DB36∕T 1415-2021 猕猴桃雌雄种质分子身份证鉴定技术规程(江西省).pdf

ICS67.080.01B05江西DB36省地方标准DB36/T1415一2021称猴桃雌雄种质分子身份证鉴定技术规程Id e n t i f i c a t i o no fm a l ea n df e m a l eg e r m p l a s mo f Ac t i n i d l ab ym o l e c u l a ri d e n t i t yc a r d2021一06一30发布2022一01一01实施江西省市场监督 管理局发布DB36/T1415一2021目次前言.H1范围.12规范性引用文件.13术语和定义.14原理.15试剂与试药.16仪器设备.17操作步骤.28结果记录与判定.3附录A（资料性）主要试剂与试药.5附录B（资料性）主要仪器设备.7附录C（规范性）称猴桃雌性种质特异性引物.8附录D（规范性）称猴桃雄性种质特异性引物.H附录E（资料性）67份称猴桃雌性种质分子身份证.14附录F（资料性）33份称猴桃雄性种质分子身份证.16DB36/T1415一2021士一口前本文件按照GB/T1.1一2020标准化工作导则第1部分：标准化文件的结构和起草规则的规定起草。请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。本文件由江西省农业农村厅提出并归口。本文件主要起草单位：江西农业大学。本文件主要起草人：廖光联、徐小彪、黄春辉、钟敏、贾东峰、曲雪艳、吴茵、涂贵庆、朱壹、魏清江、辜青青、陈明。DB36/T1415一2021称猴桃雌雄种质分子身份证鉴定技术规程范围本文件规定了分子身份证鉴定称猴桃（刀c t初f d,a）雌雄种质的术语和定义、原理、试剂与试药、仪器设备、检测及构建分子身份证方法。本文件适用于称猴桃雌雄种质鉴定与分子身份证构建。2规范性引用文件下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款。其中，注日期的引用文件，仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本（包括所有的修改单）适用于本文件。NY/T3639中华称猴桃品种鉴定SSR分子标记法3术语和定义下列术语和定义适用于本文件。分子身份证1d e n ti t yCa r d以特异性引物为探针，同称猴桃细胞核DNA的酶切片段杂交，可以获得由多个位点上的等位基因组成的长度不等的杂交带，人为将同一位置条带在不同物种上的有无标记为1 /0，构建成数字矩阵，同一物种间极少有两个品抑、系）完全相同，故称为特异性分子身份证。3.2相似度s i m il a r i t,供试品种分子身份证与对照分子身份证相似的程度。4原理利用CTAB法提取植株叶片DNA，与特异性引物混合后进行PCR仪扩增，并在丙炜酞胺凝胶进行分离和显现，采用人工读取条带的方式绘制1/0矩阵，构建成DNA分子身份证，最后统汁供试品种和对照品种的相似度，根据相似度鉴别称猴桃种质的真伪。5试剂与试药主要试剂与试药见附录A。DB36/T1415一20216仪器设备主要仪器设备见附录B。7操作步骤7.1样品制备参照NY/T3639执行。7.27.2DNA的提取准备工作水浴锅水浴加热至65，预热3裂解液、洗液，两种液体使用前均需加入2体积的p一琉基乙醇(V/V)；一ZOOC预冷无水乙醇和75乙醇（现配后预冷）。7.2.2称样称取0.109新鲜硅胶干燥后的叶片样品于Zm L管子，磨样机磨成粉状。7.2.3第一次洗涤硅胶干燥后的叶片样品需要进行的步骤。向Zm L管子中加入l m L洗液，充分混匀，65C水浴30m i n，中间轻摇数次，使其充分混匀；随后10000r p m/m i n离心5m i n。7.2.4第二次洗涤硅胶干燥后的叶片样品需要进行的步骤。弃上清液，向Zm L管子中加入l m L洗液，充分混匀，65水浴30m i n，中间轻摇数次，使其充分混匀；随后10000r p m/m i n离心5m i n。7.2.5裂解弃上清液，向2。J!J营子中为1入1m LCTAB裂解液，充分混匀，65C水浴l h以上，中间轻摇数次，使其充分混匀；冷却至室温，随后12即e印m/mi n离心10mi n。7.2.6第一次抽提取上清液（可0.80m L）于新的Zm L管了中，祀八等体积抽提液1，摇床混匀10m i n，随后12000r p m/m i n离心10m i n。7.2.7第二次抽提取上清液（可0.60m L）于新的Zm L管子中，加入等体积抽提滚之摇床汉匀10m i n，随后12000r p m/m i n离心10m i n。7.2.8沉淀取上清液（可0.50m L）于新的1.50m L管子中，加入1 /10体积Na AC,2.50倍体积的预冷的无水乙醇，一20沉淀30m i n以上，随后4OC,10000rr p m/m i n离心8m i n。此步骤肉眼可见沉淀。7.2.9漂洗DB36/T1415一2021弃上清液，加入1m L75无水乙醇，沉淀30m i n。