南瓜属EST-SSR标记的开发及在杂种纯度鉴定中的应用  

EST-SSR Markers Development from Cucurbita and Their Use in Purity Testing of F_1 Hybrid Seed

作　　者：张国裕[1] 张帆[1] 姜立纲[1] 翟伟卜[1] 李海真[1] 

机构地区：[1]国家蔬菜工程技术研究中心,北京100097

出　　处：《华北农学报》2011年第6期97-101,共5页Acta Agriculturae Boreali-Sinica

基　　金："十一五"科技支撑项目(2009BADB8B02);北京市新世纪百千万人才工程培养资助项目(2008);国家农业科技成果转化资金项目(2010GB2A000003);北京市科委项目(D101105046510001);北京市农林科学院科技创新能力建设专项(KJCX201101010-9);北京市农林科学院科研项目(2010A004)

摘　　要：为利用SSR标记进行快速、准确的南瓜杂交种纯度分析,进行了南瓜EST-SSR标记的开发。从NCBI数据库下载1 457条南瓜属作物EST序列,去除冗余序列后进行SSR位点搜索,共得到215条含有SSR位点的EST序列。这些SSR位点包含111种重复基元,其中二核苷酸(62个,占28.84%)和三核苷酸(86个,占40.0%)重复类型占主导地位;重复基元中出现最多的是CT(21个,占9.77%),其次为TC(13个,占6.05%)和AAG(12个,占5.58%)。将215个SSR位点设计成EST-SSR引物后,随机合成了其中的43对引物。利用4份遗传背景差异较大的南瓜自交系对合成的43对引物进行扩增效果评价,其中28对引物有稳定清晰的扩增产物,占合成引物的65.12%。利用这些有效扩增引物对南瓜属作物商品杂交种进行纯度检测,共获得13个杂交组合的特异性检测引物,F1杂交种的纯度在79.2%～100.0%,与田间检测结果相符。结果表明,利用南瓜EST序列开发SSR引物具有简单快速、成本低和适用性好的特点。Expressed Sequence Tags（ESTs） are a source of simple sequence repeats（SSRs） that can be used to develop molecular markers for genetic studies.The object of the present study is to develop EST-SSR markers from Cucurbita and uses these markers in quickly purity testing of F1 hybrid seed.The availability of 1457 ESTs for cucurbita,which documented in GenBank,provided a unique opportunity to develop EST-SSR markers,and 215 SSRs were identified among these non-redundant EST sequences.These SSRs contained 2-6 bp nucleotide motifs including 111 different motif types.The trinucleotide repeat is the dominant type,accounting for 40% with 86 motifs,and the frequency of occurrence of dinucleotide repeat is 28.84% with 62 motifs.Among these motifs,CT motif（21,9.77%） was the most common type,and then were TC and AAG motifs,accounting for 6.05% and 5.58%,respectively.A total of 215 primer pairs were designed and 43 of them were synthesized and used to determine their usability by amplification in 4 Cucurbita inbred lines.The results show that 28 of them could successfully amplify,accounting for 65.12% of testing primer pairs.Then these primer pairs were used to test the F1 hybrid seed purity in Cucurbita.Seed genetic purity from 13 combinations ranged from 79.2% to 100.0%,which were in high accordance with those from field grow-out trials.Taken together,this study reported an effective and feasible approach to develop SSR markers for cucurbita,and demonstrated their usefulness in purity testing of hybrid squash seed.

关 键 词：南瓜 EST-SSR 标记开发 杂种纯度 

分 类 号：S642.1[农业科学—蔬菜学]