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Improved AFLP protocol using dual‐suppression PCR and its application to species with large genomes
Author(s) -
GUAN LANHUA,
SHIRAISHI SUSUMU
Publication year - 2011
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/j.1755-0998.2011.03029.x
Subject(s) - biology , amplified fragment length polymorphism , genome , dual (grammatical number) , computational biology , genetics , gene , genetic diversity , population , demography , sociology , art , literature
Abstract To improve the amplified fragment length polymorphism assay, dual‐suppression PCR was introduced into the preamplification step of the assay. The dual‐suppression PCR blocked completely the amplification of fragments with the same sequence ( Bsp 1407I‐ Bsp 1407I or Nla III‐ Nla III) at both ends and amplified selectively fragments with different adaptor sequences ( Bsp 1407I‐ Nla III) at each end. Two protocols, referred to as A and B, were established for species with medium‐ and large‐sized genomes, respectively. Both protocols incorporated the dual‐suppression PCR. Protocol A resulted in high‐quality electrophoretic profiles for black cottonwood and rice, which have medium‐sized genomes. In protocol B, an intensely selective PCR step was added to protocol A. Protocol B yielded profiles for Japanese black pine and Japanese cedar that were improved significantly relative to protocol A: the number of strong peaks increased and that of low peaks decreased. Japanese black pine and Japanese cedar have large genomes. The optimal profiles were generated with a total of eight or nine selective nucleotides.