MORE PANICLES 3, a natural allele of OsTB1/FC1, impacts rice yield in paddy fields at elevated CO2 levels.

Improving crop yield potential through an enhanced response to rising atmospheric CO2 levels is an effective strategy for sustainable crop production in the face of climate change. Large-sized panicles (containing many spikelets per panicle) have been a recent ideal plant architecture (IPA) for high-yield rice breeding. However, few breeding programs have proposed an IPA under the projected climate change. Here, we demonstrate through the cloning of the rice (Oryza sativa) quantitative trait locus for MORE PANICLES 3 (MP3) that the improvement in panicle number increases grain yield at elevated atmospheric CO2 levels. MP3 is a natural allele of OsTB1/FC1, previously reported as a negative regulator of tiller bud outgrowth. The temperate japonica allele advanced the developmental process in axillary buds, moderately promoted tillering, and increased the panicle number without negative effects on the panicle size or culm thickness in a high-yielding indica cultivar with large-sized panicles. The MP3 allele, containing three exonic polymorphisms, was observed in most accessions in the temperate japonica subgroups but was rarely observed in the indica subgroup. No selective sweep at MP3 in either the temperate japonica or indica subgroups suggested that MP3 has not been involved and utilized in artificial selection during domestication or breeding. A free-air CO2 enrichment experiment revealed a clear increase of grain yield associated with the temperate japonica allele at elevated atmospheric CO2 levels. Our findings show that the moderately increased panicle number combined with large-sized panicles using MP3 could be a novel IPA and contribute to an increase in rice production under climate change with rising atmospheric CO2 levels.

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.

Two dominant genes in barley (Hordeum vulgare L.) complementarily encode perfect resistance to Japanese soil-borne wheat mosaic virus.

Japanese soil-borne wheat mosaic virus (Furovirus) is a damaging pathogen of wheat and barley. This virus can survive in the soil for several decades, so the deployment of resistant cultivars represents the only practical control measure. Here, a genetic analysis has identified two regions of the barley genome-one on chromosome 2H and the other on chromosome 3H-as harboring gene(s) encoding resistance to this virus. The joint presence of both loci, termed Jmv1 and Jmv2, made the plants essentially immune, with resistance being dominant over susceptibility at each locus. Phylogenetic analysis showed that the virus is not closely related to the type Furovirus species Soil-borne wheat mosaic virus. There was a difference between the RNA1- and RNA2-based phylogenies of the virus species in Furovirus implying the independent segregation of the virus subgenomes.

Genetic Elucidation for Response of Flowering Time to Ambient Temperatures in Asian Rice Cultivars.

Climate resilience of crops is critical for global food security. Understanding the genetic basis of plant responses to ambient environmental changes is key to developing resilient crops. To detect genetic factors that set flowering time according to seasonal temperature conditions, we evaluated differences of flowering time over years by using chromosome segment substitution lines (CSSLs) derived from japonica rice cultivars "Koshihikari" × "Khao Nam Jen", each with different robustness of flowering time to environmental fluctuations. The difference of flowering times in 9 years' field tests was large in "Khao Nam Jen" (36.7 days) but small in "Koshihikari" (9.9 days). Part of this difference was explained by two QTLs. A CSSL with a "Khao Nam Jen" segment on chromosome 11 showed 28.0 days' difference; this QTL would encode a novel flowering-time gene. Another CSSL with a segment from "Khao Nam Jen" in the region around Hd16 on chromosome 3 showed 23.4 days" difference. A near-isogenic line (NIL) for Hd16 showed 21.6 days' difference, suggesting Hd16 as a candidate for this QTL. RNA-seq analysis showed differential expression of several flowering-time genes between early and late flowering seasons. Low-temperature treatment at panicle initiation stage significantly delayed flowering in the CSSL and NIL compared with "Koshihikari". Our results unravel the molecular control of flowering time under ambient temperature fluctuations.

Evaluation of major rice cultivars for resistance to bacterial seedling rot caused by Burkholderia glumae and identification of Japanese standard cultivars for resistance assessments.

