CN115011720B - 用于构建橡胶树品种dna指纹图谱的snp分子标记组合及应用和方法 - Google Patents
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Abstract
本发明公开了一种用于构建橡胶树品种DNA指纹图谱的SNP分子标记组合及应用和方法。本发明筛选的50个SNP分子标记特异性、灵敏度和分辨力高,该SNP分子标记不受环境条件影响,且检测结果准确、重复性和稳定性好,能够全面揭示橡胶树遗传多样性真实水平利用;利用该套SNP标记组合建立DNA指纹图谱库,可以对橡胶树品种进行聚类分析和品种鉴定,使得检测结果更精准、高效,为橡胶树资源保护利用提供理论依据,首次有效区分出橡胶树66个品种,本发明对育成品种的区分率可达100%。利用本发明的单核苷酸标记位点进行检测具有简单、快速、成本低的优点,能够实现生产中大规模的应用。
Description
技术领域
本发明属于橡胶树品种分子生物技术领域,尤其涉及一种用于构建橡胶树品种DNA指纹图谱的SNP分子标记组合及应用和方法。
背景技术
橡胶树原产巴西亚马逊河流域,是热带地区重要的经济作物,其生产的天然橡胶是我国重要的战略资源,在军事、航空航天、海洋工程、轨道交通等领域具有不可替代的作用。橡胶树自1904年引种至中国以来,迄今国内保存的橡胶树栽培品种、野生种质、以及同属种质材料5个种、1个变种共计6165份种质材料。长期以来,橡胶树品种资源鉴定一直以形态性状鉴定为主要依据,这种经典的、常规鉴定方法一般要在橡胶树开花后才能完成其鉴定过程。然而,橡胶树属多年生异花授粉高大乔木,一般在定植后5-6年才能开花结果,具有有性世代周期长,品种高度杂合等不利因素,导致其性状鉴定过程中不仅鉴定周期较长,而且其农艺性状易受环境条件和栽培管理措施的影响。此外,传统的鉴定方法对于一些性状差异较小、亲缘关系较近品种的鉴定十分困难,给橡胶树种质资源和品种鉴定带来了诸多不便。因此,亟需准确的分子标记方法来对橡胶树种质资源和品种进行鉴定。
分子标记分析方法均以DNA的多态性为基础,如RFLP、RAPD、AFLP、ISSR、SSR、以及SNP标记等,它们均能产生具有高度个体特异性的DNA指纹图谱。早期RFLP、RAPD、AFLP分子标记技术曾被应用到多个物种的DNA指纹图谱建库上,但以上几个标记技术在使用中经常出现稳定性差、多态性低、以及数据整合困难等问题未能被广泛应用。因此,国际新品种保护联盟建议SSR和SNP作为首选的DNA指纹标记技术。由于SNP标记具有覆盖全基因组、高通量、位点特异、共显性遗传、误检率低、开发和检测成本急剧降低、数据易整合等优点,将成为未来基因型鉴定的主要标记类型。
随着二代测序技术的发展,测序通量呈现指数增长趋势,而测序成本则急剧降低,为全基因组水平SNP标记的检测提供了强有力的技术支持。每个物种上存在丰富的SNP,如何根据不同鉴定目标,制定SNP位点筛选原则、位点数目、位点组合、判定阈值等成为筛选SNP构建指纹图谱的关键。本发明基于橡胶树重测序获得的SNP,通过SNP测序质量的控制、SNP位点质量控制、SNP筛选的判定阈值设定等,以获得标准化的SNP构建指纹图谱的流程,用来建立橡胶树的分子身份证。目前,还没有建立起完整的橡胶树品种指纹图谱库从而鉴别品种的报道。
发明内容
本发明的目的在于针对现有技术的不足,提供一种用于构建橡胶树DNA指纹图谱的SNP分子标记组合及应用和方法。以解决现有技术中使用RAPD、ISSR等共显性DNA标记,重复性差、多态性低的问题;本发明的另一目的是提供橡胶树核基因组SNP分子标记的多态性引物在橡胶树品种鉴定、遗传结构和资源遗传多样性分析中的应用,从而为从分子水平鉴定橡胶树种质资源和栽培品种提供依据。
本发明的第一个方面是提供一种用于构建橡胶树DNA指纹图谱的SNP分子标记组合,所述SNP分子标记组合包括LG01_54721919、和/或LG01_96036745、和/或LG02_214754、和/或LG02_8025843、和/或LG02_57849162、和/或LG03_40084932、和/或LG03_57340517、和/或LG03_96006922、和/或LG03_104230492、和/或LG04_224562、和/或LG04_16174898、和/或LG04_33465133、和/或LG04_71146652、和/或LG04_80306944、和/或LG04_96013598、和/或LG05_56475、和/或LG05_16004126、和/或LG05_88010730、和/或LG06_32377524、和/或LG07_8072524、和/或LG07_32411839、和/或LG07_57540924、和/或LG07_72003724、和/或LG07_80004402、和/或LG08_8000585、和/或LG08_28146509、和/或LG08_35232681、和/或LG08_48608369、和/或LG09_8012416、和/或LG09_16001432、和/或LG09_46132314、和/或LG09_50708986、和/或LG09_64555870、和/或LG10_34723841、和/或LG11_54415、和/或LG11_8139610、和/或LG11_34802586、和/或LG12_284283、和/或LG12_32322540、和/或LG12_40522222、和/或LG13_32072502、和/或LG13_40000353、和/或LG14_605466、和/或LG14_33209388、和/或LG14_56176655、和/或LG15_32001166、和/或LG15_24017141、和/或LG17_24096219、和/或LG17_32001525、和/或LG18_10766;
上表中,“Sequence”为SNP分子标记的核苷酸序列;“[]”标注的为SNP分子标记位点位置,其中的碱基为该位点的多态性。
应当理解的是,在实际应用时,本领域技术人员可根据实际情况来选择合适所述的SNP分子标记进行应用。比如,实际已知待测橡胶树为海垦1或RRIM623,那么可用于鉴定的SNP分子标记可选择LG01_96036745、LG03_104230492、LG04_16174898、LG04_33465133、LG04_71146652、LG04_96013598、LG07_57540924、LG07_72003724、LG09_50708986、LG09_64555870、LG11_54415、LG11_34802586、LG14_33209388、LG18_10766等(参照下文表5)中的一种即可,也可以选择两种或两种以上进行多重验证鉴定。又比如,实际已知待测橡胶树为海垦1、RRIM623或大丰95,也可以只采用一种SNP分子标记即可,如LG03_104230492、LG04_33465133、LG11_34802586、LG15_32001166等(参照下文表5),当然也可以采用两种以上的SNP分子标记。当已知待测橡胶树为四种或四种以上橡胶树中的一种时,本领域技术人员也可以参照下文中的表5,选择适当的SNP分子标记来进行鉴定。当待测橡胶树完全未知时,本领域技术人员也可以将上述SNP分子标记分批次来进行鉴定。
优选地,所述SNP分子标记组合包括LG01_54721919、LG01_96036745、LG02_214754、LG02_8025843、LG02_57849162、LG03_40084932、LG03_57340517、LG03_96006922、LG03_104230492、LG04_224562、LG04_16174898、LG04_33465133、LG04_71146652、LG04_80306944、LG04_96013598、LG05_56475、LG05_16004126、LG05_88010730、LG06_32377524、LG07_8072524、LG07_32411839、LG07_57540924、LG07_72003724、LG07_80004402、LG08_8000585、LG08_28146509、LG08_35232681、LG08_48608369、LG09_8012416、LG09_16001432、LG09_46132314、LG09_50708986、LG09_64555870、LG10_34723841、LG11_54415、LG11_8139610、LG11_34802586、LG12_284283、LG12_32322540、LG12_40522222、LG13_32072502、LG13_40000353、LG14_605466、LG14_33209388、LG14_56176655、LG15_32001166、LG15_24017141、LG17_24096219、LG17_32001525和LG18_10766中的至少两种。
优选地,所述SNP分子标记组合至少包括LG03_104230492、LG02_8025843、LG01_54721919、LG15_32001166、LG07_72003724、LG05_88010730和LG02_214754。
优选地,所述SNP分子标记组合包括LG01_54721919、LG01_96036745、LG02_214754、LG02_8025843、LG02_57849162、LG03_40084932、LG03_57340517、LG03_96006922、LG03_104230492、LG04_224562、LG04_16174898、LG04_33465133、LG04_71146652、LG04_80306944、LG04_96013598、LG05_56475、LG05_16004126、LG05_88010730、LG06_32377524、LG07_8072524、LG07_32411839、LG07_57540924、LG07_72003724、LG07_80004402、LG08_8000585、LG08_28146509、LG08_35232681、LG08_48608369、LG09_8012416、LG09_16001432、LG09_46132314、LG09_50708986、LG09_64555870、LG10_34723841、LG11_54415、LG11_8139610、LG11_34802586、LG12_284283、LG12_32322540、LG12_40522222、LG13_32072502、LG13_40000353、LG14_605466、LG14_33209388、LG14_56176655、LG15_32001166、LG15_24017141、LG17_24096219、LG17_32001525和LG18_10766。
本发明的第二个方面是提供用于扩增如本发明第一个方面所述的SNP分子标记组合的引物组。
优选地,各个SNP分子标记的引物对如下文中表3所示。
本发明的第三个方面是提供含有本发明第二个方面所述的引物组的试剂盒或基因芯片。
本发明的第四个方面是提供本发明第一个方面所述的SNP分子标记组合、或者本发明第二个方面所述的引物组、或者本发明第三个方面所述的试剂盒或基因芯片在橡胶树品种群体遗传结构、和/或橡胶树资源遗传多样性分析、和/或橡胶树品种鉴定、和/或构建橡胶树DNA指纹图谱中的应用。
本发明的第五个方面是提供一种橡胶树品种DNA指纹库的构建方法:提取各个橡胶树品种的总DNA;采用本发明第二个方面所述的SNP分子标记的引物组进行扩增;利用本发明第一个方面所述的SNP分子标记组合进行SNP位点多态性检测,根据SNP位点的检测结果对各个橡胶树品种进行基因分型,从而构建橡胶树品种DNA指纹库;其中,所述橡胶树品种为下文表2所述的橡胶树品种。
优选地,应用SNaPshot方法进行SNP位点多态性检测,进行橡胶树品种鉴定、群体遗传结构和遗传多样性分析。
优选地,所述SNP分子标记组合至少包括'LG03_104230492、LG02_8025843、LG01_54721919、LG15_32001166、LG07_72003724、LG05_88010730和LG02_214754。
优选地,所述SNP分子标记组合包括LG01_54721919、LG01_96036745、LG02_214754、LG02_8025843、LG02_57849162、LG03_40084932、LG03_57340517、LG03_96006922、LG03_104230492、LG04_224562、LG04_16174898、LG04_33465133、LG04_71146652、LG04_80306944、LG04_96013598、LG05_56475、LG05_16004126、LG05_88010730、LG06_32377524、LG07_8072524、LG07_32411839、LG07_57540924、LG07_72003724、LG07_80004402、LG08_8000585、LG08_28146509、LG08_35232681、LG08_48608369、LG09_8012416、LG09_16001432、LG09_46132314、LG09_50708986、LG09_64555870、LG10_34723841、LG11_54415、LG11_8139610、LG11_34802586、LG12_284283、LG12_32322540、LG12_40522222、LG13_32072502、LG13_40000353、LG14_605466、LG14_33209388、LG14_56176655、LG15_32001166、LG15_24017141、LG17_24096219、LG17_32001525和LG18_10766。
本发明的第六个方面是提供采用本发明第五个方面所述的构建方法所构建的橡胶树品种DNA指纹库。
本发明的第七个方面是提供一种橡胶树品种的鉴定方法,其特征在于,采用本发明第二个方面所述的引物组对待测样品的总DNA进行PCR扩增,然后进行SNP位点多态性检测,根据SNP位点的检测结果确定SNP分子标记的基因分型,再与本发明第六个方面所述的橡胶树品种DNA指纹库中的基因分型进行比对。
优选地,应用SNaPshot方法进行SNP位点多态性检测。
本发明的有益效果:
本发明筛选的50个SNP分子标记特异性、灵敏度和分辨力高,该SNP分子标记不受环境条件影响,且检测结果准确、重复性和稳定性好,能够全面揭示橡胶树遗传多样性真实水平利用;利用该套SNP标记组合建立DNA指纹图谱库,可以对橡胶树品种进行聚类分析和品种鉴定,使得检测结果更精准、高效,为橡胶树资源保护利用提供理论依据,首次有效区分出橡胶树66个品种,本发明对育成品种的区分率可达100%。利用本发明的单核苷酸标记位点进行检测具有简单、快速、成本低的优点,能够实现生产中大规模的应用。
附图说明
图1为本发明实施例2中部分样品扩增产物的分型检测结果示例图;
图2为本发明实施例2基于SNP的橡胶树品种的UPGAM法聚类分析图
图3为本发明实施例2核心SNP对橡胶树品种的识别率。
具体实施方式
下面参照附图,结合具体的实施例对本发明作进一步的说明,以更好地理解本发明。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:橡胶树SNP位点的筛选与确定
以9个橡胶树品种PR107、GT1、IAN873、RRIM600、93-114、云研77-4、热研7-33-97、热研8-79、热垦628为样本,进行基因组重测序。具体操作参照“Du,H.,Yang,J.,Chen,B.etal.Target sequencing reveals genetic diversity,population structure,core-SNPmarkers,and fruit shape-associated loci in pepper varieties.BMC Plant Biol19,578(2019)”。
基于橡胶树基因组重测序数据,与橡胶树参考基因组(https://www.ncbi.nlm.nih.gov/genome/?term=rubber+tree)比对,挖掘SNP位点,根据以下原则进行SNP标记候选位点的筛选分析:(a)最小等位基因频率MAF>0.4;(b)缺失率<0.2;(c)杂合度<0.2;(d)上下游200bp无序列变异;(e)两等位基因位点;(f)染色体分布情况清晰;(g)在基因组上均匀分布。