每隔30m i n漂洗一次，共漂洗4次。7.2.10干燥打开盖子放入旋蒸仪，设定DAL模式，30，8m i n，干燥至无液体即可（避免乙醇影响后续PCR),不可过度干燥。7.2.11溶解往每个管子中加入60匹RNA溶解酶（按RNa s e:TE缓冲液二3睡：1000睡配制）。4OC放置过夜后用超微分光光度计测定DNA质量。OD26O/OD28O的值在1.80一2.00时，表明DNA的纯度较好。测定后用TE缓冲液将DNA含量稀释至150n g林1。7.3PCR扩增7.3.1特异性引物选用称猴桃雌雄种质特异性引物（附录C、附录D）进行PCR扩增。7.3.2扩增反应体系包含2Ta qPCRM1x s匹；上下游引物各0.80睡；模板DNAI睡；双蒸水2.40睡。7.3.3扩增程序94预变性3m i n,94OC变性305,52OC一62退火305,72延伸305，共28个一34个循环，最后72延伸10m i n于12C保存。7.4丙烯酉先胺凝胶电泳7.4.1制胶按顺序往150m L烧杯加入双蒸水60m L、SXTBE20m L、40聚丙烯酞胺20m L、APS864林L、TEMED76匹，搅拌均匀后滚入勺NA序列分析电泳仪玻璃板中，排气泡后插入梳齿，凝固l h后进行下一步。7.4.2点样拔出梳齿，用移液枪移取1睡于吹扩增产物，点入梳齿孔中；同时点入500b pDNA m a r k e r及10 xLo a di n gBu f f e r。7.4.3跑胶使用仪器为DYCZ一2OG型DNA序列分析电泳仪，先50V由压跑为，。111，随后调为200V，待10XLo a di n gBu f f e r跑到接近玻璃板底部时结束。7.4.4染色采用快速银染法，将丙烯酞胺凝胶取下放入硝酸银溶液，浸染10m i n。7.4.5显影从银染液中取出凝胶，用双蒸水冲洗干净后，放入显影液中显影，待条带全部显出，转入到清水中。DB36/T1415一20218结果记录与判定8.1结果记录拍照记录并统计清晰的SSR条带，采用人工读带的方法，在MICr o s o f tEx Ce l中 以二进制读带方式记录引物扩增的结果，同一位点上具有相同迁移率的条带记为1，无带记为O，记录位点位置（见附录E、附录F）。8.2构建分子身份证利用同一样品的不同引物条带读数绘制成1 /0矩阵，构建成该品种的特异性分子身份证，如附录E、附录F所示。8.3判定根据相似度进行结果判定，相似度二（同一位点读数相同的数量总位点数）*100%，相似度等于或大于99%，即可判定为在分子水平上是同一个样品。DB36/T1415一2021附录A（资料，l生）主要试剂与试药A.1试剂p一琉基乙醇，Tr15,HCI,EDTA,Na OH,Na CI,PVP,CTAB,Na AC，无水乙醇，RNA裂解酶，琼脂糖，DNA核染液，Lo a di n gBu f f e r，硝酸银，甲醛，Na ZCO3,Zx Ta qPCR Mi x，硼酸，过硫酸铰，丙烯酞胺，甲叉丙烯酞胺，TEMED。A.2配制试剂（灭菌后使用）A.2MTr i s一HCI称取12.119Tr i S，溶于水后定容至100m L，用HCI调节p H为8。A.2.20.50MEDTA称取18.619EDTA，溶于水后定容至100m L，用Na OH调节p H为8。A.2.3洗液1.469Na CI,25m L1MTr i S一HCI,10m L0.50MEDTA,29PVP，溶于水后定容至100m L。A.2.43裂解液称取39CTAB,8.19g Na CI,IOm LIMTr i S一HCI,4m LO.SOMEDTA,O.SOg PVP，溶于水后定容至100m L。A.2.5丁E缓冲液1m L1MTr i S一HCI,0.20n l i。，.5门MEDTA，溶于水后定容至IOOm L。A.2.63m o l/LNa AC称取40.829Na AC，溶于水后定容至100m LA.2.7SXTBETr15549，硼酸27.509,20m LEDTA，定容至1000m L。A.2.810过硫酸按过硫酸钱19溶于10m L双蒸水，4保存。A.2.940聚丙烯酉先胺丙烯酞胺1909，甲叉丙烯酞胺109，溶解后定容至500m L。A.2.10银染液DB36/T1415一202119硝酸银溶于I000m L超纯水，定容至I000m L即可，使用前加入Zm L甲醛。A.2.11显影液2OA.2.129Na OH,0.40g Na ZCO3，溶于I000m L超纯水，定容至I000m L即可。抽提液1苯酚、氯仿、异戊醇按体积比为苯酚：氯仿：异戊醇二25:24:1配制。A.2.13抽提液2氯仿、异戊醇按体积比为氯仿：异戊醇二24:1配制。DB36/T1415一2021附录B（资料，l生）主要仪器设备B.1电子分析天平精确度万分之一。B.2亘之曰产且n水浴锅最高温度100。B.3离心机最大离心速度12000r p m/m i n oB.4超微量紫外分光光度计最大离心速度12000r p m/m i n oB.5PCR仪常规。