WTPGMSF1 +15%PGMS G C C G C CG G Figure S1 Heteroduplex types and contents of PGMS gene amplicons with pre-PCR addition of 15% MT DNA. The heteroduplex content is calculated according to Palais et al. (2005). When MT reference DNA is mixed with MT samples, no heteroduplexes form after PCR. However, when MT DNA is added to DNA templates of homozygous WT and F 1 plants, heteroduplexes form after PCR: the heteroduplex content is 2x(1-x) in homozygous WT samples, and ½(1- x 2 ) in heterozygous samples. By adding 15% MT DNA, the heteroduplex content of the homozygous WT sample is about half that of the heterozygous sample, so that there is maximal separation in melting curves between WT, MT and F1 samples (Palais et al. 2005). G C C G C G G C Heteroduplexes PCR C CG G Heteroduplex content

PGMS WT G C PGMS Figure S2 A schematic representation of primers designed for CADMA analysis of the lncR (PGMS, a) and RNZ (TGMS, b) loci in rice. DNA and primer site amplicons NK-CR NK-MF NK-OLF TGMS WT TGMS A TA DNA and primer site amplicons a b G TC/GC G TA G A G TC/GC G TA GG GC G G A AG GG RNZ-R RNZ-MF RNZ-OLF

Figure S3 CADMA analysis of rice individual F 2 plants. a: Genotyping of F 2 plants of derived from cross of PGMS line DS550 and wild type line T23 at the lncR locus. DS550 and T23 are included as controls. b and c: Genotyping of the RNZ locus for F 2 plants derived from crosses of TGMS line Zhu 1S and wild type lines 08EZ01 and Z10, respectively; parental lines are included as controls. d: combination of panels b and c; Red curves show the sterile plants and all others show the fertile plants. a III P IPIP II P b ITIT II T III T c IV T VTVT ITIT d ITIT II T III T IV T VTVT

Figure S4 Hybrid rice seed purity test by CADMA analysis. a: Genotyping of F 1 hybrid seeds of DS550/T23 for the lncR locus, together with DS550 (one of the two I P lines) and T23 (II P ). b: Genotyping of F 1 hybrid seeds of Guangzhan63S/9311 for the RNZ locus, along with Guangzhan63S (one of the 4 I T lines) and 9311 (one of the 4 II T lines). a III P IPIP II P b ITIT II T III T