CN113249515A - 一种菊花叶绿体基因组ssr标记库、其获得方法及其应用 - Google Patents

一种菊花叶绿体基因组ssr标记库、其获得方法及其应用 Download PDF

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CN113249515A
CN113249515A CN202110751702.3A CN202110751702A CN113249515A CN 113249515 A CN113249515 A CN 113249515A CN 202110751702 A CN202110751702 A CN 202110751702A CN 113249515 A CN113249515 A CN 113249515A
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夏涵涵
徐玉芬
罗红辉
伍青
廖柏荣
杨敏仪
张家豪
周厚高
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Abstract

本发明公开了一种菊花叶绿体基因组SSR标记库、其获得方法及其应用，SSR标记库包括SSR标记序列SEQ ID NO.1，还包括SEQ ID NO.2‑40中的至少一个；SSR标记库的获得方法包括，将菊花品种叶绿体基因组DNA，进行SSR分析，获得SSR标记，将同一品种的SSR标记拼接为SSR标记序列，得到SSR标记库；SSR标记库的应用，其特征在于，将SSR标记库用于构建系统发育树或用于鉴别菊花的品种；SSR标记库可以用于有效而准确地鉴定菊花品种，进行菊花遗传学研究和育种亲本选择。

Description

一种菊花叶绿体基因组SSR标记库、其获得方法及其应用
技术领域
本发明涉及一种菊花叶绿体基因组SSR标记库、其获得方法及其应用，属于分子生物学技术领域。
背景技术
菊花在花类资源的探索进程中扮演着主导角色、在观赏、食用和药用生产中发挥着巨大作用。对于菊花品种及分类，在传统形态学，数量分类学、细胞学、聚类分析等领域均有相关的研究，为我们了解菊花分类差异和品种亲缘关系提供了参考，但是这些研究仅针对少部分菊花品种进行实验分析，尚不能帮助我们去正确有效地鉴别菊花。虽然通过表型鉴定菊花具有方便和直接的优点，但是由于菊花品种繁多、品种之间差异较小、形态性状的平行进化、发育变异受到自然选择、长期杂交培育情况下导致遗传情况变得十分复杂，导致鉴别效率低，阻碍了植物繁殖和国际交流，因此在菊花分类方面一直存在争议，无法单纯凭借形态学特征、细胞学等方法快速有效地鉴别菊花品种。
目前已有学者开发和利用分子标记技术对菊花进行了不同层面的研究，但是这些研究计算过程复杂、适用于样品数量少、引物不充分或研究位点少，尚不能有效、正确、快速地鉴别和区分大部分菊花品种。因此需要完善全面统一的分类鉴定方法仍是非常困难的，在品种分类和遗传关系研究都存在着不足和局限性。一是菊花的栽培育种大都从表型出发，由于各品种表型存在相似性，影响对遗传性状的接收，导致新品种育出效率低。二是菊花表型存在同化性，品种间差异小，鉴别准确率低，严重混淆菊花品种，阻碍了品种之间的交流与培育。三是栽培菊花的自交不亲和和近交导致其具有杂合性，进而致使遗传改良延迟。四是菊花分类和鉴定系统不完善，影响对其遗传关系及起源的追溯，加上菊花易杂交产生新的变异，致使其发育系统更加复杂。亟需解决的正是菊花研究现存的问题，使得菊花分类鉴别工作变得如此重要。
发明内容
为了克服现有技术的不足，本发明的第一个目的在于提供一种菊花叶绿体基因组SSR标记库，可以用于有效而准确地鉴定菊花品种，进行菊花遗传学研究和育种亲本选择。
本发明的第二个目的在于提供一种上述菊花叶绿体基因组SSR标记库的获得方法；该方法减少外源DNA的扩散，没有基因沉默现象及位置效应的影响，具有定点整合、高效表达及安全性好，实验周期短、劳动强度小、结果信息丰富等优势。
本发明的第三个目的在于提供一种上述菊花叶绿体基因组SSR标记库的应用构建系统发育树和鉴别菊花的品种。
实现本发明的第一个目的可以通过采取如下技术方案达到：一种菊花叶绿体基因组SSR标记库，SSR标记库包括SSR标记序列SEQ ID NO.1，还包括SEQ ID NO.2-40中的至少一个。
实现本发明的第二个目的可以通过采取如下技术方案达到：一种菊花叶绿体基因组SSR标记库的获得方法，包括，将菊花品种叶绿体基因组DNA，进行SSR分析，获得SSR标记，将同一品种的SSR标记拼接为SSR标记序列，得到SSR标记库；菊花品种包括野菊C.indicum，通过野菊C.indicum获得SSR标记序列为SEQ ID NO.1。
进一步地，菊花品种还包括骄阳卡萨Casa、黄草莓Gustavoorange、红乒乓Avron、黄乒乓Bonbonyellow、桔星Florange、斯特雷Stresa、绿乒乓Healing、马蒂斯Matisse、瑞多斯特黄Radostyellow、小乒夏天Mundoorange、地被菊(橙红托桂)Chenghongtuogui、地被菊(黄龙眼)Huanglongyan、菊花脑Juhuanao、国华彩Guohuacai、国华粉桃Guohuafentao、国华新星火Guohuaxingxinghuo、国华御来光Guohuayulaiguang、骏河白云Junhebaiyun、精兴之城Jingxingzhicheng、神马Jinba、太平宝Taipingbao、太平红莲Taipinghonglian、层峦碧染Cenluanbiran、彩雪春桃Caixuechuntao、东海泥金Donghainijin、东海神韵Donghaishenyun、飞云卷神Feiyunjuanshen、古寺佛光Gusifoguang、金继流霞Jinjiliuxia、南山高寺Nanshangaosi、盘龙江城Panlongjiangcheng、新星火Xinxinghuo、紫龙探爪Zilongtanzhua、粉瓣Fenban、圳紫菊Zhenziju、云山叠紫Yunshandiezi、紫瓣Ziban、紫红托桂Zihongtuogui和紫艳Ziyan中的至少一个。
