Erhard et al. (2013). Plant Cell /tpc

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1 Supplemental Figure 1. c1-hbr allele structure. Diagram of the c1-hbr allele found in stocks segregating 1:1 for rpd1-1 and rpd1-2 homozygous mutants showing the presence of a 363 base pair (bp) Heartbreaker (Hbr) MITE element in exon 2 of the c1 coding region. Red line within the Hbr element represents a premature nonsense codon with respect to the coding frame of exon 2. Gray line below gene model represents sequence coverage of the c1-hbr allele (see Methods). 1

2 Supplemental Figure 2. Representative aleurone pigmentation phenotypes. (A) Images of aleurone pigmentation classes: colorless, lightly blotched, medium blotched, heavily blotched, and fully colored (C1-like) (B) Representative F 8 ear segregating 1:1 for rpd1-1 homozygotes and heterozygous siblings (see Figure 2A for crossing scheme) showing a typical ratio of pigmented kernel classes in advanced sib-cross generations. (C) Color-converted A619 ear showing kernels with C1-conditioned fully colored aleurone phenotypes. 2

3 Supplemental Figure 3. Aleurone pigmentation in stocks containing Pl1-CML52. (A) Pl1-CML52 / Pl1-CML52 ear segregating 1:3 for rpd1-2 mutants (BC 1 F 2 S 2 generation). (B) Pl1-CML52 / Pl1-CML52 ear segregating 1:3 for rpd1-2 mutants (BC 1 F 2 S 3 generation). 3

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5 Supplemental Figure 4. Selection of pl1-r30. (A) Diagram of T Pl1-Rhoades showing recombination intervals (I, II, III) and approximate genetic distances between wx1, T6-9, and pl1 (Hollick et al., 2005). See Methods and Supplemental Table 5 for additional details. (B) + pl1-b73 / T Pl' heterozygotes were crossed by T Pl-Rh / T Pl-Rh homozygotes. Arrow indicates selection of waxy kernels and fully fertile, ACS 7, T Pl1- Rhoades pl1-b73 / T Pl-Rh recombinant heterozygote. B73 and Rhoades indicate genotype of distal molecular markers. Schematics of chromosomes are not to physical scale. (C) PCR shows that plant is homozygous for the Pl1-Rhoades coding sequence (phi031) with a recombination event distal to the pl1 locus (bnlg2249). Fully fertile pollen indicates plants are homozygous for the T6-9 chromosome. Phenotypes and genotypes of additional non-recombinant individuals (#29, #168, #172, and #229) from family are shown. f.f. fully fertile. (D) PCR results using the polymorphic marker umc2006, distal to pl1. 5

6 Supplemental Figure 5. DNA gel blot analysis of pl1-r30. (A) Hybridization analysis with indicated restriction enzymes. Pl1-Rhoades / Pl1-Rhoades (A619) and B73 were used as controls. Two individuals (pl1-r30) with sun-red anther phenotypes and B73 / B73 umc2006 genotypes are shown. (B) Map of Pl1-Rhoades with restriction sites generating the hybridizing fragments observed in (A). Open arrow doppia fragment; black boxes pl1 exons; a probe specific to pl1 is shown as a black box beneath the sequence. Repetitive sequences are shown as black arrows; arrow directions indicate orientations of the repeats relative to one another. H HindIII, N NcoI, B BspHI, A AseI. 6

7 Supplemental Figure 6. RT-PCR analysis of pl1-r30. Negative controls are, from left to right, no reverse transcriptase in the initial RT reaction, no RNA in the RT reaction, and a blank (H 2 O) PCR reaction. 7

8 Supplemental Figure 7. Cytosine methylation at the Pl1-Rhoades doppia element. (A) Schematic of Pl1-Rhoades identifying the doppia fragment (purple) and sequenced region (open box). Black boxes represent the three exons of the pl1 coding region. (B) Dot plots representing the methylation status of cytosine residues across the region highlighted in (A) based on the sequence of individual PCR amplicons from bisulfite treated genomic DNA isolated from individual aleurones. Aleurones were sampled from lightly blotched (Lt Bh) and darkly blotched (Dk Bh) kernels (See Methods) of F7 and F9 generations (Figure 2A) and a colorless kernel from an A619 inbred containing Pl1- Rhoades (see Methods). A PspGI site previously analyzed with methylation-sensitive restriction enzymes (Hale et al., 2007) is highlighted. Circles represent consecutive cytosines in CG (red), CHG (blue) or CHH (green) contexts as either methylated (filled) or unmethylated (open). (C, D) Histograms representing the mean percentage (+/- s.e.m.) of 5 -methylcytosines displayed in (B) for the indicated categories in total (C) or identified by cytosine context (D): CG (red), CHG (blue), CHH (green). 8

