgtcagtggacttaa1260ccgtcattgg ttccatgaaa tttttttgtt ttgttttgtt ttgttttgtttttagggcca1320cacccatgac atatgaaggt tcccaggcta ggagtctaat ctgagttaacagctgctggc1380cacagccaca gccatagcaa ggcaggatcc aagccgcgtc tgcagcctacaccacagctc1440acagcaactc cagatcctta acccactgag caaggccagg gatcaaaacctcaacctcat1500ggttcctagt tagattcatt tccattgtgc ctcgatagga actcccaggtttggtgaatt1560tttgtgggat atagaagcta ttatcctgac ttgccctgct ataaagcagttggatggctt1620agcagtaaaa aagttttatt gcaagttttt gagcataggg ttaggattaaaatctttctg1680
agtagaacca gtctaaacca cttgcattaa acgggagtgg tttttgaaga tgtcttttgc1740tgcagacttg ataatgcttc tcaatgaatt caactcacaa ttactcaagg caaaacagct1800ctacatgcaa attttatgct atggtaaagt cattttaaca acaatttaat aatgttgtgt1860aattcacaag taatgtcaag ctactttcta taattcccat gtagtcagaa ttttaaatga1920gaagatcttc cttcccacat gaatttcagc agacatattt tctggactta aagtacaatt1980ccagcagtgt tttgaaaact tcagtacaag agcatagaaa atttctgtat ttcaaaatcc2040
atactatgca attgaggagc agttacctaa cattcaatcg ggaatgatca atttgcaatg2100taatggcgtg tcaaaaggca actatcaata gaagaatcta atagaatgct gtaactgcct2160ttcaagcaat gagtatactt aactaaagct acatgttcat ggactgatac tagaatttgg2220cagaactacc tatgtgtatg aaaagatatc ttcaaaaatt aaatataggg agttcccatc2280atggctcagt ggttaacgaa cctaactggc atccatgagg acccaggttc aatccctggc2340cctgctcagt gggttaagaa tctggcattg cccagagctg tggagtaggt cacagacgtg2400gcttggatcc cgagttgcta tggccctggc gtaggctggc agctgtagct ccggttggac2460ccctagcctg ggaacctcca tgtgccgcat agccctaaaa aagagagaaa gacaaaataa2520aagaaatata taaatatcat tataaatctg tattaacagt tggacatttg2580tgatacattt tgatgatata gggaatctga ctctgaacct caattaagca aaatattatc2640gctccccaaa taattccatt ctctatcaat aaaccagtat caccaaatac tgtgctcaat2700actattatat tttaaatttc atcagtaaat ttttttggaa atttgtttcc tctttagtat2760acataagtgt ctacattata tcttcaattt tccctcttgg ctcacaaaac ttgaaatatt2820taatatctag gagttccctt atggcatggc aggttaagaa tctggcattg tcactgcagc2880agcttggctt gctgctgtgt ggaccgagtt tgatccctgg cctaggaact tccacatacc2940acaggcacag ccaaaaataa tttactgtct agccctttat taaaaaagtt tgctgattcc3000aatctatgtc ccagtcctac ctatgcttca gatttctaac cttagcattc atattataac3060agtaaatctt cttcatggtt ttgagttaat aagtgcaaag tgtatagcat aatggcaaag3120ggtaggtgct ttttaagtat gtttttatta atattaagaa ctatattgct atagctcttt3180tgaactatac agagttgtgg tcattacaaa agatgatctg taggcagagg gaggcctctg3240gtgaataaag atttccttta agaaaatctt ctttatcatt ttttattttt atgttttagt3300ggattatagt tcatttacag tactgtgaag acagtcctct ttaaaactgc cctggggtgg3360gggaggcttc cacttaacgc atcccagctt gctttcttaa aattttgttt ttttggtagc3420agctttactg agatacaatt cacataccat aaaattcaca cattaaaatt cactgcatca3480tcacaaccaa ttttagatta tcttatttta gccatcatct ccccatcctc ccacctcccc3540ccagccatag gcaacaccta atcctctttc tttctctgta gatttgcctg ttctggaatt3600taatgtaagt ggaattatac atgtggcctt ttatgactgg tttatttcat ttagcatatt3660actttttttt tttttttttt gtctttttgc catttctggg gccgctctca cagcatatgg3720aggttccccg actaggagtc taatcggagc tgtagccact ggcctacgcc agagccacag3780caacgcggga tccgagcctc atctgcaacc tacaccacag ctcatggcaa tgccggatcc3840ttaacccact gagcaaggcc agggattgaa cctgcaacct catgattcct agtcggattt3900gttaaccact gcgccacgac gggaactcct agcatattac tttcaagcct tctccacatt3960gtggcatgta tcagtacttc attccttttt tggtggagta gtgttccatt gtgtgagtgt4020
accccacttt gtttatccattcatcagttg atggacattg ggatatttct gtgtttgttc4080tcttgtgaat aatgaacattcacatacaaa tatttatgtg aacaatgttt tcacttcttt4140taggaatata cctagaagtg aaattgctgg gtaaaatggg aactctatgt ctaacttttt4200gagaaactgc cagatttctg tccaaagtag ctgtgtcttt gttttttttg ttgttgtttg4260 ttttttgttt gtttgtttgtttttggtctt tttagggccg cacctgcagc atgtggaagt4320tcccaggctc ggggttaaatccaagctaca gctgccaacc tgtgccacag ccacagcaac4380gccagatctg agctgggcctgcaacctaca ccctagctca cggcaacgct ggatccttaa4440cccactgagc ggagccaggg atcgaaccca catcctcatg gataccagtt gggttcctaa4500ccactgagcc tcaacaggaa ctccagctgt gccatgtttg catccttacc agcagtgtat4560gaggagtcca gttcccccacgtcgccacca acacttgaaa ttcaattatc tggctctttg4620attgtaggca tcctagtgatgataaagtga taaccagctc atttttaatc ctctgggatg4680gggtattggg gaagctattctttttaacac tccacacagg gtctacgggg gaacagccct4740ttggaaactt agccctgcaa gatcatcagg gtatttccac tgaaactgaa gtttttctca4800gagggattgt gccccattag taactcgtcg tcgatttgta acaacccaga tatttgcacc4860accctcttag tagatattattgggagatga gcacatattt ttgagggact acttacagtt4920acaaggcaga ttattacaag gcatctcgac tccacaggca ccaggacaag atcccttcta4980gagagaagtt ctgtagacacgactgcccag ccccacctcc caccacctat ttcccatccc5040tgtgatttgc agacaccaag gacccttctg gggagagcaa ccagagctgc tgggaaagtt5100tccataaaga aaggaagagtttctctgggg aggcagacac cacagcacag caagagctca5160gaagggaagt tggttcctgg tcctgccctg tgtggtggct gcttcccagg gtggaagaga5220aattgaactc tttgcaccca aagcaagtca gccagtcaag ggctgtttac tgggtgcctg5280catatgctga cgtcccttgg gtgttgcact tactgggtgc accttcagag agtgtttcaa5340agtcaggtct tcccattgca tgctcccact ccaccctgag ggtgtcttgc atagcacttt5400gcacagttat ggcagaaatg ctaatttcag ttcattgctt ggtacccaga gcctggaaca5460gcatctggca tgtgcacata ctctaaagta tttggtgaat gaaggaaaaa aatctaaacc5520tgacactggc tagtggaatccaagcacagt agtacctcag catatattat tttgtagttt5580atttgtactt agtaaatata gcgttgttct cctgttgtta ggtattccct gatggcccag5640tgcttaagaa tatggtgttg tcactgctgt gacttaggtc accattgtgg cgctggtttg5700atccctggcc taggaacttctgtaggcctc aggcgtgcag ctgaaaaaaa agatgcagta5760ttgttgaaaa tttcatttgg catagtgact atgtaacatt cttctcatgt acagtgaggt5820accacttcac actagtcaga atggccatta taacaagtca acgaataaca aatgctggag5880agagtgtgga gagaagggacccctcctttg ctgttggtgg gaatgtaaat tggtacaacc5940actatggaaa actaaacatg gaagtaccat atgatccagc aatcccattc ctgagcatat6000atccagacaa acatttgttcaaaaaaatac atgaaccctt acgttcatca catcactatt6060cacaataggc aagacatgga aacaacttaa atgcccattg atagatgaat ggatcaagaa6120gatgtggtac atatacacaa tggaatacta ctcagccatt aaaaaggaca aaataatgtc6180atttgcagca acatggccag aactagagat tctcatacta agtgaggtaa gtcagaaaga6240ggaagatatg acatcatttg tatctggaat ccaaaatatg gcacaaatgg tcctatctac6300aaaacagaag ttcccgtcatggctcactgg ttaatgaatc ttacgaggaa ccacgaggtt6360
gtgggttcaa tccctggcct tgctcagtgg gttaaggatc cagcgttgtc gtgagctgtg6420 gtgtaggtca cagacatggc tctgatcttg tgttgctgtg gctctggtgt aggccagtgg6480ctacagctcc gattcgaccc ctagcccagg aacctccata tgccacgggt gcagccctgg6540aaaagacaga aagacaaaaa aaaaaaaaaa ccaaaaaaca gaaacagatc atggacatgg6600agagcagact tgtgtttgct ggggcaggga ggggaaggag tgggatggat ggagagtttg6660gggttggaag atgcaaaata ttacatatgg aatggataag caatggggtc ctactgtaca6720gcatagggaa ccaagtctaa tgtcttgggt tagaacatga tggaagatat tatttaaaaa6780aaagaatgta catgtatgta tgactgggtc actttgctgt acaacagaaa ttgaaggaac6840attgtaaatc aactacactt taataaaaaa aattttttta aatattctcc tcacaaactg6900accaaatttg tcctgaccaa gctctggcta ttggatattt acaattgttt aaaatttttt6960cctatgaaaa aataatagtg ggcttgggct gtgggatgga aatcctgtga aatcagattg7020ttatgatcat tatacaacta cagatgtgat aaattcattt gagtaataaa aaaaatgaaa7080aaaaaatagt gggagttcct gtcgtggtgc agcggaaatg aatctgacta ggaaccagga7140gattgggcgg gttcgatcct tggcctccct cagtggattc aggatctggc gttgccatga7200gctgtgatgt aggttgcaga cgcgcctccg atctggcatt gctgtggctg tggcctagtc7260tggcagctac agctccgatg agccccctag cctgggaacc tcatatgctg tgggtgcggc7320cctaaaagga caaaagacca aaaaaaaaaa aaaaatagtg atacatcctt gtagaaatgt7380ctttgagtac atatctgact gcttccttac tataattccc ggaatggaaa tcctgggtcg7440aggaacatgc ccgttgtcaa aggtccatga tgcctatttg ccagcatccc ttctcccaag7500ggctgtggat ggggcctgtt tcccacacac cattgacctc agcaagcatc gtcattttct7560tcatgctttc agtaaaattg ccccatagtc agaatatctg attcctgata ctacacagcc7620tctcagggga ttgcctgtga atgcaggccc aagttttatg tcaaatttgc cataaagaaa7680atagttcctg agaaaaccag atcaaggaaa ccagatttcc ttcttggaaa agccatttta7740tagatgacta ataataacca ctaccaaaaa gaatgtctgg ttaaagattc ctggaaaagt7800tgcaggttga gtgtgcgcct tgaacgtcaa gttaagtgtc atacactttc cccagactgc7860cagtcagctg ggggttgggg gtgacttccc aggaagaggt gcttatctga aaataattta7920gttgctagaa agtaactcag ggtgcggctc cagcaggctt gattcaattt tctcttttac7980tacattgatg tgacgtttgt catgttactg atatatatat ttttaattta attcacctgt8040ttgcttcccc tgtccccaca cttttaaagg gcctcttccc taacatccag ccaaccatgt8100gtcctagcag attttccata caaggttctt gacacaccgt gtatctctgc tcttctgtaa8160tctttttctt gaaattgact tattttttaa agacctcaat gaatttattt ttaaaaaatt8220tattggagga cgttcccatt gtggcacaga tgaaatgaat ctgactagga accatgaggt8280ttgtgggttc gatccctggc ctcattcagt gggttaagga tccagctttg ccatgagctg8340tggtgtagtt cgcagacgca gcttggatcc tgcgtggctg tggctctggc gtaggcttgc8400cagctacagc tctgatttga cccttagcct gggaacctct gcatgctgag ggtgcggcag8460taaagagaga aaaaaaattt ttggagtata gttgactcac aatgttgcat8520tagtttcagg tgtacagcaa agtgattcag ttacatgtat acatgtatcc attctttctc8580agattctttt cccatgagct tattagagaa tattgagtag agttccctgt gctatgcagt8640aggcccttgc tgattatcta ttttagatat attagtgtgt acatgttaaa cctgaactcc8700
