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

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。

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 se-quences 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.