利用SSR分子指纹和商品信息构建水稻品种身份证

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陆徐忠, 倪金龙, 李莉, 汪秀峰, 马卉, 张小娟, 杨剑波. 利用SSR分子指纹和商品信息构建水稻品种身份证. 作物学报, 2014, 40(5): 823-829[LU Xu-Zhong, NI Jin-Long, LI Li, WANG Xiu-Feng, MA Hui, ZHANG Xiao-Juan, YANG Jian-Bo. Construction of Rice Variety Indentity Using SSR Fingerprint and Commo-dity Information. 作物学报, 2014, 40(5): 823-829] 复制到剪切板

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作物学报编辑部

利用SSR分子指纹和商品信息构建水稻品种身份证

陆徐忠¹, 倪金龙¹, 李莉¹, 汪秀峰¹, 马卉¹, 张小娟^1,², 杨剑波^1,^*

¹安徽省农业科学院水稻研究所, 安徽合肥 230031

²安徽省农业科学院棉花研究所, 安徽安庆 246003

^* 通讯作者(Corresponding author): 杨剑波, E-mail:yjianbo@263.net, Tel: 0551-62160212

第一作者联系方式: E-mail:luxuzhong100@163.com

收稿日期:2013-12-12

基金:本研究由国家科技支撑计划项目(2012BAD07B01), 国家自然科学基金青年科学基金项目(31101204)和安徽省农业科学院院长青年创新基金项目(11B0106)资助;

摘要

为方便水稻种子质量的追溯和管理, 提出基于SSR分子指纹和商品信息构建水稻品种身份证的新思路。在前期研究基础上, 进一步确定了由12个水稻SSR标记构成的核心标记组(每条染色体1个), 对127份安徽省审定(或主推)水稻品种进行荧光标记分析, 构建供试水稻品种分子指纹, 在此基础上, 再与品种基本商品信息相结合, 并进行数字化编码, 构建了127份水稻的品种身份证。该身份证号码组成的第1部分为商品码, 代表品种的基本商品信息, 包括品种类别、品种选育(或审定)的区域和时间等; 第2部分为指纹码, 代表水稻品种的分子指纹信息; 第3部分为补充码(或特异基因识别码), 代表品种的特异基因信息。还对127份水稻品种身份证进行了条码表述(一维码和二维码), 以便水稻品种种子的身份标识和溯源管理。

关键词: 水稻; SSR荧光标记; 毛细管电泳; 商品信息; 品种身份证

Construction of Rice Variety Indentity Using SSR Fingerprint and Commo-dity Information

LU Xu-Zhong¹, NI Jin-Long¹, LI Li¹, WANG Xiu-Feng¹, MA Hui¹, ZHANG Xiao-Juan^1,², YANG Jian-Bo^1,^*

¹Institute of Rice Research, Anhui Academy of Agricultural Sciences, Hefei 230031, China

²Cotton Research Institute, Anhui Academy of Agricultural Sciences, Anqing 246003, China

Fund:

Abstract

On the base of the previous research, we selected a set of 12 rice SSR markers (one marker for each chromosome) for increasing convenience of the rice seed quality tracking and management. The fluorescent marker analysis was performed on 127 released and popularized authorized (or main plant) rice varieties in Anhui Province. Then, the rice variety ID was constructed by the SSR fingerprint with corresponded commodity information. The rice variety ID included three components: part 1 was recognized as commodity code, which reflected crop species, cultivar, breeding (or approval) region and year; part 2 was recognized as fingerprint code, which contained the variety DNA fingerprint information; part 3 was recognized as complement code (specific gene identification code), reflecting specific genetic information of cultivars. We also described the rice variety ID in the form of bar code or QR code, which might be favorable for rice seed identification and traceability management.

Keyword: Rice; Fluorescent SSR markers; Capillary Electrophoresis; Commodity Information; Variety ID

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水稻优良品种的推广对保证我国粮食安全、满足人们多元化需求具有重要意义。然而, 随着水稻品种数目急剧增多和种质交流日趋频繁, 以及品种多家繁育、多元经营的既定格局, 种子市场中的“品种雷同”现象较为严重, “套牌”“冒牌”现象屡屡发生, 不仅给水稻品种选育、试验、质量和市场管理以及产权保护带来诸多困难和混乱, 更会给农业生产造成严重损失。因而, 亟需建立一套准确可靠、操作简便的品种区别和鉴定技术体系, 为水稻品种管理提供技术支撑。

随着DNA指纹鉴定技术的发展和日趋完善, 将DNA指纹(特别是SSR指纹)数字化, 构建农作物品种分子身份证, 使品种的区别、鉴定和对比更加方便直观。倪西源等^{[ 1]}首次提出利用SSR标记构建油菜等作物分子身份证和基因身份证; 高运来等^{[ 2]}利用９对SSR引物构建了83份黑龙江大豆品种的分子身份证; 陈昌文等^{[ 3]}等利用SSR标记构建了中国桃主要品种资源及其野生近缘种的16位分子身份证; 王黎明等^{[ 4]}用11对SSR引物组合构建了142份甜高粱品种资源的分子指纹图谱; 丁俊杰等^{[ 5]}利用与大豆灰斑病抗性基因连锁的SSR标记构建大豆品种(系)的分子身份证; 颜静宛等^{[ 6]}、杨剑波^{[ 7]}、辜大川等^{[ 8]}利用SSR标记分别构建了水稻杂交种或亲本材料的分子身份证; 马琳等^{[ 9]}通过逐级筛选, 最终确定7个SSR标记, 构建了130份甘蓝型油菜的品种分子身份证。

上述研究对水稻、油菜、大豆等农作物品种的资源评价、利用和保护等起到了重要作用。然而品种是育种工作的产物, 是农业生产资料, 具有商品属性, 单纯遗传学意义的分子身份证并不能完全反映一个品种包含的商品信息, 也难以区分近等基因系或代换系等。另外, 目前的SSR检测方法主要是PAGE电泳, 其条带大小依靠肉眼估测, 易出现误读, 而毛细管电泳可以得到扩增片段实际大小, 其结果更为精确、灵敏、高效, 非常适用于大批量品种的检测分析。因此, 本研究提出了一种新的水稻品种身份证构建策略, 即利用毛细管电泳SSR荧光标记分析, 获取水稻品种的指纹信息, 将指纹信息与水稻的商品信息等相结合, 构建水稻品种身份证, 为水稻种子质量的追溯和管理提供技术支撑。