Burkholderia glumae causes bacterial seedling rot (BSR) and bacterial grain rot (BGR) in rice (Oryza sativa), both of which are important diseases in Japan. We previously evaluated major Japanese cultivars for BGR resistance and selected standard cultivars for resistance assessments. Here, we assessed the BSR occurrence rate in cultivars from the World Rice Collection (WRC) and other sources and found wide variation in resistance. Next, we evaluated major Japanese cultivars for BSR resistance and found that two Japanese landraces, 'Kujuu' and 'Aikoku', showed "strong" resistance; most others were categorized as "medium" or "medium to weak". We previously developed a near-isogenic line (RBG1-NIL) by introducing the genomic region containing RBG1, a quantitative trait locus (QTL) for BSR resistance, from 'Nona Bokra' (indica) into 'Koshihikari' (temperate japonica). The resistance level of RBG1-NIL was "strong", indicating the effectiveness of RBG1 against BSR. The correlation between BSR and BGR resistance scores was low, indicating that it is necessary to introduce QTLs for resistance from different sources to develop cultivars resistant to both BSR and BGR. On the basis of the screening results, we selected standard cultivars for BSR resistance to cover a range of resistance levels.

Genomic adaptation of flowering-time genes during the expansion of rice cultivation area.

The diversification of flowering time in response to natural environments is critical for the spread of crops to diverse geographic regions. In contrast with recent advances in understanding the molecular basis of photoperiodic flowering in rice (Oryza sativa), little is known about how flowering-time diversification is structured within rice subspecies. By analyzing genome sequencing data and a set of 429 chromosome segment substitution lines (CSSLs) originating from 10 diverse rice accessions with wide distributions, we revealed diverse effects of allelic variations for common flowering-time quantitative trait loci in the recipient's background. Although functional variations associated with a few loci corresponded to standing variations among subspecies, the identified functional nucleotide polymorphisms occurred recently after rice subgroup differentiation, indicating that the functional diversity of flowering-time gene sequences was not particularly associated with phylogenetic relationship between rice subspecies. Intensive analysis of the Hd1 genomic region identified the signature of an early introgression of the Hd1 with key mutation(s) in aus and temperate japonica accessions. Our data suggested that, after such key introgressions, new mutations were selected and accelerated the flowering-time diversity within subspecies during the expansion of rice cultivation area. This finding may imply that new genome-wide changes for flowering-time adaptation are one of the critical determinants for establishing genomic architecture of local rice subgroups. In-depth analyses of various rice genomes coupling with the genetically confirmed phenotypic changes in a large set of CSSLs enabled us to demonstrate how rice genome dynamics has coordinated with the adaptation of cultivated rice during the expansion of cultivation area.

Detection of heading date QTLs in advanced-backcross populations of an elite indica rice cultivar, IR64.

IR64 is one of the world's most popular rice cultivars. To collect genetic factors involved in controlling its heading date, we developed 70 reciprocal advanced-backcross populations with a total of 6284 individuals at the BC4F2 generation from crosses between Koshihikari and IR64. We detected 29 QTLs associated with heading date on chromosomes 3, 5-8, 10, and 12. Twenty QTLs were located in the same chromosome regions as previously isolated heading date genes (Hd1, Hd6, Hd16, Ghd7, DTH8, Hd17, and Hd18). The rest were located in other chromosome regions. We found more number of QTLs than previous studies using mapping populations of IR64. Fine mapping in additional advanced-backcross populations clearly revealed that QTLs on the long arm of chromosome 7 are overlapping and seem to be a novel genetic factor for heading date because of their different locations from OsPRR37. Our results suggest that the difference in heading date between IR64 and Koshihikari is genetically controlled by many factors, and that a non-functional allele of Hd1 contributes to early heading of IR64 in the genetic background of functional alleles of other heading date QTLs and genes such as Hd6 and Hd16.

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.

Evaluation of major Japanese rice cultivars for resistance to bacterial grain rot caused by Burkholderia glumae and identification of standard cultivars for resistance.

Bacterial grain rot (BGR), caused by the bacterial pathogen Burkholderia glumae, is one of the most destructive rice (Oryza sativa) diseases in Japan; however, there are no BGR-resistant cultivars for use in Japan. We previously developed a cut-panicle inoculation method to assess the levels of BGR resistance in the World Rice Collection (WRC). Here, we evaluated major Japanese cultivars for BGR resistance and found that none showed "strong" or "medium to strong" resistance; most were categorized as "medium to weak". On the basis of the screening results, standard cultivars for BGR resistance were selected according to resistance level and relative maturity. Our results indicate that it is necessary to introduce quantitative trait loci (QTLs) from indica or tropical japonica resistant cultivars into Japanese temperate japonica to develop BGR-resistant cultivars for Japan. We previously developed a near-isogenic line (RBG2-NIL) by introducing the genomic region containing RBG2 from 'Kele' (indica) into 'Hitomebore'. In this experiment, we confirmed the resistance level of RBG2-NIL. The resistance score of RBG2-NIL was "medium to strong", indicating its effectiveness against BGR.