B.6DNA序列分析电泳价220V电压。B.7摇床最大转速200r p m/m i n o日8旋蒸仪常规。DB36/T1415一2021附录C（规范，l生）称猴桃雌性种质特异，生引物C.1雌性种质特异性引物雌性种质特异性引物见表C.1。引物表C序列雌性种质特异，生引物退火温度C等位基因长度（b P)TGGCGACATGGTCTTCTTAG2754100?150TCCAAAGCATGCTCAACTTCCCAAGCTCATACACCCAATG3l57200?250AGGTTGGCACTTGGAATAGGTTCCTTAATCCAGGGTCTCG5363150?200ACACAACAGATTGCAGGAGGGAGTCGGGAGCATAATTGGT5054250?300CGCATGGAAGGTCATAACACGTGCTCCTCCGTCCATGTATUDK97一40864100?150CGTCCTCTCTTCGCCATTTATGGTGTAAAGTCAAAAACAGCCUDK99一14359100?200AGAAGATTTCATTGTCCTCCCTAAGAGCCATAGCTTATTCACCGUDK96一03560100?150卢、产GTAAAGCCATTGTCATTGCA、CTA人G气AACGGGACCATTG62150?200TATGT台（，八TGTCCAGAAAAGCCCCTCATf G尸！”户J八i气GCATCCl l62150?200TTGTCACGGGCTf户行广。人r l一CCl 5CCGTCTCAGCAGATGTCACT竺州GCAACTGCCCATAAGATTCCI52150?200TACACCTGATGAGATGGACGAC22200?250CCTGTGGCTGATGTTGAAAGTTCGAGTTACCTAGCTACTCCGCUDK96一04062150?200CAAGGGAAGAAAATGTTGAACCGTAAGGTCATTTTTGCGAAAGGUDK96一0536450?100TTTGTTGGGAGTAACGTGAGGTACGCAGTTTACTTCCTCTTAO1760.50150?200CATTCGGCACCACTTCTAA043CCCATAGCCAACAACATC59150?200DB36/T1415一2021雌性种质特异l陛引物（续）引物表C序列退火温度。C等位基因长度（b P)CTTGACGCCTCTGAACACTCCATTCCGCCCATCCTTA05560.50100?250GTCCGACATTCTTGTGGTTCTGATCTCCATCATCTCCACCTTA06160100?150CAACGCCATCATTGTCCCGAAGCAACTCGCTACTAAGA06960.50100?200AGCCTGTAACAACCCATTTTGCTCCAGTTTCCTTCTA08060150?250TGCTATGGCACTCTATCCGAGCATACAGAGGGAAGAA10359200?300ACTGGAGTGAAGGAGGGTCCTCATTCATTACCACCTCTTA10560.50100?200ACACCGCTTCCGTCCATACACCTGCTACACCGACATA12360.50150?300CCACAGCCGAAACCATACATACCGTGGACTTCATTCA12460150?250ACATACCTTCAACGCTTCGTTTATTACACGCCTTCGA12559150?200TGACAGCCCTCAACTACGCCCTATACCCACTCAATCAA13860100?250TTCCACCTTCTTTCCATCATGGCTTGACTGAGACTTGA16758150?250TTCATCGTCGGTTGTAGC乙TCCCTCGTAGCAGTTCAA18660100?200CTCC卢（,CGATTCCTTCACCCGCATC产T一：、气GCCCAATCA19160150?300TTGTCCACC几C二C产理，TC150?250200?300!,乙U勺A193ATACCCATCATCGC、人。ACC,CAAGCCCA,ACCAC几车一CGACTAAAATCTACAAGCACEST一Ad o lGAAAACGAAATTAACACCATTAAGATTGATCGGCACGEST一Ad 036l250?500CCTTCTGGATGAGGGTTTGTTAATTTGATCGGGATGGEST一Ad 4262250?400GAGGAGCTTGAGCTGCTATATACACTTGAAGCGCCGCUDK96一01957100?200AAGCAGCCATGTCGATACGUDK96一028TCCCCACACAACAACTCCTC52100?200DB36/T1415一2021表C序列雌性种质特异l陛引物（续）引物退火温度。