1 材料与方法

1.1 试验材料

安徽省审定(或主推)水稻品种标准样品127份, 由安徽省种子管理站提供, 其中籼型三系杂交稻67份, 籼型两系杂交稻22份, 籼型常规稻19份, 粳型三系杂交稻6份, 粳型常规稻13份, 样品种子及水稻品种的特征特性等资料, 由安徽省种子协会收集整理。

1.2 SSR标记引物

用于水稻品种指纹分析的48个SSR标记引物^{[ 10, 11]}, 由Invitrogen生物技术有限公司合成, 并分别用FAM (蓝色)、HEX (绿色)、TAMRA (黄色)、ROX (红色) 4种荧光基团修饰上游引物5¢端。

1.3 DNA提取与检测

水稻种子发苗1周后(三叶期)取幼苗, 利用CTAB法提取单株水稻基因组DNA^{[ 11]}。以1%琼脂糖凝胶电泳检测DNA质量, 核酸蛋白仪测定浓度, 低温保存备用。

1.4 水稻SSR分析的荧光检测

按文献^{[ 11]}进行PCR扩增, 每样品扩增4个单株, 反应总体积为10 μL。参照文献[12-13]进行扩增产物的荧光检测: 将PCR产物稀释40倍, 在96孔板的各孔中分别加入9.05 μL去离子甲酰胺、0.05 μL LIZ-500分子量内标和1 μL稀释后的PCR产物, 95℃变性5 min, 于4℃放置10 min, 4000转 min^-1离心1 min, 于ABI3730 DNA分析仪上进行毛细管电泳。预电泳15 kV, 3 min; 2 kV电进样10 s; 电泳15 kV, 20 min。

1.5 指纹数据收集

用Data Collection软件收集原始数据, 用Genemapper软件分析收集的原始数据, 比较各峰值的位置与其泳道中的LIZ-500分子量内标, 获得SSR扩增片段的精确分子量(bp)。

1.6 水稻品种身份证的构建

将水稻品种的DNA指纹信息编码, 再结合品种的基本商品信息及可能具有的特异基因信息, 构建127份安徽省审定(或主推)水稻品种的身份证, 并利用在线条码生成器(http://barcode.tecit.com/barcodege-nerator.aspx?LANG=zhcn)和二维码生成软件Encoder2以条码表述构建的水稻品种身份证。

2 结果与分析

2.1 SSR标记组合的确定

在前期研究^{[ 10, 11]}的基础上, 分析48个SSR荧光标记在供试水稻品种中的扩增情况, 依据扩增产物在毛细管电泳中的可读性、多态性(图1), 并考虑染色体分布, 最终筛选出12个多态性较高、扩增稳定的SSR标记构成1个核心标记组(每条染色体1个标记), 对供试水稻材料进行荧光标记分析。

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	图1 水稻SSR标记RM241毛细管电泳荧光检测的特征带型Fig. 1 Capillary electrophoresis feature bands of rice SSR RM241 marker

2.2 水稻品种SSR指纹编码

读取127份水稻品种的12个SSR荧光标记的指纹数据, 并对指纹数据进行数字化编码。例如, 杂交水稻“皖稻185”(皖品审06010498)的SSR指纹编码如下, 12个标记在“皖稻185”中的扩增产物, 经由毛细管电泳其直接读取的片段大小(bp)为81/81、153/161、180/180、132/134、70/74、119/125、157/157、158/158、137/139、136/143、93/100、113/113 (按标记所在染色体由小到大排序, 水稻为二倍体, SSR扩增只有1条主带时, 须重复记录一次), 转换成24位的指纹码为3358 2278 3514 6666 4558 1322, 其中第1、2位的“3、3”表示标记RM1在‘皖稻185’中的扩增片段(81/81 bp)在其等位基因梯度(多态性片段)中排第3位, 第3、第4位的“5、8”表示标记RM6的两个扩增片段(153/161 bp)在其等位基因梯度(多态性片段)中分别排第5位和第8位(表1), 其余20位类推。

常规籼稻“早籼788”(皖稻2008001)的SSR指纹编码如下, 12个标记在“早籼788”中的扩增产物, 经由毛细管电泳其直接读取的片段大小(bp)为107/107、159/159、197/197、126/126、74/74、129/129、157/157、147/147、141/141、143/143、101/101、153/153, 转换成24位的指纹码为8877 6644 5566 6644 6688 33DD, 其中第1、第2位的“8、8”表示标记RM1在“早籼788”中的扩增片段(197/107 bp)在其等位基因梯度(多态性片段)中排第8位, 第3、第4位的“7、7”表示标记RM6的两个扩增片段(159/159 bp)在其等位基因梯度(多态性片段)中排第7位(表1), 其余20位类推。

各标记扩增片段按分子量大小排列, 扩增片段(等位基因)从小到大以阿拉伯数字1、2、3、4、……、9标注, 9个以上的等位基因, 以大写英文字母A、B、C、……、Z标注(表1)。

表1 12对水稻SSR核心标记信息 Table 1 Information of 12 rice core SSR markers

2.3 水稻品种商品码及补充码

水稻品种商品码代表品种的基本商品信息, 分为3个部分: (1)作物及品种类别码。由8位数字组成, 用以标识作物及品种类别, 其中, 第1~6位表示水稻种属(含3级分类, 水稻属于粮食作物中的禾谷类作物); 第7位表示水稻籼、粳类型; 第8位表示品种类型(杂交种或常规种等)。例如, 水稻品种‘宁粳3号’的作物及品种类别码为“01010123”, 其中, 第1、第2位的“01”代表大田作物, 第3、第4位的“01”代表禾谷类作物, 第5、第6位的“01”代表水稻, 第7位的“2”代表粳稻, 第8位的“3”代表常规种。(2)区域码。用以表示品种初次育成或审定的区域, 以各省市自治区的标准编码表示, 即北京为11、天津为12、安徽为34等, 国审品种以00表示。(3)时间码。用以表示品种初次育成或审定的年份,书如2000表示2000年审定, 2011表示2011年审定。

