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

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 lethality1 (nec1) to nec18. 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 H2O2 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 H2O2 accumulation throughout the third leaf. Mutants for late necrotic lethality (>35 days to lethality) were characterized by the incomplete spread of H2O2 accumulation within the third leaf.


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 (H 2 O 2 ), 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, H 2 O 2 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 H 2 O 2 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 H 2 O 2 accumulation and cell death.

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 Co 60 γ-ray irradiation facility of Institute for Irradiation and Analysis of Quantum Radiation, Kyushu University.Self-pollinated M 1 plants were grown and M 2 seeds were harvested and used to generate M 3 plants.Self

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).

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 F 2 generation.
Markers showing polymorphism between the two bulks were selected and used for linkage analysis of the F 2 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.

ROS Accumulation
To observe excessive accumulation of ROS in necrotic mutants, we used DAB staining to assess H 2 O 2 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 in-American Journal of Plant Sciences cubation in 96% ethanol at 80˚C for 1 h.The leaf samples were preserved in 70% ethanol and later photographed.

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.

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).

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 nec17.1, 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.

Bulked Segregant Analysis
BSA of 14 mutant lines revealed that in the F 2 population between nec1 and Hinohikari, wild-type and mutant plants segregated in a 69:27 ratio, fitting a 3:1 American Journal of Plant Sciences 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).

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 F 1 progeny of crosses between nec1 and nec13.If nec1 and nec13 are located at different loci, F 1 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 F 1 derived from the cross between the heterozygous plants in three replicates, indicating that nec1 and nec13 are located at the same locus (Table

Histochemical Characterization of Lethal Necrosis
Of the eighteen mutants, fourteen were initially characterized for their H 2 O 2 accumulation of the third leaves at 12 DAS (Supplementary Figure S2).

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]  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 lethal-American Journal of Plant Sciences ity within 21 DAS, with excessive H 2 O 2 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.

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

Figure 1 .
Figure 1.Three representative mutants for necrotic lethality.(a)-(c) Early necrotic lethal mutant, nec1.(a) nec1 (right) shows pale-green discoloration in second and third leaves at 7 days after sowing (DAS) while wild-type plant (left) possesses second and third leaves with green color; (b) Leaf-tip necrosis in nec1 (right) spread to entire leaf blade of second leaf at 12 DAS; (c) Complete death of whole plant of nec1 (right) occurs at 16 -19 DAS.(d) nec18 survived in paddy field until 82 DAS; (e)-(f) Transverse bands of necrosis in nec17.(a)-(c), (e) Left and right plants were wild types and mutants, respectively.

Figure 3 .
Figure 3. ROS accumulation and cell death in the third-leaves of necrotic mutants at 12 DAS.(a)-(e) Morphological features of wild type (left) and necrotic mutants (right); (f)-(o) Histochemical staining in the third leaf blade of wild type (left) and mutant (right) seedlings at 12 DAS by (f)-(j) DAB staining of H 2 O 2 and (k)-(o) trypan blue staining for cell death.Bar = 5 cm.Left and right plants were wild types and mutants, respectively.

nec1I
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/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/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/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/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 Dead Dead 3 rd leaf G G G G G PG PG PG PG PG PG YG/D YG/D YG/D Dead Dead , YG, D and Dead represent green, pale-green, yellow green, drying and dead, respectively.

Figure S1 .
Figure S1.Detection of DNA markers linked to nec1 locus on chromosome 2 by bulked segregant analysis.

Figure S2 .
Figure S2.Characterization of mutants for necrotic lethality in rice by DAB staining.
-pollination and screening of these mutant stocks were conducted and the resulting mutant R. S. Mbaraka et al.
DOI: 10.4236/ajps.2017.8132263352AmericanJournal of Plant Sciences stocks were maintained in the Plant Breeding Laboratory, Kyushu University.These mutants were designed as LEM (lethal mutants) (Table1

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 Identification of genes responsible for necrotic lethality in the 18 mutant lines.
a Probability in χ 2 test of a 3:1 ratio.b Observations on the third leaves of mutant at 12 days after sowing.

Table 2
. Segregation of wild-type and mutant plants in F 2 populations.a. Probability in χ 2 test of a 3:1 ratio.
a. Genotypes are represented in parentheses.

Table 1
(Figures3(k)-(o)), but not in the wild-type plants.Taken together, these results suggest that excessive accumulation of H 2 O 2 caused necrotic symptoms in the mutants.

Table S1 .
12 DASshowed 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 (nec1 and nec13) were characterized by the rapid spread of H 2 O 2 accumulation throughout the third leaf.Mutants for late necrotic lethality (nec16, nec17 and nec18) were characterized by the incomplete spread of H 2 O 2 accumulation within the third leaf.List of markers for linkage analysis of mutants induced from Taichung 65.

Table S2 .
List of markers for linkage analysis of the mutants induced from IR24.
a Position on rice pseudo-molecule build 4.0.

Table S4 .
Inferred markers linked to causal genes by bulked segregant analysis.
a Marker positions are indicated in Supplementary Table1and Table2for the mutants induced from Taichung 65 and IR24, respectively.

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

Table S6 .
Linkage analysis of nec1 and DNA markers on chromosome 2.
a Capital and lower-case letters represent the Hinohikari and Taichung 65 alleles, respectively.

Table S7 .
Linkage analysis of nec13 and DNA markers on chromosome 2.
a Capital 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.Capital and lower-case letters represent the Hinohikari and Taichung 65 alleles, respectively.

Table S9 .
Linkage analysis of nec18 and DNA markers on chromosome 4.
0 American Journal of Plant Sciences