C等位基因长度（b P)CATATGACCTGCACGTGCGGTTTGATCGGTCTTCGAAAUDK96一03957150?200ATAAATGTGTGCCAGTGCGAGAGCGTAGAATGTGGCGGUDK97一42060.50100?150CATTTGAAATGAGTCAAGCTGCAACGAAACAAAAGCCCTATTATCST一Ac d 0259200?300GCTTCAGATTCAGCCTCTGGTTCATCCCAATCCCTCATTCST一Ac d 0758200?250GACTTCTTCGTCGCCTCTGGATGGCTGACGAACAACAST一Ad O3758100?200TCTGTCACGATCACCTCCCCACTCTTCGCCTCTCTTTCAUTH03一0457150?200AGTGGTTGTGTTGGGGTCTCl 0DB36/T1415一2021附录O（规范，l生）称猴桃雄性种质特异，生引物0.1雄性种质特异性引物雄性种质特异性引物见表D.1。表0.1雄性种质特异性引物引物序列退火温度等位基因长度（b P)TGGCGACATGGTCTTCTTAG2754100?150TCCAAAGCATGCTCAACTTCTTCCTTAATCCAGGGTCTCG5363150?200ACACAACAGATTGCAGGAGGGTGCTCCTCCGTCCATGTATUDK97一40864100?150CGTCCTCTCTTCGCCATTTATGGTGTAAAGTCAAAAACAGCCUDK99一14359100?200AGAAGATTTCATTGTCCTCCCTAAGAGCCATAGCTTATTCACCGUDK96一03560100?150AAGTAAAGCCATTGTCATTGCAAACTAAGAAACGGGACCATTG62150?200TAT召f GGATGTCCCAGAAAAGGTTC一rrGA6GTGATTGTTGATG6l150?200CAGTCAGCC八AGG广、户r入AGGACTAACAGACA凡气八八CTG(.G台Gl 3200?250ATGGAAGGAGATGGCGATGTTGAGGGCTTGATGAAAACC1458lll!6I150?200GACGACGACGGAGATTACGCCGTCTCAGCAGATGTCACTATl 552150?200GCAACTGCCCATAAGATTCCTACACCTGATGAGATGGACGAC2254200?2气UCCTGTGGCTGATGTTGAAAGTGTAAGGTCATTTTTGCGAAAGGUDK96一0536450?100TTTGTTGGGAGTAACGTGAGG11DB36/T1415一2021表O雄性种质特异l陛引物（续）引物序列TACGCAGTTTACTTCCTCTT退火温度等位基因长度（b P)AO1760.50150?200CATTCGGCACCACTTCTACCCATAGCCAACAACATCA04359150?200CTTGACGCCTCTGAACACTCCATTCCGCCCATCCTTA05560.50100?250GTCCGACATTCTTGTGGTTCTGATCTCCATCATCTCCACCTTA06160100?150CAACGCCATCATTGTCCCGAAGCAACTCGCTACTAAGA06960.50100?200AGCCTGTAACAACCCATTTTGCTCCAGTTTCCTTCTA08060150?250TGCTATGGCACTCTATCCCCTCATTCATTACCACCTCTTA10560.50100?200ACACCGCTTCCGTCCATACACCTGCTACACCGACATA12360.50150?300CCACAGCCGAAACCATACGTTTATTACACGCCTTCGA12559150?200TGACAGCCCTCAACTACGCCCTATACCCACTCAATCAA13860100?250TTCC、CCTTCTTTCCATCAl行（;f rf G气CTGAGACTTGA16758150?250TTCATCCT屯GGTTCTAGCCTCCCTCGTAGC八6一l下CAA18660100?200CTCCAGCGATTCCTTCAC-斗一CCGCATCATAAGCCCAATCA19160150?300TTGTCCACCGCCCACTTCATACCCATCATCGGACACA1936l150?250CTTCAAGCCCATTACCACCGACTAAAATCTACAAGCACESTAD0162200飞UOGAAAACGAAATTAACACCATTAAGATTGATCGGCACGESTAD036l250?400CCTTCTGGATGAGGGTTTESTAD42GTTAATTTGATCGGGATGG62250?400l 2DB36/T1415一2021表O雄性种质特异l陛引物（续）引物序列GAGGAGCTTGAGCTGCTAT退火温度等位基因长度（b