进一步地，SSR分析中设置单核苷酸到六核苷酸单元的重复数最小值依次设为：10，6，5，5，5，5。
实现本发明的第三个目的可以通过采取如下技术方案达到：一种菊花叶绿体基因组SSR标记库的应用，将SSR标记库用于构建系统发育树。
进一步地，构建系统发育树的步骤包括：
1)将SSR标记序列进行多序列比对；
2)区分不同品种，对花序大小、舌状小花形态、头状花序重瓣性和花色指标进行统计分类。
进一步地，多序列比对包括步骤：
建树的源文件的获得：利用Linux shell系统命令cat对SSR标记序列进行合并，并使用MAFFT version 7.471(Katoh et al.,2002)进行多序列比对，结果作为建树的源文件；
最大似然法：jModelTest version 2.1.10对比对好的物种序列进行最适模型检测；
贝叶斯推断：使用MrBayes version3.2.7a-x86_64进行分析，利用jmodeltestversion 2.1.10对比对结果进行模型选择和参数估计，选择GTR+GAMMA+I为最佳碱基替代模型。
实现本发明的第三个目的也可以通过采取如下技术方案达到：一种菊花叶绿体基因组SSR标记库的应用，将SSR标记库用于鉴别菊花的品种。
相比现有技术，本发明的有益效果在于：
1、由于菊花是多倍体植物，利用全基因组进行研究具有一定困难和挑战性，本发明以SSR标记为材料，通过生物信息学对测序数据进行编辑和分析，对其进行测序研究；
2、本发明减少外源DNA的扩散，没有基因沉默现象及位置效应的影响，具有定点整合、高效表达及安全性好，实验周期短、劳动强度小、结果信息丰富等优势，同时避免了叶绿体DNA分离等高难度实验，也不需要设计特异性引物；
3、通过SSR标记库，有效而准确地鉴定菊花品种，显著提高菊花品种的系统分类效率，研究主栽菊花品种的杂交起源，从而为菊花的系统发育，物种起源，以及揭示系统发育问题的整个进化过程，真正打开菊花遗传学研究的大门，并且加快了遗传界研究的进度，因此，开展对SSR标记库应用研究，对探究菊属种间及种内的起源、遗传多样性以及对园艺植物进行种质创新和品种的改良都具有非常重要的意义；
4、本发明对菊花品种的亲缘关系进行研究，构建了系统发育树，对其起源和菊花品种之间的进化关系进行分析，从SSR标记水平上探索它们的亲缘关系和栽培起源问题，获得了SSR标记库，用于菊花的遗传研究并区分不同品种的多态性。针对SSR标记进行分析，鉴别菊花品种及种源的关系，从而深入了解菊花叶绿体的母系遗传规律。另外，本发明将为菊花的引种栽培和资源评价、种质资源保护提供科学依据，为完善菊花DNA条形码提供参考数据，对后期菊花品种改良及新品种杂交选育具有重要指导价值。
附图说明
图1-2为IR边界扩张收缩比较；
图3为分散重复序列比较分析；
图4-5为mVISTA比较分析；
图6为Mauve比较分析；
图7为BI和ML系统发育分析。
具体实施方式
下面，结合附图以及具体实施方式，对本发明做进一步描述：
基因组比较分析及共线性分析
以野菊Chrysanthemum indicum(JN867589.1)的SSR标记序列SEQ ID NO.1注释信息作为参考，使用mVISTA(Frazer et al.,2004)，选择Shuffle-LAGAN的对齐方式，对如表格1所示的39个菊花品种的SSR标记序列SEQ ID NO.2-40进行比较分析；通过Genious10.0.9的Mauve工具(Darling et al.,2004)在叶绿体基因组的基础上进行SSR标记序列共线性分析。
表格1菊花品种
Figure BDA0003144873080000031
Figure BDA0003144873080000041
重复序列及SSR分析
使用MISA的perl脚本(Beier et al.,2017)进行简单重复序列(simple sequencerepeats,SSR)分析，单核苷酸到六核苷酸单元的重复数最小值依次设为：10，6，5，5，5，5。
系统发育分析
利用Linux shell系统命令cat对40个SSR标记序列(FASTA格式)(如表格1所示)进行合并，并使用MAFFT version 7.471(Katoh et al.,2002)进行多序列比对，结果作为建树的源文件。
最大似然法(ML，Maximum likelihood)的发育树构建是利用jModelTest version2.1.10(David,2008)对比对好的物种序列进行最适模型检测，结果显示GTR+G+I为建树最佳模型。建树使用RAxML version 8.2.12(Alexandros,2014)进行计算，bootstrap设置为1,000次，执行快速bootstrap分析并搜索最佳的分的ML树，根据AICc(最低)，BIC(最低)，和lnL(最高)评估各树的可靠性，得到一棵带bootstrap值的最佳的分树，导入FigTreeversion 1.4.3中进行美化。