水稻补充码(特异基因识别码)表示品种可能携有的特异基因信息, 设计为字母“T”、“S”和“M”等, 加上被导入、渗入或诱变的基因名称, 分别代表通过转基因、分子标记辅助选择和诱变等育成的品种。

2.4 水稻品种身份证的构建

将上述的商品码、指纹码、补充码3个部分顺序排列, 就构成了总位数约40位的水稻品种身份证。以“皖稻185”的品种身份证01010111 34 2006 3358 2278 3514 6666 4558 1322 S为例(图2), 其作物类别为水稻(010101, 粮食作物—禾谷类作物—稻), 栽培亚种为籼稻(1), 品种类别为三系杂交种(1), 品种首次审定区域为安徽(34), 品种首次审定时间为2006年(2006); 品种DNA指纹为3358 2278 3514 6666 4558 1322; 该品种为通过分子标记辅助选择选育而成（对受体品种中控制直链淀粉含量的主效Wx基因进行了定向改良）。按上述方法完成了其余126份水稻品种身份证的构建。

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	图2 皖稻185品种身份证示意图Fig. 2 Model of Wandao 185 variety ID

2.5 水稻品种身份证的条码表述

按照中国物品编码相关国家技术标准《GB/T 18347-2001 128条码》^{[ 14]}和《GB/T 18284-2000 快速响应矩阵码》^{[ 15]}的规范要求, 利用条形码和二维码生成软件分别生成127份水稻品种身份证的条形码(一维码)和二维码。“皖稻185”的条形码和二维码如图3和图4, 部分水稻标准样品的品种身份证条码信息见表2。

将上述水稻品种身份证, 以条形码或二维码的形式标贴在种子包装上, 就可实现对品种种子质量的科学追溯、快速鉴定和规范管理, 也为品种的知识产权保护提供科学依据。

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	图3 皖稻185品种身份证条形码Fig. 3 Bar code of “Wandao 185” variety ID

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	图4 皖稻185品种身份证二维码Fig. 4 Quick Response (QR) code of “Wandao 185” variety ID

表2 部分水稻标准样品的品种身份证条码信息 Table 2 Variety ID information of some rice standard samples

序号 No.	品种名称 Name	品种审定编号 Identify number	栽培亚种 Cultivar	品种类别 Category
1	协优63 Xieyou 63	川审稻22/皖品审94010136 Chuanshendao 22/ Wanpinshen 94010136	籼 Indica	三系杂交种 Three-line hybrid
2	华安3号/皖稻79 Hua’an 3/Wandao 79	皖品审00010288 Wanpinshen00010288	籼 Indica	三系杂交种 Three-line hybrid
3	III优98 III You 98	皖品审02010333/ 国审稻2006061 Wanpinshen02010333/ Guoshendao006061	籼 Indica	三系杂交种 Two-line hybrid
4	开优10号 Kaiyou 10	皖稻2010020 Wandao2010020	籼 Indica	三系杂交种 Three-line hybrid
5	II优08/LY-8 II You 08/LY-8	皖稻2010022 Wandao2010022	籼 Indica	三系杂交种 Three-line hybrid
6	皖稻93/皖两优16 Wandao 93/Wanliangyou 16	皖品审03010372/ 湘审稻2012008 Wanpinshen03010372/ Xiangshendao2012008	籼 Indica	两系杂交种 Two-line hybrid
7	培两优98/皖稻129 Peiliangyou 98/Wandao 129	皖品审04010418 Wanpinshen04010418	籼 Indica	两系杂交种 Two-line hybrid
8	1892S×RH003/皖稻153 1892S×RH003/Wandao 153	皖品审05010463/ 国审稻2008013 Wanpinshen05010463/ Guoshendao2008013	籼 Indica	两系杂交种 Two-line hybrid
9	两优036 Liangyou 036	赣审稻2006070/ 皖品审2008007 Ganshendao2006070/ Wanpinshen2008007	籼 Indica	两系杂交种 Two-line hybrid
10	新两优香4号 Xinliangyouxiang 4	皖品审07010601 Wanpinshen07010601	籼 Indica	两系杂交种 Two-line hybrid rice
11	9优418/天协1号 9 You 418/Tianxie 1	国审稻20000009/ 皖品审02010330 Guoshendao20000009/ Wanpinshen02010330	粳 Japonica	三系杂交种 Three-line hybrid
12	金奉19 Jinfeng 19	皖品审03010385 Wanpinshen03010385	粳 Japonica	三系杂交种 Three-line hybrid
13	爱优18/爱优518/皖稻76 Aiyou 18/Aiyou 518/Wandao 76	皖品审04010415 Wanpinshen04010415	粳 Japonica	三系杂交种 Three-line hybrid
14	9201A×R-18/皖稻88 9201A×R-18/Wandao 88	皖品审06010509 Wanpinshen06010509	粳 Japonica	三系杂交种 Three-line hybrid
15	天协13 Tianxie 13	皖品审07010629 Wanpinshen07010629	粳 Japonica	三系杂交种 Three-line hybrid
16	早籼65 Zaoxian 65	皖品审03010368 Wanpinshen03010368	籼 Indica	常规种 Inbred
17	皖稻141/早籼276 Wandao 141/Zaoxian 276	皖品审05010457 Wanpinshen05010457	籼 Indica	常规种 Inbred
18	早籼788 Zaoxian 788	皖稻2008001 Wandao2008001	籼 Indica	常规种 Inbred
19	86120-5/皖稻68 86120-5/Wandao 68	皖品审03010384 Wanpinshen03010384	粳 Japonica	常规种 Inbred
20	W262/宁粳2号 W262/Ningjing 2	皖品审05010476 Wanpinshen05010476	粳 Japonica	常规种 Inbred

