**1. Introduction**

Phytic acid (*myo*-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP6) is the most common form of phosphate present in cereal kernels as well as in seeds of most plants [1,2]. Cereals' seeds contain on average 10 mg of phytic acid, which account from 65% to 90% of the total phosphorus present inside the seed [3]. Among cereals, maize possesses a grea<sup>t</sup> importance not only in human and animal nutrition but also in the use of its derivatives in many industrial sectors. As animal feed, maize can be considered one of the most important staple foods available and, in many cases, is indispensable for the formulation of animal rations. Moreover, maize is a model plant in genetic studies aimed at understanding the role of genes involved in biosynthetic pathways and in plant morphogenesis. For those reasons, maize is considered one of the most important plants studied and used in genetic improvement programs.

Phytic acid is synthesized in the endoplasmic reticulum, and then is deposited in protein bodies organized in specific structures called globoids as a mixture of phytic salts of several cations, such as potassium, iron, zinc and magnesium [4]. In cereals' seeds, the phytates are mainly localized in the embryo (80%) and in the aleurone layer (20%) [1]. During germination, the phytate salts are broken down by phytase activity releasing free phosphate, minerals and myo-inositol, necessary for seedling growth [5]. Furthermore, phytic acid, by firmly chelating iron cations, is able to counteract the formation of reactive oxygen species and is thus involved in the preservation of viability of plant seeds [6,7]. Phytic acid is poorly digested by monogastric animals, and since it is a chelator of cations, it is considered an anti-nutritional factor. Moreover, as it is not assimilated, it is expelled with manure, becoming a pollutant of cultivated land and contributing to the eutrophication of surface waters [8]. It has been estimated that nearly 50% of elemental P used in global agricultural activities is accumulated in the phytic acid fraction. For these reasons, its reduction or elimination in the seeds is a major challenge in genetic improvement programs.

The conventional breeding protocols designed for reducing phytic acid content, rely on the isolation of low phytic acid (*lpa*) mutations impairing the biosynthesis or the storage of phytic acid in the seed; the increased P and mineral cation bioavailability in the lpa mutant seeds so far isolated was confirmed by nutritional trials [9–11]. Low phytic acid mutations in maize can be classified into three categories: type 1 are mutations altering the biosynthetic pathway (MIPS, *myo*-inositol 3-phosphate synthase, catalyzing the step from glucose 6-P to myo-inositol[3]-monophosphate); type 2 are mutations altering the following phosphorylation steps (e.g., ITPKS, inositol tris/tetra kisphosphate kinases); type 3 are mutations altering the transport of phytic acid to the vacuole (MRP, multidrug-resistance-associated protein) [12,13]. In recent years, several breeding programs aimed at selecting new maize varieties with seeds characterized by a lower level of phytic acid compared to traditional cultivars, have been carried out [14,15]. The lpa phenotype has been isolated in di fferent crops, maize [16–18], barley [19–21], wheat [22], rice [23–26] and common bean [27], by physical, chemical or transposon tagging mediated mutagenesis. In maize, there are three di fferent *lpa* mutations (*lpa1*, *2* and *3*), with the *lpa1* mutation showing the lowest phytic acid content in the seed [16,17].

Due to the strong pleiotropic e ffects associated with the *lpa* mutations, the mutants thus far isolated are generally lethal; the challenge will be to isolate new lpa mutants in which the pleiotropic e ffects will be absent or dramatically reduced, allowing the *lpa* mutation to be viable.

*lpa1* mutation does not modify the total amount of seed P but reduces phytic acid content, thus leading to a proportionally increased level of free phosphate [18]. Owing to this, an HIP (high inorganic phosphate) phenotype is diagnostic for the presence of the *lpa1* mutation, making it quite easy to identify the lpa1 phenotype. The screenings so far used to identify the lpa mutants are destructive methods based on the quantification of P in the flour obtained by milling the seeds (as, for example, the Chen method [28]). For this reason, these screenings are not performed on the M1 generation but on the M2 progeny, with the consequent increase of time required to acquire the samples and number of samples to be analyzed.

Transposon tagging mutagenesis experiments [29] demonstrated that *lpa1* gene encodes a multidrug-associated-protein (MRP) named *ZmMRP4* (accession number EF586878). MRP proteins are transmembrane transporters involved in several functions such as organic ions transport, xenobiotic detoxification, oxidative stress tolerance and transpiration control [30,31].

The transposon tagging mutagenesis consists in introducing, by crossing, a known DNA sequence in the genome of a target species. If the insertion event occurs within a gene sequence, altering its expression, a mutant phenotype can be observed [32]. Because of the possibility of the transposon moving within the genome, transposon tagging mutagenesis provides the so-called mutable alleles that cannot be directly used in genetic improvement programs but that may be useful to clone the gene responsible for the mutated phenotype and to isolate stable excision events that lead to stable genomic mutations which can be used in genetic improvement programs.

The *Ac*/*Ds*transposon system of maize is often used in transposon tagging mutagenesis experiments. In fact, the elements of this transposon family can be transferred into the genome of di fferent species, determining the generation of insertional mutants.

The *Ac*/*Ds* system is made up with two types of transposons: the autonomous element, which can transpose (the *Activator* element, *Ac*), and the non-autonomous element, which cannot transpose independently (the *Dissociator* element, *Ds*). Sequence analyses have shown that the *Ds* element is

derived by deletion from the autonomous element of the family, with the loss of the function of one or more genes required for transposition. Therefore, only the *Ac* element encodes for the transposase, the enzyme required to mobilize both the *Ac* and the non-autonomous *Ds* elements [33]. Studies on *Ac*/*Ds* transposition have revealed a strong preference for insertion in regions of the genome in close genetic linkage to the donor site. In particular, it has been reported that the majority of *Ac* transpositions were within 10cM from the donor site [34]. This characteristic short-range transposition is used in regional mutagenesis studies to create multiple alleles in a target locus close to an *Ac* donor site.

In this work we present a new non-disrupting, fast and simple method to select lpa1 mutants. We describe the development and the screening of a transposon tagging mutagenized population which enabled the isolation of a new lpa1 mutant, named lpa1-5525.