贝叶斯推断(BI，Bayesian inference)使用MrBayes version3.2.7a-x86_64(Huelsenbeck&Ronquist,2001)进行分析，利用jmodeltest version 2.1.10对比对结果进行模型选择和参数估计，选择GTR+GAMMA+I为最佳碱基替代模型。利用MEGA version7.0.26(Kumar et al.,2016)将FASTA格式的多序列比对文件转为MrBayes可读的NEXUS格式序列文件。建立马尔科夫链(Markov Chain Monte Carlo,MCMC)，其中3条热链，1条冷链，运行10,000,000代，抽样频率设置为1,000，交换链的温度设置为0.2，“burninfrac”设置为0.25，表示去除最开始运行获得的2,500个老化样本(burn-in samples)。运行贝叶斯推断，将获得的一致性树用FigTree version 1.4.3进行可视化和图形美化，贝叶斯树各分支的支持率以后验概率(PP，Posterior probability)作为评估标准。
为更好地进行系统发育分析，区分不同品种，对花序大小、舌状小花形态、头状花序重瓣性及花色等4种花形态形状指标进行了统计分类，并利用Photoshop CC将性状示意标志于发育树右侧。其中，根据头状花序的大小(Flower size)分为大菊(Large>6cm)、小菊(Small<6cm)；参考UPOV(International Union for the Protection of New Varietiesof Plants)和中华人民共和国农业部颁发的菊花植物新品种特异性、一致性和稳定性(DUS，Distinctness,Uniformity and Stability)测试指南，将舌状小花的形状(predominant type of ray floret)分为平瓣(Ligulate)、匙瓣((Spatulate))、脊瓣(Quilled)、内曲(Incurved)和漏斗状(Funnel shaped)等5种类型，并将头状花序分为无舌状小花(Without ray florets)、单瓣(Single)、半重瓣(Semi double)和重瓣(Double)等4种类型；利用目测分析法(李鸿渐和邵健文,1990)将花色Flower color分为白(White)、黄(Yellow)、红(Red)、紫(Purple)、粉红(Pink)、绿(Green)、双色系(Double color)和间色系(Intermediate color)等8种系列。
结果分析
通过叶绿体基因组大小统计发现(表格2)，39个菊花品种及C.indicum的叶绿体基因组总长度为150,964bp(C.indicum)～151,106bp(Huanglongyan)，其中，LSC、SSC及IR的长度分别为82,731bp(C.indicum)～82,884bp(Huanglongyan)、18,294bp(Gusifoguang)～18,319bp(C.indicum)及24,954bp(Gusifoguang)～24,972bp(Fenban)，同时其叶绿体基因组、LSC、SSC及IR的平均长度分别为151,056bp、82,849bp、18,297bp及24,955bp。根据叶绿体基因组注释结果，进行基因数目统计，发现39个菊花品种与野菊C.indicum具有相同的基因数目及编码基因，共编码110个唯一基因，包含79个蛋白质编码基因，27个tRNA及4个rRNA。此外，它们的叶绿体基因组GC总含量均为37.5％。
表格2 39个菊花品种与C.indicum叶绿体基因组基本特征比较
Figure BDA0003144873080000051
Figure BDA0003144873080000061
叶绿体基因组IR边界分析
为探索叶绿体基因组IR边界潜在的扩张与收缩，比较了39个品种与C.indicum的IR与SC边界区域基因的分布状况(图1和图2)。如图所示，LSC-IR与SSC-IR边界分布的基因包括rps19，ycf1，ndhF，rpl2和trnH。LSC-IRB边界均位于rps19，除Huanglongyan外，rps19于LSC区域和IRB区域的长度分别为218bp和61bp。IRB-SSC边界均位于ycf1内，其中Juhuanao和C.indicum的叶绿体基因ycf1位于IRB和SSC的长度分别为556bp和4,462bp，而剩余38个品种均分别为558bp和4,457bp。SSC-IRA边界均邻近SSC区域的ndhF，除Huanglongyan，Juhuanao及C.indicum外，其余37个品种的SSC-IRA边界均距离ndhF长达66bp。IRA-LSC边界均位于IRA区域的rpl2和LSC区域的trnH之间，仅Huanglongyan的该边界距离trnH 9bp，其余均与trnH相距8bp。
如图3所示，在39个菊花品种与C.indicum的叶绿体基因组中共检测到了39(Huanglongyan)～45(Juhuanao)个SSRs，并仅检测到3种核苷酸重复类型，即单核苷酸，双核苷酸和三核苷酸，均未检测到四核苷酸、五核苷酸和六核苷酸SSR。单核苷酸SSR含量最高，具37(Huanglongyan)～43(Juhuanao)个。双核苷酸SSR的含量除C.indicum和Juhuanao大于1，其余38个品种的叶绿体基因组均只含1个。仅Juhuanao不含三核苷酸SSR，此外，C.indicum及Huanglongyan的三核苷酸类型含量为1，其余37个品种的含量均为2。
