燕麦隐性核不育cSSR标记的开发及其验证

doi:10.11686/cyxb2014232

草业学报 ›› 2015, Vol. 24 ›› Issue (7): 146-154.DOI: 10.11686/cyxb2014232

燕麦隐性核不育cSSR标记的开发及其验证

张丽君¹, 刘龙龙¹, 畅志坚², 郭彬³, 崔林^{1*, *}

1.山西省农业科学院农作物品种资源研究所,农业部黄土高原作物基因资源与种质创制重点实验室,山西太原 030031;
2.山西省农业科学院作物研究所,山西太原 030031;
3.山西省晋中市寿阳县乡镇农产品质量安全监管站,山西寿阳 045400

收稿日期:2014-05-08 出版日期:2015-07-20 发布日期:2015-07-20
作者简介:张丽君（1981-）,女,河北行唐人,助理研究员,博士。E-mail:lijun.zhang911@163.com
基金资助:
山西省农科院博士基金项目（YBSJJ209）,山西省自然基金项目（2014110303-1）,山西省国际合作项目（2014-081032-1）和国家燕麦荞麦产业技术体系（CARS-08-A1）项目资助

Development and functional verification of cSSR markers for recessive genetic male sterility in oats

ZHANG Li-Jun¹, LIU Long-Long¹, CHANG Zhi-Jian², GUO Bin³, CUI Lin^{1, *}

1.Crop Germplasm Resources Research Institute, Shanxi Academy of Agricultural Sciences;
Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture, Taiyuan 030031, China;
2.Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, China;
3.Quality and Safety Supervision of Township Agricultural Station, Shouyang 045400, China

Received:2014-05-08 Online:2015-07-20 Published:2015-07-20

摘要/Abstract

摘要： 构建燕麦隐性核不育转录组数据库,开发cSSR标记,丰富燕麦分子标记数据库,并对隐性核不育相关cSSR标记进行验证,为充分利用燕麦种质资源及深入研究隐性核不育奠定基础。对燕麦雄性不育近等基因系CAMS1进行转录组测序,利用软件对得到的EST序列进行SSR位点查找和分析,开发cSSR 引物。并用CAMS1×品燕2号F₂代群体对所开发的雄性不育相关的cSSR引物进行验证。通过转录组测序获得的EST序列中6409条存在SSR位点,736条序列中有2个及2个以上SSR位点;在所有SSR位点中三核苷酸、二核苷酸重复序列最多,分别为4340(56.66%)和1768(24.30%)个。三核苷酸重复中,CCG/CGG(26.68%)、AGG/CTT(21.29%)、AGC/CTG(18.48%)出现的频率较多,二核苷酸重复中,以AG/CT(60.63%)和AC/GT(26.24%)出现的频率较高;根据开发的cSSR设计了13344对cSSR引物,选择与雄性不育相关且能设计引物的21条序列合成45对SSR引物。PCR检测表明,32对引物(71%)有扩增产物,其中11对cSSR引物在亲本间的扩增产物具有多态性,7对引物在可育和不育性状池间存在差异。本研究对燕麦雄性不育近等基因系CAMS1进行了转录组测序,根据得到的EST序列开发了13344对cSSR 引物,并利用CAMS1×品燕2号F₂代群体进行有效性检测,32对引物有扩增产物,7个标记可用于燕麦雄性不育材料的分子检测。

Abstract: The objective of this study was to develop new cSSR markers from an oat (Anena sativa) recessive genes male sterile transcriptome database. EST sequences obtained from a RNA-Seq of oat male sterile near-isogenic line CAMS9 were used to search for SSRs using software. cSSR primers were developed and functional verification for those related to male sterility was determined with a F₂ population of hybridized CAMS9 and Pinyan 2.6409 EST sequences obtained from the transcriptome sequencing had SSR loci and 736 of these sequences had 2 or more loci. Most of the sequence motifs were dinucleotides (56.66%) and trinucleotides (24.30%). For trinucleotides, CCG/CGG (26.68%), AGG/CTT (21.29%), AGC/CTG (18.48%), AAG/CTT (8.18%) and ACC/GGT (8.11%) were the most frequent repeats. For dinucleotides, AG/CT (60.63%) and AC/GT (26.24%) were the most frequent. Based on these cSSRs, 13344 pairs of cSSR primers were designed. 45 pairs of the primers were synthesized based on male sterile-related cSSRs, and 32 (71.1%) of the pairs led to amplified products. Using the F₂ population, the polymorphism of 11 pairs between parents and the differences of 7 pairs between fertile and sterile trait pools were detected. In total, 13344 pairs of cSSR primers have been developed from the RNA-Seq database. 32 pairs were related to fertility and 7 can be used for molecular detection of oat male sterility. These new cSSR markers are beneficial for the further use of oat germplasm and for the study of male sterility.

张丽君, 刘龙龙, 畅志坚, 郭彬, 崔林. 燕麦隐性核不育cSSR标记的开发及其验证[J]. 草业学报, 2015, 24(7): 146-154.

ZHANG Li-Jun, LIU Long-Long, CHANG Zhi-Jian, GUO Bin, CUI Lin. Development and functional verification of cSSR markers for recessive genetic male sterility in oats[J]. Acta Prataculturae Sinica, 2015, 24(7): 146-154.

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燕麦隐性核不育cSSR标记的开发及其验证

Development and functional verification of cSSR markers for recessive genetic male sterility in oats

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