Neutral molecular markers support common origin of aluminium tolerance in three congeneric grass species growing in acidic soils

Research Article Neutral molecular markers support common origin of aluminium tolerance in three congeneric grass species growing in acidic soils Roberto Contreras1,4, Ana M. Figueiras1, F. Javier Gallego1, Elena Benavente2, Antonio J. Manzaneda3 and César Benito1* Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain 3 Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Paraje Las Lagunillas s⁄n, 23071 Jaén, Spain 1 2 Present address: Universidad de Atacama, CRIDESAT, Copayapu 485, Copiapó, Chile 4 Received: 23 May 2017 Editorial decision: 18 October 2017 Accepted: 27 October 2017 Published: 7 November 2017 Associate Editor: F. Xavier Picó Citation: Contreras R, Figueiras AM, Gallego FJ, Benavente E, Manzaneda AJ, Benito C. 2017. Neutral molecular markers support common origin of aluminium tolerance in three congeneric grass species growing in acidic soils. AoB PLANTS 9: plx060; doi: 10.1093/aobpla/plx060 Abstract. Aluminium (Al) toxicity is the main abiotic stress limiting plant productivity in acidic soils that are widely distributed among arable lands. Plant species differ in the level of Al resistance showing intraspecific and interspecific variation in many crop species. However, the origin of Al-tolerance is not well known. Three annual species, difficult to distinguish phenotypically and that were until recently misinterpreted as a single complex species under Brachypodium distachyon, have been recently separated into three distinct species: the diploids B. distachyon (2n = 10) and B. stacei (2n = 20), and B. hybridum (2n = 30), the allotetraploid derived from the two diploid species. The aims of this work were to know the origin of Al-tolerance in acidic soil conditions within these three Brachypodium species and to develop new DNA markers for species discrimination. Two multiplex SSR-PCRs allowed to genotype a group of 94 accessions for 17 pentanucleotide microsatellite (SSRs) loci. The variability for 139 inter-microsatellite (ISSRs) markers was also examined. The genetic relationships obtained using those neutral molecular markers (SSRs and ISSRs) support that all Al-tolerant allotetraploid accessions of B. hybridum have a common origin that is related with both geographic location and acidic soils. The possibility that the adaptation to acidic soils caused the isolation of the tolerant B. hybridum populations from the others is discussed. We finally describe a new, easy, DNA barcoding method based in the upstream-intron 1 region of the ALMT1 gene, a tool that is 100 % effective to distinguish among these three Brachypodium species. Keywords: Acidic soils; aluminium; Brachypodium distachyon; Brachypodium hybridum; tolerance. Introduction Aluminium (Al) is the most abundant metal in the crust of the Earth. This metal is toxic on acidic soils and severely limits plant growth. Under acidic environment (pH < 5) the rhizotoxic Al3+ is solubilized and the root growth is inhibited (Kochian et al. 2004, 2005). Approximately 30 *Corresponding author’s e-mail address: © The Author(s) 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. AoB PLANTS https://academic.oup.com/aobpla © The Authors 2017 1 Contreras et al. – Common origin of Al-tolerant Brachypodium hybridum % of the Earth’s total land area consists of highly acidic soils, and as much as 50 % of the world’s potentially arable lands are acidic (von Uexküll and Mutert 1995). The tropic and subtropics are important food-producing regions. These regions comprise large areas of acidic soils that are also very frequent in the Iberian Peninsula. The main abiotic stress limiting crop production is the drought and the second is Al toxicity both constituting food security threats (von Uexküll and Mutert 1995). The study of complex stress tolerance traits like Al-tolerance is difficult in important agronomical species like wheat, barley and rye, which have large genomes, abundant repetitive sequences and some, as wheat, are polyploids. Previous studies, using candidate Al-tolerance genes, have suggested that several tolerant varieties of wheat and barley were originated from acidic soils (Ryan et al. 2010; Ryan and Delhaize 2010; Delhaize et al. 2012a; Fujii et al. 2012; Garcia-Oliveira et al. 2013, 2014). Al-tolerance is common in species endemic to regions with acidic soils where the ability to cope with Al3+ stress is a prerequisite for survival. Examples include tea (Camellia sinensis), buckwheat (Fagopyrum esculentum), Melostoma and Hydrangea sp., all of which accumulate high concentrations of Al in their leaves (Ma et al. 2001; Ryan et al. 2010). However, there are no genetic studies with neutral molecular markers indicating a common origin for Al-tolerance in acidic soils. The origins of Al resistance in wheat (Triticum aestivum) are difficult to explain because all the diploid progenitors of this hexaploid species are reportedly sensitive to Al stress. The expression of an anion channel (TaALMT1) that releases malate anions from the root apices controls the genotypic variation for Al resistance in wheat (Ryan et al. 2010). This gene (TaALMT1) is not a neutral marker. This trait in wheat has multiple independent origins that enhance Al resistance by increasing TaALMT1 expression and is an example of evolutionary adaptation to a major abiotic stress (Ryan et al. 2010). Brachypodium distachyon is a diploid annual small grass with several attributes suitable for being an excellent genetic model organism for the Poaceae. For example, its morphological characteristics, like a small stature, and simple growth conditions, allow to grow large amounts of plants in a small space in growth chambers and, also, to obtain several generations in a year, with a minimum of 6 weeks from seed to seed (Garvin et al. 2008; Mur et al. 2011). In addition, as B. distachyon is self-fertile (autogamous) plant, it is easy and quick to obtain uniform pure inbred lines composed by plants with identical genotype within two generations (Vogel et al. 2009). Moreover, its small (1C = 0.3 pg of DNA), recently sequenced, genome contains mostly single or low-copy repetitive DNA (Wolny and Hasterok 2009; 2 AoB PLANTS https://academic.oup.com/aobpla The International Brachypodium Initiative [IBI] 2010). On the other hand, it is feasible to obtain transgenic plants in B. distachyon due to the existence of efficient Agrobacterium-mediated transformation protocols (Vogel et al. 2009). Finally, t (...truncated)