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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (6): 148-154.

所属专题： 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

SCoT结合克隆测序鉴别湖南甜橙变异类型

蒋巧巧 龙桂友 李武文 邓子牛   

  • 收稿日期:2010-10-13 修回日期:2010-11-24 出版日期:2011-03-20 发布日期:2011-03-20
  • 基金资助:

    优质甜橙新品种选育

Identification of Genetic Variation of Sweet Orange Clones from Hunan by Using SCoT Markers and Sequence Analysis

  • Received:2010-10-13 Revised:2010-11-24 Online:2011-03-20 Published:2011-03-20

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摘要：

本试验对SCoT反应体系进行优化，并筛选合适的引物，然后对24份试材进行SCoT标记，获得的目的片段克隆测序，通过测序结果探讨其遗传变异；结果表明，甜橙SCoT标记的20 μL优化反应体系为：DNA模板80 ng，Mg2+浓度1.6 mmol/L，dNTPs浓度0.3 mmol/L，引物浓度0.2 μmol/L，Taq DNA聚合酶用量1.6 U，扩增产物在100~2000 bp之间，扩增条带明亮清晰，反应体系具有良好的稳定性和可重复性。24份试材的测序片段的大小为1090~1091 bp，一致性达到99.84%，存在单碱基的缺失与替换，BLAST结果显示，该序列的编码蛋白质与核糖体蛋白S3的N端同源性高；利用单碱基变异可以区分其中的12个甜橙变异株系和‘安江香柚’，其他变异株系还需要结合其它的分子标记技术来进行鉴别，有待进一步研究。

关键词: 园林植物, 园林植物, 生理指标, 耐热性

Abstract:

The primers for SCoT marker analysis were designed based on the short conserved region surrounding the ATG translation start codon. However, the SCoT technique was not found yet to be applied in citrus. In the present research, we collected 24 sweet orange clones from Hunan Province and analyzed by using SCoT marker to investigate their genetic variation. The amplified SCoT fragments were sequenced. The results showed that an optimal 20 μL reaction system of SCoT included 2.0 μL 10xPCR buffer, 1.6 U Taq DNA polymerase, 80 ng template DNA, 0.3 mmol/L dNTPs, 0.2 μmol/L primer and 1.6 mmol/L Mg2+. Application of this reaction system to fingerprint the sweet orange clones produced clear bands with good repeatability and reproducibility. The PCR products were of 100 to 1091 bp in size. After being sequenced, the polymorphisms could distinguish 12 sweet orange clones and pummelo. The BLAST result showed that part of the SCoT fragments coding region had high homology to ribosomal protein S3 N superfamily. The other sweet orange clones should be studied further.