表2 部分水稻标准样品的品种身份证条码信息 Table 2 Variety ID information of some rice standard samples

3 讨论

3.1 水稻等农作物品种身份证的设计

所谓身份证, 顾名思义就是证实和区别身份的有效证明, 具有唯一性、可识别(鉴别)性、可追溯性等特点, 并可将反映身份的基本信息以数字、条码、图像等进行科学表述和系统规范, 从而被社会接受和通用。但农作物品种并不能像人类那样具有自我识别和相互识别能力, 不具备典型和突出的社会学特性。因此, 水稻等农作物品种身份证的构建, 需既借鉴人类身份证的编码模式, 又要结合农作物自身的特点。

3.2 水稻商品码和补充码设定

品种从本质上讲是一个商品概念, 它主要满足商品一般价值和农艺学基本要求, 而对遗传学纯一性要求往往不是很严格。有时为了增加适应性和对抗某些病害的需要, 还特别要求在品种内保留一定的遗传多样性; 也可在不影响其商品性和外观整体一致性前提下, 允许品种内个体间保留一定的变异度或变异残留。因此, 品种身份证设定了作物及品种类别码、区域码、时间码等商品码以反映品种的基本商品信息。同时, 为使品种身份证设计思路能够应用推广到其他农作物, 我们将作物类别设为6位代码, 如小麦为010102 (粮食作物—禾谷类作物—小麦), 油菜为144 (大田作物—油料作物—油菜)。

对于转基因育种、辐射(诱变)育种、分子标记辅助育种等选育的单一性状改良品种, 相对于原品种可以看作1个近等基因品种, 只有很少基因差异(甚至是SNP差异)。为此, 我们设定了补充码排在主体号码的后边, 用于标定这些特定类型品种。

3.3 适于荧光检测的核心引物的筛选

核心引物的筛选和确定是DNA指纹图谱^{[ 16]}和品种身份证构建^{[ 1, 2, 3, 4, 5, 6, 7, 8, 9]}的重要步骤。本研究从出入境行业标准(SN/T 3402-2012)^{[ 10]}推荐的用于水稻指纹鉴定的48个SSR标记中, 依据扩增产物在毛细管电泳中的可读性、多态性, 确定12个核心标记的组合(每条染色体1个)。按标记组合能够区分最大品种数的理论值计算^{[ 16, 17]}, 12个核心标记能够区分最大水稻品种数为1.93×10¹⁹, 再结合品种基本商品信息等, 完全能够满足现有水稻品种的区分要求。本研究构建的127份水稻的品种身份证, 其号码是唯一的, 表明所选用标记组合的可靠性、有效性。这样既保证了染色体的平衡性, 又达到了以最少标记区分品种的目的, 同时减少了指纹分析的工作量。本研究确定的12个标记能够完全区分安徽省近20年的主要水稻审定品种, 但对于来自其他审定区域的品种, 或水稻品种数量较大时, 12个核心标记未必能完全区分, 甚至出现2个品种的身份证完全相同的极端情况, 这时需用标准推荐的其余36个标记区别鉴定。

3.4 SSR标记分析方法的确定

已有的品种分子身份证构建研究中, SSR扩增产物主要经PAGE电泳检测^{[ 1, 2, 3, 4, 5, 6, 7, 8, 9]}, 整合不同批次、不同胶板的DNA指纹数据是个难题, 且容易出现误读、错读。本研究采用的SSR荧光标记毛细管电泳检测方法, 依据PCR扩增产物与同一泳道的内标比对, 直接读出目标DNA片段的准确大小, 其检测数据更为精确可靠^{[ 18, 19, 20, 21]}, 建立的分子指纹更为科学客观。

3.5 水稻品种身份证在农业物联网中的应用

本研究构建的水稻品种身份证, 通过条形码或二维码表述, 并标识于商品种子包装上, 可以用于水稻优良品种种子的防伪和溯源。但其科学使用, 还需在农业物联网^{[ 22]}的基础上组建种子物联网, 即利用传感器、射频识别、品种身份证信息、全球定位系统等现代物联网技术, 将水稻种子选育单位、种子产地、种子生产单位、包装种子、种子库、种子销售商、种植农户等相关元素通过互联网连接起来, 形成一个水稻品种种子“选育—保护—审定—繁殖—生产—流通—销售”的信息可追溯链条和全程的多功能服务网络体系, 将水稻种子的品种身份证以条行码(或二维码、芯片等)的形式, 对种子包装进行标识, 达到包装种子的可追溯性, 进而实现水稻品种和种子管理的科学化、规范化。

4 结论

构建了127份水稻的品种身份证, 并进行了条码表述。该品种身份证既借鉴了人类身份证的编码模式, 又结合了农作物自身的特点, 为水稻种子质量追溯与管理提供便利, 也为其他农作物品种身份证的构建提供了新的思路。

致谢: 127份水稻品种标准样品, 由安徽省种子管理站提供, 样品种子及水稻品种的特征特性等资料, 由安徽省种子协会李爱青、叶飞、方艳收集整理, 毛细管电泳分析得到北京农林科学院玉米中心王风格、易红梅的无私帮助, 在此一并致谢。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

参考文献

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2007

0.0

... 倪西源等^[1]首次提出利用SSR标记构建油菜等作物分子身份证和基因身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

... 4 SSR标记分析方法的确定已有的品种分子身份证构建研究中, SSR扩增产物主要经PAGE电泳检测^{[1,2,3,4,5,6,7,8,9]}, 整合不同批次、不同胶板的DNA指纹数据是个难题, 且容易出现误读、错读 ...

