1. Introduction

AJPS

American Journal of Plant Sciences

2158-2742

Scientific Research Publishing

10.4236/ajps.2017.813226

AJPS-81110

Articles

Biomedical&Life Sciences

Genetic Mapping and Characterization of Lethal Necrotic Mutants in Rice (Oryza sativa L.)

Rumanzi

Saidi Mbaraka

¹Yoshiyuki

Yamagata

¹Atsushi

Yoshimura

¹Hideshi

Yasui

¹^*

Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

* E-mail:hyasui@agr.kyushu-u.ac.jp(HY);

04122017

0813335033767, November 201715, December 2017 18, December 2017

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

The spread of tissue necrosis leads to plant death. We isolated 18 lethal necrotic mutants induced in rice ( Oryza sativa L.) by gamma-irradiation. The necrotic lethality among the 18 mutants was controlled by single recessive genes designated necrotic lethality 1 ( nec 1) to nec 1 8 . These mutants display pale-green leaves from the third-leaf stage and leaf-tip necrosis, which spreads to the whole plant, killing it. Genetic mapping and histochemical analysis of the lethal necrotic mutants were conducted. At least four independent loci on chromosomes 2 and 4 controlled necrotic lethality. Therefore, the genetic causes of lethal necrosis vary among mutant stocks. Histochemical analysis at 12 days after sowing showed that H ₂ O ₂ accumulated in the necrotic parts of leaves, and that cell death occurred throughout the leaf. Mutants for early necrotic lethality (<24 days to lethality) were characterized by the rapid spread of H ₂ O ₂ accumulation throughout the third leaf. Mutants for late necrotic lethality (>35 days to lethality) were characterized by the incomplete spread of H ₂ O ₂ accumulation within the third leaf.

Rice Mutant Necrotic Lethality Linkage Mapping Reactive Oxygen Species

1. Introduction

Necrosis of plant tissues usually shows as brown or black discoloration. Stresses such as fungal infections can cause necrosis in any tissues [1] . Necrosis is common in relation to nutrient supply; for example, deficiencies in potassium can cause severe necrotic lesions in leaves [2] [3] [4] [5] [6] . In addition, hybrid necrosis, caused by deleterious epistatic interactions, results from spontaneous activation of plant defenses associated with leaf necrosis [7] [8] [9] . Necrotic lesion formation has been reported in several mutants. The maize lethal leaf spot 1 (lls1) shows enhanced resistance to fungal infections due to necrotic lesion formation [10] . The Arabidopsis botrytis-susceptible 1 (bos1) mutant develops necrosis to resist infection by Dickeya dadantii [11] . Several lesion mimic mutants (LMMs) of rice show broad-spectrum resistance to blast and bacterial blight [12] . Excessive phosphate accumulates in the shoots of the phosphate-accumulator 2 (pho2) mutant of Arabidopsis, which develops necrotic symptoms [13] [14] . The rice leaf tip necrosis 1 (ltn1) mutant lacks a protein containing a ubiquitin-conjugating domain, which regulates phosphate accumulation, and develops leaf tip necrosis [15] . The phenotypes in most lesion mimic and necrotic mutants are often influenced by environmental factors [16] . Light and temperature affect the severity of mutant phenotypes in rice zebra necrosis [17] and faded green leaf [18] mutants and maize lls1 mutants [10] [19] . Under short-day conditions, a barley lesion-mimic mutant, “1661”, fails to reach maturity and does not produce seeds [20] . These mutants can survive to maturity. In addition, mutants in which necrosis spreads to the whole plant have been reported in maize: at least eight recessive mutants (nec1 - nec7, nec-t; [21] [22] showed leaf discoloration, necrotic leaf tips, spots, or transverse bands before death. However, rice mutants showing necrotic lethality have not yet been isolated. In plants, reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide and hydroxyl radicals are constantly produced as metabolic byproducts in chloroplasts, mitochondria, and peroxisomes [23] [24] . Under stress, ROS production exceeds the cells’ scavenging capacity, and the ROS radicals induce necrotic lesions and alter the expression of certain genes in many signaling pathways, eventually leading to accelerated cell death [23] [24] . In maize, Arabidopsis, and rice [25] [26] [27] [28] , the accumulation of ROS in particular, H₂O₂ and the presence of dead cells are studied as biochemical markers constitutively expressed in LMMs [29] . They are commonly monitored by staining using histochemical dyes: 3',3'-diaminobenzidine (DAB) and trypan blue, respectively [29] [30] . Although H₂O₂ is known to cause cell death leading to necrotic lesions in many plants [31] , little is known about ROS-induced necrotic lethality. In this study, we isolated 18 gamma-irradiated rice mutants with necrotic lethality at the seedling stage. We analyzed them to map the genes responsible for their necrotic lethality and histochemically characterized them for H₂O₂ accumulation and cell death.

2. Materials and Methods2.1. Plant Materials

Two rice cultivars; a japonica cultivar, “Taichung 65” (T65) and indica cultivar “IR24” were gamma irradiated as described by Yamagata et al. [32] . The spikelets of these cultivars were exposed to 100 - 150 Gy dosage just before anthesis using the Co⁶⁰ γ-ray irradiation facility of Institute for Irradiation and Analysis of Quantum Radiation, Kyushu University. Self-pollinated M₁ plants were grown and M₂ seeds were harvested and used to generate M₃ plants. Self-pollination and screening of these mutant stocks were conducted and the resulting mutant stocks were maintained in the Plant Breeding Laboratory, Kyushu University. These mutants were designed as LEM (lethal mutants) (Table 1). Seeds of the mutant lines were sown on 25 May 2016 on sieved soil containing 2.63 g∙m⁻² of NPK, and the seedlings were grown in a greenhouse under natural conditions at Kyushu University Farm, Fukuoka, Japan (33˚36'55''N). For phenotypic characterization, ten plants were observed per mutant line. Surviving nec18 mutant seedlings were transplanted into the paddy field and observed until 15 August 2016. For histochemical analysis, plants were grown in a phytotron maintained at 25˚C under natural sunlight in 2016 and 2017 at Kyushu University. For genetic mapping of the mutants, we crossed the wild-type segregants of twelve T65-derived mutant lines, LEM1 (nec1), LEM2 (nec18), LEM9 (nec4), LEM11 (nec16), LEM16 (nec5), LEM17 (nec6), LEM18 (nec8), LEM20 (nec14), LEM25 (nec15), LEM29 (nec9), LEM36 (nec10), and LEM37 (nec11) with japonica cultivar “Hinohikari”, and crossed normal segregants of two IR24_derived mutant lines, LEM41 (nec17) and LEM43 (nec13) with T65. The resulting F₂ populations in which necrotic lethality segregated were used to map necrosis genes.

Table 1 Identification of genes responsible for necrotic lethality in the 18 mutant lines

Gene	Mutant line	Background	DTL	Segregation for necrotic lethality			P^a	BSA	H₂O₂ accumulation^b
Gene	Mutant line	Background	DTL	Wild type	Necrotic lethality	Total	P^a	BSA	H₂O₂ accumulation^b
nec1	LEM1	T65	16	68	21	89	0.76	2L	Entire
nec2	LEM4	T65	16	87	19	106	0.09	-	-
nec3	LEM5	T65	16	88	20	108	0.12	-	-
nec4	LEM9	T65	16	82	23	105	0.46	2S and 2L	Entire
nec5	LEM16	T65	16	83	29	112	0.83	2S and 2L	Entire
nec6	LEM17	T65	16	87	20	107	0.13	2S and 2L	Entire or Tip
nec7	LEM18	T65	16	80	21	101	0.33	2L	Entire
nec8	LEM24	T65	19	71	23	94	0.91	-	-
nec9	LEM29	T65	19	87	20	107	0.13	2L	Entire
nec10	LEM36	T65	19	154	49	203	0.78	2L	Entire
nec11	LEM37	T65	19	100	27	127	0.33	2L	Entire or Tip
nec12	LEM28	T65	21	74	22	96	0.64	-	-
nec13	LEM43	IR24	21	75	36	111	0.07	2L	Entire
nec14	LEM20	T65	24	63	14	77	0.17	4	Entire
nec15	LEM25	T65	35	80	20	100	0.25	4	Tip
nec16	LEM11	T65	35	86	22	108	0.27	4	Tip
nec17	LEM41	IR24	35	83	18	101	0.10	2S	Transverse band
nec18	LEM2	T65	82	83	22	105	0.34	4	Tip

^aProbability in χ² test of a 3:1 ratio. ^bObservations on the third leaves of mutant at 12 days after sowing.

