A novel manual pooling system for preparing three-dimensional pools of a deep coverage soybean bacterial artificial chromosome library

Zhengjun Xia, Satoshi Watanabe, Qingshan Chen, Shusei Sato, Kyuya Harada

Research output: Contribution to journalArticle

Abstract

Compared with hybridization-based techniques, polymerase chain reaction-based screening of large insert libraries has been used widely as it is fast, easy and sensitive. However, various pooling strategies are needed to ensure efficient screening. It is time-consuming and labourious to prepare three-dimensional pools for a deep coverage bacterial artificial chromosome (BAC) library of soybean (1.12 × 109 bp) in the absence of robotic facility. In the present study, we describe a novel manual pooling system for preparing three-dimensional pools of a soybean BAC library. This simple technique enables a single researcher to construct three-dimensional pools for a deep-coverage (12 haploid genome equivalents) BAC library of soybean in less than 2 months without any robotic manipulation. When the prepared three-dimensional pools were screened with 29 polymerase chain reaction-based markers, an average of 9.2 clones per marker were identified. These identified clones will be useful either in quantitative trait loci gene isolation or in synteny study between soybean and other legumes including Lotus japonicus. This efficient pooling system could be applied to any other BAC libraries without the need for robotic manipulation.

Original languageEnglish
Pages (from-to)516-524
Number of pages9
JournalMolecular Ecology Resources
Volume9
Issue number2
DOIs
Publication statusPublished - 2009 Mar 1
Externally publishedYes

Keywords

  • BAC library
  • Positional cloning
  • Soybean
  • Synteny
  • Three dimensional pooling

ASJC Scopus subject areas

  • Biotechnology
  • Ecology, Evolution, Behavior and Systematics
  • Genetics

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