Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

A cell wall-localized cytokinin/purine riboside nucleosidase is involved in apoplastic cytokinin metabolism in Oryza sativa.

In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.

Duplication of a manganese/cadmium transporter gene reduces cadmium accumulation in rice grain.

Global contamination of soils with toxic cadmium (Cd) is a serious health threat. Here we found that a tandem duplication of a gene encoding a manganese/Cd transporter, OsNramp5, was responsible for low-Cd accumulation in Pokkali, an old rice cultivar. This duplication doubled the expression of OsNramp5 gene but did not alter its spatial expression pattern and cellular localization. Higher expression of OsNramp5 increased uptake of Cd and Mn into the root cells but decreased Cd release to the xylem. Introgression of this allele into Koshihikari, an elite rice cultivar, through backcrossing significantly reduced Cd accumulation in the grain when cultivated in soil heavily contaminated with Cd but did not affect both grain yield and eating quality. This study not only reveals the molecular mechanism underlying low-Cd accumulation but also provides a useful target for breeding rice cultivars with low-Cd accumulation.

Late flowering in F1 hybrid rice brought about by the complementary effect of quantitative trait loci.

Late flowering sometimes occurs in F1 hybrids between rice varieties (Oryza sativa L.), although the parental varieties show similar days-to-flowering (DTF). The genetic architecture prompting the occurrence of such late flowering is poorly understood. To clarify the genetic architecture of late flowering in F1 hybrids from a cross between rice varieties, 'Koshihikari' and 'IR64', we performed quantitative trait locus (QTL) analysis using an F2 population (selfed progeny of an F1 plant), in which heterozygous genotypes should segregate in a certain proportion in a Mendelian manner. The QTL analysis detected three significant QTLs. At one QTL (putatively Heading date 1), the 'Koshihikari' allele increased DTF, and at the other two QTLs (putatively Heading date 6 and Oryza sativa Pseudo-Response Regulator 37/Heading date 2), the 'IR64' alleles increased DTF. All alleles at these three QTLs showed partial dominance. The combination of the QTLs explained 82.2% of the total phenotypic variance of DTF in the F2 population, with contribution from epistasis between QTLs. There was no difference between DTFs of F1 hybrids and heterozygous genotypes for the three QTLs. Our results demonstrated that the complementary effects accompanied by epistasis of at least three QTLs were responsible for late flowering in F1 hybrids.

Rapid DNA-genotyping system targeting ten loci for resistance to blast disease in rice.

The fungal pathogen Pyricularia oryzae causes blast, a severe disease of rice (Oryza sativa L.). Improving blast resistance is important in rice breeding programs. Inoculation tests have been used to select for resistance genotypes, with DNA marker-based selection becoming an efficient alternative. No comprehensive DNA marker system for race-specific resistance alleles in the Japanese rice breeding program has been developed because some loci contain multiple resistance alleles. Here, we used the Fluidigm SNP genotyping platform to determine a set of 96 single nucleotide polymorphism (SNP) markers for 10 loci with race-specific resistance. The markers were then used to evaluate the presence or absence of 24 resistance alleles in 369 cultivars; results were 93.5% consistent with reported inoculation test-based genotypes in japonica varieties. The evaluation system was successfully applied to high-yield varieties with indica genetic backgrounds. The system includes polymorphisms that distinguish the resistant alleles at the tightly linked Pita and Pita-2 loci, thereby confirming that all the tested cultivars with Pita-2 allele carry Pita allele. We also developed and validated insertion/deletion (InDel) markers for ten resistance loci. Combining SNP and InDel markers is an accurate and efficient strategy for selection for race-specific resistance to blast in breeding programs.

Genetic dissection of pre-harvest sprouting resistance in an upland rice cultivar.

Seed dormancy is important in rice breeding because it confers resistance to pre-harvest sprouting (PHS). To detect quantitative trait loci (QTLs) for pre-harvest sprouting resistance, we used chromosome segment substitution lines (CSSLs) derived from a cross between the Japanese upland rice cultivar 'Owarihatamochi' and the lowland rice cultivar 'Koshihikari'. In the CSSLs, several chromosomal regions were associated with PHS resistance. Among these, the chromosome 9 segment from 'Owarihatamochi' had the greatest association with increased PHS resistance. Further QTL analysis using an advanced backcross population (BC4F2) derived from a 'Koshihikari' × 'Owarihatamochi' cross revealed two putative QTLs, here designated qSDR9.1 (Seed dormancy 9.1) and qSDR9.2, on chromosome 9. The 'Owarihatamochi' alleles of the two QTLs reduced germination. Further fine mapping revealed that qSDR9.1 and qSDR9.2 were located within 4.1-Mb and 2.3-Mb intervals (based on the 'Nipponbare' reference genome sequence) defined by the simple sequence repeat marker loci RM24039 and RM24260 and Indel_2 and RM24540, respectively. We thus identified two QTLs for PHS resistance in 'Owarihatamochi', even though resistance levels are relatively low in this cultivar. This unexpected finding suggests the advantages of using CSSLs for QTL detection.