2.2. Development of Indel Markers

To find polymorphic indel sites in temperate japonica, we conducted de novo whole-genome sequencing of T65 by next-generation sequencing on a Roche/ 454 GS-FLX Titanium sequencer (Roche, Basel, Switzerland) and de novo assembly in GS De Novo Assembler software (Roche). The assemblies were used for BLAST similarity searches of Nipponbare pseudo-molecule build 4.0 (BLAST). Polymerase chain reaction (PCR) primers for polymorphic sites with >5-bp insertion/deletion variation were designed in Primer 3 software [33] (Supplementary Table S1, Supplementary Table S2).

2.3. Genotyping

Fresh leaves of F₂seedlings and their parents were collected and ground in 1× TE buffer in a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan), and their total genomic DNA was extracted using a modified potassium acetate method [34] . The obtained DNA was amplified by PCR with simple sequence repeat (SSR) markers [35] [36] [37] [38] [39] and indel markers (Supplementary Tables S1, Supplementary Table S2). PCR was performed in 15-μL mixtures containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl₂, 200 μM each dNTP, 0.2 μM each primer, 0.75 units of Taq polymerase (Takara, Otsu, Japan), and approximately 5 ng of template DNA in a GeneAmp PCR system 9700 (Applied Biosystems). PCR conditions were 95˚C for 5 min, followed by 35 cycles of 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s. PCR products were separated in 4% agarose gels (Agarose HT, Amresco Inc., Solon, OH, USA) in 0.5× TBE buffer at 250 V.

2.4. Gene Mapping

To determine the loci responsible for necrotic lethality, we used bulked segregant analysis (BSA) [40] to detect markers tightly linked to causal genes using 43 SSR and 38 indel markers polymorphic between T65 and Hinohikari, and 84 SSR and 3 indel markers polymorphic between IR24 and T65. Bulks of wild-type and mutant plants were each composed of 10 individuals from the F₂ generation. Markers showing polymorphism between the two bulks were selected and used for linkage analysis of the F₂ populations based on calculation of recombination value by the maximum likelihood method [41] . The estimated recombination values were converted to map distances by Kosambi’s mapping function [42] to construct linkage maps.

2.5. ROS Accumulation

To observe excessive accumulation of ROS in necrotic mutants, we used DAB staining to assess H₂O₂ accumulation [43] . Because necrosis was observed in third leaf of all the mutants at 12 days after sowing (DAS), third leaves of necrotic lethal mutants and wild-type plants at 12 DAS were immersed in 1 mg mL⁻¹ DAB in distilled water and incubated for 8 h at 25˚C to allow DAB uptake, with three replicates. After staining, chlorophyll was completely removed by incubation in 96% ethanol at 80˚C for 1 h. The leaf samples were preserved in 70% ethanol and later photographed.

2.6. Detection of Cell Death

Trypan blue staining was used to assess cell death as a result of necrosis [12] . In brief, fresh leaves (third leaf) were submerged in a solution of 2.5 mg∙mL⁻¹ trypan blue, 25% (w/v) lactic acid, 23% (v/v) water-saturated phenol, and 25% (v/v) glycerol in distilled water. The samples were heated in boiling water for 2 min, placed in chloral hydrate solution (25 g in 10 mL of distilled water) for destaining, and preserved in 70% glycerol.

3. Results3.1. Identification of Mutants for Necrotic Lethality

Necrotic lethality of mutant seedlings was observed in the self-pollinated progeny of the heterozygous plants in all 18 mutant lines. Wild-type and lethal necrotic seedlings segregated in a 3:1 ratio, indicating that the lethal necrosis was controlled by single recessive genes, designated necrotic lethality1 (nec1) to nec18 (Table 1).

3.2. Phenotypic Characterization of Necrotic Mutants

Before the third-leaf stage at 6 DAS, the phenotypes of the 18 lethal necrotic mutants were similar to those of the wild-type plants except for the nec17 mutant, which had yellow-green leaves by 4 DAS (Supplementary Table S3). At the third-leaf stage, at 7 DAS, the second and third leaves of the nec1 mutant were pale green (Figure 1(a), and leaf-tip necrosis began in the second leaf. The necrosis spread to the entire leaf blade of the second and third leaves at 12 DAS (Figure 1(b), Supplementary Table S3), and the plant died at 16 DAS (Supplementary Table S3), during the forth-leaf stage (Figure 1(c)).

Similar pale-green discoloration and expansion of the necrosis to the entire plant were observed in all mutants except nec17. Different days to lethality (DTL) were recorded: 16 DTL in nec2, nec3, nec4, nec5, nec6, and nec7, 19 DTL in nec8, nec9, nec10, and nec11, 21 DTL in nec12 and nec13, 24 DTL in nec14, and 35 DTL in nec15, nec16, and nec17 (Table 1, Supplementary Table S3). nec18 seedlings transplanted into the paddy field at 19 DAS died at 82 DAS (Figure 1(d); Supplementary Table S3). In contrast to the other lines, the nec17 plants became yellow-green as early as 4 DAS and developed transverse necrotic bands at the second- to third-leaf stage (Figure 1(e), Figure 1(f); Supplementary Table S3). The necrosis in nec17 gradually spread to the whole plant, and plants died at 35 DAS.

3.3. Bulked Segregant Analysis

BSA of 14 mutant lines revealed that in the F₂ population between nec1 and Hinohikari, wild-type and mutant plants segregated in a 69:27 ratio, fitting a 3:1

ratio (Table 2). At indel markers QSTS96 and QSTS109, on the long arm of chromosome 2 (2L), the nec1 bulks showed an intensive band derived from T65, but the wild-type bulks showed heterozygous-like bands derived from T65 and Hinohikari alleles (Supplementary Figure Sl). BSA of the other 13 mutants suggested candidate regions on the short and long arms of chromosome 2 (2S and 2L) and on the long arm of chromosome 4 (Table 1; Supplementary Table S4).

3.4. Validation by Linkage Mapping

Among the 14 necrotic lethal mutants in which responsible regions were identified, we conducted linkage analysis for nec1, nec13, nec16, nec17, and nec18. nec1 was located between QSTS103 and QSTS109 on chromosome 2L (Figure 2(b); Supplementary Table S6). nec13 was located in the same region, between QSTS96 and QSTS109 (Figure 2(c); Supplementary Table S7). nec17 was located between RM3703 and RM7082 on chromosome 2S (Figure 2(a); Supplementary Table S5). nec16 and nec18 were located on different regions of chromosome 4L: nec16 tightly linked to RM3785 and RM7051 (Figure 2(d); Supplementary Table S8), and nec18 tightly linked to QSTS141 and QSTS142 (Figure 2(e); Supplementary Table S9).

Table 2 Segregation of wild-type and mutant plants in F2 populations

Cross combination		Gene	F₁ phenotype	Segregation for necrotic lethality
Female	Male	Gene	F₁ phenotype	Wild type	Necrotic lethality	Total	P^a
Hinohikari	LEM1	nec1	wild type	69	27	96	0.48
Taichung 65	LEM43	nec13	wild type	110	42	152	0.45
Hinohikari	LEM11	nec16	wild type	76	20	96	0.35
Taichung 65	LEM41	nec17	wild type	78	34	112	0.19
Hinohikari	LEM2	nec18	wild type	69	27	96	0.48

a. Probability in χ² test of a 3:1 ratio.

3.5. Allelism between nec1 and nec13

nec1 and nec13 were located in the same region on chromosome 2 L (Figure 3(b), Figure 3(c)). We evaluated allelism in F₁ progeny of crosses between nec1 and nec13. If nec1 and nec13 are located at different loci, F₁ plants derived from a cross between nec1 heterozygotes (+/nec1) and nec13 heterozygotes (+/nec13) would show only wild-type segregants. However, necrotic segregants were observed in F₁ derived from the cross between the heterozygous plants in three replicates, indicating that nec1 and nec13 are located at the same locus (Table 3).