比较分析
基于C.indicum SSR标记序列，利用mVISTA软件对测定的39个菊花品种进行了比较分析(图4和图5)，结果表明，39个菊花品种的SSR标记序列具有高度相似性，相比之下，编码区比非编码区更为保守，且差异主要存在于基因间隔区，而编码区的主要差异存在于ycf1。
通过Mauve进行比较发现，39个菊花品种与C.indicum的叶绿体所含的所有tRNA，rRNA及蛋白编码基因均表现出高度共线性，未观察到基因重排现象(图6)。
系统发育(聚类)分析
基于39个菊花品种与C.indicum的SSR标记序列，利用贝叶斯法BI和最大似然法ML分别构建了发育树，BI系统树和ML系统树的拓扑结构大致相似，本文基于贝叶斯树的结构进行系统发育分析(图7)。结果显示，40份材料分为3大类群，第Ⅰ类群由C.indicum单独形成，第Ⅱ类群由近野生种Juhuanao和Huanglongyan组成，第Ⅲ类群则包括自育新品种Ziyan、中国传统品种Zilongtanzhua、荷兰品种Mundoorange以及日本品种Taipinghonglian等37个品种，并形成两大平行结构，这表明第Ⅲ类群的品种亲缘关系较近，其中，荷兰品种Stresa、日本品种Guohuaxingxinghuo以及3个中国传统品种(Nanshangaosi，Jinjiliuxia，Cenluanbiran)形成Ⅲ-2小类群(PP＝0.99，BS＝100)，区分于小类群Ⅲ-1。
如表格3所示，根据4种花形态性状的统计结果显示，40份材料根据大小可分为大菊和小菊，其舌状小花有平瓣(Ligulate)、匙瓣(Spatulate)、脊瓣(Quilled)、内曲(Incurved)等4种类型，头状花序有单瓣(Single)、半重瓣(Semi double)和重瓣(Double)等3种状态，花色则有白(White)、黄(Yellow)、红(Red)、紫(Purple)、粉红(Pink)、绿(Green)、双色系(Double color)和间色系(Intermediate color)等8种类型。其中，发育树基部的第Ⅰ和Ⅱ类群均为小菊类型，其瓣型均为平瓣，头状花序同为单瓣，花色均为黄色。小类群Ⅲ-1中含有Ziyan、Zihongtuogui及Ziban等13个小菊品种，其余19个均为大菊品种，该小类群的舌状小花以平瓣为主，其次为匙瓣，少有脊瓣(Taipingbao和Xinxinghuo)和内曲(Zilongtanzhua)，其头状花序以重瓣为主，并含有Fenban、Zhenziju及Ziban等5个单瓣品种以及Casa、Florange和Yunshandiezi等3个半重瓣品种，同时该小类群的花色较为丰富，具有白、黄、红、紫、粉红及绿6种不同系列的花色。小类群Ⅲ-2所含的5个品种除Stresa外，均为大菊、重瓣类型，舌状小花以平瓣为主，而Jinjiliuxia和Nanshangaosi为脊瓣，该小类群花色有双色(Cenluanbiran)、黄色(Jinjiliuxia和Nanshangaosi)以及粉红(Stresa)三种
类型。
表格3 39个菊花样品与C.indicum表型结果
Figure BDA0003144873080000071
Figure BDA0003144873080000081
Figure BDA0003144873080000091
本发明鉴定菊花品种和母本来源，追踪起源，为下一步菊花育种奠定基础；取样更全面；近野生的品种菊花脑和黄龙眼和野生种野菊，在树的基部且聚到一起，说明野菊是她们的母本供体；近野生种橙红托桂，在groupIII中，和其他的近野生种距离遥远，说明他的母本不是野菊，而父本是野菊，他的母本和其他35个品种的母本是同一个，或者亲缘关系接近，因为目前缺乏对近野生菊杂交事件(或天然或人工)的记载，我们的研究可以追溯杂交事件，母本来源(更能体现本研究的价值)，区分不同近野生菊花品种之间母系的关系远近。
通过将表型和遗传联合起来分析发现，花的大小和花头数量，这两个性状比较稳定，和叶绿体的结果基本一致。比如所有的日本菊花和中国传统菊花，都是大菊且双头。这个说明，也反过来证明了分类的可靠。图7为基于39个菊花品种与C.indicum的BI(Bayesianinference)和ML(Maximum likelihood)系统发育分析，分支上的数值为后验概率/自展值，PP(Posterior probability)/BS(Bootstrap)。
对于本领域的技术人员来说，可根据以上描述的技术方案以及构思，做出其它各种相应的改变以及变形，而所有的这些改变以及变形都应该属于本发明权利要求的保护范围之内。
SEQUENCE LISTING
<110> 仲恺农业工程学院
<120> 一种菊花叶绿体基因组SSR标记库、其获得方法及其应用
<130> 2021
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 578
<212> DNA
<213> C.indicum
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<213> Gustavoorange
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<212> DNA