2009

0.0

1.667

. 2009, 35:211-218 DOI:10.3724/SP.J.1006.2009.00211

Establishment of molecular ID in soybean varieties in Heilongjiang, China

1College of agriculture, Northeast Agricultural University, Harbin 150030,China;2The Crop Research and Breeding Center of Land-Reclamation,Harbin 150090, China; 3The National Research Center of Soybean Engineering and technology, Harbin 150050,China;4College of Science, Northeast Agricural University, harbin 150030, Heilongjiang, China

A total of 1 300 cultivars have been bred in China from 1923 to 2005. To well evaluate and use the cultivars, it is necessary to identify scientifically and accurately them by DNA molecular markers due to traditional identification methods can no longer meet requirements. In the paper, 83 cultivars developed by 13 breeding units from six temperature summation zone in Heilongjiang were investigated, 157 polymorphic alleles were detected with 43 SSR primers covering 19 linkage groups of soybean genetic map, for each primer, 2 to 7 allele variations were detected in all cultivars, and 3.65 on average. According to fragment size of allele variation, the data numeralized from the PAGE bands were analyzed by the software ID Analysis 1.0. Nine primers (Satt100, Sat_218, Satt514, Satt551, Satt380, Satt193, Satt191, Satt442, and Sat_084) could be used to identify all 83 soybean cultivars. The PAGE pattern of each cultivar amplified with the 9 primers could be used as a molecular ID of the cultivars. A set of molecular ID were established for 83 soybean cultivars in Heilongjiang.

以黑龙江13个育种单位6个积温带的83份大豆品种为材料, 选择分布在大豆基因组19个连锁群的43对SSR引物进行检测, 共检测出等位变异157个, 每个引物检测到的等位变异数变化范围为2~7个, 平均为3.65个。将聚丙烯酰胺凝胶电泳得到的谱带统计结果根据等位变异的片段大小数字化, 用自行编制的ID Analysis 1.0软件进行数据分析。结果表明, 仅需9对引物(Satt100、Sat_218、Satt514、Satt551、Satt380、Satt193、Satt191、Satt442、Sat_084)可将83份参试大豆品种完全区分开。构建了一套黑龙江省大豆品种的分子ID。

... 高运来等^[2]利用９对SSR引物构建了83份黑龙江大豆品种的分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2011

0.0

. 2011, 44:2081-2093 DOI:10.3864/j.issn.0578-1752.2011.10.013

Molecular ID establishment of main China peach varieties and peach related species

1.中国农业科学院郑州果树研究所，郑州450009

【Objective】 A total of 237 peach germplasms including China landraces, bred varieties, and related species selected from national grape and peach repository of Zhengzhou were studied with SSR(simple sequence repeat) markers. Analysis was made to establish the 237 peach germplasms molecular ID and to study the methods for distinguishing the germplasms more simply.【Method】Germplasms were distinguished with selected SSR markers, bands that amplified by each marker were coded, then combined the code as a molecular ID. 【Result】The results showed that after screening of the 80 primers, 203 alleles were detected using 16 selected SSR markers located on each chromosome of peach, with a mean value of 12.7 alleles/locus. There were 123 selected alleles were coded to establish germplasms molecular ID based on strategy that alleles located both in landraces, bred and related germplasms. 【Conclusion】There were 202 out of 237 germplasms could be identified with an particular combined code numbers. Moreover, the identifying efficiency of different combinations of the primers were analyzed, 176 germplasms could be identified using 8 reduced core primers, with an average value of 22.1 germplasms/primers. The remaining primers have a higher identifying efficiency and more germplasms could be identified separately. At last, for distinguish germplasms more simply, through selection of the primer pairs according to its PIC step by step, the germplasms could be distinguished by a practical primer choice.

【目的】以国家果树种质郑州葡萄、桃圃保存的237份中国桃地方品种、育成品种及其野生近缘种为试材，进行种质分子身份证构建工作，并对构建方法进行探讨。【方法】采用筛选后的SSR标记对品种进行区分，然后根据引物对不同品种扩增条带分子量的大小进行编码、组合。【结果】从80对引物中筛选出来自桃8条染色体上的16对SSR引物，在供试种质间共检测出等位基因203个，每对引物平均检测到等位基因数为12.7个。根据等位基因既位于地方品种、育成品种，又位于近缘野生种的选择方法，筛选出123对等位基因并赋值后可用于构建种质的分子身份证。【结论】在237份种质中有202份具有的可辨分子身份证编码，继续对不同引物组合对种质的筛选效率进行分析，筛选出8对核心引物使176份种质具有可辨的分子身份证，平均每对引物区分种质达22.1份，即在保证每对引物区分效率较高的基础上，兼顾了总的区分数量。最后，为使区分品种简便化，依据引物的多样性指数先后选择引物组合，达到用最少的引物组合区分不同品种的目的。

... 陈昌文等^[3]等利用SSR标记构建了中国桃主要品种资源及其野生近缘种的16位分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2011

0.0

1.667

. 2011, 37:1975-1983 DOI:10.3724/SP.J.1006.2011.01975

Establishment of molecular identity in 142 sweet sorghum varieties

Crop Breeding Institute of Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China

A large change occurs in phenotypic characters under different environments for introducing and frequently exchanging of abundant varieties, and accurate identification is usually affected by the similar phenotypic characters performed in the field, so it is necessary to establish ID to distinguish varieties between each other. A total of 103 SSR primers covering 10 linkage groups of sorghum chromosome were screened, 41 primers showing polymorphic bands were used to establish molecular ID of 142 sweet sorghum germplasm resources. A total of 189 polymorphic bands were amplified and obtained. The number of alleles revealed by each primer was 2-11 with an average of 4.6 per primer. The polymorphism information content (PIC) value for the SSR loci ranged from 0.089 to 0.850 with an average of 0.543. There existed much difference in specific indices of varieties, ranging from 109.1 to 454.7 with an average of 189.0. The results showed that 11 primers (Xtxp329, Xtxp258, Xtxp113, Xtxp303, Xtxp61, Xtxp201, Xtxp141, Xtxp91, Xtxp47, Xtxp217, and Xtxp67) were confirmed on the basis of allele number of each pair of primers. The IDs of 142 sweet sorghum germplasm resources were established by the 11 pairs of primers, which can discriminate a variety from others.