从上述9个代表品种的基因组重测序数据中,最终筛选出50个高质量、单拷贝、高多态性的SNP标记,用于不同来源的橡胶树品种鉴定、群体遗传结构和橡胶树资源遗传多样性分析。50个SNP分子标记的序列信息如表1所示。
表1:
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上表中,“Sequence”为SNP分子标记所在的核苷酸序列;“[]”标注的为SNP分子标记位点位置,其中的碱基为该位点的多态性。
实施例2:橡胶树SNP标记的测序验证与应用
1供试材料
供试材料来源于中国热带农业科学院橡胶研究所国家橡胶树种质资源圃,选取66个橡胶树品种(表2),每个品种分别采集3株不同植株的叶片,经液氮速冻于-80℃冰箱保存,用于提取橡胶树叶片DNA。
表2 66个橡胶树品种的信息
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2橡胶树总DNA提取与检测
参照CTAB法提取所述各个橡胶树品种的叶片总DNA,后用琼脂糖凝胶电泳和分光光度计检测各品种的总DNA质量,以确保所提取的各个橡胶树品种总DNA完整性好,且A260/A280比值在1.6-2.0之间。
3 SNP引物设计与筛选
根据上述筛选的50个SNP分子标记位点序列信息,使用引物设计软件PrimerPlex2设计PCR反应引物,设计了167对SNP引物,经筛选后共得到50对特异性较好且多态性较高的SNP扩增引物,具体见表3。并使用引物设计软件Primer3plus设计单碱基延伸引物,具体见表4。
表3扩增引物
表4延伸引物
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表中,延伸引物中,大写是特异序列,与SNP区域进行结合;小写是加长序列,保证一个反应中的多个分型能够准确判读;方向代表测序方向。
4分型实验
4.1 PCR扩增反应
以前述提取获得的66个橡胶树品种的基因组DNA为模版,利用表3的引物,扩增出含SNP位点的核苷酸片段:在1.5mLEP管中配置PCR master mix,并振荡低速离心。选用8道移液器,在384孔板的每个加样孔中加入9μl PCR master mix,最后加入1μl模板DNA(20ng/uL)混匀,小心盖上384孔封板膜,并压牢每个孔,防止PCR程序时出现蒸发。1000rpm离心1min。
PCR扩增体系为:2×Taq PCR Master Mix 5.0μl、基因组DNA(~20ng)1.0μl、上下游引物的混合物(浓度10pmol/uL,上下游引物的比例为1:1)1.0μl、dd H2O 3.0μl,体系的总体积为10μL。
PCR扩增程序为:94℃预变性5min;94℃变性20s,60℃退火30s,72℃延伸30s,共35个循环;72℃终延伸3min;16℃1min;4℃保存。
4.2产物碱性磷酸酶处理
在PCR反应结束后,将PCR产物用SAP(shrimp alkaline phosphatase,虾碱性磷酸酶)处理,以去除体系中游离的dNTPs。在新1.5mLEP管中配制碱性磷酸酶处理反应液,SAPMix反应组分如下:ddH20 0.75μl,SAP(1U/uL)0.5μl,ExoI(5U/uL)0.15μl,10*SAP buffer0.6μl,PCR产物4.0μl。接着将SAP mix反应组分加入384孔PCR反应板,移液完成后,小心盖上384孔封板膜,并压牢每个孔,防止PCR程序时出现蒸发。最后离心后将384孔反应板放置于PCR仪上,启动SAP反应程序。SAP反应程序如下:37℃40min;85℃15min;4℃保存。
4.3单碱基延伸反应
在碱性磷酸酶处理结束后,进行单碱基延伸反应,反应体系为:SNaPshot Mix 0.5μl,Pooled PCR Products 3.0μl,Pooled Primers(表4)1.0μl,ddH2O 0.5μl。将反应体系加入384孔PCR反应板,移液完成后,小心盖上384孔封板膜,并压牢每个孔,防止PCR程序时出现蒸发等现象,离心后进行延伸反应程序。延伸反应程序为:95℃预变性30s;95℃变性5s,52℃退火5s,60℃延伸5s,共35个循环;72℃终延伸3min;16℃1min。
4.4上机
取2uL SNaPshot反应产物加入到8μl含有0.8%LIZ120的去离子甲酰胺中,95℃变性5分钟,然后放-20℃骤冷,然后上3730XL测序。
5引物优化调整
从ABI 3730xl仪器上导出.fsa格式原始数据,按检测反应进行分类归档后,分别导入到GeneMarker分析软件中,进行基因型数据的读取。根据各位点信号值与出峰情况,对各位点引物比例进行调整,保证各位点信号值与出峰情况基本一致。
6结果质控与分析
对正式实验结果进行分析,按样本、位点情况进行统计,得到样本各位点的分型结果,导出各反应结果的PDF图。部分样品扩增产物的分型检测结果示例如图1所示。根据结果信息进行参数分析,得出详细的分型结果,66个橡胶树品种的50个SNP位点的基因型如表5-9所示。
表5 66个橡胶树品种在50个SNP位点的基因型(1)
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表6 66个橡胶树品种在50个SNP位点的基因型(2)
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表7 66个橡胶树品种在50个SNP位点的基因型(3)
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表8 66个橡胶树品种在50个SNP位点的基因型(4)
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表9 66个橡胶树品种在50个SNP位点的基因型(5)
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根据样本间Nei氏遗传距离,使用phangorn R包进行非加权组平均法构建***发育树。对66个橡胶树品种进行UPGAM法聚类分析,结果显示一些亲缘关系较近的品系聚为一簇(如图3所示),如高产品系热研879、热研8-333、热研87-4-26聚为一簇;抗寒品系云研77-2、云研77-4、GT1聚为一簇,表明50个SNP标记能实现对66份橡胶树品种的区分。
本发明首次区分了66个橡胶树品种,对育成品种的区分率可达100%。这说明,利用本发明的单核苷酸标记位点进行检测具有简单、快速、成本低的优点,能够实现生产中大规模的应用。
为了进一步筛选核心SNP分子标记位点,从上述50个SNP分子标记的位点中选择不同数量、不同组合的位点对66个品种进行区分,统计不同数量、不同组合的位点对于66个橡胶树品种的区分率,以位点数量为横轴,以该位点数量下,不同位点组合的最大的区分率为纵轴作图,如图3所示。从图3可以看出,位点数量为7的时候,对66个品种的区分率可以达到100%。达到100%区分率的SNP分子标记组合为:LG03_104230492、LG02_8025843、LG01_54721919、LG15_32001166、LG07_72003724、LG05_88010730和LG02_214754。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
序列表
<110> 中国热带农业科学院橡胶研究所
<120> 用于构建橡胶树品种DNA指纹图谱的SNP分子标记组合及应用和方法
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<170> SIPOSequenceListing 1.0
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tagattgaaa ccttggaggg ttcatggttt tgattctagt tcacccttta gatgaacaaa 120
aactggtggt tctgggccgc ttaaaactag aaccggattg gtggctgggc tttgggctag 180
gcatatgtgg tttgcaaaca aactaatggt aaaaaaataa aaataaaaaa aaaaaaaaag 240
atactaaaag agtgaaaagt agggaagggg ggaatcttgg gaattgctct aaatatagac 300
cgctaataat tgtttggtcc attatgtttc tttccatctc ttatgctttt tcgtacaagt 360
aatattgtgg atcaacaaat ttgtgattca tttggataaa tttcacccat ggtacctaaa 420
cttagggcat tataagtagt tctttggaga agttgttatc atataactga aacaccttca 480
aattattcaa tttctaattc cctcaagtag tttcccttat atttgattca aagatatatc 540
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aaatggtaat tgggattgcc ctaacagaag aaaagctcaa agctttaaaa gaaatagaca 60
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gtggaggaga gagagaggga tttatagaga gagaagagga gggtaccatg atggcggtta 180
gtacaaatat agtagggggc gagggatgag ctgggtttgc tgatagatta atagttgtgt 240
aagaaaagga gaaagggagg gaattgaaat tggattaaag gaatcttaat tcctgaatgg 300
cacaatgaca aaggatattc ttcccaaata agcatccaga gaaacacccg agtatatgct 360
tctaaccaag cttgcagact ctgatcgctt ttactggttt tttgaaaata aaaaaataaa 420
taaatatatc gattgtgaac ctgtcactaa ttacagttga ctttttttta ttattgtata 480
tgagctaaaa ccattgataa gttgagtcaa ttttcttgtg ttctggagcg cctttttttt 540
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aaaaaaccta ctatattaaa aattcttttg aaagtaagca aaattttttg tctaatattg 60
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ttgatgaagg caacagttat gattcgcaga cttaaaaagg atgttctttc tgaacttcct 180
gtaaagcgtc gacagcaagt aaatactcta tttggttcta atatttgctg gttggatttc 240
agcaggtgtt tgtgctttgc gcatgctact agcgaacata atatatatat gctgtgaaat 300
agctgaatag tatagccatt aattatcaga gacatcaaaa taattttcaa atgctagaca 360
ttatttttta agcccccttc gggatgtgtt ttgcagtcca gctccttgag atctatgatg 420
gactggcttt gtacagttcc tttggcatta aatagctatc tactggaaaa attctcatgg 480
ggactggcaa aaattttcct aagtagtatt tttggatttt gtcatactgt agcatgcagt 540
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atatatttag gggacggcat gcctgcctac gtaatcttat aatgaaaaga aaaatgtgga 180
gaaagaccct tgaagaacga acaattgctc atatgaggtc cacgtgcatt acatattaaa 240
tatagattaa aattttaacc ttccacttct ttacatatyt gccctctttc ttgacagcta 300
gcttgtaatc ttycgctgta ctctgaccac tggtctccga tcgccatgtt gatggtgtat 360
aaactacaaa gtgcaaacca ttaatatatc tttcttttct tcacgtaaac cctaaatctc 420
ccctcttctc ttcacctgat ccgtctccaa agaagcagac tagtttactc aaaggtgcga 480
tctttttttt tttttaattt ttccccactc ttcttttgct taatgagaaa atggaagaaa 540
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tcaatactat ggagatatag atcagatccc tcacatgtct tattggtgga gagcattgat 60
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gaattaagga ataaaagaat tccattggtg aaagtacttt ggagaaacca caaagcgaag 180
gaagcgacgt gggagtgtga agagattagg agacaataat attcacagtt atttgaacta 240
ggtaaatttc gaggatgaaa tttatattaa gagggaaaga attgtaatgc cctcacttta 300
ggcagttctg tacattctac tactccgatg actaatgtct gttcagacgg ctgaaatgtt 360
tggaaccact cttaaattaa agtaaggagt cataatttaa tctaataaag atcaagtaag 420
gttgaaggaa aataaaagaa atagattata aattagttaa atgtgcataa gtcccagcga 480
tgagtaactc ccctaggaag ttggccacta agagttagag gactaaatta aattaatcaa 540
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aaaaaaaaga aattatagat tatatatata cacacacatt acttgcccag caaacgtcaa 60
aacgcatgtt ttggcaccgt ttttgtcacg aactaaaatc ctagcaccaa gaagtaaaac 120
cctttctaca aattccgcag aagcaaaaat cggaggatca aaattgccca tcaagaacga 180
agaattggcc tcagcaatgg ctgattcatc agtgaaatct gataagaaga agaagaagaa 240
gaggaagcta accagccccc aagataaacc aacgcctcca aagaagaagc acagaggtga 300
tgaaccagag aaagagagag aagtcgctgt aggtggaagc tcaaatcaga aagaatctta 360
ttctatgatt gaagcagagc ttccatggag gaatcttcaa cttattctat cacttcagag 420