taatttatccctcccccacc atgatttccc ttttggtaac tgtaaatttg attttgaaag8760ttgtgagtttgtttatgctt tataaataag ctatcttgta tcacttttga ttagattcca8820catataagtgttatcatatg atatttacct ttatcttgct gacttcactt agtatgataa8880tctctaggtctatccacgtt gctgcaaatg gcactatttc gttctttttt atggctcagt8940aatattccatcgtatacatg aaccacatct tctttatcca cttttccatc aattccacgt9000cttggctactgtaagtagtg gctactgtaa atagcagtga acattgggat gccagtatct9060ttttgaattatggttttctc tgcatagatg cccaggagtg ggattgctgg attatatggt9120acttctatatttagtttttt aaggaaactc catactgttt ttcataacag ttgcaccaat9180gtacattgccaccaacagtg taggaggatt cccttttaaa tgaatttgct tgtaaagtta9240actttacatcattacctcaa attaaaaact agtttacttt tccatgaata ggaaggcacc9300ataaaacaaatacaataaaa ttctaggtgg atctctgtga ttaccaaagt ctctttgagc9360agttttcttcctggtagtgg gaaataggca ggcattcgag aacagttaaa gatataccag9420cagtaaatgagtggtataat cagaatgatt gacataaatt tgaaaatgca attaccttcc9480ccgtcaatgtgaaccattgt tctctaacgc tatgtccaca ggccacttaa aattgtcttg9540tgtgtcacaagcacagtagc tgttatacct gagtgtgcat cagagtttcc tggagtctcg9600ttaaaacacagatcactgat taagtctggg ataaggcttg aaatgtgcat ttctaaaaag9660tgcccaagccagagttcccg ttgtgactca gtgggttaag aactgactag tacgtggagt9720tcctgttgtggcacagtggt taacgaatct gactaggaac catgaggttg cgggttcggt9780ccctgcccttgctcagtggg ttaatgatcc ggcgttgccg tgagttatgg tgtaggtggc9840agacaaggctcagatcccat gttgctgggc tgtagtgtaa gccagcagct gcagctccag9900ttccacccctagcctagaaa cttccataga ccacaaatgt ggacctaaaa9960ataaaataaaaaattaaggg cattcctgtt gtggttacga acctcactag tatctatgag10020gttgtgggttcgatccctgg cctcactcag tgggttaagg atccagcatt gctgtgagct10080gtggtgtaggtcacagatac agcttggatc ctgcatttgc tggtctgcca gcagccacag10140ctctgattcgacccttagcc tgggaacttc catattccgc aggtgcggcc ctgaaaagca10200aataataataataataataa attaaaataa aaacataagt gcctaagcca tgctggcact10260gctagttcagtgcacacact ccaagaggca gaaccctcat ttcatcacct ctcactttaa10320gaaaaactgtctggggaagg cttacagatg caaagcaaaa acaactgcag gtacaaacag10380gtacaaacctcctcccatcc cctgtgggca attttccttt caaagagcca caagcccagg10440ctaggcactaccctccctga aagcagtgct gtaatctcta gacccaccca cttcctctag10500aggaagcctgaattaaaatg tggagccggg agagagcctt ctgatcttag taaaacctcc10560ccaccataggtgagcagagg aggaagtaga tgagaaaccc aactactttc ttggaaaagc10620ccctccatataatttttccc agagctgaat ttctagaaga tcaaaaatca aatggaatta10680agaaatccacagaaattcct ttttttcttt ttctttttag ggccgcatcc acagcatatg10740gaggttcccaggctaggggt ccaatcagag ctatagcagc cagcctacac cccagctcac10800agcaatgccagatccttaac ccactgagcg aggccaggag tcgaacctgc atcctcatgg10860atgctagtcaggttcattaa ctgccaagcc acgatgggaa cttcaaaaaa gcattttttg10920agaactgactaaactcagtg aggtgagtag atgagtgatt gattgtggag tttcacacgc10980caagatgcaa acacctatgc ctcccccttt tgaaggccga ggtattggga atgcagctcc11040
agtgagtgaaggtccttgtg tcagtattag aaccttcctc tgagcccttg atatcactgg11100tgtgtgagaagagaaacatc tcaggaagac agctcaacca cgatcctgcc tcccagtaag11160tcaaagcacattaatttgtc catgtttcct gctagagctt ttctttatag aggcacattt11220ttcatgtaattagatatata atgataacct gctttttttt ttttttttgc tttttagggc11280tgcacctgcagcatatggaa gttctccggc taggggtcga atcggagcta cggtttccag11340cctacaccacagccatggca atgctgggat tgaacctgca tcctcatgga tactagtcag11400attcatttctgctgtgccac aatgggaact cccaagactc ttgcatttaa aaaaaatttt11460attgaaatagatgaagtatt gttgatttac aatattgtgt taattcctgg tgtacaaagt11520gattcggtttctcatctata ttcttttcca ttatagtttg ttacaggata ttgcatgtaa11580caggatatagtttgttgcag gatattgtgt tatacagtag gaccttctcg tttatctatt11640ttatatattgtaggggtttt tttttttgtt tttttttttt ttgctatttc ttgggccgct11700tccgcggcatatggaggttc ccaggctagg ggtccaatcg gagctgtagc caccggctta11760cgccagagccacagcaacgc gggatccgag ccgcatctgc aacctacacc gcagctcaca11820gcaacgccggatcgttaacc cactgagcaa gcgcagggac cgaacccgca acctcatggt11880tcctagtcggattcgttaac cactgcgcca cgacaggaac tcctatattg tagtttgtat11940ctgctaataccaaaccccta atttattcct cccccacccc cttttccctt tggtaaccat12000caatttgttctctatgtctg tgagtttgtt tctgtttcct aagtaagttc atttgtatca12060tattttagattccatgtata aatgatattg tacggtattt gtctttttct ttttgagggg12120tgggccacacccatggcatg cagaagttcc ctggccaggg gtcgaacctg tgccacagca12180gcaacccgagccacagcagt gacaatactg gatccttaac cttctgcacc acaagggaac12240tcctgtctttatctttccaa cttacttcac ttactataat catttctaat tgcatctatg12300tttctaggaatggcattgtt tcattctttt ttatggctga gtagtattcc attgtataat12360attatcacatcttctttatc cattcctctg ttgatgaaca tttaggttgc ttccatgtct12420tgtatattgtacataatgct gcaatgaatt ttgagatgca ggtatctttt agaactaaag12480tttttctccagatatatgcc aagagtggga tttgttggat catatggcaa ctatattttg12540gggtttttgttttgtcttgt tttttttctc ttcttagggc cacgcctgca gtatatggaa12600gttcccaggctaggggtcaa atgggagctg cagcagctgg cctactccac agccactgca12660acttaggatctgagctgcat ctacaaatat atgctgcagc ttgtggcaac atgagagact12720taacccaccgagcaaggcca gggatcgaac ctgcatcctc atggatacca gtcaggttct12780taacctgctgagccacaaca ggaactccct gtattttttt tttttttaag gcacctccat12840gctgtcttcgatagtggctg cacccattta cattcccaaa caatggtgta gagaggagaa12900
ccctgccttttccacacttt gtcacagtag aataagcact ctcttttatt gctgtaccta12960ggcttcccttgtgcgatgct gtcatatatt tcctcaagga gagtcaccac atttattcct13020ctgtccttgaatagtagcag ttgtttttaa ttgttttttg gtaataatgt aaaagtgcaa13080caaaagttattgtacatata attttccctt catttggaaa tatttcctta gggtaaagtc13140ctagaagggaatttctcagt ctaaagataa gaacattttt atggccctta atatctctcg13200ctgaattgccttttgaaggg gatgtagcat ttgcaagtgt gtgaaattat ctgaaccccc13260accagaattgaatattatcc atgtgaacat ttttaccaaa tgtaaaatat tacttcattt13320gtgttctagtcagtagcatg tcttttttgt ttgtttgctt gtttgttttt taatcttttt13380
agggccacacctgcagcata tggaaattcc caggctaggg gttgaatcgg agctgcagct13440gccagtctacaccacagcca cagcaacgtg ggatccaagc tctgtctgcg acctacaccc13500cagctcatggcaacaccgga tccttaaccc actgagtgaa gctggggatc taacccacat13560cctcatggatgct agtgggg ttcgttacca ctgagacacg aggggaactc ccagtatcat13620gtctttaaaatcagtgaaaa gtcaagcatt ttcccatata tctttactaa ttttattttc13680tctttgtgaataatttatca tgacaatcga cttttggaaa aggaattgtc caaatgcata13740gtgagtctgaagaccaggac tatatgttat ttactttgca ctccctgctc cctgtatcta13800caacaaatgggatgacgttc aactattctt ttctttattc cataaatact tactgggtgc13860ctgctatgtgccaggtttct gggtgctggg atagaggagt aaacaaggca gagacctggc13920ctcagacagctctcatgggc aacagacaac caaaccaaca aagataccaa gtcatttcat13980actgtgatgaggccatagag gaaatgaact ggggatggat agtcaggtgg cttttttttt14040tttttttttttttttttttt ttttgctgtg catgcatcat gtagaaattc ctgggccaga14100gatcaaacctgcaccatagc atctacccgg gccactgcag taacaatgct ggatccttaa14160cccactgctccacatgggaa cttaagtggc tcctttagat gagaccacca agaagagccc14220tcctgtcattctcctgctta aatgtcccca tggcttcctg gcttagcaca gaataagatc14280cagaataagacctgtgcagg accaggtcac aatctgcctc tctgacatca cctccttcca14340ctctcctcctgaatcacagt gctcctcatg tttgaatggc agcctctttc ttaataacag14400gtctgagttcagatagcagc tcccatgaga tgattctgct tactgtccta tttaaagaag14460taccgtctacccacaagtca ctatcccatt gtgttttatt ttcttcatag cacttatcac14520tctcttaaattattttgttt ggttgttttc tgctcacccc tcagcccctg taagctccag14580gagagtggagacttccatca tcttgttcac tgctttatgt tcagagcccg agagatgtac14640agaagtatctcaggcatctg atatcaattg cacagccact tgaactttat tcttgtaaag14700tatccagtgggtgaaaagtt aatttcagca gatgtgaatg ccaggattgg aaatggtttt14760gtcaacgtgtcaataattag gagtttccat tgtggctcag tagtaacgaa tccaactagt14820atccaggaggaggcaggttt gatccctggc tcactcagtg ggttgaggat ccggcattgc14880catgagctgtggtataggtc acagacaggg cttgggtcct gtgttgtggc tgtggtgtag14940gctggtatatgccgcagggc ggccctgaaa agaaagcaaa aaagcattac taattaaacg15000cttgagttggaattttgctt tcccataagc catttctctg ggaatggttt gctgaattag15060ataggaaaataactgatcaa ctttatttac tatccaaata agatattgat aattagcttt15120cgtgaatctgggaagacttt agttatcaag ggactagaat tattgagaag cagctaagag15180cagcaaaaatttcttgcaac atttgattct gtagacagaa aaatttctat ggatctagtt15240atttaatcttgagaaagatg ttagatttct aatggtgctt cccgagcctt agagccttat15300gatggtactgccatgttgca gaatcaaagt agttaaggag ggcttcttga cagatcaaat15360tccactcctaaatggctttg acacagctct ctcctgttta ctctacatct gaatagtcgg15420atgtaatttagattggtgaa cacatccttt ccctctccat agccaagaga tccttagtaa15480ggttcctgctgaagaatagt tctgtggtaa ccaggtaagc aagggttaac tttttagctg15540atgctggacacggtcaccta catgcaatat tgtttctgtg ggaaaatgga gtctgtgatc15600tcaacttacagatacatagc tagaatatag gtggtatggg acttcccatt gtggcacagt15660ggaaacgaatccgactagga gccatgaggt tgcaggttcg atccctggcc tcactcagtg15720