Table 3 Segregation in F1 population derived from nec1 × nec13

Cross combination^a					Segregation for necrotic lethality in F₁
Female		×	Male		Wild type	Necrotic lethality	Total
LEM43-4	(+/nec13)		LEM1-5	(+/nec1)	34	6	40
LEM43-8	(+/nec13)		LEM1-7	(+/nec1)	16	19	35
LEM43-12	(+/nec13)		LEM1-12	(+/nec1)	20	20	40

a. Genotypes are represented in parentheses.

3.6. Histochemical Characterization of Lethal Necrosis

Of the eighteen mutants, fourteen were initially characterized for their H₂O₂ accumulation of the third leaves at 12 DAS (Supplementary Figure S2). Five lethal necrotic mutants showed phenotypic differences in patterns of H₂O₂ accumulation and cell death at 12 DAS: nec1 and nec13 showed early necrotic lethality (<24 DTL; Figure 3(a), Figure 3(b)), and nec16, nec17, and nec18 showed late necrotic lethality (>35 DTL; Figures 3(c)-(e), Supplementary Figure S2; Table 1). Intense brown staining indicated H₂O₂ accumulation throughout the third leaves of nec1 (Figure 3(f)) and nec13 (Figure 3(g)), and in the other early necrotic lethal mutants (nec4, nec5, nec7, nec9, nec10, and nec14: Table 1; Supplementary Figure S2), as well as in the tips of the third leaves of nec16 (Figure 3(h)), nec18 (Figure 3(j)), and nec15 (Table 1; Supplementary Figure S2). The staining pattern of the third leaves in nec6 and nec11 were not clearly classified into the entire or leaf-tip types. The third leaves of nec17 showed a transverse banding pattern of brown staining (Figure 3(i)). Trypan blue staining revealed deep blue throughout the leaf blades of nec1, nec13, nec16, nec17, and nec18 (Figures 3(k)-(o)), but not in the wild-type plants. Taken together, these results suggest that excessive accumulation of H₂O₂ caused necrotic symptoms in the mutants.

4. Discussion

We identified 18 mutants characterized by necrotic seedling lethality. The necrotic lethality was controlled by single recessive genes which we designated nec1 to nec18 (Table 1). The necrosis began with leaf discoloration and necrosis formation at the leaf tip, or with transverse banding segments, during the third-leaf stage, and plants died during the fourth- to fifth-leaf stages, between 16 and 82 DAS (Figure 1; Supplementary Table S3). The initiation of necrosis at the third-leaf stage, the spread of necrosis from the leaf tip to the entire leaf, and early necrotic lethality were similar to those of lethal necrotic mutants in maize nec4, nec5, nec7, and olive-necrotic-8147 [21] [44] [45] [46] .

A number of rice mutants with disease-like lesions were morphologically categorized into spotted leaf mutants (spl: [47] [48] [49] , lesion mimic mutants (lmm: [27] [50] , cell death and resistance mutants (cdr: [51] and zebra necrosis mutants (zn: [17] [52] . DAB staining indicated that ROS accumulation was limited to the necrotic lesions. In our study, the lethal necrotic mutants showed necrosis of the whole leaves and eventually plant death. The mutants with early necrotic lethality (nec1, nec4 - nec7, nec9 - nec11, nec13, and nec14) had rapid spread of H₂O₂ accumulation throughout the leaf at 12 DAS (Table 1; Supplementary Figure S2). Those with late necrotic lethality (nec15, nec16, nec18), in contrast, had H₂O₂ accumulation only at the leaf tip at 12 DAS.

Genetic mapping by BSA and linkage analysis revealed at least four independent loci that control necrotic lethality in 14 of the mutants (Table 1). The mutants with causal gene on chromosome 2L (nec1 and nec13) showed early lethality within 21 DAS, with excessive H₂O₂ accumulation throughout their leaves (Figure 2, Figure 3 and Supplementary Table S3). These phenotypes have also been reported in a necrotic mutant of maize, nec-t [22] . nec-t has abnormal chloroplast development and lacks photosynthetic ability. The mutants with causal genes on chromosome 4 (nec16 and nec18) had late necrotic lethality (>35 DTL) and excessive ROS production progressing from their leaf tips (Figure 2, Figure 3, Supplementary Figure S2, Supplementary Table S3). Additionally, nec17 on chromosome 2S, induced late necrotic lethality (35 DTL) with distinct transverse bands (Figure 3). Rice ZEBRA-NECROSIS (ZN) encodes a thylakoid-bound protein involved in the photoprotection of developing chloroplasts during early leaf development, and the zn mutant shows leaves with transverse necrotic bands due to accumulation of light-induced ROS [17] . The above-mentioned mutants would be important resources for investigating mechanisms such as stress tolerance, photoprotection of chloroplasts, and nutrient deficiency in plants.

5. Conclusion

We identified 18 lethal necrotic mutants induced in rice by gamma-irradiation. The necrotic lethality was found to be controlled by single recessive genes designated necrotic lethality1 (nec1) to nec18. Among them, five mutants were precisely mapped on the rice chromosomes: nec17 on chromosome 2S, nec1 (nec13) on chromosome 2L, and nec16 and nec18 on different regions of chromosome 4L. Thus at least four independent loci control necrotic lethality in rice. The histochemical analysis of these mutants at 12 DAS showed that H₂O₂ accumulated in the necrotic parts of leaves, and that cell death occurred throughout the leaf. Mutants for early necrotic lethality (nec1 and nec13) were characterized by the rapid spread of H₂O₂ accumulation throughout the third leaf. Mutants for late necrotic lethality (nec16, nec17 and nec18) were characterized by the incomplete spread of H₂O₂ accumulation within the third leaf.

Acknowledgements

We are grateful to Institute for Irradiation and Analysis of Quantum Radiation, Kyushu University, for the gamma-irradiation. This work was supported by the Canon foundation and partly supported by the Japan Society for the Promotion of Science for KAKENHI Grant Number JP15H04438.

Cite this paper

Mbaraka, R.S., Yamagata, Y., Yoshimura, A. and Yasui,H. (2017) Genetic Mapping and Characterization of Lethal Necrotic Mutants in Rice (Oryza sativa L.). American Journal of Plant Sciences, 8, 3350-3376. https://doi.org/10.4236/ajps.2017.813226

Supplementary

Table S1 List of markers for linkage analysis of mutants induced from Taichung 65

