*2.1. Transposon Tagging Mutagenesis*

In order to isolate new lpa1 mutants, a transposon-mediated mutagenesis experiment was performed. The mutagenized population was generated by crossing the lpa1-1 mutant with a line carrying the *Ac* transposon (Figure 1). We chose an *Ac* line in which the transposon was inserted on the short arm of chromosome 1 in the 1.03/1.04 bin region, where *ZmMRP4* gene maps (Figure 1a).

We used *lpa1-1* homozygous plants as female and the *Ac* line as pollen donor. The mutagenized population, consisting of 4787 F1 seeds, was screened to find new lpa1 mutants.

**Figure 1.** (**a**) Diagram of chromosome 1, the position of the *Ac* element and of *ZmMRP* locus, bin 1.02/1.03 are indicated. (**b**) Genetic scheme used to generate the F1 mutagenized population.

### *2.2. Density Assay Screening of the Mutagenized Population*

As previously reported, the *lpa1* mutation showed the lowest phytic acid content in the seed in comparison to the *lpa* mutations thus far characterized [16,17]. This mutation does not modify the total amount of seed P, but reduces phytic acid content, leading to a proportionally increased level of free phosphate [18]. The screening methods to identify *lpa1* mutations were thus far based on the identification of HIP (high inorganic phosphate) phenotypes by the quantification of the phosphate level in the seeds with disruptive methods [28].

In order to isolate new lpa1 mutants inside the mutagenized populations, we developed a non-disrupting, fast, simple and cheap method. In particular, this assay was able to identify the mutants' seeds because in an highly concentrated sugar solution (1.218–1.222 g/L) the lpa1 seeds, due to their lower density [35], can float, unlike the wild controls that sink and stay on the bottom of the beaker (Figure 2a). Among the 4787 F1 seeds screened with this test, 271 were identified as low density seeds (Table 1).



Out of these 271 seeds, 50 were put aside and stored for further analyses, 41 were tested for the HIP phenotype by the Chen method [28] and the remaining 180 were sown in the experimental field (Figure 2b).

**Figure 2.** (**a**) Density assay used to isolate new lpa1 mutants: in the sucrose solution lpa1 mutant seeds float and wild type seeds stay on the bottom of the beaker. (**b**) Scheme of the experimental plan used to generate and to screen the F1 mutagenized population.

### *2.3. Confirmation of the HIP Phenotype of the Low Density Seeds*

The density assay we developed was very rapid, cheap and easy, but it also revealed a limitation, i.e., since this screening is based only on the low density of the seeds to be selected, it did not allow the exclusive identification of lpa1 seeds, but it selected all seeds characterized by low density, including seeds affected by any kind of mutations impairing endosperm/embryo development and moldy seeds present inside the mutagenized population. For this reason, 41 out of the 271 seeds selected by the density assay were chosen and tested for the phosphate content (HIP phenotype) in order to verify that the low density character was associated with the lpa1 phenotype (Figure 2b). The determination of the free phosphorus content inside the seeds was carried out using a semi-quantitative colorimetric method based on the Chen reagen<sup>t</sup> [28]. Based on the availability of the free phosphorus inside the seeds, it was possible to classify the seeds into four categories: WT (wild type—Free P < 0.3 mg/g), W (weak—0.3 mg/g < Free P < 0.5 mg/g), I (intermediate—0.5 mg/g < Free P < 1.4 mg/g) and S (strong—Free P > 1.4 mg/g) (Figure 4a). This assay allowed us to confirm the HIP phenotype for 20 out the 41 seeds selected with the density assay, with six seeds belonging to the Strong and 14 to the Intermediate categories (Figure 2b).

### *2.4. Molecular Analysis of the F1 Plants of the Mutagenized Population*

The lpa1-1 mutant was used as female parent in the initial cross we made to generate the mutagenized population. To identify and discard the contaminant seeds produced by the accidental self-fertilization of the lpa1-1 female parent, the 27 F1 plants, obtained from 180 F1 seeds sown in the open field (Figure 1), were genotyped by PCR analysis. The coding sequence of the *lpa1-1* allele is characterized, in comparison with the wild type allele, by the presence of a Single Nucleotide Polymorphism (SNP), C to T, in position 5759 with respect to the ATG on the genomic sequence. This allowed the design of two different forward primers, one specific for the wild type (ZmMRP430L) and the other specific for the *lpa1.1* allele (ZmMRP432L) [29,36]. We used the two specific forwards primers in combination with a reverse common primer (ZmMRP410R). Out of the 27 plants analyzed, 26 resulted in heterozygous *Lpa1*/*lpa1-1*; one plant was found to be a contaminant *lpa1-1*/*lpa1-1* and discarded (data not shown).

### *2.5. Screening and Selection of the Putative New lpa1 Mutant*

Among the 26 F1 heterozygous plants (*Lpa1*/*lpa1-1*), we selected the 19 more vigorous that were self-fertilized (Figure 3). The four best F2 ears were tested for the HIP phenotype: 24 seeds were collected from each ear and the disruptive assay for free phosphate was performed. All the seeds tested were found to belong to the Strong or Intermediate categories, indicating an HIP phenotype for all the four selected ears and 50 seeds from each ear were sown in the field.

**Figure 3.** Scheme of the procedure used to obtain the NIL (Near Isogenic Line) lpa1-5525, homozygous for the new *lpa1* mutation.

Among the 90 plants that survived we selected the 30 more vigorous that were genotyped with the primers specific for *Lpa*/*lpa1* alleles, to identify the *Lpa1*/*Lpa1* F2 plants, i.e., the putative new lpa1 mutants, provisionally indicated as lpa1\*.

The six F2 plants *lpa1\**/*lpa1\** were selfed and F3 seeds were tested for the HIP phenotype. The best F3 ears were selected (R4638, R4639, R4641) and generated the following six F4 families: R4889, R4890, R4891, R4892, R4893 and R4894. The F4 seeds were tested for HIP phenotype (Table 2) and the F4 ear R4893, showing high amount of S-I seeds and the best agronomic performance, was selected, sown in the field and the F4 plants were selfed. The F5 seeds (R5525) representing the lpa1\* mutant were further analyzed.


**Table 2.** Chen's assay performed on F5 progenies. The S+I/total ratio is reported. S: Strong; I: Intermediate; W: Weak; WT: Wild Type.
