《环境昆虫学报》

作者	单位
杨琼，孙娜，郑敏，罗除成，罗光华，方继朝	1.江苏省农业科学院植物保护研究所，南京210014；2.南京农业大学植物保护学院，南京210095

摘要点击次数: 1077

全文下载次数: 1175

中文摘要:

本文介绍了构建水稻二化螟和三化螟“双酶切限制性酶切位点关联DNA测序”（Double digest restriction-site associated DNA sequencing，ddRADseq）文库的方法。利用安捷伦2100生物分析仪对4种单酶切及2种双酶切的酶切产物片段大小及分布范围进行分析，筛选出MluCI和NlaIII两种限制性内切酶组合对螟虫基因组DNA进行酶切。酶切后的DNA片段两端连接上特定的P1、P2接头后，用Pippin Prep回收大小为285-435 bp的DNA片段。通过PCR扩增进行文库的富集并引入index序列。构建好的ddRADseq文库用琼脂糖凝胶电泳和生物分析仪进行质量检测。本方法所构建的文库DNA片段长度、分布和摩尔浓度能够达到Illumina平台测序的技术要求。本研究证实了利用 MluCI和NlaIII组合酶切构建水稻螟虫基因组ddRADseq文库的可行性，为在水稻螟虫中利用ddRADseq技术开展生物地理学、种群遗传学和系统发育重建等方面的研究奠定基础。

英文摘要:

Chilo suppressalis and Scirpophaga incertulas(Lepidoptera: Pyralidae). The fragment size distribution generated by a given pair of restriction endonucleases was estimated using Agilent 2100 Bioanalyzer Based on Bioanalyzer analysis, we chose two frequent cutter enzymes ( MluC I and Nla III) for genomic DNA digestion. Each individual of stem borers was labelled with a unique combination of P1 and P2 barcodes Size selection of adapter ligated DNA fragments between 285 - 435 bp was performed using a Pippin Prep Finally, size selected DNA was used as a template in PCR amplification. PCR primers will add a unique index sequence to all DNA fragments in the PCR reaction Evaluation of the final libraries was performed using both routine agarose gel electrophoresis and Bioanalyzer. Both assessments showed that the molarity and fragment size distribution were appropriate for Illumina sequencing. This method confirms the feasibility of using a combination of restriction enzymes (MluC I and Nla III) to construct the genomic ddRADseq libraries for the stem borers, which lays an important foundation for investigation into biogeography, population genetics and phylogenetics on rice stem borers.

查看全文查看/发表评论下载PDF阅读器

关闭