caaagatatt gaccttcaaa agttagtatc tttcccatct cttcttcaca caaattgaat 480
ttttgagatt atttatttga ttttgaatgg aatgtaaata aattctgtac ataaagcaaa 540
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g 601
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ttgaagttga aattgagaga ctaaaaaacg cttttcaata aaatacaatt ttagatcaag 60
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caagtgtaat acttgaaacc aagtcattaa aactacatta gaaatatggt acaccttatt 180
aagagctgaa aagaccagaa aatgaagaaa ataaggatca aaataaattc aattgtacac 240
catccagcca cttaagtgta ggcagaaccc taaccagaaa taaggtaaaa ttttcatcag 300
caatggcatt ccccaccaca tccaattgtg tcaagttcaa gcctttcaac acaaaaaagg 360
cttcccaata atttgtcatg ctgaccccat caaccaaaat tctatcttgc tcccattcat 420
ttccaatcga attagacaaa ggagatgaac acagatatct ggttgtgtca atagagcgtg 480
cacctatctc attgtatgaa atatccagcg atttcaatga cttcagctgt ctcaatggct 540
ccaatgcagt aaaactagag agtttattct cactcaaact caaatgaatc agtagctgca 600
g 601
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tatattggct tcaacagatt gtggtgtttg tctctgtaat taatactaca ttaataaaat 60
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ttgcagaagt tacagctttc tcaacaattt ctggcacatg atcgtacctt gaaaattcag 180
gacagtatgt tctgaattct cttttctatc aaactcttaa tcatgtgtct accgtactag 240
gagcagtggg taaaacgaac tcatccattc ccaagttcat taactgcttt ccaagctcat 300
ccctcaatct cctgctcatt ycaagtcttc tgcactctag ccttttgcgc aacaaatagc 360
acctcagcta ccttatctcc aaaaacgctc actttagcaa cagttcagct cagatcctct 420
gctaaatatc acttcaacaa catctcttct ggtgtagact tcttcaataa caagtgcatg 480
atagcacaaa cacagtacaa aaaaaaaaaa aaatacaaga taataaattt cataacatta 540
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atttggcaat tggttgggaa gaaaaaaaat ttattgcatc acgccgacga tgagtgcatt 60
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ctcaccgttc aaagtctgac ggctgaattg ttggacaggc taatcatgct gacttggagt 180
cctgcaattg gtactccaaa agtttattat ccagctaagt tatatataaa aaaatgcaac 240
ttaatttaat aattctcgat cgaatcacgg ccgtcatttc agagaaagga catgacgaga 300
aggaaagtga atcagagccg tcgaaataaa ataatgataa acgggattag gttaaaatgg 360
gaaaagaaac gaggtgtgtc tgccactggc aacgtctctg cacgctgctt aaaggccgaa 420
tcaatggtca gatcaaatca caggtaaatt tttgactttc aggaagaaaa taaaattaaa 480
tttaggaata aaattaaatt ttttaaataa taatcaaatt aaatttaggg ataaaataaa 540
aatttcaatc atttgatatg tgagatatac acagataagg cacatagtat atggatcagc 600
c 601
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aatcataacc ttaaaaaaag aaagtaactt cttgtatttc agttccatga tcataacact 60
tgctcgtcac acttttatgc ttcaatgaga aatctcaatg tcccaatcac tatttccaga 120
gggaattgtt attgtcacaa gattgtcctc ttctctgaat gtaaatccca cttctttgga 180
gtttaacaag caagattttg ggtttacact ggagtatgca ccaaacgtac cttcccctct 240
ccccttgatg cctattctac caccagactg ctcaattgac tctacagctc caccagaatt 300
gtacatgttt attaatccaa tagctgcgaa ctcaatattc tgattgtaaa cctgtgcata 360
tcaaacaaca ggggagaatt caaatacttg ttaatatgtg agctaaaccc caaagaactg 420
gccattccca acctaccttg ataggtgcta tggtaaagac atcacattgt agagttttta 480
acgaaatatc aaaagcttct tccttctgca atcgacatat agatcctgca aagttttaat 540
aacaaaaaaa gtataaaaat gtaatgtatc acatcaaagc aaatacattg gtcttgattt 600
t 601
<210> 11
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gtaaattcat ttttaaaatt tatgaattaa tttaattaat ttataagtta aaacaaacag 60
cctcttatga taaattatga caattaaaaa aaaactattt aattgtttta aagtttttat 120
aattaaaaca tgtcaaattt aagtttaaac tataaactat ttgttgatac cttttaatta 180
acatatttaa gaatacattt ttctcaacag aaatgttaaa taggccttgc aaattgataa 240
tgcctaacac tcgctctaac aactcgtgaa tatgcacctt cgtgaatatg caccttcaat 300
gagttcagat atttgattta ttcatgcacg taaatgtaat caaatgtatc ctttccaaat 360
ccaaatttca gaaatttctt aaatgtaaat atacagtttt catatatcgt tgaattgaac 420
ctcaatagat tttgagggaa agctacactt acttgcggga agaatgtgtc caactactgg 480
gaaatacgct ttcgaggctg gtaatcccac gtgctccaac cagcaagaac ttctaattcc 540
agcagcaacc cttcaatctt tgcaactaag ctctgatgcc actgctgtgc cattgtaaat 600
t 601
<210> 12
<211> 601
<212> DNA
<213> Artificial
<400> 12
atttcttttt agccaattcg catgtttaga atatatatat atatatatat atatatatat 60
atatatatat atatatatat atatatatat atatatatta aaagaaatag aaatcaagca 120
tgatagtatg agaattcaat taaattcttt tcttacaaat gagttattca aaaggaaatc 180
aataaggaaa tacgtatgta ttgttgcaaa tatacaagtg tagaattcct acatcggaaa 240
aatataggat gactgagaga catataaata ggaaagatcc aaaaactcat tgccttaagg 300
ttttgggtta aatgtgatgt caagctcatg agtgtgtcct tcacaagacc tatcaagaag 360
gattctccac aggcgctttg ggctccttag atttctaaca agaatatgca atcctaaaaa 420
atgcttgttt ccacttacac taaccaatca gaaaatttta tgcccaacag accataaatc 480
taaacaataa tgttctatga tatagtagta ctgttatcat tccatgcatt ggagcacact 540
aacctggtat aaatttattt aatgtagttc aaatcccaaa aaggggtata tataagttca 600
a 601
<210> 13
<211> 601
<212> DNA
<213> Artificial
<400> 13
gtgttcttct tgaaaagaga ggaaaaggtt cttaaaaatg ctagtgctaa tacaggcaga 60
gtaataaatc agcatataaa atattctcaa acaatctata tagtacgaaa acatttctgc 120
atgtccctgt gacagaagca gtaacaatgg gaaagttcag ggactttaaa catgttagtc 180
tggttttggt ataagattct cacaaaggcc ttgtccatca ttgagtataa tgacgcatca 240
tccaacttcc acaaaacagg aagttattat tcaaggcagc caaattttgg atgcagctca 300
caacaaatga agatgttgat gatgctaaga ttttggtgaa tgcaatatga gcacaaatta 360
tgttttacca acattaagcg taagggaact gcaagaatat tggaggatca aacagcaaga 420
gctttattcc tgtatgttat gttcttgtag tagtacgaat atctttcttt gtgcaaatta 480
cacaggattt aggcttcatt caggttactt ctccaaaggg cttgttaaag gtatatatct 540
cttgatgttc agcaagactg tagatcttta aatttttttc tttttcctat ttagaggaag 600
a 601
<210> 14
<211> 601
<212> DNA
<213> Artificial
<400> 14
tgcttctttt ttatttttct ttccctattt tattctttgt caaatagttt atttttgaaa 60
ttttataaat tttgctacac attattcaat ttgttgtaag tactgtccat ttgtgctgat 120
tcatgccaaa accttgcagg ctcttctttc aggatgctct gccggaggat tggcaactct 180
tattcattgt gataactttc gagatcttat gccaaaggat gctactgtaa aatgccttgc 240
tgatgcagga tttttccttg atgagtatgt ttgatatgaa tactgatact ttccacctca 300
gtatcttaag ttagcatgtc atatgagttg ggtgataccc atgtcatagt atttttgtac 360
catgtgctct taatcatctt ctaattatag aatttttatg tggatttaaa gatccccatt 420
ccccaaagtt tactttttta caccttggaa ttccaactcc cctttgcact atcctccttt 480
cttttatcat agtagaatta actacagtac gttagcaact taattcttct gcagagattt 540
tgttttccta gttttttcat caaaagttag atgtgtccgc tgaactgaat atatatatat 600
t 601
<210> 15
<211> 601
<212> DNA
<213> Artificial
<400> 15
tggaagagat cagtgaggaa gattttaata agcaggaggt taaggttgta aaagaatcga 60
aacctataga tggtaggttt tggacgatga gagatctgta caagtatcat cagatgggag 120
gaagatacgt ctccggcttg tataatcttg cttgggcaca ggcagtacag aataagcctc 180
taaatgagct atttgttgag gttgaacccg atgagaattc caagcgatcg tcgccgtcgt 240
cttctgtggc gtctgttaat agcaatagta acagcagcaa agaagacgag aagaagaaag 300
ttgataaagt tgtgattgat gatagcggcg atgagatgga tgataatatt gtggatgttg 360
acaaggagga aggtgaattg gaggagggtg agattgattt ggattttgac ccaggtgaga 420
aagctgctgg ggagggtaaa gaagggctgt ccaatagtga tgaaatgaat gttgatggct 480
tacagattga gtccaaggag aagaatttgg agaaggaaat caactccatt cgtgaggctc 540
tggagagtgt aacagtgatt gaagcacaca agtaagcaat ttgttgctgt tgtttcaata 600
a 601
<210> 16
<211> 601
<212> DNA
<213> Artificial
<400> 16
ggccaaatct cacagaccac ttccaagtcc caacaagaag gaaggccaaa agacgagctc 60