从分布在高粱染色体10个连锁群上的103对SSR引物中，筛选出41对多态性引物, 并用其扩增国内外142份甜高粱种质资源, 检测到189个多态性片段, 每对引物的等位基因数在2~11之间，平均为4.6个。引物位点的多态信息含量指数(PIC)变幅为0.089~0.850，平均为0.543。品种间特异指数差异较大，介于109.1~454.7之间，平均为189.0。结果表明, 根据引物的等位基因数确定11对引物(Xtxp329、Xtxp258、Xtxp113、Xtxp303、Xtxp61、Xtxp201、Xtxp14、Xtxp91、Xtxp47、Xtxp217和Xtxp67)组合, 并用于构建142份品种资源的分子身份证, 有效地区分了各品种。

... 王黎明等^[4]用11对SSR引物组合构建了142份甜高粱品种资源的分子指纹图谱 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2012

0.0

1.667

. 2012, 38:2206-2216 DOI:10.3724/SP.J.1006.2012.02206

Establishment of molecular ID of soybean varieties (lines) using SSR markers linked to resistance genes against Cercospora sojina

1 Jiamusi Branch of Heilongjiang Academy of Agricultural Sciences / Observation and Experiment Station of Crop Pests of Jiamusi, Ministry of Agriculture, Jiamusi 154007, China; 2 Institute of Crop, Heilongjiang Academy of Land Reclamation Science, Jiamusi 154007, China; 3 Land Reclamation Research & Breeding Centre of Heilongjiang, Harbin 150090, China; 4 College of Agriculture, Northeast Agricultural University, Harbin 150030, China

One hundred and three soybean varieties (lines) developed by 29 breeding units from six accumulated temperature zones in Heilongjiang were investigated. The resistances of 103 soybean varieties (lines) were identified with three races of Cercospora sonija. Eighty-six polymorphic alleles were detected with 19 SSR markers, for each primer, two to six allele variations were detected in all varieties (lines), with an average of 4.42. The diversity index of the 19 SSR loci ranged from 0.198 to 0.751 with an average of 0.606. The results of calculating specific index and genetic similarity among varieties (lines) showed that specific index of varieties (lines) ranged from 46.592 to 481.541, and the average was 87.415. According to fragment size of allele variation, the data calculated from the PAGE bands were analyzed by the software ID Analysis 1.0. Seven markers (Satt565, Satt547, Satt431, Sct_186, SOYGPATR, Satt244, and Sat_151) linked to the gene controlling resistance to Cercospora sojina were used to identify all 103 soybean lines, so that a set of molecular ID of soybean varieties (lines) was established.

以黑龙江省29个大豆育种单位的103份已鉴定大豆灰斑病3个生理小种抗性的大豆品种(系)为材料，选择与大豆灰斑病抗病基因连锁的19个SSR标记检测，获得等位变异数86个，每个标记检测到的等位变异数分布在2~6个之间，平均为4.42个。应用遗传统计软件(genetics statistics 3.0)分析表明, 标记的多样性指数介于0.198~0.751之间，平均多样性指数为0.606。品种(系)特异指数差异较大，介于46.592~481.541之间，平均为87.415。根据标记的等位基因数，使用ID Analysis 1.0软件分析表明，利用与大豆抗灰斑病基因连锁的7个SSR标记(Satt565、Satt547、Satt431、Sct_186、SOYGPATR、Satt244、Sat_151)就能有效区分各品种(系)，因此利用这7个标记构建了供试品种(系)的分子身份证。

... 丁俊杰等^[5]利用与大豆灰斑病抗性基因连锁的SSR标记构建大豆品种(系)的分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2011

0.0

0.7902

. 2011, 26:148-152

Constructing SSR molecular database for identity system of hybrid rice parents

利用前期构建的基于明恢86的SSR电子遗传图谱,筛选得到分布于水稻12条染色体长短臂上的24对多态性好的SSR标记,利用这些标记对139份水稻品种材料进行多态性分析,从而得到142个等位位点,24个标记位点的分布范围在2～10之间,平均值为5.9.这些标记对杂交稻组合的鉴别能力高于对杂交稻亲本的鉴别能力,这在对目前生产中数目繁多的杂交组合的识别鉴定具备突出的优势.对18个杂交稻组合与其亲本材料进行了标记匹配性分析,在确定了母本高精确性遗传的同时,剔除了2个假杂交稻.此外,构建了137个水稻品种材料×24个标记的分子身份证数据库,本库可直接应用于生产中相关的亲本鉴定及种子验杂,从而为杂交水稻品种纯度和真伪鉴定提供科学数据.

... 颜静宛等^[6]、杨剑波^[7]、辜大川等^[8]利用SSR标记分别构建了水稻杂交种或亲本材料的分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2012

0.0

... 颜静宛等^[6]、杨剑波^[7]、辜大川等^[8]利用SSR标记分别构建了水稻杂交种或亲本材料的分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2012

0.0

0.5398

... 颜静宛等^[6]、杨剑波^[7]、辜大川等^[8]利用SSR标记分别构建了水稻杂交种或亲本材料的分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2013

0.0

0.95

. 2013, 35:231-239 DOI:10.7505/j.issn.1007-9084.2013.03.001

Establishment molecular identity of 130 Brassica napus varieties

1. Institute of Rice Research, Anhui Academy of Agricultural Sciences, Hefei 230031, China; 2.Institute of Cotton Research, Anhui Academy of Agricultural Sciences, Anqing, 246003, China

Germplasm accurate identification is usually affected by the similar phenotypic characteristics performed in the field, it is necessary to establish ID to distinguish varieties between each other. 463 SSR primers covering 19 linkage groups of rapeseed chromosome were screened, 33 primers showing polymorphic bands with 191 alleles were amplified and obtained. The number of alleles by each primer was 3 to 9 with an average of 5.7879 per primer, the effective number of alleles (Ne) is 1.9068 -7.4790 with an average of 4.730 7, the diversity (Nei’s genetic diversity index, He) was 0.773 8 to 2.045 1 with an average of 1.551 5, the discrimination power (DP) was 0.389 2 to 0.862 9 with an average of 0.742 8, polymorphism index content (PIC) was 0.4756-0.8663 with an average of 0.764 0. The principal coordinate (PCO) analysis indicated that all material could be clearly distinguished by 33 pairs of core primers, which divided into 3 subfamilies based on genetic similarity. After gradually screening, 7 pairs core primer (BrGMS075，Na14E08，BrAS084，CB10545，Na14G10，CN57 and Ra2E04) were selected, and their bands amplified by each marker were coded, and then the codes were combined as a molecular ID for Brassica napus.