ggttaaggatccagtgttgc catgagctgc ggtgtagttc gcagatgcgg cttggatcct15780gtgttgctgtgtctgtggtg taggctggca gttgtagctc cgattggacc cctagcctgg15840gaacctccatatgccacggg tgtggcccaa aaaataaaaa ataaaaaaaa ttttttttaa15900atagaatataggtagtgtgg agttaataaa tatcacctct tttcctcgcc caggtgttga15960agcatgtgagaaatgaaaca aagcatatgg cagatggaga caaaatagca ctttcagttg16020aagctagttttggagactat gttttaaggc ttcctaatac tgaatcctaa ccctccccag16080 ctacaaccatcctgaatacc caggttctct ccccaaaggg acaaggcaag atcgagaggt16140gagtatttccagtttattcc tctgagagcc ctcagtaccc gtcacttagg gggcagtgaa16200taaaggcaggaagggaggcc aggagcctgg cgctcacaga gctgcagcca cgtgaacaca16260gcagcaacgcggagcaagca tctcccctac tgagaggctc tgcgtacatt gtctctacag16320ccctggagtggtgcagtcca gtgtggactg agttaaaagg ggcctaagag aggagggctg16380tggattcacactttggggac tgcctaacgt cgttgcccat ggttttccag ggaaggccag16440ggaagagtgagaaggcaggt gcctagcaga cctgcatccc ttcccggtga ctcttccctt16500ccatgtgtcctcagcagaga agtggagtgg aaagccaggg taggtatcaa caccttcata16560aaaaatagcatgagcacaca ctggcaacag ggccctgtgg atgttactct cttgatatct16620tgaacccaggaaaagggaga gccgtgtgct cttcctgatt tagaagatag gatttgagac16680tctgcagctaattatgtgac cccaggccac tgtcccaatt ttcctaaatt cctgggtagc16740cttttataaaatgaatgtaa taggttgaaa cgccatgtaa aaatggcagg aagggagaat16800atatttcccatacatatgta aaggacactg gaaactcaaa aataagaaaa taactccact16860aagaatggataatgattgaa acaaatcttt ttttaaaaga agagatatga gtggctaata16920ggcacatgaaaagatgctga atgtcattcg tcatcaggag atgcaaatta aaaccctaat16980gggagactactaaataccct ttagaatggt taatattaga aagactgatg atgtggagtt17040gccatcatggctcggcagaa acgaatctga ctagcatcca tgaggatgca ggttcaattc17100ccggcctccctcggtgggtc aaggatctgg tcttgccatg agctgtggtg gagaccacag17160acaaggctcagatctgacgt tgctgtagct gtggtgtagg ccagcggcta caactccaat17220ctgacccctaacctgggaac ctccatatgc tgcgggtgcg gccctaaaaa gacaaaaatg17280aaaagaaagactgacaataa caaagtgtta gcacaggtga ggagcagctg gagtgcatat17340gttgctgatgggaatgtaaa acgggacagc tttggaaaac agtctgagag tttatgaaaa17400gttaaccatgcaccagggtt cccaagtggc ctaccagcta aagatttggc cttgtcacta17460ccagagctcaggttctaccc ctggccctag aatttctgca ggccatggat gcagctacca17520aaaacagtattaagcatata cctaccatat gatgcagcca ctccatgact aagtatttac17580ccaagagaaatgaaagcata taaccacaca aagacttaca catgaatatt ctcatacctt17640tataataatcaaacactaga tcaagccagg ccaaaaaata tcccctccca aatccccata17700acattctacatatataaaat tctagaaaat gcaaactaat ctatagagac ggaaagcaga17760tcaggggttacctcagactg agggtgcgag gaaggatgaa cacaggaaca cttgggaatt17820atgagaatgtttgctatcct gactatggtg atggttccat ggtgtttgcg catgccaaag17880ctttaaacaggtgcagtttg catacattac acttcactat aaaagttgta aaagaaagaa17940aggacgcaaggagggaagaa gaactgatac ccaggaattt gtatgaaatt tcacaatcat18000cctgctgagggagaaaaacc aggcataagc cagcacctac tgtatgattc cacttctatg18060
caattctagaaaaagtgaac tgatcaccgg tgacagagag caggtgaggt gttgcccaga18120gttgggggtgggggagggga tggactacag aaaggcaaga ggaaatttgg ggaagtgata18180gaagcattgtgtatcttgga gatgggatca aaacggcgga atagtaggat gggagctcaa18240 cttctctcctaaatttacaa ctacatgctg agcaatcttc aaccaaattg18300actggaaaccttcaaaaaga tatcctactc caaaggcaaa gaggaggcca catcaagagg18360tatgaggggcgattacatga tataaacaac cccatacctc ctgggtggga agccccccac18420agagtagaaactaactggtt cacagagact cacctacagg agtgagagtg ctgagcccca18480catcaaaccctcaccagcgg ggatctggca ctgggagaag gagcccctgg agcatctggc18540attgaagaccagtggggatt gtacgcagga gccccacagg actgggggaa acggagaccc18600cattcttaaaaggcgcacat ggattttaac gtgcactggg tcccagggca aagcaaagtc18660tccataggaatctgggtcaa accagactgc agttcttgga ggacctcctg ggaaaatagg18720ggtgaatgtggcttgttgtg ggggaaggac attggaagca aatctctcgg gaatattcag18780cagctgcttttctctggaag tggccatttt gggaaaatct ggcctcaccc atcagtcagt18840cagcactgagaagccccagg gcaaacaaca acccaggtgg gatcacagcc ctgcccctca18900gtaaacaggctacctaaaga cccctcaggc actcggctgc ctctaacccc atccagagac18960taagccccacccaccagagg gattagaagc agctccacct accagtgggc aggcatcagc19020ccctcccatcaggaagccta cagcaagccc ccacacagac ttcagccaca aagggggcag19080acaccacaaggaagagaggc tacaactcta ttatctggaa aaaggtcacc acaccaaaaa19140cctataaaaatgaaaagaca gagaactata actcagatga gggagaaaga aaaaccccca19200gaaaatcagctcagcgatga gaagattctc agcctccagg aaaaagactt cagactgtcg19260atgctgaagatgatgcaaga cattgaaaat aaactggagg caaagatgga taacttacag19320gaaacactgagcaaagagat acaagataca caacatttct taagttttat atcttgaata19380taatggtgatttgacatgac tatacatcta tcaaagctaa ttgaatttta cacttttttt19440ttcttcttagggctgcacct gtggcatatg attacaaagt tgctaaaagt gaccctcaga19500gaggcccagagcggtggtcc acagcgaaag acgacaaagc tcagcagcgc gccttgtggg19560gaagcacagtctcctcagag attctcaaac taccgctgga ggagttccca ttgtggcgca19620gtggttaacg aatccgacta ggaaccacga ggttgcaggt tgatctttgg ccttgctcag19680tgggttaagg atccggcgtg gctgtgagct gtggtgtagg tggcagacgc ggctcggatc19740ccgcattgctgtggctgtgg tgtaggctgg cggctacagc tccgatgaga cccctagcct19800gggaacctccacatgccaca ggagccgccc tagaaaaggc aaaaaactac cgctggagga19860aacaggatgtcctgcatccc agtggagtga ctggagcaca aactctgtga ttgggtattt19920gaaggaattg tgacttccaa gaagatggta gcttggggtg tttggaatgt gaagagatta19980gctgacacccccgccccctt ccgctgtccc ccaaccccca tcctcccaag gacgtttggg20040ctgtgttgag ttattttata ctatgagaca gaaaaataat ctataagact agttttgcct20100cttccatcacaaaagctcag aagagaccta cgctctatga cccaacccta actagcagtt20160tcctttgtctggtttgacgt cagttaatga ctggactttg aacctgaaag aacattaaaa20220ggaaagatag ctgaagggaa aattttctct ctggattacc tggcaccaac atccaaaatg20280aaagtcttca ttcacttcac tctgcttgcc ctccttttcc taaaatcagg taaggaacat20340ttacacatttttaattcttt ccgtcatatt tgtctttgag atagaattag caggcttaag20400
ctaccaatcc cctctgcccc aactagtggg ctattttgta gatttcctcg ccaaccaggc20460tcaaagatcc aagccaggga gttcctgttg tggctaagtg ggttacaaac ctgactagta20520gtctatgagg acatggattc aatccatggc ctagctcagt gggttaaaga tctggcattg20580ctgtgagtgg cagtgtaggt tgcagacatg gctcggatgc tgcattgctg tggctgtggc20640gtgggccagt agctgtagct ctgattcaac ctggagcctg ggaacctccg tatgtgaccc20700 taaaaagaaa aataatgaag aagaagaaga cacaaggcat ttcaacctgt gatgaaagca20760ggtagaacat tagtaagagg ggcttctgga ctaagatttg gtgttcagca aggcagacat20820tttcttgagt gtcctgaggc cagacaggaa cagcttagta attccagggc ccaatgcctt20880agagtctcac taaccactag atatcacttg ggttctaatg cagagtaggg atctggtttg20940atgcctgaat cttgcatcaa gagttttctg cccttgctgc cctgttgtgg cgcagaagaa21000actcatccga gtttgatgcc tgaatcttgc atcaagagtt ttctgccctt gctgccctgt21060tgtggcacag cagaaactca tccgacttgc aaccatgagg ttgagggttc aatccctggc21120ttcgctcagt gggttaagga tctggcattg ctatgagctg tggtgtaggt cagagatgca21180gctcggatct ggcgttgctg tggctgtggc gtaggctggc agctgtagct ctgattagac21240ccctagcctg ggaacctcca tatgctgcag gcatggtcct aaaaagacaa aaaacaaaac21300gtgttttctg ccctttacac gtgggaattg aggcttatct ctggacaaat21360cctttagttc ctctgagtat tggtgggcat ttctgtcaaa tgtggatgct cacatccatt21420ccataggcac tgttgggcac agggtaagtc cttgataaat gataggagga gtgggaacag21480gggcagcagc agtcaaggtg ctcagctggt gaactatttt ctagccccta gaagaaaact21540ggcttatgaa tgatggttta agccagaccc tgttcaaggt gaagagccaa attctgctac21600ccagaggctt ccacacccaa gggggaatca ggtagcttca gcattttttc tatgtctgaa21660agtttaggga tagcttcctc attaaaactt atcttttaat attttaaacc caccttcatt21720gtcagaattc ttagagacaa agtttctaaa aaagtgactg gatcacaaaa agcttattac21780cccatatgtc caggggttcc taagagttga aaaaaggcaa ctttggaagt tcccactgtg21840gcgcaatggg ataggtggta tctctgcagc tctgggacac aggttcgatc ccccccagcc21900accgtgggct aaggatctgg cataggttgc aactgcagct tggatcttat ccctggcctg21960ggaactccat ttgccatggg ggaccctcaa aaaaggcaag tttgaggatc accatagccc22020ttagagtaaa gatcatctct atcattttac tctttaaaaa cttcatgtgt ctagaattat22080aaatgggtaa ataacactag aaaaaaattt gaaagaagtt taatacacac ttaatgtatt22140aacgattttt atttaatcaa tctttaaaca catccccttc cctaccatcc ctcagctatt22200atattgtgta atggaaaatt ggggcaaatg ccttaaagtg taaaaggaca aaaggaggcc22260atttccaggt agtaacagtg ccaccttgtg gcattttcaa ttttcctcac tgagaatcat22320ggaggaatat aaactcagat tgacagatct gcaactatag gcataaatga tactacatct22380tgtgtcaaca tctgatatgt tgatattaat atgtatatat aattatactt tatatatcat22440aatatcatat atcatattat aaccaataat taatacaatt aataataaga taaatgatgt22500taatacttct ttatccagtt gagttaacag accaggtacc atcatatttt ccagtaaccc22560ttttccagtt ttgtctgttt aattttttct ttgtctttgc cttttctagg gccgctctgg22620cgacatatgg aggttcccag ggtaggggtc gaataggagc tgtagccacc ggccttcgac22680agagccacag caacacggga tccaagccat gtctgcaacc tacaccacag ctcacggcaa22740
tgctggatcc ttaacccactgaacaaggcc agggatggaa ccctcaacct catggttcct22800agtcggattc gttaaccactgccccacgac gggaactcca attttttatt tttctttatg22860tgtttttttt tttagatgta gtcatgtatt ttcattttta atgtactcca ctacatctac22920ttttcagaaa aaaaaaggattgggtaccaa ttagtattac atagtagcct ctgccattat22980
