双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定
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第 5 期
刘平武等 :双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定Fra bibliotek6 41
were fertile1 The hybrid purity was 96107 % and 91133 % respectively ( Table 3 and Table 4) 1 Compared wit h t he results of t he observation of fertility in t he field , SSR marker can detect more false hybrids ,
In t he investigation reported in t his paper , t he fingerprints of 39 B1 napus hybrid parents including 18 restorers and 21 sterile lines were constructed wit h SSR1 106 SSR primer pairs were screened on 3 materials ( Y04 , Y12 , Y29) wit h great genetic diversity , and 16 primer pairs were selected for furt her analysis1 A total of 73 bands were amplified ( Table 2) , and t he certain band patterns were just t he fingerprints for each B1 napus hybrid parents , which could be used in identifying t he factuality of t he parents and t he purity of hybrid1
杂种优势的利用是提高作物产量的重要途径 , 目前生产上利用甘蓝型油菜杂种优势的主要途径有 细胞质雄性不育 ( CMS) 、细胞核雄性不育 ( GMS) 、 自交不亲和 ( SI) 和化学杀雄等途径 ,其中细胞质雄 性不育是油菜杂种优势利用最主要的途径 。但在杂 交制种中 ,由于不育系不育性不彻底或由于温度 、光 照的影响导致不育系产生微量花粉 ,进而导致 F1 杂 种中混杂不育株 ,比例过高会严重影响杂交种的产 量 。因而在杂交种应用于生产之前 ,需要鉴定杂交 种的纯度 。另外由于杂交种具有较高的科技含量和 商业价值 ,少数杂交种生产经营者为谋取私利常掺 假或以非法手段获取亲本生产杂交种 ,从而严重侵 害了育种工作者和合法的种子生产经营单位的正当 权益 。为了更好地保护杂交种亲本和进行杂交种纯 度鉴定 ,需要建立一套准确 、简便 、快速 、高效的体 系 。这在油菜杂交种生产中具有重要的实际意义 。
Ξ基金项目 : 国家 863 计划资助 (2001AA241111) 。 作者简介 : 刘平武 (1976 - ) ,男 ,博士 ,讲师 。Tel : 027287281713 ; E2mail : HZAUL PW @1631com
3 通讯作者 :杨光圣 。Tel : 027287281507 ; Fax : 027287280009 ; E2mail : rapelab @public1wh1hb1cn Received (收稿日期) :2004203222 , Accepted (接受日期) :20042082231
(华中农业大学作物遗传改良国家重点实验室 ,国家油菜改良武汉分中心 ,湖北武汉 430070)
摘 要 : 利用 SSR 分子标记构建了 18 份恢复系和 21 份不育系共 39 份甘蓝型油菜杂交种亲本的 DNA 指纹图谱 ,筛选 出 H9901 和 H9905 两个杂交种亲本的共显性 SSR 标记 Na10 G08 ,并用其鉴定了 H9901 、H9905 两个大田生产 F1 杂种群 体的纯度 。对比 SSR 和田间调查的鉴定结果表明 ,SSR 标记鉴定可以识别更多的非杂交种单株 ,如母本因微粉导致的自 交 、花粉污染 、机械混杂等带来的非杂交种单株 。本文还就 SSR 标记用于 DNA 指纹图谱构建和杂交种纯度鉴定的可行 性和优点进行了讨论 。
Abstract: Utilization of heterosis is an important way of increasing yield potential in crop breeding programs1 Application of cytoplasmic male sterility ( CMS) , compared wit h ot her ways of utilizing heterosis in rapeseed , is t he main way1 However , wit h t he effect of temperature and light , t he male sterile lines of polima cytoplasmic male sterility (pol CMS) can produce small amount pollens , which will lead to self2pollination of t he sterile lines in hybrid seed production1 And t he high ratio of sterile plants in F1 populations will reduce t he yield1 In order to test purity of hybrid and protect t he parents of hybrid , an effective and accurate system must be set up1 Many molecular markers , such as RAPD , SCAR and AFL P have been widely used in constructing t he fingerprints of crop varieties1 However , t he fingerprints analysis and hybrid purity test in B rassica napus has rarely been reported by using SSR1
L IU Ping2Wu , ZHOU Guo2Ling , YAN G Guang2Sheng 3 , FU Ting2Dong
( N ational Key L aboratory of Crop Genetic I m provement , Huaz hong A gricult ural U niversity , Research Center of N ational Rapeseed I m provement , W uhan 430070 , Hubei , Chi na)
目前构建杂交种亲本的指纹图谱是鉴定和保护 杂交种亲本及检测杂种纯度的有效手段 。用于构建 品种指纹图谱的遗传学标记主要包括形态学标记 、 生化标记和分子标记 。分子标记是近年发展起来