P)ATACACTTGAAGCGCCGCUDK96一01957100?200AAGCAGCCATGTCGATACGUDK96一039GGTTTGATCGGTCTTCGAAA57150?200ATAAATGTGTGCCAGTGCGAUDK97一420GAGCGTAGAATGTGGCGG60.50100?150CATTTGAAATGAGTCAAGCTGCUDK96一013ACGTGACTTGGTTTTTGAAGG55150?200CACTCCGATCAGCTCTCCTCSTACD02AACGAAACAAAAGCCCTATTATC59200?300GCTTCAGATTCAGCCTCTGGSTACD07TTCATCCCAATCCCTCATTC58200?250GACTTCTTCGTCGCCTCTGSTAD037GATGGCTGACGAACAACA58100?200TCTGTCACGATCACCTCCUTH03一04CCACTCTTCGCCTCTCTTTCA57150?200AGTGGTTGTGTTGGGGTCTC13DB36/T1415一2021附录E（资料，l生）67份称猴桃雌性种质分子身份证E.1雄性种质特异性引物67份称猴桃雌性种质分子身份证见表E.1。表E.167份称猴桃雌性种质分子身份证编号品种金丰庐山香赣金3号宜黄2号袭金1号袭金2号袭金4号袭金3号武宁2号宜黄1号云海2号金果金 艳井黄1号井真三月一一一斗一井黄3号井黄4号奉黄1号奉黄2号奉黄3号奉黄4号宜黄1号宜黄2号武宁1号武宁2号武植 3号武植5号山维1号云海1号UDK96一035FOR一13EST-Ad 42UDK96一040000000000000000000000000000000101000000000001 1 10000000000000000000000000000100000010100000000101111011000000000000000000000000000000000000000000000001 101000000000001 10001100101 1 1 1 1111001111100001000001 1 101010000000000000000000000001000000000000000000000000101000000000100101000001 1001110000000100000001 1 1 1 1 101010000000001000011000001 101100000010100001000001 1 100100000000001001010001 1000010010111101000001 10001 1 10101000000000000000000000001 10000000010000000000011001010000000001000010001010001100000100100000001 1 101001010000000001010010001000001001000101100000000101 1001010000000000000001000000000000000100000000000001 1 101010000000001001010001 1 1001 1000000101000000001 1 1 1 101 101000000000100001000001 1001011000111100000101010101 1 110000000001001 11100001 100101100011110000000111010100000000000100000000001 10010100001 1 1 1000000010001001 1100600(010110111000001 10010100001 1 1 100000001 10010000100000001！、11Cl1100101 1 1 1 101一一十一:00001 1 10100-弓-100)!飞匀j o l 001011111111001 1 1 1 1 101 101010000001 101001二10001 1 1 1 1 11110000000110110111000001011011101011111111000110000110101010000010100111100户001100If,G00001 1 1 1000000001010101011000000001001 110001 1 1 10011jG:,00000111001001 1 1 1 101 1010000000001 100010001001 1 1 1110，少七q1 1 116户000000001010101000000000000001000000000100000010160Gn 300000010100000000000001010011000010101011010111101j、011 1 1 1 1 1 1000000000000010100100001 10001001000111100000001 10001 1 1000000000001001 11100000110001101101110110110010111111110000000000000000000000000010001000000000000000001 100000000000000000001 1000010000001 1 1 100001 100001 1 101 110);1.