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<212> DNA
<213> Healing
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<212> DNA
<213> Matisse
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<210> 10
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<212> DNA
<213> Radostyellow
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<212> DNA
<213> Mundoorange
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<210> 12
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<212> DNA
<213> Chenghongtuogui
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<210> 13
<211> 540
<212> DNA
<213> Huanglongyan
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<210> 14
<211> 627
<212> DNA
<213> Juhuanao
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<210> 15
<211> 606
<212> DNA
<213> Guohuacai
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<210> 16
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<212> DNA
<213> Guohuafentao
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tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 17
<211> 606
<212> DNA
<213> Guohuaxingxinghuo
<400> 17
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 18
<211> 606
<212> DNA
<213> Guohuayulaiguang
<400> 18
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 19
<211> 606
<212> DNA
<213> Junhebaiyun
<400> 19
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 20
<211> 606
<212> DNA
<213> Jingxingzhicheng
<400> 20
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 21
<211> 606
<212> DNA
<213> Jinba
<400> 21
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 22
<211> 606
<212> DNA
<213> Taipingbao
<400> 22
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 23
<211> 606
<212> DNA
<213> Taipinghonglian
<400> 23
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 24
<211> 606
<212> DNA
<213> Cenluanbiran
<400> 24
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 25
<211> 606
<212> DNA
<213> Caixuechuntao
<400> 25
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 26
<211> 606
<212> DNA
<213> Donghainijin
<400> 26
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 27
<211> 606
<212> DNA
<213> Donghaishenyun
<400> 27
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 28
<211> 606
<212> DNA
<213> Feiyunjuanshen
<400> 28
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 29
<211> 604