的 ,它反映了 DNA 水平上生物个体间的遗传差异 , 目前广泛应用的主要包括限制性片段长度多态性 (RFL P) 、随机扩增长度多态性 ( RAPD) 、特征序列 扩增标记 ( SCAR) 、扩增片段长度多态性 (AFL P) 、 简单重复序列 ( SSR) 、单核苷酸多态性 ( SN Ps) 等 。 它们具有表现中性 ,鉴定不受时空限制和环境影响 等优点 ,因而发展很快 。RAPD 、SCAR 、AFL P 已广 泛用于品种指纹图谱构建和杂交种纯度鉴定[1～5] , 但利用 SSR 标记在甘蓝型油菜中进行指纹分析和 杂交种纯度鉴定方面的研究还未见报道 。本文即试 图开展这方面的研究 。
According to t he DNA fingerprints , primer Na10 G08 was chosen to test purity of F1 hybrids H9901 (8086A × Lun31R) and H9905 (8110A ×8759R) , and 3 bands were produced by t his primer1 That was“010”in t he female parents 8086A and 8110A ,“101”in t he male parents Lun31R and 8759R , and“111”in t he hybrids ( Fig11 and Fig12) 1 A total of 178 hybrid plants in H9901 and 173 hybrid plants in H9905 were investigated1 PCR amplification result showed t hat 168 F1 hybrid plants in H9901 and 152 F1 hybrid plants in H9905 were true hybrids1 The hybrid purity was 94138 % and 87186 % respectively1 In addition , t he fertility investigation results of t hese two F1 populations in field showed t hat 171 F1 hybrid plants in H9901 and 158 F1 hybrid plants in H9905
关键词 : 甘蓝型油菜 ;杂交种亲本 ;指纹图谱 ;杂交种纯度 ;SSR 中图分类号 : S565
Fingerprints Construction of Hybrid Parents in B rassica na pus and Its Util ization in Hybrid Purity Test
第 31 卷 第 5 期 2005 年 5 月 640～646 页
作 物 学 报 ACTA A GRONOM ICA SIN ICA
Vol131 , No15 pp1 640 - 646 May , 2005
双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定
刘平武 周国岭 杨光圣 3 傅廷栋 Ξ
which might come from self2pollination of female parent , biological contamination of ot her pollens or machanical contamination wit h ot her seeds1 Meanwhile , t he feasibility and merits of constructing DNA fingerprints and identifying hybrid purity were discussed also1 Key words : B rassica napus ; Hybrid parents ; Fingerprints ; Hybrid purity ; SSR
1 材料与方法
111 材料 所用的材料包括 18 份恢复系和 21 份不育系共 39 份双低甘蓝型油菜杂交种亲本材料 (表 1) 和 2 个 大田生 产 F1 杂 种 群 体 H9901 ( 8086A ×轮 31R) 、 H9905 (8110A ×8759R) 。
第 5 期
刘平武等 :双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定Fra bibliotek6 41
were fertile1 The hybrid purity was 96107 % and 91133 % respectively ( Table 3 and Table 4) 1 Compared wit h t he results of t he observation of fertility in t he field , SSR marker can detect more false hybrids ,
In t he investigation reported in t his paper , t he fingerprints of 39 B1 napus hybrid parents including 18 restorers and 21 sterile lines were constructed wit h SSR1 106 SSR primer pairs were screened on 3 materials ( Y04 , Y12 , Y29) wit h great genetic diversity , and 16 primer pairs were selected for furt her analysis1 A total of 73 bands were amplified ( Table 2) , and t he certain band patterns were just t he fingerprints for each B1 napus hybrid parents , which could be used in identifying t he factuality of t he parents and t he purity of hybrid1
杂种优势的利用是提高作物产量的重要途径 , 目前生产上利用甘蓝型油菜杂种优势的主要途径有 细胞质雄性不育 ( CMS) 、细胞核雄性不育 ( GMS) 、 自交不亲和 ( SI) 和化学杀雄等途径 ,其中细胞质雄 性不育是油菜杂种优势利用最主要的途径 。但在杂 交制种中 ,由于不育系不育性不彻底或由于温度 、光 照的影响导致不育系产生微量花粉 ,进而导致 F1 杂 种中混杂不育株 ,比例过高会严重影响杂交种的产 量 。因而在杂交种应用于生产之前 ,需要鉴定杂交 种的纯度 。另外由于杂交种具有较高的科技含量和 商业价值 ,少数杂交种生产经营者为谋取私利常掺 假或以非法手段获取亲本生产杂交种 ,从而严重侵 害了育种工作者和合法的种子生产经营单位的正当 权益 。为了更好地保护杂交种亲本和进行杂交种纯 度鉴定 ,需要建立一套准确 、简便 、快速 、高效的体 系 。这在油菜杂交种生产中具有重要的实际意义 。
Ξ基金项目 : 国家 863 计划资助 (2001AA241111) 。 作者简介 : 刘平武 (1976 - ) ,男 ,博士 ,讲师 。Tel : 027287281713 ; E2mail : HZAUL PW @1631com
3 通讯作者 :杨光圣 。Tel : 027287281507 ; Fax : 027287280009 ; E2mail : rapelab @public1wh1hb1cn Received (收稿日期) :2004203222 , Accepted (接受日期) :20042082231
(华中农业大学作物遗传改良国家重点实验室 ,国家油菜改良武汉分中心 ,湖北武汉 430070)