，厂7；孙nUJI今工伪J月任丈J乙U工OOC,nUJee气乙j月任气乙U工OnC,23456789l(l仁厂卜Fhr州朴2(2乞工冬之入了川办14DB36/T1415一202167份称猴桃雌性种质分子身份证（续）编号表E品种金什1号金桃金梅早 鲜魁蜜翠玉华优金桃红华赣红楚红脆皮红香红阳东红奉野1号红什2号脐红仙红晚 红早红金魁徐香翠香米电；r J海沃德贵长美实1号武枣1号长叶1号华特赣称6号麻毛4号麻毛7号赣绿2号赣绿1号麻毛13号麻毛28号低酚6号UDK96一035FOR一13EST一Ad 42UDK96一04000000000100001000001100101000011110000010000000011100000000010000100000110010110001111000000001100011000000000001001010000111001001011111111000001110110101000000000100101000010010101001 1 1 1100101100000011001010000000000000000000000000000001110000000000000000000000000000010000001100011001001110100100001111111000000000000100111000110001100001 1 1100110000011111000000000000001000010000011001010000010100000000110000100000000000100001100000000100000000010000000001000101000000000000000000000000010100001101000000000000000000000000001000010000000001001000101100000001100010010100000000100111000001110101100010110000000000011001010000000010000100000000010000000000000000000000010000000000000000000000000001000000000000000000000000000000000000100001000001100100100011110000000000001001010000000000000010000100000000001001000000000000000000000000001000010000000001001000101100000000000001010000000000000000000000000000000010110000000000000000000000000000000000000000000000001010000000000000000000000000001000110000000001000000000000000000000000000000000000100001110110000100001 11010000000000011100000000000001100010001100011001 1100001010000000011100000000000001000110000000011000000101100000000000101111100000000100011000000000100001 11011000000000000110000000000001000000000000001000000000000000000000000000000Je 0O勺000010100011100010011001111000000011111100101OOCCO(001(:e公0601110010010000110010000000000001000000I00000100Jn J日0000010101100001000100000000000010100000000000000000OC000f JC，00000001100100000000000001000001001001011011101 1101010110000000011111100100100101101010d 6o1口0010、一一一一丰0001I0001CI110010101101 1 10000111010000111001011110100000000010011八01010110000000011101100011100101111010000000101001 11010，口10000011111100000100100101101111000000101000010101000O、00000001001100长00010010110101000000000100101010101101 1 101 1 1 