<212> DNA
<213> Gusifoguang
<400> 29
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc cccccccata ataataataa taatattttt tttttttttt 120
tttttttttt tttttttaaa aaaaaaaaaa aaaaaaaaaa aaaaaaattt tttttttttt 180
tttttttttt tttttttttt tttttttttt ttttttttta aaaaaaaaaa attttttttt 240
tttttttttt tttaaaaaaa aaaaaaaaaa aaaaaaaaaa aaattttttt tttatatata 300
tatatttatt taattaaaga atagaatttc aaataactgt aaaatattat agaacataac 360
gatgaatcta gcgatataga actagtaata aaaaaaaaaa aaattttttt tttaaaaaaa 420
aaattttttt tttttaaaaa aaaaaaattt tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttaaaaa aaaaattttt tttttttttt 540
tttttttaaa aaaaaaaaaa aaaaaaaaaa aaaatttttt ttttaaaaaa aaaaaaaaaa 600
aaaa 604
<210> 30
<211> 606
<212> DNA
<213> Jinjiliuxia
<400> 30
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 31
<211> 604
<212> DNA
<213> Nanshangaosi
<400> 31
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc cccccccata ataataataa taatattttt tttttttttt 120
tttttttttt tttttttaaa aaaaaaaaaa aaaaaaaaaa aaaaaaattt tttttttttt 180
tttttttttt tttttttttt tttttttttt ttttttttta aaaaaaaaaa attttttttt 240
tttttttttt tttaaaaaaa aaaaaaaaaa aaaaaaaaaa aaattttttt tttatatata 300
tatatttatt taattaaaga atagaatttc aaataactgt aaaatattat agaacataac 360
gatgaatcta gcgatataga actagtaata aaaaaaaaaa aaattttttt tttaaaaaaa 420
aaattttttt tttttaaaaa aaaaaaattt tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttaaaaa aaaaattttt tttttttttt 540
tttttttaaa aaaaaaaaaa aaaaaaaaaa aaaatttttt ttttaaaaaa aaaaaaaaaa 600
aaaa 604
<210> 32
<211> 606
<212> DNA
<213> Panlongjiangcheng
<400> 32
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 33
<211> 606
<212> DNA
<213> Xinxinghuo
<400> 33
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 34
<211> 606
<212> DNA
<213> Zilongtanzhua
<400> 34
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 35
<211> 606
<212> DNA
<213> Fenban
<400> 35
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 36
<211> 606
<212> DNA
<213> Zhenziju
<400> 36
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 37
<211> 606
<212> DNA
<213> Yunshandiezi
<400> 37
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 38
<211> 606
<212> DNA
<213> Ziban
<400> 38
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 39
<211> 606
<212> DNA
<213> Zihongtuogui
<400> 39
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606
<210> 40