atatacagtg ttttgtcttctgaaactttg cctttcagca agtatggctc agaacaagac23040aggaggcacc acaaagctga catccacaga aaacaccagc tccccgaacg cggcggaaag23100caccagcgct ccttccgctg caagaaacag tgctccgggc acggcagcaa gcaccagcgc23160tccgtctgcg gcagcaagca ccagcgctcc ggccgcaggg gcaagcacca gcgctccggc23220cgcaggggca agcaccagcg ctccgtctgt ggcggcaagc accagcgctc cggccgcagg23280ggcaagcacc agcgctccttccgctgcaag cacgagcgca ccggccgcag gggcaagcac23340cagcgctcca gccgctgcgg caagcaccag cgctccagcc cccgcggcaa gcaccagcgc23400tgccgcggca tcaagcacca gcgctccggc cgtggcggca agcaccagcg ctccggcccc23460cgcggcaagc accagcgctccctccgctgc ggcaagcacc agtgctccgg ccgcaggggc23520aagcaccagc gctccggaccccacggcaag caccagcgct caggccgcag aggcaagcac23580aagcgctccg tcggcggcgg caagcaccag cgctccggcc gcaggggcaa gcaccagcgc23640tccgtctgtg gcggcaagca ccagcactcc ggccgcaggg gcaagcacca gcggtccggc23700cgccacggca agcaccagcg ctccggccac aggggcaagc accagcgctc cgtctgtggc23760ggcaagcacc agcactccgg ccgcaggggc aagcaccagc ggtccggccg ccgcggcaag23820caccagcgct ccggccgcag gggcaagcac cagcgctccg tctgtggcgg caagcaccag23880cactccggcc gcaggggcaa gcaccagcgg tccggccgcc gcggcaagca ccagcgctcc23940ggccacaggg gcaagcacca gcggtccggc cgcggcggca agcaccagcg ctccggctgt24000aggggcaagc accagcggtccagccgcgtc agcaagcacc agcgctctgt ctgaggcgga24060aagcaccagc tctccagccg caggggcaag caccagcgct ccgtctgtgg cggcaagcac24120cagcgctcca gccgcggcgg caagcaccag cgctccttcc gctgcaagca ccagcagtcc24180ggaccccacg gcaagcacca gcgctccggc cgcaggggca agcaccagcg ctccctccgc24240tgcggcaagc accagcgctccggccgcagg ggcaagcacc agcgctccct ccgctgcggc24300aagcaccagc tctccggccg caggggcaag caccagcgct ccttctgctg caagcaccag24360cgctccgtct gtggcggcaa gcaccagcgc tccggcccct gtggcaagca ccagcgctcc24420ggccccaggg gcaagcacca gcgctccttc tgctgcaagc accagcgctc cggccacagg24480ggcaagcacc agtgctccctccgctgcggc aagcaccagc gctccgtcca cggcggcaag24540caccagcgct ccggccgcag gggcaagcac cagcgctccg gcccccgcgg caagcaccag24600cgctccggcc cccgcggcaa gcaccagcgc tccagccccc gcggcaagca ccagcgctcc24660ggctataggg gcaagcacca gcgctccgtc cgctgcaagc accagcgcac cgtctgctgc24720aagcaccagc gctccggccg caggggcaag caccagcgct ccgtctgtgg cggcaagcac24780cagcgctccg gcccctgcgg caagcaccag cgctccggcc gcaggggcaa gcaccagcgc24840tccttccgct gcaagcacca gcgctccagc cgcaggggca agcaccagcg ctccggccgc24900ggcggcaagc accagcactccagaccccac ggcaagcacc agcgctccgg ccgcaggggc24960aagcaccagc gctccggcccctgcggcaag ccccagcgct ccggccgcag ggacaagcac25020cagcggtcca gcccccgcgg caagcaccag cgctccggcc gccgcggcaa gcaccagccc25080
tctggacccc acggcaagca ccagcactcc ggccgcaggg gcaagcacca gcgctccatc25140ggcagcggca agcaccagcg ctccttctgc tgcaagcacc agcgctcctt ctgctgcaag25200caccagcgct ccggccgcag gggcaagcac cagcgctccg gccgtggagg caagcaccag25260cgctccggcc cccgcggcaa gcaccagcgc tccggctgca ggggcaagca ccagcgctcc25320 gtccgctgca agcaccagcg ctccggccgc aggggcaagc accagcgctc cggccgcggc25380agcaagcacc agcgctccgg cccccacggc aagcaccagc gctcccgccg cggtggcaag25440caccagcgct gcggcccccg cggcaaccac cagcgctctg gccgcggtgg caagcaccaa25500ccctccggcc gccggggcaa gcaccagctc tccggccgca ggggcaagca ccagcgctcc25560gtctgtggcg gcaagcacca gcgctccggc ccctgtggca agcaccagcg ctccggccgc25620aggggcaagc accagcgctccttccgctgc aagcaccagc gctccggccg caggggcaag25680caccagcgct ccggcccccg gggcaagcac cagcgctccg gcccccgcgg caagcaccag25740cgctccttcc gctgcaagca ccagcgctcc ggccataggg gcaagcacca gcggtccagc25800ccccgcggca agcaccagcg ctccggccgc cgcggcaagc accagcgctc cggaccccac25860ggcaagcacc agcactccgg ccgcaggggc aagcaccagc gctccgtcgg cagcggcaag25920caccagcgct ccggcccccg cggcaagcac cagcgctcct tctgctgcaa gcaccagcgc25980tctggacccc acggcaagca ccagcgctcc gtccgctgca agcaccagcg ctccggccgc26040aggggcaagc accagcgctccggcccccgc ggcaagcacc agcactccgg ctgcaggggc26100aagcaccagc gctccttctg ctgcaagcac cagcgctccg gccgcagggg caagcaccag26160cgctccggcc cccacggcaa gcaccagcgc tcccgccgcg gtggcaagca ccagcgctgc26220ggcccccacg gcaagcacca gcgctccggc cgcggcagca agcaccagca ctctggcccc26280cacggcaagc accagcgctccggctcccgc ggcaagcacc agtgctccgt ccgcggcaag26340cacaagcgct ccggccgcgg cggcaagcac cagcgctccg gcccccgcgg caagcaccag26400cgctccggct ggggcggcaa gcaccagcgc tccggctgca ggggcaagca ccagcgctcc26460atccgctgca agcaccagcg ctccggccgc ggcggcaagc accagcgctc cggccgcagg26520ggcaagcacc agcgctccgg ccgccgcggc aagcaccagc gctccggccc ccgctgcaag26580caccagcgct ccggcggcgg tggcaagcac cagcgctccg gccccagggg caagcaccag26640cgctccatcc gtggcaagca ccagcgctcc ggcccccgct gcaagcacca gctctccggc26700cgcaggggca agcaccagcg ctccatccgc ggcaagcacc agcgctccgg tccccgctgc26760aagcaccagc tctccggccg caggggcaag caccagcgct ccggcccccg ctgcaagcac26820cagctctccg gctgcagggg caagcaccag cgctccggcc gcgggggcaa gcaccagcgc26880tccggccgca ggggcaagca ccagcgctcc agcccccgct gcaagcacca gctctccggc26940cgcaggggca agcaccagcg ctccggcccc cgctgcaagc accagcgctg cggccgcggg27000ggcaagcacc agcgctccatccgcggaaag caccagcgct ccggctgcag gggcaagcac27060cagcgctccg gctgcagggg caagcaccag tgctccggcc gcgggggcaa gcaccagcgc27120tccatccgcg gcaagcacca gcgctccggc cgccgcagca agcaccactc ctgcctctgc27180agcaaatgct tcttctgcta tttcgagtac acctattgcc tccacaccca cctcaggtaa27240gagagattcc ctcacatctcctcactgagg catcagggcc ttttggcagt tgacaggtac27300gtgtgttgtt cgggaatggg gaagtgcata taaagggacc caagaggacc ttggttacga27360aaccctggag tcacaaatgctaatagtgat ggcaaagatg ctaaaagatt acctgcagtt27420
ccatgctattcccctttcctcaagcatttg ccgtatgtgg aatattccta tgagttggac27480tttatgaaagctcctgttccagcttccttc tccctaggag atttttggaa ataatgtcag27540cttctttgagaatatcaggtccaaataaac actgactctt cagccccact gatctagtga27600ctgaaggagagagtcacttctactctgatt ccacatcacg tgttattgaa aaatatgaca27660
ttaaatggtaggtgatcaca atttggtatt tgagctaatt catggaattt gaggctaagg27720aataaaagccaaacaagagcattgctcaaa gagtcctaga catgaccaag gccatggtcc27780aaaagacggatgtttgggga tacttatcta agacaagatg attgatctca agccaaagtt27840gaaaccaaaaccacttgcgtcatttcccaa agtttggtgt agagcaagtg tcaataaagt27900ttatcagtgaaaggaaacta ataagtagtt taggttttat gggccgtagt aaggtctttg27960ttacaactactccactctgtcctgttaaca tcaaaacagc cataggcaat atacaaatga28020atgtgcctgactgtgtgcca ataaaacttt atttgcaaaa tcagaaaact ggtcagaatt28080ggcactcaagtcatcatttg ccaaaccctt gtctaaagga atgctgaaac ctaggcttca28140agggtctatataaaccctgttattccaatt taggggaact gaagtccagt aagggcagca28200acttgcccaaggccaaaagtcaagtcgatg tctccctgag cttatttctt gtggcttccc28260caatgttctttgatagacctgtgaaatgaa tgattaagca aatggagcca gagcctgtat28320caaaacttggttcccaggcttcttttgtgt ttgaagggtc cactctgaag gccaggtcaa28380gagaaccacagctgcacaatcagttctcca gtagggagct tatctaagct gttttgcttc28440cctagacactcttgcagagtaaggcagtct tcttgtgttc tggccctaca ctaactaact28500ctggggcacaaggcaaatca cttctgtctt ggcttcaatg gactcatctg taacaaggtg28560tgactgaactgaatagccatcccggaattc ctgttgtggc tcaacaggtt aagaacccaa28620ctagtatccacgaggatgtg ggctctatca gtgggttaag gatctggcat tgctgcaagc28680tgtggcataggatgtggcta ggatcctgtg ttgctgtggc tatactttaa gccagcagct28740acagctctgattcaacccccatggcttggg aacctccata tgccatgagt gcagccctaa28800aaagacaaaaaaaaatagccacccaaatcc ttttcccact gtgacatcct acgactccat28860tcctggcacacagaaatgttaacaagtctg ttctttattg ccagggagaa agcacaccgg28920gagttatctgtctggtatattcagaatgga gctgtactaa atttcttccc tagaattttc28980caatgtgactgaggcttttttaaaaaagac atttacttta cttagaatgt tggtttacat29040cttctcaaaggggtgaccctcacatggttt atgagatctt gtgccatgcc tcttatgtcc29100catagcatggggtttgggatttgggggttg attttgtgtg ggtttttttt cccccttaaa29160atagagcaattttgggttca cagcaaagtt gagaggaagg tacatagatt tcccatgttt29220cccggtccttgtgcctgtacagcctccctt atcatcaaca tccctcgcca gatggcacat29280ttgtgacaactgatgactctacattgacat attataatcg cccaagtcca gtttacgtta29340gggtttacagttgatgttgtatgttctata ggtttcaaca agcatatggt gacatgcatc29400catcattacgctatcgtaaa gactgttttc acctgcttat tcatcctccc tgctaaccct29460tggcaaccactaatcttttttactattgcc atagttttgc ctttttcaga atgtcatgta29520gttggaatcccacagtatgcagccttttca gattggcatc tttcatttag caatggcatt29580tgagtcctccatgtcagtttcaaggcttga gcactgagta atattccatt gtctgattat29640accagtttgtttatccactcacctactgaa ggacatcttg gttgcttcca aattttggca29700gttatgaatacaatgctcta agtgtttgta tacagggttt tgagtggata taagttggta29760
aatacttagt tacatcaggtaaattacaag acaggcactt gctaggtcat gtggttaaga29820gtgtgtttag ttttgtaaga aactgccaac atgtcttcta aagtggctga accatttcac29880