摘 要 : 利用 SSR 分子标记构建了 18 份恢复系和 21 份不育系共 39 份甘蓝型油菜杂交种亲本的 DNA 指纹图谱 ,筛选 出 H9901 和 H9905 两个杂交种亲本的共显性 SSR 标记 Na10 G08 ,并用其鉴定了 H9901 、H9905 两个大田生产 F1 杂种群 体的纯度 。对比 SSR 和田间调查的鉴定结果表明 ,SSR 标记鉴定可以识别更多的非杂交种单株 ,如母本因微粉导致的自 交 、花粉污染 、机械混杂等带来的非杂交种单株 。本文还就 SSR 标记用于 DNA 指纹图谱构建和杂交种纯度鉴定的可行 性和优点进行了讨论 。
Abstract: Utilization of heterosis is an important way of increasing yield potential in crop breeding programs1 Application of cytoplasmic male sterility ( CMS) , compared wit h ot her ways of utilizing heterosis in rapeseed , is t he main way1 However , wit h t he effect of temperature and light , t he male sterile lines of polima cytoplasmic male sterility (pol CMS) can produce small amount pollens , which will lead to self2pollination of t he sterile lines in hybrid seed production1 And t he high ratio of sterile plants in F1 populations will reduce t he yield1 In order to test purity of hybrid and protect t he parents of hybrid , an effective and accurate system must be set up1 Many molecular markers , such as RAPD , SCAR and AFL P have been widely used in constructing t he fingerprints of crop varieties1 However , t he fingerprints analysis and hybrid purity test in B rassica napus has rarely been reported by using SSR1
L IU Ping2Wu , ZHOU Guo2Ling , YAN G Guang2Sheng 3 , FU Ting2Dong
( N ational Key L aboratory of Crop Genetic I m provement , Huaz hong A gricult ural U niversity , Research Center of N ational Rapeseed I m provement , W uhan 430070 , Hubei , Chi na)
目前构建杂交种亲本的指纹图谱是鉴定和保护 杂交种亲本及检测杂种纯度的有效手段 。用于构建 品种指纹图谱的遗传学标记主要包括形态学标记 、 生化标记和分子标记 。分子标记是近年发展起来
的 ,它反映了 DNA 水平上生物个体间的遗传差异 , 目前广泛应用的主要包括限制性片段长度多态性 (RFL P) 、随机扩增长度多态性 ( RAPD) 、特征序列 扩增标记 ( SCAR) 、扩增片段长度多态性 (AFL P) 、 简单重复序列 ( SSR) 、单核苷酸多态性 ( SN Ps) 等 。 它们具有表现中性 ,鉴定不受时空限制和环境影响 等优点 ,因而发展很快 。RAPD 、SCAR 、AFL P 已广 泛用于品种指纹图谱构建和杂交种纯度鉴定[1～5] , 但利用 SSR 标记在甘蓝型油菜中进行指纹分析和 杂交种纯度鉴定方面的研究还未见报道 。本文即试 图开展这方面的研究 。
According to t he DNA fingerprints , primer Na10 G08 was chosen to test purity of F1 hybrids H9901 (8086A × Lun31R) and H9905 (8110A ×8759R) , and 3 bands were produced by t his primer1 That was“010”in t he female parents 8086A and 8110A ,“101”in t he male parents Lun31R and 8759R , and“111”in t he hybrids ( Fig11 and Fig12) 1 A total of 178 hybrid plants in H9901 and 173 hybrid plants in H9905 were investigated1 PCR amplification result showed t hat 168 F1 hybrid plants in H9901 and 152 F1 hybrid plants in H9905 were true hybrids1 The hybrid purity was 94138 % and 87186 % respectively1 In addition , t he fertility investigation results of t hese two F1 populations in field showed t hat 171 F1 hybrid plants in H9901 and 158 F1 hybrid plants in H9905
关键词 : 甘蓝型油菜 ;杂交种亲本 ;指纹图谱 ;杂交种纯度 ;SSR 中图分类号 : S565
Fingerprints Construction of Hybrid Parents in B rassica na pus and Its Util ization in Hybrid Purity Test
第 31 卷 第 5 期 2005 年 5 月 640～646 页
作 物 学 报 ACTA A GRONOM ICA SIN ICA
Vol131 , No15 pp1 640 - 646 May , 2005
双低甘蓝型油菜杂交种亲本指纹图谱构建和杂交种纯度鉴定
刘平武 周国岭 杨光圣 3 傅廷栋 Ξ
which might come from self2pollination of female parent , biological contamination of ot her pollens or machanical contamination wit h ot her seeds1 Meanwhile , t he feasibility and merits of constructing DNA fingerprints and identifying hybrid purity were discussed also1 Key words : B rassica napus ; Hybrid parents ; Fingerprints ; Hybrid purity ; SSR
1 材料与方法
111 材料 所用的材料包括 18 份恢复系和 21 份不育系共 39 份双低甘蓝型油菜杂交种亲本材料 (表 1) 和 2 个 大田生 产 F1 杂 种 群 体 H9901 ( 8086A ×轮 31R) 、 H9905 (8110A ×8759R) 。