9 Supplemental Tables Supplemental Table 1. c1-n co-segregation with aleurone pigment phenotype Genotype of F 1 parent c1-n / c1-hbr; + / rpd1-1 No. of No. of F 2 individuals grown from darkly pigmented ear kernels with indicated genotypes progeny c1-n/c1-n c1-hbr/c1-n c1-hbr/c1-hbr Total

10 Supplemental Table 2. Aleurone pigmentation phenotypes of ear progeny resulting from outcrossing individuals grown from pigmented kernels Filial Female No. of F 1 F 1 Progeny Female Parent Male Parent Generation Parent pl1 Ear Kernel Background 1 Background 2 (Male allele Progeny Phenotype Parent) Pigmented B73 (~50%) pl1-b73 rpd1-2 F 3 1 kernels 3 Mo17 (~94%) Pl1-Rh + / rpd1-2 F Mo17 (~94%) Pl1-Rh + / rpd1-2 or + / + BC 1 2 rpd1-1 * Pl1-Rh A / rpd1-1 Pl1-Rh A Male parents were homozygous Rpd1 / Rpd1 from stocks that are genetically null for aleurone color. Exceptions were male parents (*), which were rpd1-1 homozygotes grown from darkly colored kernels, and ( ), which were + / rpd1-1 heterozygotes grown from lightly colored kernels. 2 Male parents were homozygous for the rpd1 allele designated in undefined backgrounds and all were homozygous for Pl1-Rhoades. Exceptions were male parent ( ), which was a + / rpd1-2 heterozygote grown from a lightly colored kernel and ( ), which were color-converted A632 inbreds (see Methods). 3 Graded classes of aleurone pigmentation (see Supplemental Figure 2) were quantified: 306 kernels had lightly blotched phenotypes and 79 kernels were colorless. 10

11 4 Graded classes of aleurone pigmentation (see Supplemental Figure 2) were quantified: 62 kernels had lightly blotched phenotypes and 48 kernels were colorless. 11

12 Supplemental Table 3. Pl1-CML52 co-segregation of aleurone pigment phenotype Genotype of F 1 parent T Pl1-Rhoades / Pl1- CML52; + / rpd1-2 No. of Wx/-/- F 2 individuals grown from No. of ear pigmented kernels with indicated pollen progeny phenotypes Semisterile Fully fertile Total of the 28 fully fertile individuals were confirmed to be Pl1-CML52 / Pl1-CML52 homozygotes by PCR-based genotyping. Amplification of DNA from fully fertile plants using pl1 primers homologous to sequences 5' of the doppia fragment and the intron 1 exon 2 boundary (described in Methods) allows detection of a 32 bp polymorphism that distinguishes the Pl1-Rhoades doppia fragment from the Pl1-CML52 doppia fragment. 12

13 Supplemental Table 4. Progeny phenotypes resulting from cross of pl1-cml52 / T Pl X T Pl-Rh / T Pl-Rh Inbred Progeny No. of individuals with specific anther color scores (ACS) and indicated pollen phenotypes Semi-sterile Fully fertile or or 6 7 CML