Chr.	Position^a	Marker name	Marker type	Forward primer sequence	Reverse primer sequence	Reference
1	548,641	RM3148	SSR	GCTTTGGTATTTGCAGGTTCACG	CTATTGCTCGAACACTTTGCTTCTCC	McCouch et al. (2002)
1	2,575,209	QSTS60	Indel	AAAGCTCTGAGTGGCTTTGC	TTGGTTTGTCGATATGTTCAGC	This study
1	4,635,844	QSTS76	Indel	GCGAAAACACAATGCAAAAAG	GTGTGGGGGAAGCAAGAG	This study
1	23,968,523	RM5	SSR	TGCAACTTCTAGCTGCTCGA	GCATCCGATCTTGATGGG	Panaud et al. (1996)
1	26,035,809	RM3475	SSR	GTCGGTTTGCCTAGTTGAGC	TTCCTCGGTGTATGGGTCTC	McCouch et al. (2002)
1	30,718,595	QSTS82	Indel	TAACAACGGGGGCCTAGATG	GCCGGTGGTGAAGACGAC	This study
1	40,246,974	RM3520	SSR	GAGGCTATATGCTCATGCTC	AAACCTGCAAATGCACAG	McCouch et al. (2002)
2	1,878,328	RM7562	SSR	AGACATGCCAATGTGATGGC	TCGGTAGTATGGGGCTTGTC	McCouch et al. (2002)
2	4,414,049	RM3865	SSR	AACCATGGACAGTTGAACAC	CTCCGACAAGAACTTCCTC	McCouch et al. (2002)
2	5,477,492	RM6378	SSR	CTGATCATCTCATGCCTCCTACG	TCCATCTCCCAATATGACCAACC	McCouch et al. (2002)
2	5,852,749	QSTS88	Indel	GCCATGGAAAAGAAAAGCTG	TGATCGATGGATAGCCACAC	This study
2	7,112,707	QSTS89	Indel	TTTTGCACGGTTTTGTATGG	AACCGATGTCTGCATCCAAG	This study
2	9,984,944	QSTS90	Indel	GAGGAGGAGGAACGAAGAGG	GTTCGCTCCTGACCTTCG	This study
2	9,998,681	QSTS91	Indel	GAGGAGGAGGAACGAAGAGG	GTTCGCTCCTGACCTTCG	This study
2	10,186,627	RM3501	SSR	CTACAATGATTCCATGCCTGTCC	TCCGGCTCAAGCTACAGTTAAGG	McCouch et al. (2002)
2	17,297,690	QSTS95	Indel	TGAGGACAAGCATGTGAAGG	CAATGGACGCTAGTGGAACC	This study
2	18,744,542	QSTS96	Indel	GCTCGTGTTCGTGCATCTATC	GCAACAACGAGAACGAGAAC	This study
2	20,751,269	QSTS103	Indel	TTCGTTTCGAGTAACAAAGCAC	GTGTTAGGGAAGCACATTCG	This study
2	24,254,973	QSTS105	Indel	AATATTGTAAACAAAAACTCAAAATCC	TTCTTCTCTCTCCGCCTTCC	This study
2	24,518,130	QSTS109	Indel	AATATAGTTTGGTCAAAAGAACAAAAG	CGGATAATCTTGATGAGTGAACC	This study
2	24,531,197	QSTS110	Indel	TGTTGGTGGTCAGTTCAACTTC	TCCCGCCTTCCATTTATTAC	This study
2	24,656,209	QSTS111	Indel	CGGAGGCAGAGTAAGAATCG	CAATCCCGATTGTCGGATAC	This study
2	32,812,348	QSTS114	Indel	GAAAATTTGCATTCACTCCTACC	TTGAAAAACACGGGAATTTTATAG	This study
2	33,019,362	RM6733	SSR	TCCATGTGCACAATCCAATTCC	GTGACGGCATGAGAGTGTTTGTAGG	McCouch et al. (2002)
2	33,254,219	QSTS115	Indel	CGCAGTGGCTATAACCCAAG	CCGCATTAGAATCCTTCTCG	This study
2	34,676,703	RM213	SSR	ATCTGTTTGCAGGGGACAAG	AGGTCTAGACGATGTCGTGA	Chen et al. (1997)
2	35,424,303	RM2265	SSR	AACTGACCGTATATTAGCCA	TGACCGCCTCTATTATATTG	McCouch et al. (2002)

2	35,450,160	RM3850	SSR	AAGTTGAGAATGAGGGACAA	TTCGGAAGTGAAAAGGTAAT	McCouch et al. (2002)
2	35,729,140	RM3274	SSR	GCTGCTGCTGCTACAGTTTG	GCGAGCTCCAGCATTTAAAC	McCouch et al. (2002)
2	35,160,161	RM208	SSR	TCTGCAAGCCTTGTCTGATG	TAAGTCGATCATTGTGTGGACC	Chen et al. (1997)
3	8,170,550	RM5639	SSR	GGAAGAACAGAGTTGCTCGG	GTGCCATTTATTTCCGTCCC	McCouch et al. (2002)
3	1,437,206	RM3372	SSR	GAGCGACCAAAGAATCCAAG	CCACGGGGAGCTGATGAAG	McCouch et al. (2002)
3	5,971,990	RM3467	SSR	ATAATGGCAGGGTTGTCTCG	CTCGGTGAGCCTCCTACAAC	McCouch et al. (2002)
3	16,285,889	QSTS125	Indel	TTGAATCAATCACACATCTTATTCAG	TCCATATGGCCCAACATAAAC	This study
3	28,479,745	RM1350	SSR	CGCCCTAGTAGATAGGTAATTG	AAATCAGCAAGAAAGCTCTG	McCouch et al. (2002)
4	24,222,895	RM3785	SSR	ACCTTTTCTTGGCTTGAGGG	GCTTTTGCTACTTTTGGGGG	McCouch et al. (2002)
4	24,277,170	RM7051	SSR	CTCGATGAGCTTGGCGTC	TTCAGTGTTCATCGCCTCTG	McCouch et al. (2002)
4	5,275,427	QSTS132	Indel	TTTCGATGCTGTCAATCTACG	TCGATCCATCCACTTCCTTC	This study
4	13,164,721	QSTS134	Indel	CTTGTATTGCGTGCAGGATG	TTGGCTTCAATGCATACACC	This study
4	31,466,041	QSTS141	Indel	CCATGCATCCTCCTGTTCC	CTGTTGATGGGTCCCAATTC	This study
4	31,485,856	QSTS142	Indel	AGCTTTTTGGGTGATGTTGG	GGATGCTCTTTCTCCACCAC	This study
5	9,320,757	RM6836	SSR	TTGTTGTATACCTCATCGAC	AGGGTAAGACGTTTAACTTG	McCouch et al. (2002)
5	82,104	RM3529	SSR	CGCGCCACCTCGATATATAC	GCTCAGGTTAACCAAGGTGG	McCouch et al. (2002)
5	4,311,860	QSTS145	Indel	GAGACGGTCCCAACAAAGAC	CGTTTTAAACCATCCTTAGACG	This study
5	17,292,400	QSTS152	Indel	GAAACTCGGCCAATTGTAAC	TGACATGGCTGTCTTCAACC	This study
6	3,168,368	RM204	SSR	GTGACTGACTTGGTCATAGGG	GCTAGCCATGCTCTCGTACC	Chen et al. (1997)
6	10,151,590	QSTS157	Indel	TGCTGTCATGCCAACTTACC	AAATGGCAGCATCTTCAAGC	This study
6	23,054,814	QSTS171	Indel	TGAAAACCGTGTCACTGTCC	GGGGCTCTAATCACCTCCAG	This study
6	25,612,879	RM6395	SSR	GGCTTCGGCTTCTGAACTAGC	CGACTAAGCAGCAGTAACAATCTCG	McCouch et al. (2002)
6	28,563,631	RM3307	SSR	CAGTGCTCTCGAACATGGAG	CTGCATTGTAAACGGTCGAG	McCouch et al. (2002)
7	1,186,748	RM6697	SSR	GCAAGATCCAGTCGATTTGG	ATAACATGAGCATCTCCCCG	McCouch et al. (2002)
7	23,596,003	RM5847	SSR	TGAGATGAGAGATAGACTCC	AACAGATGAAGGCTATTTTA	McCouch et al. (2002)
7	4,692,609	RM6872	SSR	CACCACGATATCCACCTCTAGC	CCTAGGATGAACACTGATGATGG	McCouch et al. (2002)