atttgagagt tcctcaccag cagtatttta gtccataggt tataagaaaa gaagcttaaa29940gagggcaaaa gtatatgaaa taaaaatgaa atcttcctcc tatcctaaac tcctaggacc30000cagacttcag tttcaacttctgcagccaat tttttgtgtt tgtgcaaata caaacgaata30060tacttgtatg tttctgtgta ttcctctcct tttttttaat catgaaaaag tgcagggtat30120ttctggccat atacccaaga gtggaattgc tggatcatag ggtaaaacta aaaataaact30180accctgtgat ccagcaattccattccttgg tatatagcca ggaaaaaaaa atgaaaacac30240taacttgaaa agatccacgcacccccatgt tcataagagc actacttaca atagccaaga30300catgggagca acccaagtgcccaccaatag actggataaa gaaaatgtga tatgtgtgtg30360tgtgtgtggc tgtaatgata gtacttatat gtagaatcta aaaaaataat actggagttc30420ctgttgtgac tcagtggtta acgaatccga ctaggaacca tgaggttgca ggtttggtcc30480ctggccttgc tcagtggtttaaggatccgg tgttgccgtg aactgtagtg taggtcgcag30540atgcggctcg gatcccacattgctgtggct ctggcgtagg ccggcagcta cagctcagat30600tagaccccta gcctgggatcctccatatgt ggtggggaaa ggccctagaa aagacaataa30660ataaataaat taataataca aatgagtgta tatacaaaac aaaaacagac tcacagatag30720agaaagtaaa cttgtagtta ccaaagggaa gacagacaaa ttaggggtat gggattaaca30780gaaacaaact actatgcata aaatagagaa gcaacaaagg taaattgtat agcacaggaa30840attttagrtg ttatcttata ataacttgta rtatartctg caaaaatact taatcctatg30900ctgtatgctt gamactrata magtactgta ratcaatgat acttcaataa aaagagacca30960tgttggagtt cccatcgtgg ctcagtggaa acaaatctga ctagcatcca tgaggatgca31020ggttaaatcc ctggccttgctcaatgggtt aaggatccgg cattgccgtg agctgtggtg31080taggtcacag acacggcttg gatttggcgt tgtcgtggct gtgacataga ccagtgttta31140tagttccgat ttgaccccta gcccaggaac ctctgtatgc cactggtgag gacctaaaaa31200aaaaaaaaaa aagagagaga gggagaccat gttataccca ttttagttta tgtatagctt31260tttttttttt aagcaatgatttccacccat aggatcctaa gaatcttaag acmgcactga31320gggttgtcag tcacttgattctatcctgaa accaaagttc agaatcattt cacttaacaa31380agataaagaa aatggctaaccaagctcatc ttttggccac tgccctaact tttccttctc31440agatcccatc aattctccag agacaggcaa gatgctgcat cctgcctggt ctaggagtcc31500cagcccatgg tgtagtaaccattgtaatgt tcttctcacc atttattcag gtcatccacc31560gcgatcccctcatcacttcc tcttagaatc ttcactggat taatccactt31620tgttccctta ctatccctttcttttcttcg taggtctgta taccatgcat actaaccaaa31680atataacacc taatytatttttggcagctt cttgcctagt cctctctgta agtcagttgt31740tcacaaaata tgatgctttctctgaaatgg ttttagggaa cactacctag tgttagaaaa31800ccagatgtca aggttctgga ggatccttga ggtccaaatt atttccttat tcctcagaat31860tagaagatgc cctctctctctctgcacaga ctcatccacc tctgatcttt cctgtttgac31920aagtgcttgg ctccaaaattttagcaaaaa actttccaga gacataagaa aatatgtact31980aacagaaatt gacagaacattgtaaatcaa ctgtaacaaa aaaagaaaaa gaaaaagaaa32040gaaaatatgt atttgtttctagctgattta agggtgtgcc atggagtaag acagctgaag32100
aatagaaagt gttcaggaga ggaggcatga atatttaaaa gcatgtttct gggtgctgtt32160tgtttgttgt cattctgtgtatttttaacg tttttctact ccattgcagg ttccaggaac32220ccttgccagc aagattcctg taagggtggt tcttcatgtg ttagtctgaa taatacgttt32280ttttgcctgt gttcagaagg gtattactat aactcttcca cctgcaataa aggtgagtta32340
gaagggatga atcaggtaga gctgcatctc agacctaaaa tagaagagaa aacttaaact32400aatagcaaga agaacttcta tagtattaga ggcagatttg gaggcaacaa agcccaggag32460agtacagaaa agagtcccgtcttatttcag tgcaaactga tgggagagga aatggactaa32520gtcagatttg tgagctagtcctaaaggtgt gcgtgtggat gggtcccttc acctgacctg32580ttcaagggaa tggtgccatg ggatcaaagt cagaatgata ggtgttgacg tccctggaca32640ggtacaaagg tgtataaata ttaagtgatg ctgttacatg gggacactga tgcagaatta32700aggtgatttc ctaaaaccacacagaccagc cggaaacctg gtctctgacc ttggttcagt32760tctctgtcct aagatttgca taggaaatga aagcattgta ttggccacaa ggattagata32820tcccaaaagc caaaatagatgttcatrttt ttagcactaa taatctcatt ttggggatcc32880acactaagtc aatgacccta aatacgggaa gttttatgca cgaagaggtt cattgcagtt32940ttttctcttt tragtagtta aatgcactga aaacaagaaa tgactgcatt ggagytcctt33000ggtggctcag caggttaaag atctgttgtc actgctgtgg ctcaggtcra tgcagtggtg33060crggttcagt ccctggtctg ggaatttcct cttrttatgg gtgtagccaa aagagaaaaa33120atgaaacaaa acaaaayaaa aatgacttca tcgataaagg aaacatgtac ttcctagaat33180gttttgtagc atttaaaatg ataattatga agtctaagta atgatttggg aaatgcttat33240gtaatatacr aagacatgatataaacttgg arttataatg atgtaaacta gggacacaaa33300aaagctggga gaaatatgccaaaatgagga tagttgtggt aggttaggtg rgggttgggg33360tcatgttttt cmtattcmca gacttcctgt agtatcatca gacatttaga atttttaaaa33420aattatctgt tttaaagaaa agagaaatgc ctctcttcac attaaatatc acagaaagat33480tgtgtttgaa tttcaggaaa gattttccct ggtaaaatta cggtcaaaat atcagaaaca33540actgatctag aacatgaaaa ttctgtgacc tatcaaaatt tataccttca agttacaaat33600tttgtaagta ttttccaaccatccttccta tacaaatatt ctttatatat atatatataa33660tgatttctak ttttttccca ttatagctgg tttacagtgt tctgtcaatt ttctgctgta33720cagcatggtg acccagttacacatacacgc atacgttctt ttttctcaca ttatcatgct33780ccatcataag tggccagaca tagttcccag tgttacacag aagtagctta ttgcttatcc33840attccaaagg caatagtctg tgtctgttaa ccccaagcac ccatccatcc tactccctcc33900cctgatacta tatatagaaa accctaagga ctcaacccag aaactacctg aactgatcaa33960caaattcaga aaagtagtag ggtataagat taatattcat aaatcagtca catttctgta34020tactaacaat gaaatattag aaaaggaata taaaaatata atacctttta aaattgcacc34080ccaaaaaatc aaatacctgg ccaagaacrt aaaagactta tatgctgaga gctataaaac34140atgaatcaag gaaatcaaataagatgtgaa gaaatggaaa gctattccrt gctcctgggt34200tggaaaaatt aatatygtaa aaatggccat actacccaaa gcaatctaca gatccaatgc34260atccctatca aattacccatgayatttttc mcagaactag rrsraacaaa caatccaaaa34320acttatgtgg aaccacaaaa gaccyagaat taccaaggca attccgarga acaaaaacca34380agkaggaggc ataactctcccagacttcag gcaataaata ttctttaact gacttgttag34440
actgatattc ggrtggtaca ggctggtacc agtgattcam ataggtgaaa tgcatcctta34500ttggttggtg aagagcagctgttccaaact cttgggtgcy gtggcttcct ggttgcctag34560actccacctc tgggaccctg ggacctcccc rtgatcctac acctagagtg taacacccag34620cactgcagag agcctttaga agcctgctat agccatgcta tacttgtttt tatggaggct34680attaaaatat ttttkaatgg acagataaat taattatact trrraataag ttatttccat34740agatttgaag agaaaaaaattatttgatta ctcaatgaat ttattrcatt tatagttgat34800catcacaatc cagttttata gtgtttccat cccayacccc cagcgcatcc ccccaccctc34860caacctctct cctttggaaa ccataagttt ttcaaagtct gtgagtcagt acctattctg34920caaagaagtt ccttgygtccttttttcaga ttcccgamct gtcagtgaaa gcattcaatg34980ttggtgtctc attgtctgactgacgtcact yagcatgatc atttctaggt ccgtccatgt35040tgstwaaaat gccggtatttcattcctttt aatggctgag taatattcca ttgtgtatat35100gtaccacmtc ttctkgatccactcctctgt ygatggacat ttaggttgtt yccatgtctt35160ggctattgca aacagtgctg caatgaacat cggagtacat gtgtcttttt gagtcatggt35220tttctctgga tagatgccca ggagtgggat tgctggatca aatggtagtt ctatgtttag35280ttttctgagg aatctcccca ctgttttcca cagtggttgc accaatttac aatcccacca35340agagtgtact agggttcctttttctccaca ccctctccag cacttattgt ttgtagactt35400tttgatgatg gccattctgrcyrgtgtaag gtggtacctc atagtggttt tgatttgcat35460tttgaagaga aaaaaattta aaggtgttgt tttttccatt ttaccatcca atttgcatac35520atttctagac tagtccagcttttttaatgt cygaaggaac tccttctaaa ttaacatccg35580ttacttcttt ttcccattgtcaaaaatgtc caaatgagca acagcttcat ttctttgtga35640ttattgtgag tgaatrtgtcgatgtgagag aggtggagat ctctattcta aaaccacagc35700tgtcagacag gaaggcctta gagaaggtgt cycatcrgtg tttctcaaar tttagttttg35760aaggaaaaaa tgttagcttg gaaatatgtc taagcacata attctaggtg aagaaatgct35820ttttaaaack rrsatcttactctatgatac tgcctttccg tttgacatta tgtcattgta35880aagagtttta ttgmtagaattggctaaaag tgaaaartta ttatccaaat ggtcttagag35940attgtacata taaagtgagtgctcacaacc aaacagccag tgcctgacac tgctctgata36000aaacccatgt ccactgcatcccggacttgc cctggcccct ttgctgttyc acctgcacat36060gaggggcrca tggccatgacttagcytctt accctgggtc tgcattccag ttccccactc36120agaagatatt cagtacattg aagatagcaa cacratgttc ctgtctccag ggaacccctc36180aattgtctta tgtracttccccaakccctg gtccctggag ccactgcatt tcccaacttg36240ctttggatct gaatgtccaa agcccygtct ggcttcaggc caacgccctg gtcttcggag36300ataagtgtag caacytgatctcagatagga cttgatggtc ccaggggtac cagggctggg36360atggaacact ttcataatttctgagagtct ttgaatctga aagggatctg ccagaaygcc36420tttgcagctc ctagagagag gaaaagagag acaattgact ttcttcaagg ycccctttgc36480agactctgta gctgtggcag tgccagctca ggccctgagg ggtctcccag ggcccatttt36540ctccccawgc agagccaaatggacagtcca catagggakt ttttttttaa tgagtcatat36600tggagtatag ttgacttaca aagagttaat ttcaggtata cagcaaagtg aatcagccat36660acatatacat atacatatatatccattcct tttcagattc tttccccaaa taggtcacca36720cagggtactg agtaaattccctgtgctgta cagtagatcc tggttactta ctgattttgt36780