gctaaaacag ctggttatgt cggcgtagaa ccaagcaccc cattagtcct ctgggttgta 120
ctcgagggga gagagagatg ccaacccact tggtgggtgt tcttagggga gtgatagatt 180
cttaaacaag taaatcaaat agagtagatt gataaaatat ttaaataatt aattatatat 240
ataacaaaac aaagaatatg gtatacaagt attatattac catataaact aagtagaccc 300
ataatagtct gttgaatatt ttacgcttgg ggtaactggt agactaacca cgttcctaaa 360
acaggatata aaggtagttg aacaaaaagg catttttttt taattagaga gtgagaaaga 420
gggataaagg caagaaaaac acattgaaac gcaagcatct tgctccaaaa gaagtgaatc 480
tcatatttct ctggtgcttt attgactgca gataaaacac aagaaagccg ggaatcaaat 540
gattccatct caagagaaga aatctcaact cgacgccatt gctggtgaaa agagtcgatt 600
g 601
<210> 17
<211> 601
<212> DNA
<213> Artificial
<400> 17
ttatatattc ttttggattt tggaagtcaa gattcaactt ttgatttcat ttcctgctta 60
tattctggga tttgttttcc atagtttggt tgattttagt aattgaatat gggttcttga 120
gttgaaagtt attgagatct gagaattggg aaagctcagt gatttggata ggtgtcaatt 180
gccagtgtga aacttaagtt tgcttgctgt agctattcat gttcttggtg ctttgcttaa 240
ttagagtaca gcatgtattg agtgaagctc ttatttggct aagtgctcct ttgtcgttgg 300
atttcttgga tgtattacat tcttagttat gcttttattc actattcttt tctgtctttt 360
tcttggaatt cttggaatgc agcaagcaga tatttaaata tggtttaaaa cgactgattc 420
taaattctgt ttggtctcct aggcttttca ttttagcaat gaaactcttg ctttgaattg 480
attatgatgc tgcacctctt ctttgtcctt gaaattttga actgttttta atggtttttt 540
tttttttttt ttttgtttta tacatgcaat gaaaatttaa tttttttttt ttctgccata 600
t 601
<210> 18
<211> 601
<212> DNA
<213> Artificial
<400> 18
attattggtt tgaaatttgt ttctcggctg ttttttatct ctctatacgc cgtggtcttt 60
tgcatttata gttgagcttt ggataatttc ttatgtatct ctatctgtag gtctctgcat 120
atgaatagga tttatttttc cagttcaatt tctttattct gctgacattg cttttagttc 180
tcaaaatcaa cctttgagca catgtgaata tattttgcct aaaacttcac cattctgcat 240
gtattcaccc ctacacttga tttcctggtt catatatttg acatgattat aatatcggta 300
tttttggctc ccatgttgac atattgttgc aaactatagg ttgtattctt gttcatgctg 360
ggttgtgctt ctcttcatta ttaaagtctg tcttttctgt tttgcgtgat gaactctaat 420
tcatttttcc atttaaattt ctttgacttc attatggata attaacccag tttgatttga 480
atgctggtga gagttaagga aaactgaaaa ttactcttat gctgttgtga ttttcagaaa 540
atatcaactc aggccaggct aaatgatctt tttgggtaga atatatcccc tagaatgata 600
t 601
<210> 19
<211> 601
<212> DNA
<213> Artificial
<400> 19
ataaaaataa ttttcggttg gtttttattg aaaaactaaa attgaactaa attatcaaaa 60
aaatttggtt cagtatggtt cttcagttcg gttcaggatc cctaagaact gtgcacagct 120
ctgcttatac tatgtcaata ataatttaat tttaaagttc aattttgtag ataatgaccc 180
aaaaatagat tttcatttct ctaacaatcc ttattcgcat tattcttcag aaatatgaaa 240
tatgtggagc ataaaaaaga taacatttca ttaagataat caattgtaaa taaggtgaat 300
agtgagaaca tttactcaag aattagtttg gttgaacata tactcaagaa ttagtttggt 360
tgaagtaaga ggcttgcctg gcgttgatga aatccattat gcttgatcca tcaccgacct 420
tcaccactct tccattttcc atataaaaag ctccatcagc atactcgagt tcttccaatc 480
gatgggtcac ccataaagct gtaatttcat cagatgcaac caaggagttc ttgactgctt 540
taatcacccc aatctggcaa tcaatagcca acatgagtaa ataaatatta tcaccacatg 600
a 601
<210> 20
<211> 601
<212> DNA
<213> Artificial
<400> 20
gatatggaac caccaatcct gcataaactt gattctaact acaactaaga cgatgaacga 60
caatgacaat aattctgatg gcagcaaagg aaattttctt tgatattaga ttctgctttg 120
ttttcacatc tttttctgat tacacaggct tgaacgtctc aatcctggat gttcattaat 180
gtacttacct cattcaagac cgaaagacaa ctgcttttca agaatcaaga aaattcaaga 240
ttcacttatg aagaaacaga aatgaagaaa caacaaacat taaatccata cataaaggac 300
cagccaacat tgcataccta aggttggttg gttatattgt tgtatccgca ccatgaaatc 360
tttaatatca aaatggtttg aagccctcta atctatttcc agctatttct ttgctgcctt 420
ctctctctct ctctctctct ctctctatat atatataatt gaatatataa ttgaatcatg 480
atttggacaa accaccaaac ttcacttatg aagaagcaga aattattgag caccacatta 540
gttccactac tttgctgata ttcaaccggc ttttgtggct tttgtgatca aaaagttgct 600
g 601
<210> 21
<211> 601
<212> DNA
<213> Artificial
<400> 21
ttcctcccat atcaaatttc actattccct tgggatccaa atcagttgct gtcaggtcct 60
acttcttaga aagaatttgg tgacaaagtg acgactagtg tgtctgatca agtgatggga 120
gggtttaatt agatcttccc ttcaacacaa taagagggct ttacttcatt agttttattt 180
tttttagtta acttttccaa gttcttttgt tatttctctt atttcttcat taataaaata 240
tttatcttgt ttgagaacaa ggcttgaatg ttaaggattg agaacttcat gttgagaagt 300
taatgttctg caggacattg atgagtttaa gagaatgaag caaaggagaa aaaaaaatgc 360
tttttgaggc tcagattgat aaagcaatcc caagggatca gatgagatgg taattgtatt 420
gtagggaagt ggggtagagc ctagaggacc tggatcacct ataccagaag gcaccagaag 480
gatctgcagc agtatgagaa ttcaaatggt tctctggaag ttatccagct acgagctgat 540
aaggtggaga aatttctgaa aggccatgtg atcacggcaa ctgaatcttc tataaaaagg 600
g 601
<210> 22
<211> 601
<212> DNA
<213> Artificial
<400> 22
tagacttttg cccaataatg cttttgcgtt tggttttatg gatcccttct tgcttcatta 60
cactggtaaa aatgaacagt ttactgagac cttatataat attggtatca gtattatttc 120
cagtgttgag ggcaaatctt tgtcctttta gtgattttat ttgtttatac tgtggcaaat 180
tatgctcaag caatgatgtc atgtaactta atcaatgtca ttgcctgtgt catagtggtc 240
atatgttatt gcatattatg ttgtattcat ttacgccaag caactgtcat cagtagttct 300
catctatcat tagctcatgc ccatgctttg cttttgtatc ctacagaaat tttatgctct 360
tcaaaattag aacttgatca caagtttttg atcaataagt tagttgtcaa gatatgatac 420
tattttaatg tttgttttag ccagtgatag tataatgtgc ttttgaatca tctatactaa 480
tgtcattatt ccaggttggt ggtaatttaa ctgtggatgg tattgactat tatttatctg 540
aaacttttgg agaagggctg tacaagtcat gcaaagatgt taagtttggg acaatgaata 600
c 601
<210> 23
<211> 601
<212> DNA
<213> Artificial
<400> 23
ttaccaacta aatataacaa taatgatatt ataatcaaaa tgcttctgtt aaactagagc 60
ctcaatctct aaaaagttta ccctccctct catgccctct aaacgctaaa cattataccg 120
ttttagcgta ttgtttcttg tctctcttct ctatgtcctt tctgtcgata ttttcttagc 180
tagacttgca gggaaggaaa agtagatgtg cagtgcataa accctaatct ttctcggcag 240
tggcccgatc aactcatttt ctctctcaat tcaaggtcaa ttttactctc gttttcattt 300
tattatcaat tttgttatca ttgataatcg caatctttaa gttgtttttg cttcagtaac 360
ctacaatatg actgaagacg tcgagatgaa ggaacttcct gctccctcca actccgccac 420
ctcctcttcc ccttccacac tcaaccgtaa gcctatttct gggttttctt caattttttg 480
atttcttgct tgatttctga tttgggtatg tgagtttttc tgttttgtag atttgaagga 540
gatcgcttca ctgattgaga ctggtgccta tgcccgggag gtgcggcgga tagtgcgggc 600
t 601
<210> 24
<211> 601
<212> DNA
<213> Artificial
<400> 24
tattgcagaa gactgagaaa tcaatgaaag atcactcctt ccaaatccca taagacctga 60
agcccctcca aagagacctc tattattcct gccacatcca aatatgaaat tgtccacagg 120
ggttgttcct aaattgagat gctccgtccc tagctcaccc ctggtatagg aaccatcacc 180
atagttaaca acatagttac aggttggtgg gttgctccca cagactcctg aattcccagt 240
tgcaaattgg agggattggc aaattgatga attacataaa actgtttcgt acgaagggga 300
tactgaaggg ttgaaaagag ggtcttgttg attataacat agtctgcaag gttgacattg 360
aacccatgtt aaatcacttc ctgtgtctac tatcactgtc atgtttcgac cacctaattc 420
tactgtaaca atgtagttca atgtctgaag tcttacacca gaaactaatg ggatttgact 480
atgtacagaa tcttcacttt tgccatgaac actctttttt attcgagatt gaagtgaccg 540
aacttgaaaa ttgtcaagaa tcaaactttc ttgtagcttc ttgttccagt cagtaatctg 600
g 601
<210> 25
<211> 601
<212> DNA
<213> Artificial
<400> 25
ggccaacata ttaggtactt aaaaaaaaaa aaaaaatcac cttgtcataa ccccgcccca 60
ccccccaacc cccatccccc acccacgcca ccccacatgc cccccaccca taggcttgga 120
cgcttattgc cctatatcct caggcatatc ttaagaagac cttagcaata aaagcgtcct 180
atatatgagt tcccctccat ttcaggccct tctatctatc catttatggt gccataatcc 240
