title: A novel manual pooling system for preparing three-dimensional pools of a deep coverage soybean bacterial artificial chromosome library.
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 × 10(9)  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.
authors.affiliation: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8510, Japan Soybean Research Institute, Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University Harbin, Harbin 150030, China Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Xia
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.forename: Qingshan
authors.initials: Q
authors.lastname: Chen
authors.forename: Shusei
authors.initials: S
authors.lastname: Sato
authors.forename: Kyuya
authors.initials: K
authors.lastname: Harada