本研究从分布于油菜19个连锁群上的463对SSR引物中，筛选出33对扩增清晰、稳定的多态性引物，并用其分析国内外130份甘蓝型油菜材料，共检测到191个等位变异，每对引物的等位变异数变幅为3-9个，平均为5.787 9；有效等位变异数（Ne）变幅为1.906 8-7.479 0，平均为4.7307；多样性指数（He）变幅为0.7738-2.0451，平均为1.551 5；引物个体识别能力（DP值）变幅为0.389 2-0.862 9，平均为0.742 8；多态信息含量（PIC值）变幅为0.475 6-0.866 3，平均为0.764 0。主坐标分析表明：33对核心引物可以将供试材料明确区分，并按照遗传相似性归为3类。通过逐级筛选，最终确定7对引物（BrGMS075、Na14E08、BrAS084、CB10545、Na14G10、CN57和Ra2E04），将每对引物的扩增产物进行数字标识，构建了130份甘蓝型油菜的品种分子身份证，达到用最少的引物组合区分不同品种的目的。

... 马琳等^[9]通过逐级筛选, 最终确定7个SSR标记, 构建了130份甘蓝型油菜的品种分子身份证 ...

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

2012

0.0

... 2 SSR标记引物用于水稻品种指纹分析的48个SSR标记引物^[10,11], 由Invitrogen生物技术有限公司合成, 并分别用FAM (蓝色)、HEX (绿色)、TAMRA (黄色)、ROX (红色) 4种荧光基团修饰上游引物5#cod#x000a2 ...

... 1 SSR标记组合的确定在前期研究^[10,11]的基础上, 分析48个SSR荧光标记在供试水稻品种中的扩增情况, 依据扩增产物在毛细管电泳中的可读性、多态性(图1), 并考虑染色体分布, 最终筛选出12个多态性较高、扩增稳定的SSR标记构成1个核心标记组(每条染色体1个标记), 对供试水稻材料进行荧光标记分析 ...

... 本研究从出入境行业标准(SN/T 3402-2012)^[10]推荐的用于水稻指纹鉴定的48个SSR标记中, 依据扩增产物在毛细管电泳中的可读性、多态性, 确定12个核心标记的组合(每条染色体1个) ...

2012

0.0

. 2012, 26:853-861

Establishment of DNA fingerprinting database for parental lines of hybrid rice

Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, Anhui 230031

In this study, we performed SSR (simple sequence repeats) analysis of 133 hybrid parental rice varieties with 48 SSR core primer pairs recommend by GB/T20396-2006. Based on polymorphism among the materials, we built molecular ID and fingerprint for each variety. The ID was constructed by 48 digit numbers and letters, which represent the detail polymorphism on certain variety. Combined the SSR polymorphism and anatomical information, a searchable SSR fingerprinting database was built. This database could be used to identify the basic information, molecular ID and similarity of varieties. Furthermore, it also could be applied in seed purity examining, parental similarity testing to support the rice hybrid breeding. This database not only sets a sharing platform for examination institutes or breeders, but also technically supports the breeding, testing, managing and judging on rice varieties.

本研究利用GB/T20396-2006推荐的48对SSR核心标记,对133份杂交水稻亲本(包括部分常规品种)进行SSR分析,根据引物在133份材料中扩增的片段大小,构建了各品种的分子身份证和指纹图谱,该身份证由48位数字或数字与字母组合组成,代表了48对引物在该品种中的扩增情况。将品种的身份证信息与品种的形态学特征特性等信息相结合,构建了具查询功能的SSR指纹网络数据库。该SSR指纹数据库除具有品种基本信息查询、品种分子身份证查询、品种间相似率查询和身份证核对等基本功能外,还可指导杂交水稻品种真实性和纯度鉴定,分析亲本间遗传相似性,为杂交稻新组合的选育提供亲本选配的参考。该数据库的建立,不仅为检测单位和育种工作者提供了一个便利的网络数据交换和共享平台,也为品种的选育、试验、管理、评价、仲裁提供技术支撑和科学参考。

... 3 DNA提取与检测水稻种子发苗1周后(三叶期)取幼苗, 利用CTAB法提取单株水稻基因组DNA ^[11] ...

2006

0.0

. 2006, 21(5):64-67

Comparison of two maize SSR detection methods: capillary electrophoresis with fluorescence detection method and denaturing PAGE silver staining detection method

以192份玉米品种为材料,用7对SSR引物对毛细管电泳荧光检测和常规的变性PAGE银染检测两种SSR标记检测方法进行比较分析.结果表明,两种方法检测的SSR标记片段大小一致.毛细管电泳荧光检测法检测的结果更为精确、灵敏、高效,更适用于高通量材料的检测分析.对少量材料进行检测分析以及SSR标记的筛选时,使用常规的变性PAGE银染检测法更经济适用.

2011

0.0

1.494

. 2011, 25:672-676 DOI:10.3969/j.issn.10017216.2011.06.016

Application of capillary electrophoresis detection with fluorescent SSR markers in rice DNA fingerprint identification

China National Rice Research Institute, Hangzhou 310006, China

With 16 hybrid rice male sterile lines, restorer lines and combinations as materials, a capillary electrophoresis detection method with fluorescent SSR markers in rice was primarily developed. The accurate size of DNA fragment could be detected by the capillary electrophoresis detection method with fluorescent SSR markers. The capillary electrophoresis detection has the advantages of data accuracy and efficiency compared with polyacrylamide gel electrophoresis detection. It provides a feasible and reliable method for rice DNA fingerprint identification.