acaagcttct caggcaatta gaggaaatta acgtgaaatg acaacttgca aaaatgccga 300
aaatgaattt caccaataac aattattagt ttggcaagta aattagtgcc tcctgcagtt 360
agatgcaagc gactacagaa gattcaagat cgatgtcaaa ttgccagatc tggcttcaca 420
tattcaaaca ttgaagtaag aaggttcatt tattagattt tctagatgat gatgatgatt 480
ggatggatca taatgacatt gaattgtatg atcataatca tggctgctgt ctggttcgga 540
gttgaaagct gaccgatcta actcagaatc caaaccggta aactttgatt gaccaaaacc 600
a 601
<210> 26
<211> 601
<212> DNA
<213> Artificial
<400> 26
gtgcatctat tatggtcatc atgttggaaa aacatagaat gatcaacttc actcctagac 60
attccaaatt gttagactac attactgaat cgactaaatc atgctctcgg aattacttta 120
aaccatacag tgattgtcat agatgacata ataagccaga cttctccctg aacaacaaac 180
cctggtggtt gctttttata tatttccttg gctagttcac catgtaaaaa ggtattttta 240
atatccaatt gatagagtgt tcagtgatta attgtagcta atgaaatggg tagtcgaacc 300
aaggcaatct tagccactgg agagaaagta tcactatagt caagcccaaa gatctacgta 360
tatcctttgg caacaagatg agctttaagc ttatcaattt tttcgttagg acccacctta 420
actgtaaaaa tccatcgaca tccaacaata gatttatcta gtggtaaaag taccaagtac 480
caagtctcat ttgtatggag catagtcatc tcctcaatca ttgcatctcg ccaccctgga 540
tggtccaatg cttctctaac cttctttggt attgttggaa tctcaatccc tacattaaag 600
g 601
<210> 27
<211> 601
<212> DNA
<213> Artificial
<400> 27
taaaaatgga aaaccatcta ttctttttaa ttgaaattgt ttttgacaag cagggagagt 60
gcaatatgga aggcaacatt caaaagatta gatttatcta tgatattagc ctatgtttta 120
tataatcaat gatgtaatat aatataatta taatttttat tttaatattt attttatttg 180
tgatggtaat tataacaaag gagttataat tgcaatgata gtgtaatctc ctcaacaaac 240
cattgccatc ggcatccaaa attagagcag actagccaat tttgataaat aattctaagt 300
tggtttctat caaagttgat tcatatcgac tttaatttca aattagactg atctgattct 360
gggctaaaac caaattgtga tcatgattac cataggtatt accatgattc aagttagacc 420
taaaattgaa tcaaaaccaa attgatcaag ctgaagatca aaattagtca atattttatg 480
agctcaaaca ttgtgggagg gtcggtctct ctccccttag ccgaattgcg ttgcaaccaa 540
tggtttagac cgatgatata attaagttaa aaccaccttt ttaaaaaaaa aattatttta 600
a 601
<210> 28
<211> 601
<212> DNA
<213> Artificial
<400> 28
gagaaaatag tgatgtatct ttaagtgtct ctcatatgac ttcaaataag cagttggtcg 60
acatgtttac tctaagtagc aaattattcc atactagagt ttgcaagtta ggcatgtgta 120
atatacatta accaacttga aggggagtat tagagtccct aacttgtagg atttaattgt 180
ttattctttc ctatttgtat ttattcttct cgattgtatt tattagtttc ctattgtttt 240
aatttcccta aaatacttgt gtataagtag gatccatgga aaagtttgaa ttacagagaa 300
atacataaaa tttcctccaa attagtggtg tccacaactc atttaacgag ttggcaagtc 360
aagatgcaca taaaacaacc attgaagatg gataacataa ttatttattt ggttaatatt 420
ttttatcatt ttatcttcaa aatcacaaat ttaaatctga cattttttaa aaaattactc 480
ttaagtagaa taattaaata tttatatggg gttaagttag agtttagtgg tgaacctgta 540
acacaatata atatccatgt tccaacataa taaccatgtg aaaacacaaa agagagggcc 600
a 601
<210> 29
<211> 601
<212> DNA
<213> Artificial
<400> 29
cgcaggttgg taatccaagc aaagcaaatg gcaggaaatc caaacctcat ctctctgaag 60
tttagtacca ttgaagcttg tgcctatatt ctagttacta tgttctcata ctgttaccta 120
catagggaaa aatgccaaat tatgatcaat cctgacagct tcctagaagc acgcaccatc 180
ctgtaacctg cgcatgctcc aacatgccaa attcaatcaa catcatcacc agaaaagggc 240
aatggtaatg cacgtggtag aggctggagc tccacaaaca tcacataatt caacagaatg 300
tcaagaatct catccactta tatcaacatt agattaatca tatcattgcc attaattatt 360
atgcttaatg aacataaatt gaaatatcca agcaccagga aagatccata gttgtatcca 420
agcaccgttt attataaagc agaaagttgc tgtacatggc acatgaaaaa tacctatagc 480
caactgaaat caagtgagac gttccttgtt tcccaaatcc cttttaaatg tgctctacat 540
ggcaaaaagg ctaatttcac gcataaaatc aaaatatgat agagctttga gaagagttat 600
t 601
<210> 30
<211> 601
<212> DNA
<213> Artificial
<400> 30
caaggggctt gcaaaaagaa taagtcttaa taacatccca aaattgagct tcaagttcag 60
tatagccttc agggcccatc tcctatgcca cacacacaca cacacacaca cacacagaga 120
aatagagaga tactgctatt gaccgctcca ccgcctgaat gaaatcaatt ttttccacta 180
ataatttgtt ttcccttcca gccaacaaag cagcttcatt tactagattt gcaagatcag 240
ccctgcaaaa tataaaatgg aaaaagtcac ttccgataca ttagctaaaa gtaggacatt 300
gaagtctctt aatcaggagg aaatttaccc agtaaaacca gtggtcatag atgcaatgtc 360
actaaggtca acatcctcgc caagaggaag ctctttcttg gaagcatgca cttttaaaat 420
ggcttctctt ccattcctat caggcgtttc cacctataga gtatagacca aatgagataa 480
atgtctgaga caactattta gtggaactag cttgcctgtc aatcttgacc atccaaaatg 540
tgaagaagct gaaatacaaa ccataaccac acgatcaaat cttcctggtc ggcgaagtgc 600
a 601
<210> 31
<211> 601
<212> DNA
<213> Artificial
<400> 31
attgtaatta aactttgaga ttataagtaa acttgtaatt tattatttat gaatttctat 60
tatgaatgca atagatgata ccttttggag atcgcataaa taattgtgaa tgatgtgata 120
cagtagaata tgatatggaa aagtgtgaaa tgaatataaa cataaaaaca ggtaactagt 180
ggaactcgct agatgctaac aaaacagagg aggctctgtc tgggtttcta taaaaaaaaa 240
aaattctaca tatagatttt ctgttttaaa tgatattaaa atttacaatg gacatgataa 300
gacaagatag ggtgctccag caccgaatgt ggcacttctt gctcgactat acagtagaca 360
agtaaggggc gtcacacatt atgagttgcg tgagtaatgc tctttgtgag cttcaccttg 420
tgtcaagtag tattagagta ggaaaatcac cccaactgag atgacttacc ttgatggagg 480
tggcaactaa tagtggttct tctgatggtg atcgggaagc taaaaccgtg atcattagaa 540
caaaatgttg cgattaagga agtgacattt ggagcaacat atggatctca aattcatgca 600
t 601
<210> 32
<211> 601
<212> DNA
<213> Artificial
<400> 32
aatttagtaa gcagaattaa acaatggaaa ttcaatatat atttatttat atgtaaataa 60
aaaaataact atagacgaac ttacgagtat gtcagagaca attctgggct gctttagagg 120
cttgagaata aaacgaacta ctcgagaaat taaaacaatc aatataatct caaacaagac 180
gaggctgaag gagtaatcca aagggttttc tccataaaaa acacctaatg gatggggagc 240
atggattttt tgacatatta gtctaactgg agaaatgact tgctctacat gcaaagaggc 300
tcgaggctta gcttccatcg tattgtgcca ttcatctctg atttgagaat gataaactgg 360
gaaactccta accatgacaa gtacctgtaa tttcgaaacc atagaaaaaa aaaatgaaga 420
agaaaagaat agaaccacag atagaggcga gagtcattgc tgttaataat ggacaagcgt 480
atttttgggg gtgcttagca gtcttcaacg tttaccattt tccccctatt tttagtctat 540
catccacgtc aaggttgttg ggtttctcaa ctcagatgca cgcagcaaaa gtacaattta 600
a 601
<210> 33
<211> 601
<212> DNA
<213> Artificial
<400> 33
agtattctct gactgtgtct ctactttttt tttatttttg ttttaaaact aatccacgta 60
ttattatgtg gttgggttgc cataccaact atgattctta attggtttag cagcttcact 120
ataaaatttg tcattatata ataaggaaaa agatgtaatt ataataaatt aacataaaat 180
ttttgcaata aggatcatat atttataatt ttaaatttga agtttcttct aaaaataaat 240
atactcaaga attttaagaa agaaacaagt aatcttattt ctggtggcaa ataatatcat 300
ttaccggttg gctaataata taaaaacaaa taccgcaatt gatatataac cgaatggtat 360
atggatgaag ctataaaatt cgtagggttg caaacagcaa tgttgcttaa gattagtagt 420
ataagaaatg gttaattaag agaatacatt aattagaatt aaaacaacaa agcagtgtag 480
aataattggt tccaagtatc aggaagccca ggaaggcacc aatggctaca gtctgctgat 540
cggtaagggt tagccctctg ctcagggctc aagtcaccac tatatatgga tggatggcaa 600
t 601
<210> 34
<211> 601
<212> DNA
<213> Artificial
<400> 34
aatatagatt acaaaataat gtggataata ttccaagcta tagacaaagt ctcctatcat 60
taaattcaag gaaattttag aatttttcta ctaaacttat ttttataaag taaatattat 120
tttatcaata attattcttt tacaattgta aaaaatttaa cctaatagtg aaatagataa 180
aattatagtt tttaaatttg gttcaatatg agagaatcaa tgtgatgcat ctggttgatg 240
agccatgtac aaaggttcgc tgatgttctt ggagagaggt tggggtgaac gagttatgac 300
atttgttgct tcacgaggac atttagtcta aacgggtcgg aaatgatgca tcacacattg 360