7	11,713,561	QSTS176	Indel	GTGGCAGCTTGAGAGCATTC	TTTAATCCAAGCTTAGGCAACC	This study
7	13,552,392	QSTS180	Indel	GGAGCTCGTACAGAAAAATCG	CCGGAGGTATATTCCTTGTTTG	This study
7	17,831,145	QSTS182	Indel	GGCAATGGAACTTTTTGATTTG	ATGCACTTTCTGTCTCAGTTTG	This study
7	21,243,983	QSTS191	Indel	CAGTTTGGCACTGGCCTATC	GCATTTCACTCAAGCAATGG	This study
7	28,894,378	RM1306	SSR	TGCCAATTACCTTCCCGTAC	TGCTCCGTATTGCTGCTATG	McCouch et al. (2002)
8	20,645,571	RM223	SSR	GAGTGAGCTTGGGCTGAAAC	GAAGGCAAGTCTTGGCACTG	Chen et al. (1997)
8	26,294,879	QSTS215	Indel	TCCCTTAACTTGTCAACAAATCC	GCCACGTCAGCTAAAACCTC	This study
8	27,898,042	RM3155	SSR	GAAAAGGACAGGGGAAAAGC	GATCGTTCGTGTTCGTGTTG	McCouch et al. (2002)
9	5,048,789	QSTS222	Indel	CAACCAGCGAAACATACTGC	ACGGGAGGAAGATGACCAC	This study
9	11,045,262	QSTS227	Indel	TTTTTGCTTCTCACCCATTC	TTGCATGAACAGAGGTTTGC	This study
9	12,583,087	QSTS228	Indel	ATATCATTCCGGTGCCTACC	GTGCGCATGTGTGTGTACTG	This study
9	14,644,334	QSTS229	Indel	AGTGGATGGATGGATGGATG	CGTCGTCTCTTCCGTTTTTC	This study
9	20,246,472	RM3808	SSR	CGTTAGCGAAACGAACAGTG	CAGTGGCTCGGTAATCGC	McCouch et al. (2002)
9	17,054,142	RM3600	SSR	TGCCCACACATGATGAGC	AACGGGCAAGAGATCTTCTG	McCouch et al. (2002)
10	2,522,494	QSTS237	Indel	GACCAGAGACTGCCCCATC	TCACTTCAAGTCTGTGGTTTCAG	This study
10	22,133,218	QSTS248	Indel	TGCAATTGACTACTACTCCAAGG	TCAAACAAACAATCGTGAAGC	This study
10	17,490,576	RM6704	SSR	AATCGAATCTGGATATCTTG	CTTCTACCTAGCTACCGAGA	McCouch et al. (2002)
10	15,903,727	RM5304	SSR	CATCTTGAATCCTCCTTCGACTCC	GGCAGCGATAGCAGGAAGAGG	McCouch et al. (2002)
11	794,611	QSTS250	Indel	CGTTGGATAATTAATGGAGATCG	CACAAGCACCAAACCCAAC	This study
11	4,484,051	QSTS255	Indel	CATTGCTACGAGGCACAATC	CCGCGCACGTAGGTATATG	This study
11	7,007,889	QSTS263	Indel	ATAAAATCCGGCGAGACAAG	GAAGCGAGTGCTCCTAGCTC	This study
11	21,626,773	RM206	SSR	CCCATGCGTTTAACTATTCT	CGTTCCATCGATCCGTATGG	Chen et al. (1997)
11	28,173,902	RM144	SSR	CATGTTGTGCTTGTCCTACTGC	AGCTAGAGGAGATCAGATGGTAGTGC	Temnykh et al. (2000)
12	905,092	RM1080	SSR	AGAGCCCTCGTAAGCCAAAG	GGTCGTGAATCTCCTCCAAG	McCouch et al. (2002)
12	4,919,671	RM3455	SSR	TGAATCCACACTCGCAGATC	GCCAGTCCACGATTGGTC	McCouch et al. (2002)
12	25,036,187	RM3739	SSR	AGTTGCGCAGCTAATCGATC	AAGATCCAACGGGTTCTGTG	McCouch et al. (2002)
12	8,828,165	RM101	SSR	GTGAATGGTCAAGTGACTTAGGTGGC	ACACAACATGTTCCCTCCCATGC	Temnykh et al. (2000)
12	19,156,079	RM1246	SSR	AGCTCGATCCCCTAGCTCTC	TTGGAGAAGGTCACCTGCC	McCouch et al. (2002)
12	25,067,717	RM5715	SSR	GCAGAAGAGAGAAATGAAAG	CTTGTCTACGTAGCATGACA	McCouch et al. (2002)

^aPosition on rice pseudo-molecule build 4.0.

Table S2 List of markers for linkage analysis of the mutants induced from IR24

Chr.	Position^a	Marker name	Marker type	Forward primer sequence	Reverse primer sequence	Reference
1	6,273,454	RM8111	SSR	AGGTAACTAAGCTAGGTGTT	TAGGTACAGTAATACCAAGC	McCouch et al. (2002)
1	8,915,941	RM8083	SSR	GATGTGCAAATTATCA TGTGTTTTGTGAGG	ATAGTAGGCCCACA CCTGACAGGTTGTA	McCouch et al. (2002)
1	9,938,600	RM8046	SSR	AGTACGATTTCTGT CAGCGTTGCTTAGT	GGATGAAAGTTGATGG ATGATCTACTTGTT	McCouch et al. (2002)
1	10,703,670	RM23	SSR	CATTGGAGTGGAGGCTGG	GTCAGGCTTCTGCCATTCTC	Chen et al. (1997)
1	18,973,690	RM24	SSR	GAAGTGTGATCACTGTAACC	TACAGTGGACGGCGAAGTCG	Chen et al. (1997)
1	23,968,523	RM5	SSR	TGCAACTTCTAGCTGCTCGA	GCATCCGATCTTGATGGG	Panaud et al. (1996)
1	30,732,007	RM128	SSR	AGCTTGGGTGATTTCTTGGAAGCG	ACGACGAGGAGTCGCCGTGCAG	Temnykh et al. (2000)
1	38,002,959	RM6333	SSR	AGAGAAGACACGGTGGATGG	CAAACTCCTCATTTCGCTCC	McCouch et al. (2002)
1	40,161,142	RM104	SSR	GGAAGAGGAGAGAAAGATGTGTGTCG	TCAACAGACACACCGCCACCGC	Temnykh et al. (2000)
1	42,919,515	RM8136	SSR	ATGTAAGCTAGGTAGAGCTG	GCGTACGTACGTAAGTAATA	McCouch et al. (2002)
2	346,475	RM6800	SSR	CAAGCCTACATGGCCTAGACTCC	ATCCATGATCCATCATCCATGC	McCouch et al. (2002)
2	1,083,895	RM154	SSR	ACCCTCTCCGCCTCGCCTCCTC	CTCCTCCTCCTGCGACCGCTCC	Temnykh et al. (2000)
2	1,660,838	RM7033	SSR	AGAATAACTCCAGCCCACACTGG	GCGGTGATTTCTGATGACATTCC	McCouch et al. (2002)
2	3,863,789	RM3703	SSR	GAGAGAGAGGGAAGGGAAGG	GCTCCCCGACATTTAAACTG	McCouch et al. (2002)
2	4,264,267	RM4355	SSR	GGGATGAGAGTAGAAGGCA	TATATGGCAAGCCTAGCG	McCouch et al. (2002)
2	5,100,932	RM7082	SSR	TCTCCAACAGCAGCGAGG	GACCCGGCCTTCTACCTAAC	McCouch et al. (2002)
2	5,204,941	RM3294	SSR	TTACACACACTACGGACGCG	CCTGGTGGTACCTCTCTTAATC	McCouch et al. (2002)
2	5,314,413	RM1347	SSR	AACAAATTAAACTGCCAAG	GTCTTATCATCAGAACTGGA	McCouch et al. (2002)
2	6,732,637	RM5897	SSR	GGCATCTTCCCCTCTCTCTC	CCAACCCAAACCAGTCTACC	McCouch et al. (2002)
2	7,541,911	RM3505	SSR	GATGAGGTGGGACGACGAC	TCTTCACAGTGACGAAACCG	McCouch et al. (2002)
2	8,759,533	RM71	SSR	CTAGAGGCGAAAACGAGATG	GGGTGGGCGAGGTAATAATG	Temnykh et al. (2000)

2	8,980,509	RM5699	SSR	ATCGTTTCGCATATGTTT	ATCGGTAAAAGATGAGCC	McCouch et al. (2002)
2	10,186,627	RM3501	SSR	TCCTAGTGCATCAGCACAGC	GTCCGTTTCAGCAAGCAAAC	McCouch et al. (2002)
2	15,999,806	RM5812	SSR	CGCTGACATCTTGCCCTC	GTAGGACCCACGTGTCATCC	McCouch et al. (2002)
2	17,297,690	QSTS95	Indel	TGAGGACAAGCATGTGAAGG	CAATGGACGCTAGTGGAACC	This study
2	18,744,542	QSTS96	Indel	GCTCGTGTTCGTGCATCTATC	GCAACAACGAGAACGAGAAC	This study
2	17,946,057	RM6611	SSR	CACACACGCACGGTTAGATC	CTCCTCACCTCTTCCCCTTC	McCouch et al. (2002)
2	18,169,378	RM6844	SSR	CAGAGCAGGAACAGATGCTG	GTCCAAGAAAGGCACGAGAG	McCouch et al. (2002)
2	19,361,410	RM341	SSR	CAAGAAACCTCAATCCGAGC	CTCCTCCCGATCCCAATC	Temnykh et al. (2000)
2	20,996,870	RM6023	SSR	AAGGAAGCAGCGATGTGAAG	GAGCTAGAGATCACCTGGCG	McCouch et al. (2002)
2	24,518,130	QSTS109	Indel	AATATAGTTTGGTC AAAAGAACAAAAG	CGGATAATCTTGATGAGTGAACC	This study
2	26,260,867	RM3730	SSR	TGCGAGTATCTTCAAGGCAG	ATTGAGGGGGCTAATCATCC	McCouch et al. (2002)
2	27,172,434	RM5470	SSR	CATGGATTGTCTGGGCCTAG	AAGACATACCCTGAGTGTGGG	McCouch et al. (2002)
2	28,291,970	RM5631	SSR	CGTCCAAGAAATATTGCAGT	GTGAGACAGAATCCTTACGC	McCouch et al. (2002)
2	29,604,207	RM6	SSR	GTCCCCTCCACCCAATTC	TCGTCTACTGTTGGCTGCAC	Panaud et al. (1996)
2	29,655,949	RM318	SSR	GTACGGAAAACATGGTAGGAAG	TCGAGGGAAGGATCTGGTC	Temnykh et al. (2000)
2	34,709,116	RM3789	SSR	TTCCCGAATTAAGCAGATATA	CTGTAGACCATTGACTGGTG	McCouch et al. (2002)
3	8,170,550	RM5639	SSR	GGAAGAACAGAGTTGCTCGG	GTGCCATTTATTTCCGTCCC	McCouch et al. (2002)
3	12,901,320	RM5551	SSR	GACTAGTCCGGCCGTACATG	AGTTTCTTGTGTGACGCGTG	McCouch et al. (2002)
3	22,203,310	RM6832	SSR	GTTGTAAATGCCTGAGTGC	AAAGAGCTAAACCGCTAGG	McCouch et al. (2002)
3	26,547,224	RM347	SSR	CACCTCAAACTTTTAACCGCAC	TCCGGCAAGGGATACGGCGG	Temnykh et al. (2000)
3	31,185,979	RM448	SSR	TCTGATCTTGATGCAGGCAC	TCTCCCGATTTGGACAGATC	Temnykh et al. (2001)
3	33,589,206	RM7000	SSR	CCCTTCTTTTCAACTGAATA	TTGTAACAATGAACTCGTTC	McCouch et al. (2002)
4	19,913,395	RM6679	SSR	TTTAGGCCGTAAGAGCGAAC	GAATTTGAGTAGCTGGCTCC	McCouch et al. (2002)

4	28,733,259	RM303	SSR	GCATGGCCAAATATTAAAGG	GGTTGGAAATAGAAGTTCGGT	Temnykh et al. (2000)
4	32,869,409	RM348	SSR	CCGCTACTAATAGCAGAGAG	GGAGCTTTGTTCTTGCGAAC	Temnykh et al. (2000)
5	456,843	RM3796	SSR	ATTAGCCTTTAATTCCACTG	ATACAAACAAACAGCTTGTG	McCouch et al. (2002)
5	2,084,750	RM4710	SSR	AACTGGTTACAAAGACATGG	TCATCTACATATGGGGACAC	McCouch et al. (2002)
5	8,799,525	RM6082	SSR	AACCCTAGAATCGGCGCTG	CACCGATGACAACGAGGAC	McCouch et al. (2002)
5	19,107,688	RM163	SSR	ATCCATGTGCGCCTTTATGAGGA	CGCTACCTCCTTCACTTACTAGT	McCouch et al. (2002)
5	22,817,739	RM3870	SSR	TACATCTCCGGCGTTTACAC	CCAAGGTTGAAACAGGAAGC	McCouch et al. (2002)
5	26,712,707	RM2357	SSR	CCTCCGTTTCACAATGTAAC	CTGATGCTACCAGAATCCTC	McCouch et al. (2002)
5	29,161,099	RM6346	SSR	ACTTTGATCGATCAGCCACC	AGGTGGTGGAGATGAAGCAG	McCouch et al. (2002)
6	3,168,380	RM8125	SSR	CTCGTACCATCGGCTGT	AACTTACTGTGACT GACTTGGTCA	McCouch et al. (2002)
6	5,108,560	RM111	SSR	CACAACCTTTGAGCACCGGGTC	ACGCCTGCAGCTTGATCACCGG	Temnykh et al. (2000)
6	9,320,757	RM6836	SSR	TTGTTGTATACCTCATCGAC	AGGGTAAGACGTTTAACTTG	McCouch et al. (2002)
6	19,152,497	RM3	SSR	ACACTGTAGCGGCCACTG	CCTCCACTGCTCCACATCTT	Panaud et al. (1996)
6	28,216,527	RM340	SSR	GGTAAATGGACAATCCTATGGC	GACAAATATAAGGGCAGTGTGC	Temnykh et al. (2000)
6	30,349,942	RM345	SSR	ATTGGTAGCTCAATGCAAGC	GTGCAACAACCCCACATG	Temnykh et al. (2000)
7	2,711,818	RM427	SSR	TCACTAGCTCTGCCCTGACC	TGATGAGAGTTGGTTGCGAG	Temnykh et al. (2001)
7	3,174,334	RM5711	SSR	GTCCATGCATCCATCTCTAG	ACGGAAGGAATACGTCTGTA	McCouch et al. (2002)
7	5,762,184	RM6728	SSR	GGGTATGTGTCGCTATTTTA	GAAATCTGGAATTTTCCCTA	McCouch et al. (2002)
7	17,132,877	RM3795	SSR	CATTTGCATGGAGAGGATAG	TCATCTTCATTTCATTTCACC	McCouch et al. (2002)
7	22,135,427	RM10	SSR	TTGTCAAGAGGAGGCATCG	CAGAATGGGAAATGGGTCC	Panaud et al. (1996)
7	25,599,925	RM18	SSR	TTCCCTCTCATGAGCTCCAT	GAGTGCCTGGCGCTGTAC	Panaud et al. (1996)
7	28,871,445	RM1362	SSR	AAACAGGCCCTTAGTGCATG	CTACCATGGCGGCTTATGTC	McCouch et al. (2002)

8	12,288,044	RM331	SSR	GAACCAGAGGACAAAAATGC	CATCATACATTTGCAGCCAG	Temnykh et al. (2000)
8	19,002,002	RM4815	SSR	AGTAAATTTCACAAAACTTC	GTGATACAATGCATTAAATA	McCouch et al. (2002)
8	20,971,647	RM5887	SSR	CAATGATGGTGGTGAAAATC	GCTCATCTAGAAATCACCGA	McCouch et al. (2002)
8	26,140,941	RM5493	SSR	GCAGGACACAGTCACACAGG	AGATTCTTTCACCGGTGACG	McCouch et al. (2002)
8	27,834,839	RM3496	SSR	CGCTGAAAATACTGAATTGA	AGATGCATTTATTCCGAAAG	McCouch et al. (2002)
9	7,259,847	RM5526	SSR	TCAGCCTGGCCTCTCTTATC	ATGATCCTCCACCCACTAGC	McCouch et al. (2002)
9	11,694,729	RM3769	SSR	TGCATGCTTCGTTCAGCTAG	GTCTCCGAGCTCCTCAGGTC	McCouch et al. (2002)
9	14,313,180	RM5657	SSR	TATGTGCATTTGTAAGGTGA	GCTTTAGATTATTGAGCGAG	McCouch et al. (2002)
9	16,818,078	RM7175	SSR	ACAGTAAACGTGGTGCCTCC	AGAAGTAGCCTCGAGGACCC	McCouch et al. (2002)
9	21,906,494	RM7306	SSR	TCGATCCAACGCTAGCTACC	CGGAATGGGGAGGAGATC	McCouch et al. (2002)
10	17,490,576	RM6704	SSR	AATCGAATCTGGATATCTTG	CTTCTACCTAGCTACCGAGA	McCouch et al. (2002)
10	21,122,837	RM3451	SSR	CGGCGAGATAACAATTCTCC	GCGTGATGATATGGTATCGG	McCouch et al. (2002)
10	21,982,081	RM496	SSR	GACATGCGAACAACGACATC	GCTGCGGCGCTGTTATAC	Temnykh et al. (2001)
11	2,327,413	RM7557	SSR	GTGTACTGCCATGAAAGGCC	GAAGTGCCTTTGCAGGAGAG	McCouch et al. (2002)
11	8,908,333	RM202	SSR	CAGATTGGAGATGAAGTCCTCC	CCAGCAAGCATGTCAATGTA	Chen et al. (1997)
11	23,829,201	RM7277	SSR	GCTGAACGTTTCAATATGTA	GTTTGTAGGGAGTTTAATGG	McCouch et al. (2002)
11	26,668,576	RM6688	SSR	GTGCCGTTTAATACGTAGAC	AAGGAAACTTTTTTTTGCTC	McCouch et al. (2002)
12	2,181,948	RM7315	SSR	CACAAAGGCGTGTGGGTTAG	GAGTCACGGGATGTTGCC	McCouch et al. (2002)
12	7,566,745	RM2529	SSR	CATTAAAATCAGTGGGACTG	AGGCATTTCCTGATATGATC	McCouch et al. (2002)
12	19,156,079	RM1246	SSR	AGCTCGATCCCCTAGCTCTC	TTGGAGAAGGTCACCTGCC	McCouch et al. (2002)
12	21,282,401	RM1986	SSR	TAACGGAGGGAGTAGTTTTC	GAACCTACATATCGAGAGCA	McCouch et al. (2002)
12	27,420,423	RM2197	SSR	ACTGAGAACTTTAATCATCG	GAACAACTTTGAAGAGAAAC	McCouch et al. (2002)

^aPosition on rice pseudo-molecule build 4.0.

Table S3 Phenotypic characterization of necrotic mutants in rice (2016)

Mutant line	Organ	Days after sowing (DAS)^a
Mutant line	Organ	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	32	35	82
LEM 1 nec1	I^st leaf	G	G	G
	2^nd leaf	G	G	G	PG	PG/D	PG/D	PG/D	PG/D	PG/D	PG/D	PG/D	Dead	Dead
	3^rd leaf	G	G	G	PG	PG	PG	PG/D	PG/D	PG/D	PG/D	PG/D	YG/D	Dead
	4^th leaf							G	G	PG	PG	PG	PG	Dead
LEM4 nec2	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	PG	PG	PG/D	PG/D	PG/D	PG/D	PG/D	Dead	Dead
	3^rd leaf	G	G	G	G	PG	PG	PG	PG	PG	PG	PG	PG/D	Dead
	4^th leaf							G	G	PG	PG	PG	PG	Dead
LEM5 nec3	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	G	PG/D	PG/D	PG/D	D	D	D	Dead	Dead
	3^rd leaf	G	G	G	G	G	PG	PG	PG	PG/D	PG/D	PG/D	PG/D	Dead
	4^th leaf							G	G	G	G	G	PG	Dead
LEM9 nec4	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	PG	PG	PG	PG	PG	PG	PG	Dead	Dead
	3^rd leaf	G	G	G	G	PG	PG	PG	PG	PG	PG	PG	Dead	Dead
	4^th leaf									G	G	G	YG	Dead
LEM16 nec5	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	G	PG/D	YG/D	YG/D	YG/D	YG/D	YG/D	Dead	Dead
	3^rd leaf	G	G	G	G	G	PG	YG	YG	YG/D	YG/D	YG/D	YG/D	Dead
	4^th leaf							YG	YG	YG	YG	YG	YG	Dead
LEM17 nec6	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	G	PG/D	PG/D	PG/D	PG/D	PG/D	PG/D	Dead	Dead
	3^rd leaf	G	G	G	G	G	PG	PG	PG	PG/D	PG/D	PG/D	PG/D	Dead
	4^th leaf							G	G	G	G	G	PG	Dead
LEM18 nec7	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	YG/D	YG/D	YG/D	YG/D	YG/D	YG/D	YG/D	Dead	Dead
	3^rd leaf	G	G	G	G	YG	YG	YG	YG	YG	YG	YG	YG/D	Dead
	4^th leaf							G	G	G	G	G	YG	Dead
LEM24 nec8	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	PG	PG	YG/D	YG/D	YG/D	YG/D	YG/D	YG/D	Dead	Dead	Dead	Dead
	3^rd leaf	G	G	G	G	PG	PG	YG	YG	YG/D	YG/D	YG/D	YG/D	YG/D	YG/D	Dead	Dead
	4^th leaf							YG	YG	YG	YG	YG	YG	YG	YG	YG	Dead
LEM29 nec9	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	PG	PG	PG/D	PG/D	PG/D	PG/D	PG/D	Dead	Dead	Dead	Dead	Dead
	3^rd leaf	G	G	G	G	PG	PG	PG	PG	PG	PG	PG	YG/D	YG/D	YG/D	Dead	Dead
	4^th leaf							G	G	G	G	G	PG	PG	PG	PG	Dead
LEM36 nec10	I^st leaf	G	G	G
	2^nd leaf	G	G	G	G	G	PG	PG	PG	PG	PG	PG	YG/D	Dead	Dead	Dead	Dead
	3^rd leaf	G	G	G	G	G	PG	PG	PG	PG	PG	PG	YG/D	YG/D	YG/D	Dead	Dead
	4^th leaf									G	G	G	PG	PG	PG	PG	Dead

^aG, PG, YG, D and Dead represent green, pale-green, yellow green, drying and dead, respectively.

Table S4 Inferred markers linked to causal genes by bulked segregant analysis

Gene	Original cultivar	Chromosomal locations	Markers positively suggested^a
nec1	Taichung 65	2L	QSTS91, QSTS95, QSTS96, QSTS103, QSTS109
nec4	Taichung 65	2L and 2S	QSTS89, QSTS91, QSTS95, QSTS96, QSTS103, QSTS109, RM3850,
nec5	Taichung 65	2L and 2S	RM7562, RM3865, QSTS88, QSTS89, QSTS91, RM3850
nec6	Taichung 65	2L and 2S	QSTS88, QSTS89
nec7	Taichung 65	2L	QSTS89, QSTS91, QSTS95, QSTS96, QSTS103
nec9	Taichung 65	2L	QSTS91, QSTS95, QSTS96, QSTS103, QSTS109, QSTS114
nec10	Taichung 65	2L	QSTS91, QSTS95, QSTS96, QSTS103, QSTS109
nec11	Taichung 65	2L	QSTS91, QSTS95, QSTS96, QSTS103, QSTS109
nec13	IR24	2L	QSTS95, QSTS96, QSTS109
nec14	Taichung 65	4L	RM3785, RM7051, QSTS141, QSTS142
nec15	Taichung 65	4L	RM3785, RM7051, QSTS141, QSTS142
nec16	Taichung 65	4L	QSTS134, RM3785, RM7051, QSTS141, QSTS142
nec17	IR24	2S	RM3703, RM4355
nec18	Taichung 65	4L	QSTS134, QSTS141, QSTS142

^aMarker positions are indicated in Supplementary Table 1 and Table 2 for the mutants induced from Taichung 65 and IR24, respectively.

Table S5 Linkage analysis of nec17 and DNA markers on chromosome 2

Gene pair^a		Number of plants										Recombination value (%)	Map distance (cM)
A(a)	B(b)	AABB	AABb	AAbb	AaBB	AaBb	Aabb	aaBB	aaBb	aabb	Total	Recombination value (%)	Map distance (cM)
nec17	RM3703	26	44	1	_	_	_	1	0	26	98	3.1	3.1
nec17	RM4355	17	57	0	_	_	_	0	0	29	103	0.0	0.0
nec17	RM7082	20	50	2	_	_	_	0	5	23	100	6.7	6.7
nec17	RM3294	21	53	1	_	_	_	0	6	20	101	6.9	6.9
nec17	RM1347	18	54	3	_	_	_	0	7	22	104	9.2	9.3
RM3703	RM4355	16	8	1	1	43	0	0	1	26	96	6.5	6.5
RM3703	RM7082	16	8	1	4	36	2	0	6	20	93	12.6	12.9
RM3703	RM3294	17	9	1	4	38	1	0	7	17	94	13.1	13.5
RM3703	RM1347	14	11	1	4	37	3	0	8	19	97	15.7	16.3
RM4355	RM7082	15	1	0	3	49	2	0	5	23	98	5.8	5.8
RM4355	RM3294	16	1	0	3	52	1	0	6	20	99	5.8	5.8
RM4355	RM1347	14	2	0	3	51	3	0	7	22	102	7.7	7.8
RM7082	RM3294	20	0	0	0	53	1	0	3	19	96	2.1	2.1
RM7082	RM1347	17	2	0	0	54	1	0	2	23	99	2.6	2.6
RM3294	RM1347	18	2	0	0	57	2	0	1	20	100	2.5	2.5

^aCapital and lower-case letters represent the Taichung 65 alleles and IR24 alleles, respectively.