acatagtagc gtgtctgggccaatcgcaac ctcccagttt atccctcccc tcaatgtttc 36840cccataatca gagggatttg aagtttccct gttgcatgcc atacctggca ttgcatgtgt36900catatttcta attctcaaaa taggcacctg ggggagacgg tactttgata caggtttgca36960ggtgaggtag gtagtcaagg atcaggaagg ctatgtaact gaataactta cggaaggtca37020cccagctgac gaatgaggatggggagatgg gagcccagga cctgcctgac cacagagccg37080cttctycttc tctcctgctg gctctcttgt gccatggact ccagacaggc ctgtttggat37140agttcctgcc ctcctgtcca accctgcagc ccttcacatt gaaaggtgta tagccttaaa37200tcctcagagg gtkacctgga agagggggaa aaaaaggaaa tcttttcttc cctgtgcccg37260atgctaattt trcaatcccttctttggaat taagaaaact aaatgggcaa tggtcagtag37320gsgagctgta aggaaaacttggggtggaac ttgttccagt taatttgttc ctaagactcc37380cagggccctg agggtttata ggaataccag ctttccctga tattkaaaag aaktaaaatg37440aatatggaca tattctggatacaaggctkc ccagactaac taaatttatt tgaatttgac37500taaaataaaa aagaaatgatttgatatctt tttaaatgac attggctgat cttctaaaat37560rtggggtcct tgtgggagaa aaataataaa attcttgggt tataaaaaat ttgcgccaac37620catgttcaac attgacataa taaatatggr gaccakggcc caaagagaag acatttccaa37680agtcatgtct ttcatagtta attcattcag tcatcagtca actggtacct gttgagtgtt37740tcatggggat tgccctgtgctcagtgctat gggtgggaat gggtgacaga caggtgtatg37800catgtggggg gtgtgtcatg agaaagagga aattaactcc agcctgacat caagaaactt37860atgttccaac tgggacccctgccctacctc cacctcaccc ctactagaag tcccagctcc37920gtctctggcc atgtctttataagtatctag agttgtctct gccttttttt ttagggccac37980acttgcagca tacggaagatcccttgctag gggtcgaatc ggagctgtca ctgccagcct38040acaccacaga cacagcaaca ctagatccaa gccacatctg caacctacac tgcagctcac38100agcatygtca gatccttaacccactgatca aggccaggga tcgaactctt atcttcatgg38160ayactagtca ggtccwctaccaccaagcca caatgggaac tcccaagttg tctctgctct38220tgaatacaac tcaatcccaa taagatcaac ttgtgctgga gcccttccca gttcccattt38280cacaaagcca tgtatcatatataccatctc atttcctaca tcgtatagcc ctatgtggtg38340ggaattactg atctctcaga ttttccaaga ggaagtgaag cccaaagaag ttaaatttwg38400carcaaagat gacaagttgtgtaatgtgac actccagcct gctaccttcy gagtggcact38460gtttcccagt gttggtgtgg gtggctcctt ctwctgcttc ctggccccac atgaytctcg38520aaatgcttta agtcccatctcctttatgaa gccctctcyg gtttkctaca actggaagtg38580agtttctttg aacttccaca ttagattgta ctctctgtgg cacttacctc tctcagctcc38640atggtgtaac ctcctatttg atcacacgct gggtctgccc atcttggtta tctctatgtt38700tctggcatga tgcctcagca aggtaggcaa ctgagtcttc ctttggtaaa ctgatgagcg38760tatgaagtac gcrtaaagataaattaacaa atgtgcagaa cttcacagtg gctttgaaaa38820gacaaaagtt agtcacatta tttttaagac tgtgatcatt gatttggctg agtgctttga38880gtttccccct tcttcctggg gagctctgca ttgtatcagg atcktactac tttgaagcaa38940aaaaatactg aacgttktta gcgtgtgtgt gaagagtaat ttgctgtwaw awaaawgtta39000atatrwawaw waatgwwaaw awrwawawrw awawwrwwaw awaaatgttt agaaatarat39060ttatgttaaa attaaaatatagcatcacgt ttccccctct ttgtagttac taaatctgtt39120
cttcttctcc acagtttaatgacacattta gcaattctga gtacggacag accgtaattc39180ttaaagtaag gtaagtttactgtggaccga tgrgktactt tggaagcagg tgtgagtttg39240cctaacttcc tgtcttctcttgcattcccc atgcagcacc aaggccctag ggtggctgct39300cacatgttaa gaagaagtag tcagtcagag gactgcaggg gsccctgcct ctgatgtcac39360attgacccca gcctgggtgg gtggggtgaa gcctgggaca gcaagttgct gtctgtaatt39420ggctgctcag cgacttacacacatggtcta gggcatcacc tggatcggtt tggttggttt39480
gatcccttgg ctacaaacca cacctagtcc acccctggaa tgtgcattcc aggaggtcag39540gaggcgaatg aatggctcacggccatctct tccctcatag cggggagtat ggcagacgac39600aggaaggtat cagtcatgca cctggctact gaaggccctg tcgtgagctc tttctgccat39660gagagtaagc atgcaatgag cccagcaaca ttcagtgacc aggactgcat gctatttggg39720gacccaaaaa ggcagaggactctcaagaag tcagtgattc catctcagcc trccctgaag39780casattgata cacaagggctgcagtctgag gaaagaatgt cctaggatrc atggagtcat39840ctagtactgc agccctaaca tttctgtttt acggttttac tactgcatct ctctttgtaa39900actgtgtcga atcctattgtggaaaaagat aatctgtata tacttagata tagtatatat39960ctatatasag aaggcctcctaaatatggtg agytttgatc ttgrtgrtga asgaggggca40020cttgtgattg ggagccagga ggccagcakg gagcgtartg ggatgcacag akcaaacatc40080aggcagggag gctgggctctgactgcagag gctggaagcg ggagggatag cagaccactg40140tcatgtagtg tttaaggacg ttagtttggt tgaggggacg acagggtgtt catacatcgg40200gttgaccagg taaaaacagg aatggttagg tgtgggtgtg tccgctaaat ctgtacttga40260atcctggcct cagcattttcttgctgtggg accgggcagg cattttcacc tctctgaact40320ttcgatacct cttctgcatg actgggatag tctgatcaac tttcccggaa ccctgggaga40380agcgaatgag ataccattga aaagatgccc tgtgtaaagt ttctggcggg tagtcagttt40440cagtatgtgt tcatttccatcagtttccct tccagaatac cagatcctac aagtgtgatg40500gagagctggt cccacatggtcacttgggga gccggggggg ggggggtcct taaaatagtg40560tccctacttt cattcttaag gagttactac tgaaaggacg cccatggagt ttggaagcaa40620ccccttgcct gtgagctcag ggtcctggag aacaccacct tcccccagct tgggggctga40680ggagggcaac gagacctcag tctcacctca gaagacattt ctttgactcc agaccagtgt40740cctctcctct gactccaaag ctctttattt acttcccaaa tgctgcaatg ggaaggatgc40800tcagaaattc tctaaccttcaaatctctct ctggacagag gaggacactc agactgcggg40860aagctgggac gggtcttggg tcacggaagg tggtgaccat cctgggttct taactcccag40920gctatgctct agccatttacactgtacctc tttgtcactt agtacacagc tgctccatcc40980tttccctgaa caattctgtg attctaacgt cttgcctctt gaatcagagt atatgttagt41040aagttcttca aacgaatgaa tgatggacgg catttctttc gtagccccac tgcagaatta41100taccatagat ctctagataa agaatagctt atgtgaaata cccctttctc actgccctgt41160acattccact accacctcca aatcccatca tgaacaagtc atgataggaa gcttgaaatt41220agaattcagc cctaaagaga ccaaaagcag aaacaaagca cacggccaca aatcagagaa41280ggaagggggt tttgttttgttttttttgtt tgtttgtttt tggtcgtttg tctttctagg41340gccacacccg cagcatatgg aggttcccag gctaggggtt gaatcagagc tacagctgcc41400ggcctatacc acagccacag caatgcggga tctgagccaa gcctacaacc tacaccacag41460
ctcatggcaa tgctggatccttaacctgct gagcaaggcc agggattgaa cccacaacct41520tatggttcct agtcggatttatttccgctg agccatgatg ggaactccag gagttctctt41580ctttaagccc tttctctctctctctctctc tctctctctc actcactcag tggttggatc41640atttgactcc ttttggataa tgaaagaatt agaacttggc tggtcctagc gccctgcttt41700gcagtgctga ttagagagag actcctataa tgtcttgytc atcacaagcc attagtgtct41760ggtggtgtga tttggtatca gatgctgatg ttcaccacag ccccatcgct ccttggaagg41820cagctatgct taccatgtaccaccaacacc ccatccctcc ttggaatcaa ctctcctctg41880
ggtcaggtgg gcccagaccccatcatacaa gcattttgaa gctgcttgct ggcttcctag41940gccctttctg ggagtgacccagacaccata tggtcagctg ccgccctctc tgtgcccaaa42000gacaaacacg cacctcccaa cccccatctc tctcaaagca caagtctggc attttacagc42060tcatccagag acctattcttggaagttccc tggtggctta gcagttaagg rtccagcgtt42120gtctcttgat tttctgcaga aagtgacaac ttactatttc agatgtcatt tattctactt42180ctggttcact ccaggctcctaggtcctaaa aataagcatc catgattttc ctaagtgtcc42240tccaacagga ccttgttgta cccctagatt tcctcagaat gcattctagg ccagagatct42300ctaggcctat aaatgtccttcttatggcaa ccccaagcct ccctgggatt tgggcctcaa42360ctgtggctat agaacagtgg gttgggacaa gcgtggggag attggagagt ctgggaggtc42420tgaagatcaa tttgttgatg cttcaattca acttaagtga tgggttcatt tccattttta42480gcacaactcc ttcagcaaga tatgaaatgc gtgccagcaa ccagcttgtt gaggtagaag42540tagtaaacat ttttagagaa actacaacag aaaatgagag ttccatattt aatgccattg42600accaagcaat taaaagcaacccaagtgact ttacagaata tatccgtaag tactaaatca42660ttttattcaa catttacctg aagatatgtc tacctttttg tgaacttttc tattatatgt42720gtgaaagagg ccagaagaactgaatcactg aatccacaga tgtctctgag tttcctttga42780acccgccctt tcctctctta attggcttct gctcctttat aaccattctc caagccgtca42840gactgactag tttgttcctcttcttggaag gttgtcttaa taaacaacct ctggacaggg42900aaagatgaag aaaagagaaa caagggggct ctatgctccg ttatcacttt ccacatgctt42960atagaaagtt ttcctaggctgttttccttt taggcagaca cacaagtcca cagcagagct43020gtaacaattc attttgctgtgttaccaata ccccaaaact tagtggccca aaacattaac43080catctgttta ttacaataccataattctct gggtcaggat ttgggctggg ctcagctggg43140tggttctgct gatctaggca gggcttggct gatctctgct gagctcactt aagctgctgg43200gtgggtccac tggaggtgaa tgatctcaga tgctcacctg gatagtttgt ctctgctcca43260cacagtatct catcctctag caggttagtc caggtttcca aggtggcaag agggtgcgtt43320tcaattcaca ggtgtttctcactctccgct tgtgtcacat ttacaactgt cttcatggcc43380ggagcaattc acatagctga gcactgtgtg gcaggcccca ccacggggca tggatacagg43440aaggtaggaa aaaattgacg tgattagcacgcagtcatcg ggagtttctc43500ttctcagagg taccttggccagccctgggg tcggttctgt gcaactggtt atcagtcctg43560gttgccaaac ccaaaggcctctgttgagac acaatttatc tgacagttct gtagcgtttg43620cactggtacc ctccccttcctaaagtcctc ttctctctgg ccctccatcc accattctcc43680tgatcccctt catcccgcctccctcatcct tcattttgag acttccaaga ctcctgcttt43740accctctcac gttttgctttttgatgattg taacctccat gtcctcattt gcaaaacata43800
ccactctttc taaaaataca cttaaatcct gtttgtatat agtgttcaaa catctttctc43860tctttctaag atgtcacaga gattctcacg gttctctcag gaacttggaa tataaatgct43920ctggaataaa acatgatgctgccagaaaag atggggctct cctgcaccat cccctctccg43980tggctgtggt ccccttagca atgtccatcc aagtatcctt tcctggcaga agagactgga44040 gcaaaatagt tgcagagcca ttcgattttt ctcttgtcat tcatattact ccagccaggc44100ctgacctttc catgagcccctgtagcatagcaccccttca ttcggccatt44160tttcttttct ttctcacttttcatgtttaa ctctttgctc attgccatca gattttcttc44220ttcttgttat tgttttcggttttgtttgtg gcaaacctat gctcatttgg actctagacg44280gtctctgtca ttcttttaatattccatctt tggttaagag accttctttg catattctgt44340ctatatctct cacctgagag tttcctgtgc gaaccatgcc agtttcttaa attaactcac44400cctctcccat ggtttttttg tagttcattg ggaccatatg cagctgcaca gtgagaatta44460cattttcaga gcttcttactgttttaacct attttcatct tcctgagtct catgccatgg44520aactgtacct taccctttctgaacacttca aaatgtatct ctcaaagtca tacttctcta44580tctggctatc acttggtccttatcttcaaa aggacccatc gcttctgtct gccaatatat44640ttcttcttgg ggtcataatttgctccagag tagtaattca tgtagtcaga acctctgccc44700cctggaagat gaaactgtca atgaggcaga caaaacctta tcatacgctc cgtttggggc44760tggatgagac ttctaacaag tgtgaggaag ttgagtgtcc ccgtcagcat ggtcaccaga44820tgaaaacaac tggtgcattcttcaagavaa agttggggtt ccctttgtaa ttctctcatt44880tgactctact tagtaaccaccttggctctg