caa a22 4500 475796675 CHVBK 20180406123726.0 cr unu---uuuuu 170329e20000101xx s 000 0 eng 10.1007/s001220050043 doi (NATIONALLICENCE)springer-10.1007/s001220050043 The applicability of consensus PCR primers across species and genera: the use of wheat Em sequences to develop markers for orthologues in rye [Elektronische Daten] [S. Van Campenhout, R. M. D. Koebner, G. Volckaert] Abstract : Two wheat consensus primer sets, directed to ”early-methionine-labelled” (Em) gene sequences, were tested for their ability to amplify beyond their original source. A range of widely diverse templates, including other Triticeae species and sample monocot and dicot species, was assayed. Primer set EMC5/EMC3, amplifying the entire coding region with its intron and part of the 3' untranslated region, targets Triticeae and sorghum Em sequences. The other set, EMC5/EMCO31, directed to the coding region and its intron, amplifies templates from all the grass species. Both primer sets fail to amplify Em sequences from more distant monocots and the dicots. Using set EMC5/EMC3, we isolated and sequenced ten members of the rye Em gene family from five different rye sources. Significant DNA sequence variation between wheat and rye sequences in the non-coding regions was found, and this was used to develop seven sequence-specific primers. Twelve primer combinations were analysed, 7 of which were Em-R1-specific, amplifying a product in at least one of the tested rye or rye-carrying genotypes but not in wheat. Four sets exhibited clear amplification length polymorphisms which allowed discrimination between and within the rye sources. The primers also discriminated between wheat-rye recombinants with proximal 1RL rye chromatin and those carrying distal 1RL rye chromatin. These results show that wheat consensus primer sets can be used to isolate orthologous sequences, especially from species that are used for alien gene transfer in wheat. Subsequently, species-specific assays can be designed that are useful tools for this application. Springer-Verlag Berlin Heidelberg, 2000 Key words Early-methionine-labelled protein multigene family nationallicence Heterologous PCR primers nationallicence Grasses nationallicence Sequence-tagged-sites nationallicence Wheat-rye recombinants nationallicence Van Campenhout S. Katholieke Universiteit Leuven, Laboratory of Gene Technology, K. Mercierlaan 92, B-3001 Leuven, Belgium Fax. +32-16-321965 e-mail: steven.campenhout@agr.kuleuven.ac.be, BE aut Koebner R. M. D. John Innes Centre, Norwich Research Park, Colney NR4 7UH, UK, GB aut Volckaert G. Katholieke Universiteit Leuven, Laboratory of Gene Technology, K. Mercierlaan 92, B-3001 Leuven, Belgium Fax. +32-16-321965 e-mail: steven.campenhout@agr.kuleuven.ac.be, BE aut https://doi.org/10.1007/s001220050043 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s001220050043 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Van Campenhout S. Katholieke Universiteit Leuven, Laboratory of Gene Technology, K. Mercierlaan 92, B-3001 Leuven, Belgium Fax. +32-16-321965 e-mail: steven.campenhout@agr.kuleuven.ac.be, BE aut NATIONALLICENCE 700 1- Koebner R. M. D. John Innes Centre, Norwich Research Park, Colney NR4 7UH, UK, GB aut NATIONALLICENCE 700 1- Volckaert G. Katholieke Universiteit Leuven, Laboratory of Gene Technology, K. Mercierlaan 92, B-3001 Leuven, Belgium Fax. +32-16-321965 e-mail: steven.campenhout@agr.kuleuven.ac.be, BE aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer