Identification and isolation of Mu-flanking fragments from maize

Yijun Wang; Guangming Yin; Qin Yang; Jihua Tang; Xiaomin Lu; Schuyler S. Korban; Mingliang Xu

doi:10.1016/S1673-8527(08)60029-6

Volume 35 Issue 4

Apr. 2008

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Article Navigation > Journal of Genetics and Genomics > 2008 > 35(4): 207-213

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Identification and isolation of Mu-flanking fragments from maize

doi: 10.1016/S1673-8527(08)60029-6

a
Agricultural College, Yangzhou University, Yangzhou 225009, China
b
National Maize Improvement Center of China, China Agricultural University, Beijing 100094, China
c
Corn Research Institute, Henan Agricultural University, Zhengzhou 450002, China
d
Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana IL61801, USA

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Corresponding author: E-mail address: mxu@cau.edu.cn (Mingliang Xu)
Received Date: 2007-12-11
Accepted Date: 2008-01-08
Rev Recd Date: 2008-01-05

Available Online: 2008-04-24

Publish Date: 2008-04-20

Abstract

Abstract

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertionMutator (Mu) is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2) enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Top1. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.
- Mutator transposon,
- flanking fragment,
- AFLP,
- maize

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References(28)

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