agctttggca gagttgaaac catgtggcat44940cagcactgag aagtgctctttttttttttt tttgatatct cctcttaaca cgtctctact45000ctgaggccag gcatgtgctctcagatacat taacaaattc ctgaatgatg ccaactagag45060gtggccacaa gagcatgaatcacccagcaa atagaagtga gagcttagga cttttagaga45120aaaacacctt ttgctgctctcttgtttctg ctgcaaatac ggactctctc ctttgtgtca45180aaagctgtag aatctctttctctaggtttt tacttggagg catttttaaa tggcaggtaa45240tgaaggaagg ttagttacag ctttgggatg tttagaagag agcaaccaca tctctgttct45300acctgctcct gctctctgctcagattcatc ttttgtccct aatgccttcc attatgcttc45360tacactattt ttaaaagatg gctttctmgt gccatgtttc tgctcagaat ccaccaattg45420attcttaggc ttacaggata aaagtcagtg ctcttagcct ggacccaaac cctgctatac45480caacgaatga acaagagaatgattatttgt tgagtttcct gtagtggaca cttgcattta45540tatgagctca ttcagttcta acartaatga tgtgaaacaa gaatcattag ccccatttca45600tagagggagg gaacttggacctaccacaaa tcaaataatt gaacaaaaag cccacagcca45660atggggacgg caaagccaag aagtctcagt gttcatcccc ctcattgcat tgcctctccc45720ctgcccaagg acaactccatcactcctgga caggaatcct ttacttcttt ccataccttg45780tttattccac ataggcagactctgcctccc ctttgactgt cacatcttcc ttatctttca45840agacttagcc caattcctgcctcctccaca atgttacctt taggtagctg agcccctgaa45900ttcccattgt acttaaagtctgtgttacaa gttgctggat gcaactggct atgggaacca45960acacgtaact ctccaaagatggggcccaaa acttaaccaa gtactgtgtc ccttgtggaa46020cctagtcaag agctaggttcagaagagatc ttgaacttga tcgtaattgc gttgcactgg46080attgaattca tttgaattgg ataatgtaag gaaattgttc tagaagagat gattggggag46140[1031]ttgagtttca aaagcccaga tttctattgg ttgaccccca agacatgtta aaaggcaacc46200gtaacatcag tggaatgggg tgctttcaca agctaccctg tagtgtcttg ctaaatacgt46260ctttgaattc tttgtgcctctcagagcaag atcggtgtga ttattatgga tgtaagaggc46320aaaatgagca agatgagtgccccagtggtt tactgtgtga gtgcaaagag gggctggaga46380gaccaaaccc acagatccctctgtgtgttg gtaagtgttc cacctgcttc tgaatggtcc46440tcacacctcc tgccttttctccattcacat ttgcaaagga ctcattttct ggctcaagtt46500 [1037]tccatgtaca tttcagagtctgagggatgt gaattttggc tataggcctg tggtcctgga46560tggggcgtgg gctaaggagctggtgaggag gcccatgtgc tgcctcacag gggacaagtc46620acatgcagag ccatggagcg cctcattcta cccagaaaaa gcaatgtgaa aaaattgtat46680tctgtgacat gtcattttgg ggttgaacgg gttgaactgg tcgtttctga aaattaccca46740gaaatcgctc tgttttgaag ctttgggtcc agcatgctct gatacctgca atgcagagaa46800caataggcaa tgcctcgtga ggagcgctgg gaacgccgaa tgcttgtgcc tgcctggcta46860caaggaggat aaaaacaaga tctgccaagc gtaagagaaa tcactccttt ttcatcttgt46920catagatgaa gctgagagag aagttagcac gtgtcacata catatgggtt tagggtcagg46980aagctggtac gctggcatccgttaggaagt tgcaaagcgt caccaccttc atcatcacca47040ttcccatcac catcgggttttgaaccttct ctatgtgctg ggaaccaccc aagcaatact47100tgtcacacag cagcactgtcgggcctgtgt tcgttccctg tcttcaggtg aggaagctga47160agttcggaga atataaacattcccagatca cagggcagtg aataagtagc agagccatgt47220ggcctcacag gctgaggggg tcccacaatg ctccatgcct ctggggctct gctccgccca47280aactgctgaa tgttcctgatgctcggaggg yggagcgttc agtcagtagc aattggaacc47340acaggagatt ggggagcacctccctcaacc ttccggaggc cgtgaaaggt actcccctga47400agaatgggag tgtaaggaag aaaagagaaa gaggagatgg aacagggagg agagagtgaa47460agaaagatgc aaggatggca agttcccatt gtggcacagt ggaaacaatc caactaatat47520gcatgagaat gcgggttcca tccctggcct tgctcagtgg tttaaggatc tggcattgcc47580cggagctgtg gtgtaggtcg cagactcagc tcagatcttg agtggctgtg gctgtggctg47640tggttgtggc tgtggcatag gctggcagct gtagccctgg tttgactcct agcctgggaa47700tttccatatg ctacaagtgcagccgtaaaa aaaaaaaaaa aaaaaagaaa agaaaaatgc47760caggatgaca tggaggttgg gtgggacctc tgatgtgctc ctccaaggct ctttaccatg47820gtttccttct cattgcattttaggtgtcca tttggctaca gtggagttgg ctgtgaaaac47880cgtgagtaga aaagtcttgctctgttattc agcttctgaa aaccatggac cacattgaga47940attctaccca gtcccccaaa ggccctggct tgtttcataa agagagagaa aatctgcctg48000tagtttgcct tctgggtcag ccatcccatg tccaccctta ggtcccagac agagcccctt48060cagtgtagga aataacagaa gtccacccaa ggggtgcact ggtgcctctg gacttgtctc48120aggccaaaga agtttgtcccaacattatat ttgtttccta cgttagcttc aatctgcatt48180gtctgccact ggaaaggata caagactrta ttccaaaact gtgttcatgg gcgagcagaa48240ctagaaactg gacttttaactgctcaagtt ttctttttgt ctcagagctc tttgctagtt48300tgggatgtgt tattgcaaattcagttgctt tgttccctcc tgccagattg ggacaatact48360aacttcatat tttacattattctttttttt tttttttrtc tttgtccttt ttagggctgc48420accctcggca tatggaggttcccaggctag gggtctaatc agagctgtac ccaccggcct48480[1070]aagccacagc cacagcaaca ccagatctga gctgcgtctg cgacctacac cacagctcac48540 [1071]ggcaacgccg gatccttaatccgctgagcg agaccaggga ttgaacctgc aacctcctgg48600ttcctagtcg gatttgtkrrstaaccactg agccacgaca ggcactcctt aaattattct48660atatttaaaa ttgtacgtaa cgcaccaaaa gatcaatgac ccaaaatgaa cgaaatattc48720ctattcacct tccagccagccacaaagtta gtgatgagaa atgytttcag tttccgctgt48780attttttttt tttcttttttggccgcccca cagcgtatgg agttccctgg gccaaggatc48840agatccaagc tgcagtatccacttacgctg cagctgcagc aatgctggat ccttaaccct48900cctgcatccc agtgctcccg acacactacc tctcccttcr cgccacttca ggaactccaa48960tttccactgt attttgtttcaaggctgggt ttgctaagtc atttcttcct tctggctttc49020agtgtccaac ctataaagtg agaggtttgg atcagataac rtttgaagtt tttccagacg49080gaacagtctg tggcccatga acccaggtta ggggtggcat acgtggcagg catgtagcag49140aagaatcaag gggatctaga gtcgtgctgt ccaatagaaa tatgacgtga acttcagatt49200tcatttcaca tttaaaagttctaagcaaaa ttttaaaggt acagagaaat agatgaaatt49260gatttttaac attttgtttcattcartaca ttgaaaatag tatcatttca acaggtaatc49320aatataagaa tattaataag gggagttccc gtcatggcgc agtggttaac aaatctgact49380acggacyatg gggttgaaga ttcgatccct ggccttgctc agtgggttaa ggatccggtg49440ttgccgtgag ctgtggtata ggtcgcagac gcggcttgga tcccaagttg ctgtggctct49500ggcgcaggct ggcagctaca gctccaatta aacccctagc ctgggaacct ccatatgctg49560tgggaacgrc cctagaaaag gcaaaaagac caaaaaaaaa aaaaaagaag agaatattaa49620taagatattt tacatttttttcatactatg tgtttggaat cactgcgtat gttacgcttc49680caggccatcc cacagccaca tttcgagtgc tcagtaacat tagagcagat ctagaggaaa49740aagggtggta atgaacctccagacttttcc ctccccaatt ccatctggac atcacccggc49800tcctctatca gggacaaatttaactcccag ccaggtcccc tggtttatcg gcaaggtcag49860ttgtggcagg cargtgagag cagcagaagc agggagatgg catggacctc ggaygagatc49920ccatccccat gaagcccagg aaggtcctgc caccctggcc tactttgtgt ttaggtcctt49980gattatctgg caaggctgcctcttctgaac acacagttgg gggtgagcag acagagggtt50040agagaaggtg agccacccacctatcctggc cctggagacc agagcctgat ggtcctcagg50100gccaggagcc actaggaacg cgtgtacctg tgcagtagac agtggaggtc tgcttggagg50160aggctgttcc tactcttctcttctcccttc cagcatatca gctgatcctc accatcgtgg50220gcaccatcgc tggcatcctcattctcagcc tggtgattgc attcgtcctc tcattaaggt50280atggaatggt gtctctttcctgggacacag cgycactggc ctcacactct ggttcgcaaa50340cacacccaag ccttttccca ggcaagagga gaaaaataag gcgaggcctc tctagcacca50400ttgccctttt ttcccgaccttaattttctc ctgattctat aagctacaca tagtccctga50460agaaaayagg gaaacgtgca aagaaacaga ataaaaaatc acccacaaaa tccactaccc50520tgaagcaacc aacattttgg catatatctt tccaggtttt ttcctacctt ttaaaacaat50580attgtatatg taactttttttttcawtttg tgcctttttt tttcattttt tttaagtttt50640attgaggtag agtggattta caatgttgtg gtgatttctg ctttacaaat aaagtgattc50700tgttatgcat gtacccacatccattctctt tcagattctt ttcccacatg gattatcaca50760aaatattggg tcaagttccctgtgctatac agcaggttcc tgttggccaa tcattctata50820[1109]tacctcagtg agcaaaggccaatcccaaac cccccctctg ttccattccc tcccgcctcc50880[1110]ccctccccct cagcaaccacaagtctgttc tctaggtctg tgagtctgtt tctgtttttt50940gcagataggt tcatttgtgccatattttag attccacgta taagtgatat catatctgta51000tacatagttt ttatcttttg cgtttgtaaa atttgttgtc gaggtttgat tttgcttaat51060actgcaatgt gatgtagcag tcagttttct gatgtcatta aaaaactcta gttagcggta51120tttaactcca ctgatgttgtcaaatgtaat ttacaagtca ctcccctgct caagtccctc51180caaagactcttcaactgagc gccctgggct ttaagtagaa atgttatctc51240cgtgtggcca tttctcagcttgatgcctta aatgctctct ttaaaaactt aagtagggtc51300cattcattta ttcagcaaatatttcgatat aataaaattc cctccattaa gagggatgct51360atcatctatt attatctattatttattcta ttctaaatgt gccaagtgag gatctacttt51420gaccaaaaaa aaaaaaaaaa aagctacttt tggaattccc gtcatggcac agcatgttac51480caaatccgac taggaaccatgaggttgcag gttcaatccc tggccttgct cattgggtta51540aggatctcgc attgctgtgg ctgtggtgta ggccagcagc tacagccctg attagacccc51600tagcctgaga acctccatatgccatgggag cagccctaga aaaggcaaaa agacaaaaaa51660