以16个杂交水稻不育系、恢复系及组合为材料，初步建立了水稻品种SSR荧光标记毛细管电泳检测方法。与常规凝胶电泳检测方法相比，荧光标记毛细管电泳检测方法可以读出目标DNA片段的准确大小，检测数据更为精确，检测效率更高。常规凝胶电泳检测方法验证表明，利用SSR荧光标记毛细管电泳检测方法进行水稻品种DNA指纹鉴定，方法可行、结果可靠。

2001

0.0

... 5 水稻品种身份证的条码表述按照中国物品编码相关国家技术标准《GB/T 18347-2001 128条码》^[14]和《GB/T 18284-2000 快速响应矩阵码》^[15]的规范要求, 利用条形码和二维码生成软件分别生成127份水稻品种身份证的条形码(一维码)和二维码 ...

2000

0.0

2003

0.0

0.965

... 3 适于荧光检测的核心引物的筛选核心引物的筛选和确定是DNA指纹图谱^[16]和品种身份证构建^{[1,2,3,4,5,6,7,8,9]}的重要步骤 ...

... 按标记组合能够区分最大品种数的理论值计算^[16,17], 12个核心标记能够区分最大水稻品种数为1 ...

2009

0.0

1.23

. 2009, 7:1160-1168

Screening of SSR core primer pairs for identificating cotton cultivar

本研究利用来自Cotton Marker Database(CMD)数据库中的2 000个引物对32份审定棉花品种基因组DNA进行了PCR扩增,筛选出26个多态性核心引物,其中11个引物被推荐为棉花品种鉴定的首选核心引物,同时提出了核心引物理论上可区分的最大品种数的计算公式:N=G1x…xGn,理论推测结果表明:利用11个首选核心引物进行棉花品种鉴定是可行的.建立在SSR指纹数据基础上的不同组合品种(系)间聚类分析以及对25份区试品系和A品种的真实性鉴定,结果表明:所有参试品种(系)遗传基础狭窄,32份审定品种间DNA水平上的差异与品种来源的系谱分析结果基本吻合,首选核心引物不仅可以对品种(系)的真实性做出鉴定,而且对遗传距离狭窄的品种(系)具有很好的鉴别力.

Abstract：

In this study, genomic DNA from thirty-two approved cotton cultivars were amplified with 2 000 primer pairs selecting from cotton marker database (CMD) by using polymerase chain reaction (PCR), twenty-six core primer pairs with polymorphism were selected, of which eleven core primer pairs were proposed as preferred primer pairs for identification of cotton cultivars, at the same time a formula for calculating the maximum number of cotton cultivars that the eleven core primer pairs can identify in theory was advanced: N=G1x...xGn, and according to theory, it has deduced that eleven core primer pairs were effective. Genetic clustering analysis of different combinations of cotton cutlivars (lines) used in this study and authenticity identification of A approved cutlivar and twenty-five cotton varieties in Shandong provincial regional trial were performed based on SSRs fingerprint database, the result showed that genetic basis of the tested materials were limited, difference of thirty-two approved cotton cultivars at DNA level was in accordance with the result of pedigree analysis of them, the preferred eleven core prime pairs not only can identify the authenticity of cotton cultivars (lines), but also had good power in identification of cotton cultivars (lines) with limited genetic basis.

... 按标记组合能够区分最大品种数的理论值计算^[16,17], 12个核心标记能够区分最大水稻品种数为1 ...

2005

0.0

1.667

. 2005, 31:144-149

Comparison of fluorescence and silver staining detection systems of microsatellite markers

Key Laboratory of Crop Germplasm and Biotechnology, Ministry of Agriculture, Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences, Beijing 100081

Four-hundred and fifty-one wheat cultivars from Northern Winter Wheat Region were analyzed by fluorescence based genescan with multiple PCR microsatellite markers and SSR marker technology based on silver staining. In the same cultivars and the same set of SSR primers, the detection efficiency of th

应用多重PCR简单重复序列（SSR）荧光标记分析技术和常规的SSR银染技术，对北方冬麦区451份材料（中国小麦初选核心种质的一部分）进行分析，用相同材料和引物，对这两种方法的检测效果进行评价。用24对引物扩增，银染法共检测出235个等位变异，每个位点检测到的等位变异为3～20，平均为9.8个，多态性信息指数PIC为0.22～0.93

... 本研究采用的SSR荧光标记毛细管电泳检测方法, 依据PCR扩增产物与同一泳道的内标比对, 直接读出目标DNA片段的准确大小, 其检测数据更为精确可靠^{[18,19,20,21]}, 建立的分子指纹更为科学客观 ...

2008

0.0

1.889

2009

0.0

. 2009, 21:540-543

Application and evaluation of fluorescent EST SSR markers detection technique with capillary electrophoresis in pea

试验采用MegaBACE 1000 DNA分析系统,利用荧光标记豌豆EST-SSR引物,建立豌豆EST-SSR荧光标记毛细管电泳检测法,并对该检测法的特点进行了分析和评价.结果表明,试验所建立的毛细管电泳荧光标记检测法能准确地识别片段大小、重复性好,可以明确区分不同等位基因;同时,通过采用两种不同颜色的荧光标记,可使检测192个样本的时间缩短在2 h之内.可见,本试验所建立的毛细管电泳荧光标记检测法具有高通量、高效率、高精确性等优点.

Abstract：

To detect EST-SSR markers in pea,a capillary electrophoresis with fluorescence detection was developed and evaluated based on fluorescence labeled EST-SSR primers and MegaBACE 1000 DNA analysis system. The result showed high precision and reproducibility in determining pea EST-SSR alleles by this detection system. Furthermore,the detection and analysis of 192 samples could be automatically completed within two hours by two different-color fluorescences labelled EST-SSR primers. Therefore,it is suggested that the capillary electrophoresis with fluorescence detection developed in our study with high throughput,efficiency.

2012

0.0

1.194

2012

0.0

... 但其科学使用, 还需在农业物联网^[22]的基础上组建种子物联网, 即利用传感器、射频识别、品种身份证信息、全球定位系统等现代物联网技术, 将水稻种子选育单位、种子产地、种子生产单位、包装种子、种子库、种子销售商、种植农户等相关元素通过互联网连接起来, 形成一个水稻品种种子#cod#x0201c ...