gttatgcaag gagattttgg gtgttatata taggtttgta aacctcttgt gagctagctt 420
ttgaggtgaa tttaagtaga tgtatatcat ttggtatcag agccccactc tgtatgggtt 480
gaaaggttcc ctatgttagt gtatgctcta gagcatatca ttgtattgta tcttgttgta 540
gactcttatt ttatgatgag tatgtgcatt gaggccgtga gaaacccgat gatgaaaaat 600
t 601
<210> 35
<211> 601
<212> DNA
<213> Artificial
<400> 35
gctgcttttg tccgctgtca tgtgcgcatt ctcagccaaa caatgcccca actgtggcct 60
tacctcagtt ccctacccac tcagcacaca ccccacctgc ggagaccaat catacaagat 120
acggtgcaac gcaggtgtac tcattttcga cacgcttaac aattcctatc cgatattttc 180
catcaatccg tcgattcaac gattggtaat acaacctgca aatctgttat caaatacctg 240
catttcatct gattttatcc accaaggcat ccagttgagc tcttccctac cgttcaacat 300
taccggcgag aacactgtca tgctttttaa ctgctccgag tcgatacttg atcaaccgtt 360
gaattgttcg ccgagtagct tgtgccactt atacgttgag ggagaagagt actctcggtg 420
cagagattca tcgttgtgtt gtacattcaa agcgggcggg tcagcgtcgg cctataggat 480
acgcgtccta gaaaacgggt gtagggctta tagaagtttc attggtttgg attggggttt 540
gccagtggat aagtggccaa agccgggtgt ggagatccag tgggtgcttc ctctggaacc 600
a 601
<210> 36
<211> 601
<212> DNA
<213> Artificial
<400> 36
gcaaagctta gaaagcttag atttatcaca caataatttt ttggtgaaat tccttgcaca 60
ttggcaaatc tctttgagct gagttactta gacttgagta acaataaact agagggtcgc 120
ataccaagtg gtcctcagat ggatcggatg aacgacccaa attcttatgc caataacagt 180
ggattgtgtg gaatgcaaat taaagtatca tgtgagaagg tgccttctga accaaagctg 240
aaagaggaaa agaccaagaa aagcaatagt tgggagatat ggttttcatg ggaaatggca 300
atgattggat atccttctgg atttttgtca acagttttag ttatgtatgt cattggctac 360
tttaatatta caccacagcc aggtagaaga aaaagaagaa gaagccctgt taggcatggt 420
ctttttggtt tttgacgcat ttgatatatg ttcttagtgt ttgttgttat aaaaaaaggg 480
cttacaaggt cttcatgcta tgtccttaat tgttgcactg ctgcttccct atttcctttg 540
ttacctcaga agattgttgc ccttttgttt ttgtatgttg tgatgtatct cctaccaccc 600
c 601
<210> 37
<211> 601
<212> DNA
<213> Artificial
<400> 37
aaattttgaa ttgtagtagt tatgatgtaa tcctcttaat tgtaggaaga cttgttcttt 60
tgaaattttg taagcttgct ttaaaaaggt ggagcttatc attaataaaa taagcaaaat 120
ttctatacaa aagtgttctt atgagattgg aggattctct ctcaacgagc actcaattaa 180
ttcttcacaa taggtttctt taagaaagca ggaacgtgaa tcagggaaag tccaagtact 240
tgaggtagat ttctaggtca ttgtcctgtg atgagatcct attcaagtgc acataacttg 300
atatcagagt tggttgtcat gggtgaacct tagttaccta atatgctgta ttcctaagtc 360
atggtatggg tattgggtac gcggtatgca gtacaagaat taatgaaata taaggtactt 420
taggggtaga cttaattggt gcattaattt gcttcgatgt ttgtcaaatt atatttattc 480
agtatataaa ttatatttat atagaattat taatttttta attttattta atataatgtt 540
tataaaaaaa ttaaaaatta attagtatga taatttttta acataatata cacattattt 600
a 601
<210> 38
<211> 601
<212> DNA
<213> Artificial
<400> 38
cattcttgcc gaaacttaac aatacaacgg ttcttagtaa ctctagatgt gtaatgcaat 60
tggtaaggtt gtgttgctct ttcgtcaatg gtcatcggtt tagatcgtga gaataagggg 120
aaggccgcgt acccttgccc gtctttcaca aaagttactc tataaagtta aaattttgag 180
ttagattgct ctgcaaaatc ctccattagg ttagctggtg gttcagggta atttggagag 240
ggacgcatag agtccagttg tactgaagac aggtgaagca attgaatcca cttgctcttg 300
caattgagag attatgtcaa tctcttctac acctgtctac ccaatgtcaa acgtaagtaa 360
tcaaagacct agattttaac gtcccaatta taatttctaa tctatctcta tttctcagaa 420
ccaatggctc aaggaattca ccttatacaa aactaaagag ctgacccaca catacacttg 480
taaataaaat tctgcaatta caattttttc gctcgattca aatcacgaac acgcaaccca 540
tcgatctgtg atttttagca gaggctctgc gtttggtttt gctgcttatt gtagcacctg 600
g 601
<210> 39
<211> 601
<212> DNA
<213> Artificial
<400> 39
tagaaatatt ctttataatt gaacaatata ttcttattat atagtatagt tttatgttat 60
aaattatgaa cttgtagaca atcattatca ttgtgcatgt gttgaaactc acttaaattt 120
aatgaaaaaa ttaaataata caaaggggta gcgggttaaa ttaaaaggtt agaatatatt 180
aatataaaat gtcataatct gacaactata ataagaggag taaattaacc aaaaagaaaa 240
aaaagaaaaa agaaagaaaa ggatggagac aaagaatgac cggtggaaga aaaatagtta 300
tgcgaagtat taggtgcact tctcatcgta aaaataaaat aaatccttaa attttaaagc 360
tgtagggtct aacaagtaac aaccctttag aaaaagaatc gaccgaatcg gtcaatttgt 420
atcgattctc ctgattcact atcaaattgt aagattcgat cacgattcag gtacatttgg 480
gatttacccc atagacttga atcattgtga tagagtgaag aacagcaatt ataaacaatc 540
agaatagaaa tggaactgag ttagtattat gaaacataga atgctcaaac agcaaataca 600
a 601
<210> 40
<211> 601
<212> DNA
<213> Artificial
<400> 40
tctaggcaac acacagaaag ctaaagctag gccaagagaa gataacagaa acaagatggg 60
ttctctatcc ggtgaatcaa agtatcatgt agttctgttc cctttcatgt caaaaggcca 120
cactatccct attctccagc tagcccgtct ccttcttcgt cgccagatct ctgttaccat 180
ctttactact ccagccaatc gtcccttcat agctaaatat ctgtccaaca cagctgcctc 240
aatcattgag ctgtccttcc ctggaaacgt ccctgaaata ccttctggca ttgaaaacac 300
agataagctc ccttccattt ctctgtttcc ttattttgcc ttctccacca agctcatgca 360
acctgagttt gaacgtgcac ttgcgtatct tccatctgta aatttcatgg tttctgatgg 420
attcctgtgg tggactctgg agtctgcaat caaattcggt tttccaagat tggttttctt 480
tggcatgtcc aatcatgcta tgtgtatggg caaagctatt aatgagaaca ggcttttttt 540
tgggcctgag tcggatgaag agttaattac agtgattcca tttccatgga taaaggtcac 600
t 601
<210> 41
<211> 601
<212> DNA
<213> Artificial
<400> 41
tatgatgttt attattgttc acaaatccac aattttcctt cgcattacta attatgaatt 60
aagagtaatc aaatattaca aaggtcaatc caaaagcatt aatttgcatt aataaagaaa 120
actaatacaa aatttgatag agtaaggaac ttaagcgaat ggaaagaaaa taatctaaaa 180
ttgtattaag aacttgagat tacagtaatg gaagtacacg agcaattctc ttactctcct 240
ctctaattct caatgcaaat atagcaagaa aagggggaaa aaaccttcca aacactctag 300
ggagaatgct tggcccagca ataatgccaa aataaaagag ctaagccctc ccgccattaa 360
gaattttgcc cccaagttag tattatagtt ggaaaataca attactctct catgcccccc 420
attctacctt ttctctccca tatataattc tcgtagctgt tgcatcacat gcacccgaga 480
attgctccaa aggagttgag ctaagctggc caaccaactg caatccttcc agaactttct 540
tgcttcatct agaatacacc atatccctaa caatagtacc cgctcaaaat agccttgtcc 600
t 601
<210> 42
<211> 601
<212> DNA
<213> Artificial
<400> 42
tggcaaagtc ccaacttctt tgcatacttc caagcaaatg caagcaatgc tggctttctt 60
ggagagatgc tttgttcagg cctgaatatt gttggcttca actggatttc atctccagcc 120
gcaactgagc tagaatccct tgtcatagat tggatgggaa aactgttgaa gcttccgccc 180
tcatttctct tctctggtaa tggaggcggt gtcttgcatg gcagcacatg tgaggctgta 240
gtgtgcactt tggctgcagc aagagataag gccttgaaaa ggatgggatg ggataaaatt 300
acaaagttgg tggtttatgc ctctgatcaa actcatgcca ctctcttcaa gggtaccaaa 360
atagtaggta tcccatcctc taatatccgt tccctcccta cttcattttt atctgggttt 420
tccctgccac ctcaaacact tcaagaagca atcgaaaatg atataaaatc tgggttttat 480
ccattgtttc tttgtgccac tgtgggaaca actgcctgcg gagcagttga tcctatacga 540
gaactagggg aaattgctac gaaatacaac ttatggttcc acattgacgc agcttatgct 600
g 601
<210> 43
<211> 601
<212> DNA
<213> Artificial
<400> 43
atgcgatagc gtcgtcaatg tcgatgaggg accatcggca acagccctgc attacctgta 60
ccaccttcaa catcttagcc ccgatataca agcgcctcaa ccttgacaac aatcaaaatt 120
cccgtgaaag cgattgcaga gcctattggc ttgccagaaa caaccggatt ttggattcct 180
tattgcacga gagatcttcc atcatttgtc tccaggtttt tcttggcttt atcaattcaa 240
atcctttact atcgttcaat cattattatt tctttattgt tgtttattcc tttttggcgg 300
ggatgacaaa ggaattctgg ttggggaacg aagagctggt gaacatgtac gagaagagac 360
tgggtgacgc tggttatgtc aatttccagc ttgcgcgcac caacaaccgc ggtgatggta 420
ctcttctttt cgccatcttt ttttcctcct tttaattttt tttaatctct tcttgtgaca 480
tataagcgat cgaatttttt tgttttattt gcttttttgg atgttttgct ttcttttaaa 540