Fine Mapping of QUICK ROOTING 1 and 2, Quantitative Trait Loci Increasing Root Length in Rice.

The volume that the root system can occupy is associated with the efficiency of water and nutrient uptake from soil. Genetic improvement of root length, which is a limiting factor for root distribution, is necessary for increasing crop production. In this report, we describe identification of two quantitative trait loci (QTLs) for maximal root length, QUICK ROOTING 1 (QRO1) on chromosome 2 and QRO2 on chromosome 6, in cultivated rice (Oryza sativa L.). We measured the maximal root length in 26 lines carrying chromosome segments from the long-rooted upland rice cultivar Kinandang Patong in the genetic background of the short-rooted lowland cultivar IR64. Five lines had longer roots than IR64. By rough mapping of the target regions in BC4F2 populations, we detected putative QTLs for maximal root length on chromosomes 2, 6, and 8. To fine-map these QTLs, we used BC4F3 recombinant homozygous lines. QRO1 was mapped between markers RM5651 and RM6107, which delimit a 1.7-Mb interval on chromosome 2, and QRO2 was mapped between markers RM20495 and RM3430-1, which delimit an 884-kb interval on chromosome 6. Both QTLs may be promising gene resources for improving root system architecture in rice.

Improvement of seed shattering and dormancy in Oryza sativa L. 'Hokuriku 193' based on genetic information.

In this study, we investigated the genetic basis of seed shattering and dormancy in Hokuriku 193 and bred an NIL improved these traits. Analysis of an F3 population from Hokuriku 193 × Koshihikari revealed a general correspondence between seed shattering and genotypes at the qSH1 locus, suggesting a strong influence of this locus on the seed shattering in Hokuriku 193. An F2 population from [ms-bo] Nekken 2 × Hokuriku 193 was also analyzed to identify quantitative trait loci (QTLs) for seed dormancy as measured by germination rate in the first December and March after seed harvest. The results revealed a concurrence QTLs of on chromosomes 1, 3, and 6 (qSDo1, qSDo3, qSDo6). In particular, qSDo1 and qSDo6 were considered regions worthy of active modification because they were QTL regions that promoted seed dormancy when carrying Hokuriku 193 genome regions around. SSDo_NIL, a near isogenic line (NIL) derived from Hokuriku 193 by introgressing Nekken 2 alleles only at the qSH1 locus and qSDo1, did not shatter, and its germination rate was significantly higher than that of Hokuriku 193. Yield performance was similar between SSDo_NIL and Hokuriku 193, suggesting that improvement of seed shattering and dormancy does not affect yield.

Quantitative trait loci analysis of blast resistance in Oryza sativa L. 'Hokuriku 193'.

To investigate the genetic background responsible for blast resistance in Oryza sativa L. 'Hokuriku 193', QTL analysis was conducted using the F3 lines from the cross [ms-bo] Nekken 2 × Hokuriku 193 that were artificially infected with rice blast fungus (Magnaporthe grisea). QTLs were detected on chromosomes 1, 4, 6 and 12 that correlated with greater blast resistance in the Hokuriku 193-type lines. Notably, the QTL on chromosome 12 had a major effect and localized to the same region where Pi20(t), a broad-spectrum blast resistance gene, is positioned, suggesting strongly that the blast resistance of Hokuriku 193 was controlled by Pi20(t). Also, QTL analysis of the lines found to have no Pi20(t) detected two QTLs on chromosome 4 (qBR4-1 and qBR4-2) and one QTL on chromosome 6 (qBR6), of which qBR4-2 and qBR6 correlated with higher percentages of resistant plants in the Hokuriku 193-type lines. The blast susceptibility of BR_NIL (a NIL of Hokuriku 193 from which Pi20(t) was eliminated) was greater than that of Hokuriku 193, suggesting that elimination of Pi20(t) may markedly increase blast susceptibility. The disease severity of BR_NIL was mild, which might be the effect of qBR4-2 and/or qBR6.

Precise estimation of genomic regions controlling lodging resistance using a set of reciprocal chromosome segment substitution lines in rice.

