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。
关键词:
南瓜,
EST-SSR,
标记开发,
杂种纯度
Abstract: 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 Cucurbitaand uses these markers in quickly purity testing of F1hybrid 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， a nd 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 Cucurbitainbred 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 F1hybrid 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．
Key words:
Cucurbita,
EST-SSR,
Marker development,
Hybrid seed purity
中图分类号:
张国裕, 张帆, 姜立纲, 翟伟卜, 李海真. 南瓜属EST-SSR标记的开发及在杂种纯度鉴定中的应用[J]. 华北农学报, 2011, 26(6): 97-101. doi: 10.7668/hbnxb.2011.06.019.
ZHANG Guo-yu, ZHANG Fan, JIANG Li-gang, ZHAI Wei-bo, LI Hai-zhen. EST-SSR Markers Development from Cucurbitaand Their Use in Purity Testing of F1Hybrid Seed[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(6): 97-101. doi: 10.7668/hbnxb.2011.06.019.