tctcattgcc taacggtaat tttagaaaaa gaaggtacag agggtttaaa tttaaataaa 600
g 601
<210> 44
<211> 601
<212> DNA
<213> Artificial
<400> 44
aggaagtctt caaagacaat atcgacatta ttgatcaaga ggtagaagca ataattctgg 60
aatttttgaa ctaatccaaa taaatgggtt gccttcgagg agagtccctt gaaacttttt 120
acactactgc ataaagaata ttattatggg tatgtgtcaa tgtcatatca tttaactttt 180
tcttctgcct ttgtgtccct ttctcttcta tttctccttc tctttgcttc ttattttttc 240
tcttttttgt aactttaatt tttgtcttgt gttcaggctt tgttagttat gacaagcgcc 300
cgatacaagg ctgtttggga ggtttatcac acaaatgggg atctggtaag ggtggtgagt 360
acaaaggaga ttgacatgaa agagagatat cttctgaccc ttattgaggc aaaaaggagg 420
actactgaac caatacgcta agcagatggc cttcaattat agatcaatgc attagaggag 480
atggctgaga ggcaccgaaa gaggattgta gaattggaaa tagaatgggg ggatatgatg 540
agtaaataag tgtggatgga caagaagata caaagaacac gaggtcttct caacgagagc 600
c 601
<210> 45
<211> 601
<212> DNA
<213> Artificial
<400> 45
tattgataga tgagaattgg gtggccaagg tttcagactt tggtttgtct agatgggtcc 60
caccagcgac tcccaaacgc acgttagcac tgtagttagg ggaagttttg ggtacgtgga 120
tccagagtac taccgtcggc aacacctcac ggaaaaatct gatgtgtact catttggagt 180
agttctgttt gagttgctct gtgctaggcc gccagtgatg ccaggactac caaaagagca 240
agtcaattta gctgattggg ctcggatttg ctgtagaaga ggagccattg atcagataat 300
ggacccctat ttgaggggta atattgaccc tgtctgtttg gagaaatttg gagagattgc 360
tgagagttgc ctgcgtgacc aaggaacaca aaggccaaca atgagcgatg tggtttgtgg 420
gcttgagttt gctttgcagc ttcaagagac tgctgagaac actagaaata gtattgatag 480
agtgagtgag gagcaggaaa gtccattgtt gttgcgtgga gaagctataa ctactgatga 540
tgatgacttg tttagcgtct caggtggaca caagcatgga tccaagagca cgattagtag 600
t 601
<210> 46
<211> 601
<212> DNA
<213> Artificial
<400> 46
aaagaacatt attacttcat ttttctgctt attggtatat ttgttatttg attcaaacta 60
tggaattggc tgctgcaaat tgatatccgt tagttctctg ttaagtgctt ttgttggact 120
gcgagaatca agatttcttg cttcttgcat cttcaagcta tagaatactg tttgaaatct 180
tcaccatgtg ttgcccctaa tcccattgaa ctcagcttcc tggaaaattg ttgttccata 240
ttctaatgaa ttcaaatcaa aatgttcgtg ttttcttgcc tcgccctttt tccttcaaat 300
gtcaacagga ctgtttttca ttcattttcc tcagataggg ttcacatact gttttctttt 360
ctactaataa tcaatctttt ctttttttgg ttaagagaat tttgtctggc aggctgtagc 420
ttgttactct cttggtacat ttatgtgttt tactgggatc atcctttaac ttctgaatta 480
tgttgctaat ccaaaatcat gacatgtaac tactgaagtt tctttatcct acttgattcc 540
attgaaatga aactatagtt agaatattgt caattacgtg ttttatacct catatattct 600
g 601
<210> 47
<211> 601
<212> DNA
<213> Artificial
<400> 47
atgcttagtc aacgtgggac tgtggcatca agtacattgt ttgttcctta tcttttgtta 60
tgtgctacgc ataagaaatt ggagatattc gttttgtttg aggaaaaatg ataagagtac 120
aattcattga gccaattata tttgtgcata ttggctgatc ttctattatt ttctgtgtca 180
tgttgaaatt attttttgta attacttgct gtttgggatg aaatatattt gtggcttcat 240
agtttttaag atgttgcaat attttatgta attactacaa ttttgcttta gagtttgaaa 300
agttgttatg ttatattcta ctttacagtc cactatgtgg aaataagacc ttgggtttgg 360
ctactaggta ctggtgtatc ttctctactc ctactcacaa ttcgcttact gaatttcaga 420
tggagtggat gcatgaagcg atcgtatgat aaaggtgtta cttggacaaa gagagaacaa 480
cttcctcctg gtatattagg accaataaag aacaaggtat aatttgaaaa tgaaagccat 540
cataatggat gtttatatca ggaaggatgc agctttattg ttactgatgg gatacccctg 600
a 601
<210> 48
<211> 601
<212> DNA
<213> Artificial
<400> 48
taaaaataaa aaaattaaat aattaatttt attaaattga agggactaat taatatttaa 60
aaaaaaaaag ggggacaaag cttaaaccag ctaaaatgtc atatcctgcg ccgaccttgt 120
actactgttt tgcgtaaaaa actattccgc gtgtcagata acataagccg tagtcttgtc 180
ggtttgtagt gtcttatcat agtcggtgtt tttacaggcg acacttgcaa gcgacccctc 240
acaccaaacg tgtccgtcaa ttcttctctt caagatactt ctctctcttt ggatacggtc 300
aaatttcggc ttctctcttt tcaacctttt ttttcaagta cttccataaa ctaacaatga 360
cttgagcata gacttttctt tcttggctac tactactgtt gaagcggaca ttgaggtgaa 420
agctagtgca cactgtgtga tattttcttc tttcctattt ttggtttttt atttttttta 480
gcttaaattt gatcttgaac tcttagcaga agagaggaat accatcgtcg tttggttagg 540
agctgggtgg ttaggtggtc ggtcctcttg ctttcaaaga gcacttcttt acaagttggc 600
t 601
<210> 49
<211> 601
<212> DNA
<213> Artificial
<400> 49
ggccaattct ctcagacact cttcttgcct atattttgca cttatcatca aagctgtgtt 60
accagtatct gtcctggaat ttatatcaca acctgaatct attagacatt taaggatagt 120
tggtaagcct aaacgggcag ccatgtgtat gggccggaac tcggtttttt gagttttgac 180
agggtactcc acattggcac cacagctcag tagcacattg acagctccag cattaccaca 240
gagaatggcg tggtggagga gagttcttcc atggtgagta gtgtttgggg aggtgtgttc 300
gagaagcatg cgcaatatag cacccgtagt ttcaaaatac tccactgcac accaggtaat 360
tgtgtagggc tcagctagtc ctgcgcccac tcgaaactct tctccagtag tagtgtccca 420
tgaccatgct cctagactca ctttgacatc tgtcctggca ccatcctgtt ttatcatttt 480
gataccaatc agaataacca ttagtaaagc aatattgaga gaaggatagg gattcctttt 540
ttcaaaaaaa aaaaaaagaa gaactaacat aataacaagg agagaagtac ggctctatta 600
t 601
<210> 50
<211> 601
<212> DNA
<213> Artificial
<400> 50
tttaatgctc taatgaacta attaccatgc tttcgctcct acaattgata tttagggtta 60
atattcattg cattttatta gtgttttgtc tcttttatgt ttattttatg tgtttttagt 120
aatattttag aaaatttctt attccaaccc aattttttga tttttatatt ttattatgtg 180
tttttatcta gttcttgaac taaggacaaa tttggaagag atctgtaagt caaaaagtga 240
agaacggaaa gccagaagaa ttacacaggt catgtaacct accctatgta acctcctgga 300
ccccgtgcaa ccttctagac aaaagaaaat taaaaaattc aagccataaa aaattatacg 360
aggtacccag tgtaacgtta caaggcctgt gtactgtccc cggaaaaaat tctaacttaa 420
tttctcttct ttctaactcg atccggatgg acttataaga tttttaggac tctgaaataa 480
ggatttttac actagatata aatataataa gtcaggattt aggggttagg aaccttatta 540
cattaaaaag gaccaattta cattcaagag tgtgcattaa cttcaagaaa ggcataggag 600
a 601
<210> 51
<211> 20
<212> DNA
<213> Artificial
<400> 51
gccgcttaaa actagaaccg 20
<210> 52
<211> 20
<212> DNA
<213> Artificial
<400> 52
aagtttaggt accatgggtg 20
<210> 53
<211> 20
<212> DNA
<213> Artificial
<400> 53
gtggttgaga aaagtggagg 20
<210> 54
<211> 20
<212> DNA
<213> Artificial
<400> 54
atcagagtct gcaagcttgg 20
<210> 55
<211> 20
<212> DNA
<213> Artificial
<400> 55
tgtaaagcgt cgacagcaag 20
<210> 56
<211> 20
<212> DNA
<213> Artificial
<400> 56
ggaactgtac aaagccagtc 20
<210> 57
<211> 20
<212> DNA
<213> Artificial
<400> 57
aagacccttg aagaacgaac 20
<210> 58
<211> 20
<212> DNA
<213> Artificial
<400> 58
agtctgcttc tttggagacg 20
<210> 59
<211> 20
<212> DNA
<213> Artificial
<400> 59
gacatatgag gaggaaccag 20
<210> 60
<211> 20
<212> DNA
<213> Artificial
<400> 60
gccgtctgaa cagacattag 20
<210> 61
<211> 20
<212> DNA
<213> Artificial
<400> 61
tgcccagcaa acgtcaaaac 20
<210> 62
<211> 20
<212> DNA
<213> Artificial
<400> 62
gcgacttctc tctctttctc 20
<210> 63
<211> 20
<212> DNA
<213> Artificial
<400> 63
attgtacacc atccagccac 20
<210> 64
<211> 20
<212> DNA
<213> Artificial
<400> 64
agccattgag acagctgaag 20
<210> 65
<211> 20
<212> DNA
<213> Artificial
<400> 65
gcttagagtg cttgcagaag 20
<210> 66
<211> 20
<212> DNA
<213> Artificial
<400> 66
tggagataag gtagctgagg 20
<210> 67
<211> 20
<212> DNA
<213> Artificial
<400> 67
tctgacggct gaattgttgg 20
<210> 68
<211> 20
<212> DNA
<213> Artificial
<400> 68
tttaagcagc gtgcagagac 20
<210> 69
<211> 20
<212> DNA
<213> Artificial
<400> 69
tctccccttg atgcctattc 20
<210> 70
<211> 20
<212> DNA
<213> Artificial
<400> 70
cgattgcaga aggaagaagc 20
<210> 71
<211> 20
<212> DNA
<213> Artificial
<400> 71
tgcctaacac tcgctctaac 20