110011001001111011010110000001010010111010110000011011101000000111001101011000000101001011101011000000100100100000010100100010100000010100101110101100001 11001101100n曰jlo乙qo月任尸匀匕门了00QdC口11C乙qo月任叹J(b门1QOQ口门Uj1Q乙几j月任尸匀八匕门了OOQ口n曰jlo乙gd月任尸匀n匕门了gdgdqoqoqoqoqoqoqoqo4444444444叹d叹d叹d叹d叹d叹d叹d叹d叹J叹J(b(bbbbbbb15DB36/T1415一2021附录（资料，l生）33份称猴桃雄性种质分子身份证1雄性种质特异性引物33份称猴桃雄性种质分子身份证见表F.1。表133份称猴桃雄性种质分子身份证编号UDK96一053UDK96一035Fo f l 3UDK96一0400001 10010100000001 1 100000000000000001 1 100101 1010101001 101 1000000000000000000001 100101000000010000000000000000001 1 100101 1010101001 101 1000000000000000000101010100000100101 101 100000001000001 1 10000010101 1100101000001 1001 1000000000010010000001000101 100100000001 100001010000010101 10001010000001000100000000001 1 1 10100010101 10100000000000000000101000001010000001 1 10000001 1001000100000001 10010100000001 1 100000000000000001 1 1 10101 1010101001 101 100000100000010000001 1010000010001000100000000000000001 1 10000010101 11000100000001 100000010000001 1010000000001000100000000000000001 1 10000010101 11000100000001 1000000100000010010000000001000100000000000000001 1 10000010101 11000100000001 1000000100000001 1 1000000000010010000001010011111000100100000001010000000101000010000000001 1 101000000000101 100100000001 10000101000001010000001 10000001 1010100000100100100001000001001000010000000001010101000000011100110101000001011101001000011000010100000000101010100000001110011010100000101110100100001100001010000001001p O00J01000101 101 101010001 100001010000010101 10001010000001000100000000000110(l忆）,000900101001101010010000T001 1 100101 1010101001 101 1000000000000000000001 1 1 10000n 100001 1 1 100000101001几1110001001000000010100000001010000100000000010101000001000又0二01 101010001 100001010000010101 10001 10000001 10101000001000001 1 1 1000001000101100JOGO,601100001010000010101 10001 10000001 10101000001000001 1 10100001000101 101 1010100011600001 1 1 1000001000101 101 10101000110与101000001010110001 101 100000000000000000；乏，*000001010110001 10000001 10101000001000000000000000000000000000000000000户0110066, IU二01110000000000000000000000000001 1 1010000100001 1 1 1000001000101001 101010010000001 111心l,101010二G01 10