aagctacttttaaaaaataa cattgagcac atatcttata ttttagctaa51720ataggttttt ttttcactataatatggaca aatagatttt aggttcctaa atatttgact51780tcctcttctt ttttttctctttaatccatt ttgatatgaa ctctccccag agacttgatt51840acaagggcac cacactggga actggaaagg caactttccc tttccagtca gagggccagg51900atgccaggct ctggagcagg tctgccctct tggcaggagg actgactcac tgccctggtc51960tccagggtcc aggggctgca gcagaacttg caagcccgtg acagccacag ttttcagtcc52020ccccttcctg ctgcagagccaggtttgctc ttaattcagc cttagggtca tgattcttgg52080gagcacaccc rcaattgtca tttcttgccc atttcaaagg gaaggaagcc tctcctcagt52140cacmctagat gggagtgttg tcacagcarg aaccctataa tgggctggrc tgaccatcag52200gtcaccgggt gaggggrtcttgacgacatt ccagtgtctg agagacacct gagamtactc52260tgtggccatc acccatgcctgtggccccac tgcccactct ccagggccag gaccatacct52320gggggccaga ggacagaaag aagggttaga ggatcagagt tacttggggg cagatgccty52380cctcacctgc aagtccttcyacttggcttt gaayrcttac wcttgagggc ccatagcaga52440acctgggaaa ctcctatttytccttttaag ayctttatwt cttgaattca ttttattcak52500ggagskgytt ctgtagatactgactttgcc mraatgaaaa atwygtcaca awtatyttct52560ttctttgact tttattatca tttccataag taattcaaag gcatacttaa raatactrac52620aatattagca aaatataaacaggactggtg gaattagagg gttttagttt tgatggtttc52680atggagtttc tcaggagagttagggaatct acactgaatg aagcaatgaa gtagaaccgg52740taatgggcat caggtctgca acttactctt aaatgrttca aaaatgttaa cagttgggga52800atctgggtga gagatgtatg ggagctctat gtaatttgca tgtgtgtgac ttacgtgtaa52860atttgaaatt atttcaacatttttaaacat tttgaaaaaa gcctattaac atcagtttca52920agggacttgc tgccattgtcagatcctaat tcctgtgact gattacagat
52980aaacaagaat gtggaagaacagaacttgat tgagaaygac tttcaaaacc tgaaactgca53040ggagactggc tttagcaacctaggagcagg aaacctcttt cctaagatca aggtggacat53100ttccagaggt ggtcagccacagaatcccta tataacccmg ggtagtatac cccgccctga53160[1148]ctattaggtg agtctttgggtgctttctgt ttgcttttgc tgagtgaaat gccccagagc53220agacttggaa cctgcctagtgtatcatcac cagccctggc tgccctgtgg gtttgggttt53280tgggttttkg ggggtttgtttttttgaatc aaaagttact ggagttyccg tcctggctca53340gtggttaaca aatccgactaggaaccatga ggttgcgggt ttgatccctg gcctcactca53400gtggggtaag gctccggcattgccgtgagc tgtggtgtag gttgcatact cggcttggat53460cccacgttgc tgtggctctggcgtaggccg tcggctacag ctctgattag accccttgtc53520tgagaaccgg ctacagctytgattagaccc cttgtytgag aacctccata tgctgtgggt 53580gcagctctwg aaaagacaaaaaaagacaaa agaaaaaaaa aaaaagttwt tgtaatascc53640mccgcttgat ccgtagcactagccatctct gtgactttar acacacaact taatttctct53700gaccatcgat ttcctcagctgtaaaaaggg aagcttacat taracaccct taaratctct53760ttacatcctc tgttcctatggctgtaacgc tggkggttgt gatgacaatt ccaaractgc53820tgaagggccc aggacctaggccagaaatgg gaaggactgt gggatgtaaa cacagttttg53880ttcacgtggw tcctttccagccascccttc taaagraaaa tctctccttt cttycctccc53940tcagacctca aggcaactatatcaatttct aaaagaacaa gacaagtgag gctggtaata54000ctttctttcc cctggaggam tttcaaggag catagcttgt cccgttagat ttgtcttgga54060cagtcatggc cagcaatgctgtatgacccg atggaatgct gaaagcagaa agcccatcag54120agagcactcc tgaaggaaagcgtgacaaga tgaaggcggg agagatggca aaggaggagg54180aggggccaga gaggagaggggggaaggaag agaggcgagg agagcagcga cttgtttgaa54240aaaggaaatg ctggcattccagtcgtggcg cagtggttaa cgaatccact aggaaccatg54300aggttgttgg ttcgatccctgtcctcgctc aatgggttaa ggatctggca ttgccgtgag54360ctgtggtgta gctcgcagacgtagctcgga tcccgtgttk ctgtggctgt ggtgtgtagg54420cctgcagctg tagctccaattagaccccta gcctggaaac ttctgtatgc catgggtgca54480gccctaaaag gacaaaagaaaaaaaagaaa aagaaaaaaa aaggaaatat tcagagaaaa54540ggaaagaagg agggccaggagtcaggcatt cccaggagag aagaaaaata aagagtctga54600aagaggaggg gacccagaagggtggtagag aggctgggga agccccccta ccccggcccc54660caacatccac actctctgtgtgcatatttc acttccatgg gctgccctca gcagatggat54720cgtcctttga ggatcttaatgggaaaggct gacttttatt ttgaaatccc agggttgatt54780ttagacattg tttcctttagtcatgaaatc taagtgttac atgtgtgcac aaagggatga54840cttgtaccac ctgggccatgtggggtcaga cacagagtct ggtagcagga gctgagagtg54900gatgtcctgt tgggtctacctgaggtgaac cttaaccatc aaactcattt cagacttccc54960ttctccacat cagtggagcccgtacttcag gtataacctc tgaagaaagc ccttgagtgg55020ctttcccatg cttataggatttagaattgg acagacctag gttcatagcc aggctcttcc55080tgccactagc tgtgtgatctccaatgagtt gtttgacttc tgtgggtttg ttttctcatc55140tgtgaaacgg acaaaatgaaacctacctca caggataaag atcaaataag caaatatgca55200aaaagaactc agcaccatgatgggttctaa tttaaataac agtattcaaa taatggtaat55260taaaataatg ttaatcatttatgaatttaa attttctcat aggcttctgt tggaatgttt55320ctaagagttt ctctctctctttctctcttt ttgtctagaa tggtaaaaat ttggaaccat55380ccatggccgt caacccagcacatgagattt ttgtgtgagg gtttgataga ctgatggaga55440aaagagatct ggcctttagtatttttcctc catccaacat ctggtctctc tgaatggaag55500[1187]ctgtaactgt ttgcattgaa ttcagcgtgt ttgaatctaa gtacctatga agttaaatgc55560acacataaat gctattagca cttgtaagat agttgatttg ctccagtaca gtggttaggt55620gtttgtttgt tttttcctgg tgggacaaca ataaccattt ccaaactaga gggaagctcc55680 [1190]ccaggattgt tcattcctgg aagagtctaa gtgttgcttt gagttgatag catgcagata55740agtgacctaa ttctcggaaa ttcttacaca tcagctgtgg aattccagta gacgggagga55800gacaccatcc ttaaaggtac tcagtggacc ctgaagattc attctctagc aagttaaaat55860aaggatgcta cttctggtcc tcaaatgtgg gaatttcatt acccttaaac tgcttgcgac55920ctggcttcac accaagagga cagaaaacaa gacatgtctc ccagctttca ggaaggagtt55980cctgggaatc caggaccttt gagatgttaa ttaaaagatg tggacttggg agttcccgtc56040gtggcgcagt ggttaacgaa tccgactagg aaccatgagg ttgcgggttc ggtccctgcc56100cttgctcagt gggttaatga tccggcgttg ccgtgagctg tggtgtaggt tgcagacgcg56160gctcggatcc tgcgttgctg tggatctggc gtaggccgga ggctacagct ccgattcgac56220ccctagcatg ggaacctcca tatgccgcgg gagtggccca agaaatagca56280aaaaaaaaaa aaaaaaaaaa gatgtggact tgagatgtct tccatgagga tacattctag56340agtctttgaa atgctggtgg gtgagaccaa gagtaggagc aacagaggca gaaggaagct56400gagctgggag gaagaaggta cccaagaccc accttcatcc tcagattccc ctccccaata56460aacaggtggg cactttctct aatgcatgcc ctacctatga ctcaagacct atgaaaggat56520gtgaagataa aaaccctgga gcaagagttt ggagtcactt tgaaattgta gacattctgt56580ctggctgatg gtggagaagt gatgcagatt ccagaggtac ttcaccaaag tctcagcctt56640ccctggggac agaggcttag cagtggccac caacacacac acgcacatac acagccaaga56700atggggcagc cacctgccgc acaagctt56728SEQ ID Ν03<210>3<211>1796<212>PRT<400>3MKVFIHFTLL ALLFLKSGKE HLHIFNSFRH ISSAIIYTVF CLLKLCLSAS MAQNKTGGTT60KLTSTENTSS PNMESTSAP SMRNSAPGT MSTSAPSM ASTSAPMGA STSAPMGAS120TSAPSVMST SAPMGASTS APSMSTSAP MGASTSAPA AMSTSAPAP MSTSAAMS180STSAPAVMS TSAPAPMST SAPSAMSTS APMGASTSA PDPTASTSAQ MEASTSAPS240AMSTSAPM GASTSAPSVA ASTSTPMGA STSGPMTAS TSAPATGAST SAPSVMSTS300TPMGASTSG PAAMSTSAP MGASTSAPS VMSTSTPM GASTSGPAM ASTSAPATGA360STSGPAAMS TSAPAVGAST SGPMSASTS ALSEAESTSS PMGASTSAP SVMSTSAPA420AMSTSAPSA ASTSSPDPTA STSAPMGAS TSAPSAMST SAPMGASTS APSAMSTSS480PMGASTSAP SMSTSAPSV MSTSAPAPV ASTSAPAPGA STSAPSMST SAPATGASTS540APSAMSTSA PSTMSTSAP MGASTSAPA PMSTSAPAP MSTSAPAPA ASTSAPAIGA600STSAPSMST SAPSMSTSA PMGASTSAP SVMSTSAPA PMSTSAPM GASTSAPSM660STSAPMGAS TSAPAAMST STPDPTASTS APMGASTSA PAPMSPSAP MGTSTSGPA720PMSTSAPM MSTSPLDPT ASTSTPMGA STSAPSAMS TSAPSMSTS APSMSTSAP780[1226]MGASTSAPA VEASTSAPAP MSTSAPMG ASTSAPSMS TSAPMGAST SAPAAMSTS840APAPTASTSA PMVASTSM APMTTSALA AVASTNPPM GASTSSPMG ASTSAPSVM900STSAPAPVAS TSAPMGAST SAPSMSTSA PMGASTSAP APGASTSAPA PMSTSAPSA960ASTSAPAIGA STSGPAPMS TSAPAAMST SAPDPTASTS TPMGASTSA PSAMSTSAP1020 [1230]APMSTSAPS MSTSALDPT ASTSAPSMS TSAPMGAST SAPAPMSTS TPMGASTSA1080PSMSTSAPA AGASTSAPAP TASTSAPMV ASTSAMPTA STSAPAAMS TSTLAPTAST1140SAPAPMSTS APSMSTSAP AAMSTSAPA PMSTSAPAG MSTSAPMG ASTSAPSMS1200TSAPAAMST SAPMGASTS APAAMSTSA PAPMSTSAP MVASTSAPA PGASTSAPSV1260ASTSAPAPM STSSPMGAS TSAPSMSTS APVPMSTSS PMGASTSAP APMSTSSPA1320AGASTSAPAA GASTSAPAAG ASTSAPAPM STSSPMGAS TSAPAPAAST SAAAAGASTS1380APSAESTSAP MGASTSAPA AGASTSAPAA GASTSAPSAA STSAPAAMS TTPASAANAS1440SAISSTPIAS TPTSGSRNPC QQDSCKGGSS CVSLNNTFFC LCSBGYYYNS STCMGKIFP1500GKITVKISET TDLEHENSVT YQNLYLQVTN FFNDTFSNSE YGQIYILKVS TTPSARYEMR1560ASNQLVEVEV VNIFRETTTE NESSIFNAID QAIKSNPSDF TEYIQQDRCD YYGCKRQNEQ1620DECPSGLLCE CKEGLERPNP QIPLCVALGP ACSDTCMEN NRQCLVRSAG NAECLCLPGY1680KEDKNKICQA CPFGYSGVGC ENPYQLILTI VGTIAGILIL SLVIAFVLSL RSNNKNKNVE1740EQNLIENDFQ NLKLQETGFS NLGAGNLFPK IKVDISRGGQ PQNPYITXGS IPRPDY179權利要求
一個決定仔豬F4ac腹瀉易感性/抗性的MUC13基因，其特征在于其cDNA序列特征如序列表SEQ ID NO 1。
2.如權利要求1所述的決定仔豬F4ac腹瀉易感性/抗性的MUC13基因，其特征在于 其DNA序列特征為如序列表SEQ ID NO 2。
3.如權利要求1所述的決定仔豬F4ac腹瀉易感性/抗性的MUC13基因，其特征在于 其編碼蛋白氨基酸序列如序列表SEQ ID NO 3。
4.如權利要求2所述的決定仔豬F4ac腹瀉易感性/抗性的MUC13基因，其特征在于 其序列表SEQ ID NO 2的第32813bp處為C32813-32813T的堿基突變；第37618bp處為 C37618-37618T的堿基突變；第42785bp處為A42785-42785C的堿基突變；第46531bp處為 A46531-46531C的堿基突變；第53019bp處為A53019-53019C的堿基突變。
5.根據權利要求2所述的決定仔豬F4ac腹瀉易感性/抗性的MUC13基因，其特征在 于分離MUC13基因所用的引物序列為分離MUC13基因引物信息引物名稱 引物序列5’-3’_退火溫度
6.根據權利要求4所述的決定仔豬F4ac腹瀉易感性/抗性的MUC13基因，其特征在 于檢測MUC13基因的堿基突變引物序列為用于判定MUC13基因5個關鍵SNP標記的引物信息
7.決定仔豬F4ac腹瀉易感性/抗性的MUC13基因及其SNP位點在選育種改良豬抗腹 瀉病性狀中的應用。
全文摘要
本發明公開了一種決定仔豬F4ac腹瀉易感性/抗性的MUC13基因及其在種豬遺傳改良中的應用，它是利用大規模白色杜洛克×二花臉資源群體和15個中外豬種的遠緣群體，通過全基因組掃描連鎖定位、目的區域高密度SNP多點連鎖精細定位、斷點重組分析、基于連鎖不平衡的遠緣群體關聯性分析、最后鎖定了編碼ETEC F4ac受體的目的基因-MUC13基因，并分離克隆了MUC13基因。在MUC13基因中鑒別到5個關鍵性突變位點。這五個突變位點在來自5個省份核心群的144頭純種杜、長、大仔豬中與ETEC F4ac體外黏附表型基本共分離，最強相關的標記判定抗性/易感個體的準確性達97％(P＝1.59×10-21)。本發明為種豬ETEC F4ac腹瀉病的抗病育種提供了一種新方法。
文檔編號C12N15/12GK101864423SQ20091018671
公開日2010年10月20日 申請日期2009年12月15日 優先權日2009年12月15日
發明者任軍, 張志燕, 晏學明, 艾華水, 黃路生 申請人:江西農業大學