Supplementary material Article title: Chromosomal locations of a gene underlying heat-accelerated brown spot formation and its suppressor genes in rice Journal name: Molecular Genetics and Genomics Author names: Atsunori Fukuda, Kazuhiko Sugimoto, Tsuyu Ando, Toshio Yamamoto, Masahiro Yano Corresponding author: A. Fukuda Agrogenomics Research Center, National Institute of Agrobiological Sciences, Kannondai , Tsukuba, Ibaraki , Japan

Supplementary Table 1 Grain yield analysis of Koshihikari and SL518. Values are means ± S.D. Asterisks indicate significant differences between Koshihikari and SL518 (*P < 0.05; **P < 0.01, Student’s t-test). Fertility rates were analyzed after logit transformation Year LineN Total spikelet number (plant -1 ) Fertility rate Panicle number (plant -1 ) Spikelet number per panicle (plant -1 ) 1000-grain weight (g) 2010 Koshihikari ± ± ± 3 77 ± ± 2.37 SL ± ± ± 2 63 ± ± Koshihikari ± ± ± 2 83 ± ± 0.55 SL ± ± ± 3 74 ± ± 1.08 ** * *

Supplementary Fig. 1 Scheme of growth condition for the analysis of effect of high temperature on the spot formation Seeds were imbibed Plants were grown under a 14-h photoperiod at 28˚C/25˚C (day/night) Plants were grown under short-day conditions (10-h photoperiod) Evaluation of brown spots Treatment at high temperature 33˚C/27˚C 36˚C/30˚C 38˚C/32˚C Treatment at standard temperature 28˚C/22˚C 2 days 22 days 5 weeks 1 week 2 weeks

a x xy z b x y z Supplementary Fig. 2 (a, b) Severity of blast disease was evaluated as (a) the percentage of diseased leaf area or (b) the amounts of Magnaporthe grisea genomic 28S rDNA relative to rice genomic Rubq1 DNA. Values are means ± S.D. (a, n = 6; b, n = 3 or 4). Different letters indicate significant differences (P < 0.05, Tukey’s HSD test)

* * * P < 0.05 P < P < ab cd Supplementary Fig. 3 Effect of brown spot formation on leaf photosynthesis in Koshihikari and SL518. (a) Brown spot evaluation scores, (b) SPAD values, (c) leaf photosynthesis rate (P n ), and (d) stomatal conductance (g s ) in each topmost expanded leaf of the main culm of Koshihikari (Ksh) or SL518 were determined 72 and 105 days after sowing in a paddy field in Values are means ± S.D. (n = 6). Asterisks indicate significant differences between Koshihikari and SL518 (P values are shown in the graph, Student’s t-test)

KoshihikariSL518 Supplementary Fig. 4 Frequency distribution of evaluation scores of the brown spots on the leaves of SL518 × Koshihikari F 2. The F 2 population showed three genotype classes of the SSR marker RM3170, which was found to be linked to the QTL for brown spot formation (Table 1). Genotypes of RM3170 are represented as white bars (homozygous for Koshihikari allele), grey bars (heterozygous allele), and black bars (homozygous for Nona Bokra allele). Evaluation scores were determined at 3.5 months after sowing. Arrows indicate the mean values for the scores in Koshihikari and SL518.

cd a b Supplementary Fig. 5 Temporal changes in evaluation scores of the brown spots on the leaves of Koshihikari, SL518, and KBSNIL1. (a) Brown spots on a flag leaf blade of a 4-month-old KBSNIL1 plant. Scale bar = 2 cm. (b) Graphical genotype of KBSNIL1. White box, Koshihikari; black box, Nona Bokra. (c) Evaluation scores were determined as in Fig. 2. Values are means ± S.D. (n = 21). (d) Percentage of plants at heading stage (n = 21)

ab Supplementary Fig. 6 QTL locations on chromosomes 2 and 9. (a) Log-likelihood curve indicating a putative QTL position on chromosome 2 in the F 2 population of KBSNIL1 × SL505. (b) Log-likelihood curve indicating a putative QTL position on chromosome 9 in the F 2 population of KBSNIL1 × SL531. LOD, logarithm of odds; a, additive effect of the Koshihikari allele on the evaluation score of brown spots; d, dominance effect of the Koshihikari allele on the evaluation score of brown spots; PVE, percentage of variance explained. Dotted horizontal lines indicate the significant threshold of LOD score for detection of putative QTLs