Note that I is the same image as Fig.?6A. pathways are distinguished, however, by the cell layer in which they operate C mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature. mutants have subtle alterations in vascular, BS and M development (Slewinski et al., 2012). In (hereafter referred to as orthologue radially patterns cell-types in the root (Di Laurenzio et al., 1996; Wysocka-Diller et al., 2000); AtSCR prevents movement of AtSHORTROOT (AtSHR) beyond the cell layer adjacent to the vasculature, which ensures specification of endodermal cells in that layer (Cui et al., 2007). However, an organized GDC-0834 endodermal cell layer is present in mutants (Slewinski et al., 2012), suggesting that gene function may have diverged between maize and mutants precludes an understanding of the precise role played during Kranz development. GDC-0834 Both gene and whole-genome duplication events are highly prevalent throughout the plant phylogeny (Adams and Wendel, 2005; Blanc and Wolfe, 2004) and if retained in the genome, duplicated genes are free to sub- or neo-functionalize (Moore and Purugganan, 2005; Ohno, 1970). Perhaps more commonly, however, gene duplicates function redundantly. Indeed, there are many examples illustrating the importance of genetic redundancy in plants, and without understanding phylogenetic context, loss-of-function data can be difficult to interpret (Strable et al., 2017; Yi et al., 2015). This is particularly important in maize, which, in addition to undergoing three ancient whole-genome duplication events common to monocots, has also undergone a more recent event not shared with its close relative (Messing et al., 2004; Schnable et al., 2009; Swigonova et al., 2004). It is thus likely that acts redundantly with a duplicate gene to pattern cell types in maize. To better understand the role of ZmSCR1 in maize development, we first constructed a phylogeny of has a previously overlooked homeologue duplicate double mutants, with endodermal defects observed in the root. Intriguingly, however, M rather than BS cell development was primarily perturbed in the leaf. We present a quantitative analysis of single and double mutant leaf phenotypes, plus expression data for both genes in developing wild-type maize leaf primordia. The results are discussed in the context of how SCR function has diversified in flowering plants. RESULTS is duplicated in maize To determine phylogenetic relationships between genes are present in both eudicots and monocots, with the underlying duplication event inferred after the divergence of and vascular plants. In clade contains a single gene (C In contrast, has independently duplicated in at least four monocot genomes (maize, and is likely an annotation error. The maize duplicates reside on syntenic regions of chromosomes 4 (orthologues. (A) Maximum likelihood phylogeny of SCR genes. Bootstrap values are indicated below branches. Light-blue shading indicates the clade, light-orange shading indicates the clade. sequences were included as an outgroup. (B) Cartoon depiction of transposon insertions in and and cause loss of function To test the hypothesis of functional NSD2 redundancy, we first identified transposon insertion alleles for each gene. Two alleles (and alleles (and and (insertions in the genes of interest are documented for the and lines, whereas the line contains four additional elements inserted at other loci (Fig.?S1A). Insertion positions were confirmed by PCR amplification of genomic DNA, using primers in the transposon and in the adjacent genic region (Fig.?S1B-D). In all cases, the size of the amplified product was consistent with the predicted insertion site. Primers flanking the element enabled homozygous mutant individuals to be identified (Fig.?S1B-D). To confirm that the transposon insertion alleles compromised gene function, transcripts were amplified and sequenced, using RNA GDC-0834 extracted from homozygous mutant leaf primordia as a starting template. Reverse transcriptase (RT)-PCR revealed that in all cases, the element was present in the or transcript, at the position predicted by the insertion site (Fig.?S1E). As such, even if transcripts were translated, a non-functional protein would be produced. Loss-of-function mutants do not exhibit cell-type patterning defects To determine whether mutants display similar defects in Kranz patterning to those reported in mutants (Slewinski et al., 2012), leaf traits were compared between and single mutants, and corresponding wild-type siblings segregating in each line. There was no qualitative difference between wild-type and either or single mutants in overall plant growth (Fig.?S2A-D), or in general Kranz patterning (Fig.?S2E-H). Quantification of the number of M cells between veins (Fig.?3A), vein density across the leaf (Fig.?3B), and the ratio of rank-1:rank-2 intermediate veins (Fig.?3B), failed to confirm previous reports of altered vein density and interveinal M cell number in mutants, which may reflect.