<210> 72
<211> 20
<212> DNA
<213> Artificial
<400> 72
gaagttcttg ctggttggag 20
<210> 73
<211> 20
<212> DNA
<213> Artificial
<400> 73
gtgtagaatt cctacatcgg 20
<210> 74
<211> 20
<212> DNA
<213> Artificial
<400> 74
agatttatgg tctgttgggc 20
<210> 75
<211> 20
<212> DNA
<213> Artificial
<400> 75
caatgggaaa gttcagggac 20
<210> 76
<211> 20
<212> DNA
<213> Artificial
<400> 76
gctcttgctg tttgatcctc 20
<210> 77
<211> 20
<212> DNA
<213> Artificial
<400> 77
tcgagatctt atgccaaagg 20
<210> 78
<211> 20
<212> DNA
<213> Artificial
<400> 78
aaggaggata gtgcaaaggg 20
<210> 79
<211> 20
<212> DNA
<213> Artificial
<400> 79
taatcttgct tgggcacagg 20
<210> 80
<211> 20
<212> DNA
<213> Artificial
<400> 80
tctcacctgg gtcaaaatcc 20
<210> 81
<211> 20
<212> DNA
<213> Artificial
<400> 81
tggttatgtc ggcgtagaac 20
<210> 82
<211> 20
<212> DNA
<213> Artificial
<400> 82
ggttagtcta ccagttaccc 20
<210> 83
<211> 20
<212> DNA
<213> Artificial
<400> 83
aggtgtcaat tgccagtgtg 20
<210> 84
<211> 20
<212> DNA
<213> Artificial
<400> 84
aagcctagga gaccaaacag 20
<210> 85
<211> 20
<212> DNA
<213> Artificial
<400> 85
ctctatctgt aggtctctgc 20
<210> 86
<211> 20
<212> DNA
<213> Artificial
<400> 86
tgaagagaag cacaacccag 20
<210> 87
<211> 20
<212> DNA
<213> Artificial
<400> 87
gtgcacagct ctgcttatac 20
<210> 88
<211> 20
<212> DNA
<213> Artificial
<400> 88
tttcatcaac gccaggcaag 20
<210> 89
<211> 20
<212> DNA
<213> Artificial
<400> 89
tacacaggct tgaacgtctc 20
<210> 90
<211> 20
<212> DNA
<213> Artificial
<400> 90
ggcagcaaag aaatagctgg 20
<210> 91
<211> 20
<212> DNA
<213> Artificial
<400> 91
cttcaacaca ataagagggc 20
<210> 92
<211> 20
<212> DNA
<213> Artificial
<400> 92
ccatctcatc tgatcccttg 20
<210> 93
<211> 20
<212> DNA
<213> Artificial
<400> 93
ttgcctgtgt catagtggtc 20
<210> 94
<211> 20
<212> DNA
<213> Artificial
<400> 94
gtacagccct tctccaaaag 20
<210> 95
<211> 20
<212> DNA
<213> Artificial
<400> 95
tcttagctag acttgcaggg 20
<210> 96
<211> 20
<212> DNA
<213> Artificial
<400> 96
ttacggttga gtgtggaagg 20
<210> 97
<211> 20
<212> DNA
<213> Artificial
<400> 97
atcccataag acctgaagcc 20
<210> 98
<211> 20
<212> DNA
<213> Artificial
<400> 98
caagaccctc ttttcaaccc 20
<210> 99
<211> 20
<212> DNA
<213> Artificial
<400> 99
taggcttgga cgcttattgc 20
<210> 100
<211> 20
<212> DNA
<213> Artificial
<400> 100
cttctgtagt cgcttgcatc 20
<210> 101
<211> 20
<212> DNA
<213> Artificial
<400> 101
cgactaaatc atgctctcgg 20
<210> 102
<211> 20
<212> DNA
<213> Artificial
<400> 102
cgtagatctt tgggcttgac 20
<210> 103
<211> 20
<212> DNA
<213> Artificial
<400> 103
ctcctcaaca aaccattgcc 20
<210> 104
<211> 20
<212> DNA
<213> Artificial
<400> 104
cgaccctccc acaatgtttg 20
<210> 105
<211> 20
<212> DNA
<213> Artificial
<400> 105
gttggtcgac atgtttactc 20
<210> 106
<211> 20
<212> DNA
<213> Artificial
<400> 106
ggacaccact aatttggagg 20
<210> 107
<211> 20
<212> DNA
<213> Artificial
<400> 107
aagggcaatg gtaatgcacg 20
<210> 108
<211> 20
<212> DNA
<213> Artificial
<400> 108
ggaaacaagg aacgtctcac 20
<210> 109
<211> 20
<212> DNA
<213> Artificial
<400> 109
gatactgcta ttgaccgctc 20
<210> 110
<211> 20
<212> DNA
<213> Artificial
<400> 110
gcttccaaga aagagcttcc 20
<210> 111
<211> 20
<212> DNA
<213> Artificial
<400> 111
aaaacagagg aggctctgtc 20
<210> 112
<211> 20
<212> DNA
<213> Artificial
<400> 112
gatgccagga ctaccaaaag 20
<210> 113
<211> 20
<212> DNA
<213> Artificial
<400> 113
acgaggctga aggagtaatc 20
<210> 114
<211> 20
<212> DNA
<213> Artificial
<400> 114
atgactctcg cctctatctg 20
<210> 115
<211> 20
<212> DNA
<213> Artificial
<400> 115
gtaatcttat ttctggtggc 20
<210> 116
<211> 20
<212> DNA
<213> Artificial
<400> 116
taccgatcag cagactgtag 20
<210> 117
<211> 20
<212> DNA
<213> Artificial
<400> 117
gatgcatctg gttgatgagc 20
<210> 118
<211> 20
<212> DNA
<213> Artificial
<400> 118
gggaaccttt caacccatac 20
<210> 119
<211> 20
<212> DNA
<213> Artificial
<400> 119
tacaagatac ggtgcaacgc 20
<210> 120
<211> 20
<212> DNA
<213> Artificial
<400> 120
taagtggcac aagctactcg 20
<210> 121
<211> 20
<212> DNA
<213> Artificial
<400> 121
agatggatcg gatgaacgac 20
<210> 122
<211> 20
<212> DNA
<213> Artificial
<400> 122
cctaacaggg cttcttcttc 20
<210> 123
<211> 20
<212> DNA
<213> Artificial
<400> 123
gattctctct caacgagcac 20
<210> 124
<211> 20
<212> DNA
<213> Artificial
<400> 124
gcaccaatta agtctacccc 20
<210> 125
<211> 20
<212> DNA
<213> Artificial
<400> 125
ccagttgtac tgaagacagg 20
<210> 126
<211> 20
<212> DNA
<213> Artificial
<400> 126
ggttgcgtgt tcgtgatttg 20
<210> 127
<211> 20
<212> DNA
<213> Artificial
<400> 127
gagacaaaga atgaccggtg 20
<210> 128
<211> 20
<212> DNA
<213> Artificial
<400> 128
gctgttcttc actctatcac 20
<210> 129
<211> 20
<212> DNA
<213> Artificial
<400> 129
gtcaaaaggc cacactatcc 20
<210> 130
<211> 20
<212> DNA
<213> Artificial
<400> 130
acgcaagtgc acgttcaaac 20
<210> 131
<211> 20
<212> DNA
<213> Artificial
<400> 131
agtaatggaa gtacacgagc 20
<210> 132
<211> 20
<212> DNA
<213> Artificial
<400> 132
attctcgggt gcatgtgatg 20
<210> 133
<211> 20
<212> DNA
<213> Artificial
<400> 133
ctggctttct tggagagatg 20
<210> 134
<211> 20
<212> DNA
<213> Artificial
<400> 134
gatcagaggc ataaaccacc 20
<210> 135
<211> 20
<212> DNA
<213> Artificial
<400> 135
cttagccccg atatacaagc 20
<210> 136
<211> 20
<212> DNA
<213> Artificial
<400> 136
tcgtacatgt tcaccagctc 20
<210> 137
<211> 20
<212> DNA
<213> Artificial
<400> 137
ttcttctgcc tttgtgtccc 20
<210> 138
<211> 20
<212> DNA
<213> Artificial
<400> 138
gccatctgct tagcgtattg 20
<210> 139
<211> 20
<212> DNA
<213> Artificial
<400> 139
gatgccagga ctaccaaaag 20
<210> 140
<211> 20
<212> DNA
<213> Artificial
<400> 140
tgctcctcac tcactctatc 20
<210> 141
<211> 20
<212> DNA
<213> Artificial
<400> 141
tgttgcccct aatcccattg 20
<210> 142
<211> 20
<212> DNA
<213> Artificial
<400> 142
ggatgatccc agtaaaacac 20
<210> 143
<211> 20
<212> DNA
<213> Artificial
<400> 143
gtgcatattg gctgatcttc 20
<210> 144
<211> 20
<212> DNA
<213> Artificial
<400> 144
tcatgcatcc actccatctg 20
<210> 145
<211> 20
<212> DNA
<213> Artificial
<400> 145
cataagccgt agtcttgtcg 20
<210> 146
<211> 20
<212> DNA
<213> Artificial
<400> 146
atcacacagt gtgcactagc 20
<210> 147
<211> 20
<212> DNA
<213> Artificial
<400> 147
gagagttctt ccatggtgag 20
<210> 148
<211> 20
<212> DNA
<213> Artificial
<400> 148
ggaatcccta tccttctctc 20
<210> 149
<211> 20
<212> DNA
<213> Artificial
<400> 149
acaggtcatg taacctaccc 20
<210> 150
<211> 20
<212> DNA
<213> Artificial
<400> 150
ggttcctaac ccctaaatcc 20
Claims (4)
1.一种用于构建橡胶树品种DNA指纹图谱的SNP分子标记组合,其特征在于,所述SNP分子标记组合包括LG03_104230492、LG02_8025843、LG01_54721919、LG15_32001166、LG07_72003724、LG05_88010730和LG02_214754;
其中,“Sequence”为SNP分子标记的核苷酸序列;“[]”标注的为SNP分子标记位点位置,其中的碱基为该位点的多态性。
2.如权利要求1所述的SNP分子标记组合在橡胶树品种群体遗传结构、和/或橡胶树资源遗传多样性分析、和/或橡胶树品种鉴定、和/或构建橡胶树DNA指纹图中的应用,所述橡胶树品种为下表所述的橡胶树品种:
3.一种橡胶树品种DNA指纹库的构建方法,其特征在于,提取各个橡胶树品种的总DNA;采用引物对进行扩增;利用权利要求1所述的SNP分子标记组合进行SNP位点多态性检测,根据SNP位点的检测结果对各个橡胶树品种进行基因分型,从而构建橡胶树品种DNA指纹库;所述的引物对为:
;
所述橡胶树品种为下表所述的橡胶树品种:
4.一种橡胶树品种的鉴定方法,其特征在于,采用引物组对待测样品的总DNA进行PCR扩增,利用权利要求1所述的SNP分子标记组合进行SNP位点多态性检测,根据组合中各SNP位点的检测结果确定SNP分子标记的基因分型,再与权利要求3所述构建方法所构建的橡胶树品种DNA指纹库中的基因分型进行比对;所述的引物组为:
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