<211> 606
<212> DNA
<213> Ziyan
<400> 40
aaaaaaaaaa aatttttttt ttttttagct cttctttatt taattattat tatattatta 60
ttattataaa aaaaaaaccc ccccccccca taataataat aataatattt tttttttttt 120
tttttttttt ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaat tttttttttt 180
tttttttttt tttttttttt tttttttttt tttttttttt taaaaaaaaa aaattttttt 240
tttttttttt tttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaattttt tttttatata 300
tatatattta tttaattaaa gaatagaatt tcaaataact gtaaaatatt atagaacata 360
acgatgaatc tagcgatata gaactagtaa taaaaaaaaa aaaaattttt tttttaaaaa 420
aaaaattttt tttttttaaa aaaaaaaaat tttttttttt tttttttttt tttttttttt 480
tttttttttt tttttttttt tttttttttt tttttttaaa aaaaaaattt tttttttttt 540
ttttttttta aaaaaaaaaa aaaaaaaaaa aaaaaatttt ttttttaaaa aaaaaaaaaa 600
aaaaaa 606

Claims (8)

1.一种菊花叶绿体基因组SSR标记库，其特征在于，所述SSR标记库包括SSR标记序列SEQ ID NO.1，还包括SEQ ID NO.2-40中的至少一个。
2.一种如权利要求1所述的菊花叶绿体基因组SSR标记库的获得方法，其特征在于包括，将菊花品种叶绿体基因组DNA，进行SSR分析，获得SSR标记，将同一品种的SSR标记拼接为SSR标记序列，得到SSR标记库；所述菊花品种包括野菊C.indicum，通过野菊C.indicum获得SSR标记序列为SEQ ID NO.1。
3.如权利要求2所述的菊花叶绿体基因组SSR标记库的获得方法，其特征在于，所述菊花品种还包括骄阳卡萨Casa、黄草莓Gustavoorange、红乒乓Avron、黄乒乓Bonbonyellow、桔星Florange、斯特雷Stresa、绿乒乓Healing、马蒂斯Matisse、瑞多斯特黄Radostyellow、小乒夏天Mundoorange、地被菊(橙红托桂)Chenghongtuogui、地被菊(黄龙眼)Huanglongyan、菊花脑Juhuanao、国华彩Guohuacai、国华粉桃Guohuafentao、国华新星火Guohuaxingxinghuo、国华御来光Guohuayulaiguang、骏河白云Junhebaiyun、精兴之城Jingxingzhicheng、神马Jinba、太平宝Taipingbao、太平红莲Taipinghonglian、层峦碧染Cenluanbiran、彩雪春桃Caixuechuntao、东海泥金Donghainijin、东海神韵Donghaishenyun、飞云卷神Feiyunjuanshen、古寺佛光Gusifoguang、金继流霞Jinjiliuxia、南山高寺Nanshangaosi、盘龙江城Panlongjiangcheng、新星火Xinxinghuo、紫龙探爪Zilongtanzhua、粉瓣Fenban、圳紫菊Zhenziju、云山叠紫Yunshandiezi、紫瓣Ziban、紫红托桂Zihongtuogui和紫艳Ziyan中的至少一个。
4.如权利要求2所述的菊花叶绿体基因组SSR标记库的获得方法，其特征在于，所述SSR分析中设置单核苷酸到六核苷酸单元的重复数最小值依次设为：10，6，5，5，5，5。
5.一种如权利要求1所述的菊花叶绿体基因组SSR标记库的应用，其特征在于，将SSR标记库用于构建系统发育树。
6.如权利要求5所述的菊花叶绿体基因组SSR标记库的应用，其特征在于，所述构建系统发育树的步骤包括：
1)将SSR标记序列进行多序列比对；
2)区分不同品种，对花序大小、舌状小花形态、头状花序重瓣性和花色指标进行统计分类。
7.如权利要求6所述的菊花叶绿体基因组SSR标记库的应用，其特征在于，所述多序列比对包括步骤：
建树的源文件的获得：利用Linux shell系统命令cat对SSR标记序列进行合并，并使用MAFFT version 7.471(Katoh et al.,2002)进行多序列比对，结果作为建树的源文件；
最大似然法：jModelTest version 2.1.10对比对好的物种序列进行最适模型检测；
贝叶斯推断：使用MrBayes version3.2.7a-x86_64进行分析，利用jmodeltestversion 2.1.10对比对结果进行模型选择和参数估计，选择GTR+GAMMA+I为最佳碱基替代模型。
8.一种如权利要求1所述的菊花叶绿体基因组SSR标记库的应用，其特征在于，将SSR标记库用于鉴别菊花的品种。
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