Severe lodging has occurred in many improved rice varieties after the recent strong typhoons in East and Southeast Asian countries. The indica variety Takanari possesses strong culm characteristics due to its large section modulus, which indicates culm thickness, whereas the japonica variety Koshihikari is subject to substantial bending stress due to its thick cortical fibre tissue. To detect quantitative trait loci (QTLs) for lodging resistance and to eliminate the effects of genetic background, we used reciprocal chromosome segment substitution lines (CSSLs) derived from a cross between Koshihikari and Takanari. The oppositional effects of QTLs for section modulus were confirmed in both genetic backgrounds on chromosomes 1, 5 and 6, suggesting that these QTLs are not affected by the genetic background and are controlled independently by a single factor. The candidate region of a QTL for section modulus included SD1. The section modulus of NIL-sd1 was lower than that of Koshihikari, whereas the section modulus of NIL-SD1 was higher than that of Takanari. This result indicated that those regions regulate the culm thickness. The reciprocal effects of the QTLs for cortical fibre tissue thickness were confirmed in both genetic backgrounds on chromosome 9 using CSSLs.

Hd18, Encoding Histone Acetylase Related to Arabidopsis FLOWERING LOCUS D, is Involved in the Control of Flowering Time in Rice.

Flowering time is one of the most important agronomic traits in rice (Oryza sativa L.), because it defines harvest seasons and cultivation areas, and affects yields. We used a map-based strategy to clone Heading date 18 (Hd18). The difference in flowering time between the Japanese rice cultivars Koshihikari and Hayamasari was due to a single nucleotide polymorphism within the Hd18 gene, which encodes an amine oxidase domain-containing protein and is homologous to Arabidopsis FLOWERING LOCUS D (FLD). The Hayamasari Hd18 allele and knockdown of Hd18 gene expression delayed the flowering time of rice plants regardless of the day-length condition. Structural modeling of the Hd18 protein suggested that the non-synonymous substitution changed protein stability and function due to differences in interdomain hydrogen bond formation. Compared with those in Koshihikari, the expression levels of the flowering-time genes Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and Rice flowering locus T1 (RFT1) were lower in a near-isogenic line with the Hayamasari Hd18 allele in a Koshihikari genetic background. We revealed that Hd18 acts as an accelerator in the rice flowering pathway under both short- and long-day conditions by elevating transcription levels of Ehd1 Gene expression analysis also suggested the involvement of MADS-box genes such as OsMADS50, OsMADS51 and OsMADS56 in the Hd18-associated regulation of Ehd1 These results suggest that, like FLD, its rice homolog accelerates flowering time but is involved in rice flowering pathways that differ from the autonomous pathways in Arabidopsis.

The Sorghum Gene for Leaf Color Changes upon Wounding (P) Encodes a Flavanone 4-Reductase in the 3-Deoxyanthocyanidin Biosynthesis Pathway.

Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L.) Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP) and tan Greenleaf (pp) cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol) to flavan-4-ols (apiforol or luteoforol) in vitro Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway.

Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat.

Detection of QTLs for cold tolerance of rice cultivar 'Kuchum' and effect of QTL pyramiding.

KEY MESSAGE: A QTL for cold tolerance at the booting stage of rice cultivar 'Kuchum' was detected and delimited into a 1.36 Mb region, and a cold-tolerant line was developed by QTL pyramiding. Low temperature in summer causes pollen sterility in rice, resulting in a serious loss of yield. The second most widely grown rice cultivar in Japan, 'Hitomebore', has been developed as a cultivar highly tolerant to low temperature at the booting stage. However, even 'Hitomebore' exhibits sterility at a temperature lower than 18.5 °C. Further improvement of cold tolerance of rice is required. In the present study, QTLs for cold tolerance in a Bhutanese rice variety, 'Kuchum', were analyzed using backcrossed progenies and a major QTL, named qCT-4, was detected on chromosome 4. Evaluating cold tolerance of seven types of near isogenic lines having 'Kuchum' alleles around qCT-4 with a 'Hitomebore' genetic background, qCT-4 was delimited to a region of ca. 1.36 Mb between DNA markers 9_1 and 10_13. Homozygous 'Kuchum' alleles at qCT-4 showed an effect of increasing seed fertility by ca. 10 % under cold-water treatment. Near isogenic lines of 'Hitomebore' having 'Silewah' alleles of Ctb1 and Ctb2 and a 'Hokkai PL9' allele of qCTB8 did not exhibit higher cold tolerance than that of 'Hitomebore'. On the other hand, a qLTB3 allele derived from a Chinese cultivar 'Lijiangxintuanheigu' increased cold tolerance of 'Hitomebore', and pyramiding of the qCT-4 allele and the qLTB3 allele further increased seed fertility under cold-water treatment. Since NILs of 'Hitomebore' with the 'Kuchum' allele of qCT-4 were highly similar to 'Hitomebore' in other agronomic traits, the qCT-4 allele is considered to be useful for developing a cold-tolerant cultivar.

