CROP SCIENCE, VOL. 54, MAY– JUNE 2014 WWW.CROPS.ORG 1133 NOTE T he transfer of a transgenic trait from a lab genotype into a cultivated plant variety is accomplished in several steps. First, single-locus T 0 plants are selected to avoid complications from multilocus transgene segregation; then a backcross procedure is performed that requires crossing the transgenic trait donor with tens of di ferent plants of the variety of interest at every backcross cycle, followed by screening a large progeny for transgene pres- ence and zygosity (Micallef et al., 1995; Samac and Temple 2004). Polyploidy and outcrossing add signifcant complications to this process, as in the case of alfalfa (Medicago sativa L., 2n = 4x = 32), the most important forage legume worldwide. One of the factors that can limit the use of genetic engineering in these species is the amount of work required to introduce a transgenic trait from a lab genotype into a cultivated variety. In tetraploid tetrasomic plants, a single-locus transgenic event will have a simplex (T---, where T is the transgene construct) condition at the transgene locus. When crossed with a non-trans- genic plant, 50% of the progenies will be T--- and 50% will be non-transgenic. For stability of the introduced trait, the triplex (TTT-) or the quadruplex (homozygous, TTTT) conditions are Copy Number Estimation of a Plant-Derived Selectable Marker Gene by High Resolution Melting Analysis: A Tool to Simplify Transgenic Plant Breeding Sara G. Milner, Nicoletta Ferradini, Alessandro Nicolia, Fabio Veronesi, Silvio Salvi, and Daniele Rosellini* ABSTRACT Transferring a transgenic trait from a lab gen- otype into a cultivated plant variety requires selection of single-locus T 0 plants, a backcross- ing breeding program with transgenic progeny selection at each cycle and, generally, attaining the homozygous state for the transgene locus. Polyploidy and outcrossing complicate this pro- cess, as in the case of alfalfa, an autotetraploid, cross-fertilizing forage legume. Recently, a new, ef fcient selectable marker gene, MsGSAgr, derived from the alfalfa glutamate 1-semial- dehyde aminotransferase (GSA) gene was described. Since MsGSAgr differs from the wild- type GSA by one nucleotide, high resolution melting (HRM) analysis could be used to screen transgenic plants for MsGSAgr , and linked transgene(s), copy number. An HRM assay was developed by simulating different copy num- bers with mixes of plasmids containing mutated and wild-type GSA sequences. The assay was validated by analyzing transgenic alfalfa plants containing one or multiple MsGSAgr loci. HRM enabled us to clearly discriminate transgenic from non-transgenic plants, and the single copy from the multicopy state, thus providing a tool to streamline molecular plant breeding. S.G. Milner, and S. Salvi, Dipartimento di Scienze Agrarie, viale Fanin, 44- 40127 Bologna, Italy; and N. Ferradini, A. Nicolia, F. Veronesi, and D. Rosellini, Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo XX giugno 74, 06121 Perugia, Italy. Received 24 Sep. 2013. *Corresponding author (daniele.rosellini@unipg.it). Abbreviations: GSA, Glutamate 1-Semialdehyde Aminotransferase; HRM, High Resolution Melting; PCR, Polymerase Chain Reaction, SMG, Selectable Marker Gene, SNP, single nucleotide polymorphism, and wt, wild type. Published in Crop Sci. 54:1133–1138 (2014). doi: 10.2135/cropsci2013.09.0631 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. Published March 21, 2014