*4.5. lpa1 Locus Molecular Genotyping*

A molecular analysis was performed using *ZmMRP4* sequence-specific amplification polymorphism (S-SAP) markers that allowed the identification of the *lpa1-1* versus *Lpa1* allele. The allele-specific forward primers were designed on the single nucleotide substitution polymorphism in the *ZmMRP4* 10th exon [29,36]. The *Lpa1* wild type–specific forward primer was ZmMRP430L (5'-GTACTCGATGAGGCGACAGC-3'), whereas *lpa1-1* mutation-specific forward primer was ZmMRP432L (5'-GTACTCGATGAGGCGACAGTG-3'). The reverse primer ZmMRP410R (5'-CCTCTCTATATACAGCTCGAC-3') was used to amplify both wild type and *lpa1-1* alleles.

The reaction mixture of the *Lpa1*/*lpa1-1* allele-specific amplifications contained an aliquot of genomic DNA, 1 × Green Go Taq buffer (Promega, Madison, WI), 2.5 μM MgCl2, 0.2 μM each of dATP, dCTP, dGTP, and dTTP, 0.3 μM of forward ZmMRP430L/ZmMRP432L-specific primer, 0.3 μM of reverse ZmMRP410R primer and 1.25 unit of Go Taq Flexy DNA polymerase (Promega), in a final volume of 25 μL.

The reaction mix underwent an initial denaturation step at 94 ◦C for 2 min, 37 cycles of denaturation at 94 ◦C for 45 s, annealing at 62 ◦C for 1 min, extension at 72 ◦C for 1.5 min. Extension at 72 ◦C for 5 min was performed to complete the reaction. The *Lpa1* and *lpa1-1* amplicons were 468 bp long. The amplicons were loaded on 1% (*w*/*v*) agarose gels and visualized by ethidium bromide staining under ultraviolet light.

### *4.6. Structural Analysis of ZmMRP4 Locus in Putative New lpa 1 Mutant*

*ZmMRP4* locus was amplified in wild type and in putative new lpa1 mutant using an high fidelity long range DNA polymerase (Platinum Super Fi DNA Polymerase, Invitrogen) with the primers 5L (5'-TGGTGAGGGGATCAGAGACG-3') (forward primer, position -313) and ZM 2R (5'-GAACTTCCAAAGGCAAGGGACA-3') (reverse primer, position +3413), ZM2F (5'-GGAAAAGTGAGCTCCAAAGTTTA-3') (forward primer, position +3218) and 51R (5'-AAGCATCAGCTTCGGGTAATGT-3') (reverse primer, position +6460). The primers positions are referred to the ATG on the genomic sequence.

The amplicons obtained were run on 1% agarose gel and visualized by ethidium bromide staining under UV light.

**Author Contributions:** Conceptualization, R.P.; methodology, R.P. and M.L.; data curation, G.B., C.R. and E.C.; writing—original draft preparation, G.B. and C.R.; writing—review and editing, R.P. and M.L.; funding acquisition, R.P.

**Funding:** This research was funded by Regione Lombardia, BIOGESTECA project, gran<sup>t</sup> number 15083/RCC.

**Acknowledgments:** We thank V. Raboy (USDA ARS, Aberdeen, ID, USA) and T.P. Brutnell (Shandong Agricultural University, China) for the generous gifts of lpa1-1 and *Ac* line seeds, and Davide Reginelli for his hard work in the field.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