14 Supplemental Table 5. Frequencies and descriptions of genotypes and phenotypes of wx1 / wx1 seedlings and plants resulting from pl1-b73 / T Pl T Pl-Rh / T Pl-Rh Recombination interval pl1 genotype phenotypes Expected Frequency seedling pollen anther Parental Type T Pl / T Pl-Rh Pl f.f. Pl ~96.2% I 1 + pl1-b73 / T Pl-Rh Pl-Rh s.s. Pl-Rh ~2.3% Recombination events flanking pl1 II (A or B) 2 T pl-b73 / T Pl-Rh Pl-Rh f.f. Pl-Rh ~1.5% II (A) 3 T (Pl1-Rhoades pl1-b73) / T Pl-Rh Pl f.f. Pl rare III (B) 4 T (Pl1-Rhoades pl1-b73) / T Pl-Rh Pl-Rh f.f. Pl-Rh rare III (A or B) 5 T Pl / T Pl-Rh Pl f.f. Pl n.o. Recombination intervals are indicated in Supplemental Figure 4A. (A) Scenario A in which paramutagenic elements are centromere-proximal of the pl1 coding sequence. (B) Scenario B in which paramutagenic elements are centromere-distal of the pl1 coding sequence. Expected frequencies are based upon genetic distances between wx1, the T6-9 breakpoint, and the pl1 locus on the T Pl1-Rhoades chromosome (Hollick et al., 2005). Abbreviations: f.f., fully fertile; s.s., semi-sterile; n.o., not observed. Notes: 1. Recombination breaks linkage between the wx1 marker and the T6-9 breakpoint. 2. Recombination is proximal to any paramutagenic elements in scenario A or B. 3. Recombination creates an allele with proximal Pl1-Rhoades paramutagenic elements and a pl1-b73 coding sequence if scenario A is true. 14

15 4. Recombination creates an allele with proximal Pl1-Rhoades elements, a Pl1- Rhoades coding sequence, and non-paramutagenic pl1-b73 elements if scenario B is true. 5. Recombination is distal to pl1 and any paramutagenic elements in scenario A or B. This is indistinguishable from parental type chromosomes. 15

16 Supplemental Table 6. Anther phenotypes of genotyped progeny resulting from outcrosses of (T pl1-r30 / T Pl-Rh) to indicated testers pl1 allele of No. of umc2006-linked No. of individuals with indicated anther female parent ear genotype of progeny phenotypes progeny ACS color- sun- less red pl1-co159 2 pl1-co159 / T pl1-r pl1-co159 / T Pl-Rh pl pl1-987 / T pl1-r pl1-987 / T Pl-Rh Pl-Rh (A619) 1 Pl-Rh / T pl1-r Pl-Rh / T Pl-Rh Pl (A632) 1 Pl (A632) / T pl1-r Pl (A632) / T Pl-Rh T Pl 2 T Pl / T pl1-r T Pl / T Pl-Rh

17 Supplemental Table 7: Anther phenotypes of progeny resulting from outcrosses of (T pl1-r30 / T Pl-Rh) to indicated testers Female parent No. of individual progeny with indicated ACS genotype T Pl-Rh Pl-Rh (W23) Pl-Rh (A632) Pl-Rh (A619) T Pl Pl (A632)

18 Supplemental Table 8. Anther and seedling phenotypes and umc2006 genotypes for F 2 progeny of (T pl-r30 / T Pl-Rh) Anther Seedling Diploid genotype for No. of individual Phenotype phenotype umc2006 progeny sun-red n.d. B73 / B73 7 sun-red sun-blush B73 / B73 11 dark sun-red n.d. B73 / B73 1 sun-red n.d. Het 3 sun-red sun-blush Het 1 ACS 7 n.d. Het 18 ACS 7 Pl-Rh Het 12 ACS 5 Pl-Rh Rhoades / Rhoades 2 ACS 4 Pl-Rh Rhoades / Rhoades 1 Abbreviations: n.d., not determined; het, heterozygote (Rhoades / B73). Dark seedlings were sampled for RT-PCR analyses, thus accounting for the under-representation of Rhoades / Rhoades homozygotes in this population. 18

19 Supplemental Table 9. pl1-r30 and Pl1-Blotched anther phenotypes in rpd1 mutants Genotype of F 1 parent pl1-r30; + / rpd1-1 Pl1-Blotched; rpd1-1 / rpd1-1 No. of No. of individual rpd1 / rpd1 tassels with anthers of ear the indicated ACS 1 progeny ACS1 Var- Var- Var- ACS (5) ACS (6) ACS (7) ACS7 Total The majority of tassels produce anthers with a homogenous ACS score (Hollick et al., 1995), as represented in the first and last data columns, ACS 1 and ACS 7, respectively. We used a Var-ACS class to describe tassels that had anthers with a wide range of ACS, the highest of which is noted in parentheses. 19