Table S6 Linkage analysis of nec1 and DNA markers on chromosome 2

Gene pair^a		Number of plants										Recombination value (%)	Map distance (cM)
A(a)	B(b)	AABB	AABb	AAbb	AaBB	AaBb	Aabb	aaBB	aaBb	aabb	Total	Recombination value (%)	Map distance (cM)
nec1	QSTS95	26	33	10	_	_	_	5	7	14	95	30.1	34.8
nec1	QSTS96	27	33	8	_	_	_	4	10	12	94	29.7	34.2
nec1	QSTS103	20	44	5	_	_	_	0	6	21	96	11.1	11.3
nec1	QSTS109	18	47	3	_	_	_	0	5	22	95	8.0	8.0
nec1	QSTS110	18	47	4	_	_	_	1	4	22	96	10.0	10.1
QSTS95	QSTS96	28	1	1	1	36	3	0	5	18	93	6.6	6.7
QSTS95	QSTS103	17	11	3	7	30	3	0	8	16	95	20.4	21.6
QSTS95	QSTS109	11	14	5	8	25	7	0	12	12	94	31.9	37.8
QSTS95	QSTS110	11	13	7	8	25	7	1	12	11	95	35.6	44.7
QSTS96	QSTS103	18	11	2	10	32	1	0	7	13	94	19.4	20.5
QSTS96	QSTS109	12	14	4	10	28	5	0	10	10	93	29.9	34.5
QSTS96	QSTS110	12	13	6	10	28	5	1	10	9	94	33.8	41.1
QSTS103	QSTS109	13	6	1	6	38	5	0	8	18	95	15.5	16.0
QSTS103	QSTS110	13	6	1	8	37	5	1	8	17	96	17.9	18.7
QSTS109	QSTS110	18	0	0	1	50	1	0	1	24	95	1.6	1.6

^aCapital and lower-case letters represent the Hinohikari and Taichung 65 alleles, respectively.

Table S7 Linkage analysis of nec13 and DNA markers on chromosome 2

Gene pair^a		Number of plants										Recombination value (%)	Map distance (cM)
A(a)	B(b)	AABB	AABb	AAbb	AaBB	AaBb	Aabb	aaBB	aaBb	aabb	Total	Recombination value (%)	Map distance (cM)
nec13	QSTS95	31	60	19	_	_	_	2	10	29	151	21.6	23.2
nec13	QSTS96	30	66	13	_	_	_	1	13	28	151	18.4	19.3
nec13	QSTS109	33	61	14	_	_	_	5	6	31	150	20.0	21.2
QSTS95	QSTS96	26	5	2	5	63	2	0	11	36	150	9.5	9.6
QSTS95	QSTS109	19	12	2	15	36	17	4	19	25	149	28.5	32.3
QSTS96	QSTS109	21	8	2	13	44	20	4	14	23	149	25.6	28.3

^aCapital and lower-case letters represent the Taichung 65 alleles and IR24 alleles, respectively.

Table S8 Linkage analysis of nec16 and DNA markers on chromosome 4

Gene pair^a		Number of plants										Recombination value (%)	Map distance (cM)
A(a)	B(b)	AABB	AABb	AAbb	AaBB	AaBb	Aabb	aaBB	aaBb	aabb	Total	Recombination value (%)	Map distance (cM)
nec16	QSTS132	28	27	16	_	_	_	2	8	9	90	34.2	41.8
nec16	QSTS134	28	27	11	_	_	_	1	8	9	84	28.2	31.9
nec16	RM3785	21	50	0	_	_	_	0	0	19	90	0.0	0.0
nec16	RM7051	21	52	0	_	_	_	0	0	19	92	0.0	0.0
nec16	QSTS141	18	37	17	_	_	_	3	6	9	90	37.8	49.3
nec16	QSTS142	19	38	16	_	_	_	3	7	9	92	36.9	47.3
QSTS132	QSTS134	26	4	0	1	27	2	2	4	16	82	9.5	9.6

QSTS132	RM3785	9	18	2	6	21	8	6	9	9	88	39.5	53.7
QSTS132	RM7051	9	19	2	6	21	8	6	10	9	90	39.9	54.7
QSTS132	QSTS141	7	14	8	7	13	14	7	14	4	88	53.2
QSTS132	QSTS142	7	15	8	8	14	13	7	14	4	90	53.2
QSTS134	RM3785	9	18	1	6	20	8	2	9	9	82	33.6	40.8
QSTS134	RM7051	9	19	1	6	21	8	2	9	9	84	33.7	40.9
QSTS134	QSTS141	9	13	7	7	12	14	3	14	3	82	48.3	101.1
QSTS134	QSTS142	9	13	7	7	14	14	3	14	3	84	48.2	100.2
RM3785	RM7051	21	0	0	0	50	0	0	0	19	90	0.0	0.0
RM3785	QSTS141	12	7	2	6	28	15	3	6	9	88	29.9	34.5
RM3785	QSTS142	12	7	2	7	29	14	3	7	9	90	30.0	34.7
RM7051	QSTS141	12	7	2	6	30	15	3	6	9	90	29.4	33.7
RM7051	QSTS142	12	7	2	7	31	14	3	7	9	92	29.5	33.8
QSTS141	QSTS142	21	0	0	0	43	0	1	0	25	90	1.1	1.1

^aCapital and lower-case letters represent the Hinohikari and Taichung 65 alleles, respectively.

Table S9 Linkage analysis of nec18 and DNA markers on chromosome 4

Gene pair^a		Number of plants										Recombination value (%)	Map distance (cM)
A(a)	B(b)	AABB	AABb	AAbb	AaBB	AaBb	Aabb	aaBB	aaBb	aabb	Total	Recombination value (%)	Map distance (cM)
nec18	QSTS132	16	33	11	_	_	_	5	10	7	82	43.0	64.6
nec18	QSTS134	11	30	18	_	_	_	4	10	7	80	48.8	109.7
nec18	RM3785	22	36	1	_	_	_	0	12	9	80	18.0	18.8
nec18	RM7051	23	37	1	_	_	_	0	14	9	84	19.6	20.7
nec18	QSTS141	30	30	0	_	_	_	0	3	20	83	4.0	4.0
nec18	QSTS142	30	31	0	_	_	_	0	3	19	83	4.0	4.0
QSTS132	QSTS134	10	6	3	5	28	9	0	4	13	78	21.8	23.4
QSTS132	RM3785	7	10	2	15	22	5	0	15	2	78	40.2	55.4
QSTS132	RM7051	8	11	2	15	23	5	0	16	2	82	39.5	53.5
QSTS132	QSTS141	8	9	4	16	16	10	5	8	5	81	45.9	79.0
QSTS132	QSTS142	8	9	4	16	17	9	5	8	5	81	45.8	78.0
QSTS134	RM3785	9	4	1	9	23	7	4	17	2	76	40.5	56.4
QSTS134	RM7051	9	5	1	10	23	7	4	19	2	80	41.5	59.4
QSTS134	QSTS141	4	8	3	15	14	10	10	10	5	79	53.9
QSTS134	QSTS142	4	8	3	15	15	9	10	10	5	79	54.1
RM3785	RM7051	22	0	0	0	48	0	0	0	10	80	0.0	0.0
RM3785	QSTS141	14	7	0	14	25	9	0	1	9	79	21.9	23.5
RM3785	QSTS142	14	8	0	14	25	9	0	1	8	79	22.8	24.6
RM7051	QSTS141	15	7	0	15	25	11	0	1	9	83	22.9	24.8
RM7051	QSTS142	15	8	0	15	25	11	0	1	8	83	23.7	25.8
QSTS141	QSTS142	30	0	0	0	33	0	0	0	19	82	0.0	0.0

^aCapital and lower-case letters represent the Hinohikari and Taichung 65 alleles, respectively.

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