Advanced backcross QTL analysis reveals complicated genetic control of rice grain shape in a japonica × indica cross.

Grain shape is an important trait for improving rice yield. A number of quantitative trait loci (QTLs) for this trait have been identified by using primary F2 mapping populations and recombinant inbred lines, in which QTLs with a small effect are harder to detect than they would be in advanced generations. In this study, we developed two advanced mapping populations (chromosome segment substitution lines [CSSLs] and BC4F2 lines consisting of more than 2000 individuals) in the genetic backgrounds of two improved cultivars: a japonica cultivar (Koshihikari) with short, round grains, and an indica cultivar (IR64) with long, slender grains. We compared the ability of these materials to reveal QTLs for grain shape with that of an F2 population. Only 8 QTLs for grain length or grain width were detected in the F2 population, versus 47 in the CSSL population and 65 in the BC4F2 population. These results strongly suggest that advanced mapping populations can reveal QTLs for agronomic traits under complicated genetic control, and that DNA markers linked with the QTLs are useful for choosing superior allelic combinations to enhance grain shape in the Koshihikari and IR64 genetic backgrounds.

Detection of QTLs for white-back and basal-white grains caused by high temperature during ripening period in japonica rice.

There is increasing evidence that global warming affects the development of rice. High temperatures during ripening increase the ratio of undesirable chalky grains followed by deteriorating grain appearance quality. In order to detect quantitative trait loci (QTLs) controlling the occurrence of white-back and basal-white chalky grains of brown rice, QTL analysis was performed using recombinant inbred lines derived from a cross between two strains, 'Tsukushiroman' (sensitive to heat stress) and 'Chikushi 52' (tolerant of heat stress). The F7 and F8 lines were exposed to heat stress during the ripening period in two locations, Fukuoka and Kagoshima, in Japan. QTLs for white-back grains and basal-white grains were detected on chromosomes 1, 3, and 8, and those for basal-white grains were detected on chromosomes 2, 3, and 12. QTLs on chromosome 8 for white-back grains were shared in the plants grown in both locations. Near-isogenic lines (NILs), which harbored a segment from 'Chikushi 52' on chromosome 8 with the genetic background of 'Tsukushiroman', showed relatively lower ratios of white-back grains than 'Tsukushiroman'. Therefore, insertion of the 'Chikushi 52' genomic region of the QTL on chromosome 8 can improve the quality of rice when it is grown under heat stress conditions.

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar 'Koshihikari'.

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultivar 'Koshihikari' was performed to extract regions containing large indels (10-51 bp) shared by diverse cultivars. To design indel markers for the discrimination of genomic regions between 'Koshihikari' and other diverse cultivars, we subtracted the indel regions detected in 'Koshihikari' from those shared in other cultivars. Two sets of indel markers, KNJ8-indel (shared in eight or more cultivars, including 'Khao Nam Jen' as a representative tropical japonica cultivar) and C5-indel (shared in five to eight cultivars), were established, with 915 and 9,899 indel regions, respectively. Validation of the two marker sets by using 23 diverse cultivars showed a high PCR success rate (≥95%) for 83.3% of the KNJ8-indel markers and 73.9% of the C5-indel markers. The marker sets will therefore be useful for the effective breeding of Japanese rice cultivars.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism.

Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait locus (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding.

Chromosomal locations of a gene underlying heat-accelerated brown spot formation and its suppressor genes in rice.

Brown spots on mature leaves from the heading to ripening stages in rice are considered to be lesions induced by heat stress. However, there are few studies of lesions that are induced by heat stress rather than by pathogen infections. To understand the genetic background underlying such lesions, we used the chromosome segment substitution line (CSSL) SL518, derived from a distant cross between rice cultivars Koshihikari (japonica) and Nona Bokra (indica). We observed brown spots on mature leaf blades of the CSSL, although the parents barely showed any spots. Spot formation in SL518 was accelerated by high temperature, suggesting that the candidate gene for spot formation is related to heat stress response. Using progeny derived from a cross between SL518 and Koshihikari, we mapped the causative gene, BROWN-SPOTTED LEAF 1 (BSPL1), on chromosome 5. We speculated that one or more Nona Bokra genes suppress spot formation caused by BSPL1 and identified candidate genomic regions on chromosomes 2 and 9 using a cross between a near-isogenic line for BSPL1 and other CSSLs possessing Nona Bokra segments in the Koshihikari genetic background. In conclusion, our data support the concept that multiple genes are complementarily involved in brown spot formation induced by heat stress and will be useful for cloning of the novel gene(s) related to the spot formation.