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.

Quantitative trait loci analysis of grain appearance in Oryza sativa L. 'Emi-no-kizuna'.

A quantitative trait locus (QTL) analysis was conducted on grain appearance in Emi-no-kizuna, a rice cultivar that has superior and stable appearance of the brown rice grain and high tolerance to high temperature stress, by using F3 lines derived from Emi-no-kizuna and Tomohonami. The investigation was performed 2013 and 2015. In summer 2013 the air temperature was higher and the larger differences in grain appearance were observed. In the QTL analysis, a highly contributing locus, qGA8, was detected at the end of the short arm of chromosome 8. Because trends of reduced the occurrence of white immature kernel and increased the percentage of perfect grain were observed in Emi-no-kizuna genotype in both years, qGA8 is likely to be an important QTL that is dominant in the superior grain appearance of Emi-no-kizuna. Also, qGA8 was linked to a QTL associated with days to heading. Another QTL, qGA7, associated with grain appearance was detected on chromosome 7 in 2013. Because no negative correlation was found between the genotype of qGA8 and thousand grain weight, it should be possible to breed cultivars that possess Emi-no-kizuna genotype qGA8 without a decrease in thousand grain weight.

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.

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.

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.

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 to reduce cadmium content in rice grains using LAC23/Koshihikari chromosome segment substitution lines.

To advance the identification of quantitative trait loci (QTLs) to reduce Cd content in rice (Oryza sativa L.) grains and breed low-Cd cultivars, we developed a novel population consisting of 46 chromosome segment substitution lines (CSSLs) in which donor segments of LAC23, a cultivar reported to have a low grain Cd content, were substituted into the Koshihikari genetic background. The parental cultivars and 32 CSSLs (the minimum set required for whole-genome coverage) were grown in two fields with different natural levels of soil Cd. QTL mapping by single-marker analysis using ANOVA indicated that eight chromosomal regions were associated with grain Cd content and detected a major QTL (qlGCd3) with a high F-test value in both fields (F = 9.19 and 5.60) on the long arm of chromosome 3. The LAC23 allele at qlGCd3 was associated with reduced grain Cd levels and appeared to reduce Cd transport from the shoots to the grains. Fine substitution mapping delimited qlGCd3 to a 3.5-Mbp region. Our results suggest that the low-Cd trait of LAC23 is controlled by multiple QTLs, and qlGCd3 is a promising candidate QTL to reduce the Cd level of rice grain.

A MAPKKK gene from rice, RBG1res, confers resistance to Burkholderia glumae through negative regulation of ABA.

Burkholderia glumae causes bacterial seedling rot (BSR) of rice and is a threat to a consistent food supply. When previously screening for resistance against B. glumae in the resistant cultivar Nona Bokra (NB) versus the susceptible cultivar Koshihikari (KO), we detected a gene, Resistance to Burkholderia glumae 1 (RBG1), at a quantitative trait locus (QTL). Here, we found that RBG1 encodes a MAPKKK gene whose product phosphorylates OsMKK3. We also found that the kinase encoded by the RBG1 resistant (RBG1res) allele in NB presented higher activity than did that encoded by the RBG1 susceptible (RBG1sus) allele in KO. RBG1res and RBG1sus differ by three single-nucleotide polymorphisms (SNPs), and the G390T substitution is essential for kinase activity. Abscisic acid (ABA) treatment of inoculated seedlings of RBG1res-NIL (a near-isogenic line (NIL) expressing RBG1res in the KO genetic background) decreased BSR resistance, indicating that RBG1res conferred resistance to B. glumae through negative regulation of ABA. The results of further inoculation assays showed that RBG1res-NIL was also resistant to Burkholderia plantarii. Our findings suggest that RBG1res contributes to resistance to these bacterial pathogens at the seed germination stage via a unique mechanism.

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.

Relationship between transmission ratio distortion and genetic divergence in intraspecific rice crosses.

The strength of reproductive isolation often correlates positively with parental divergence in both animals and plants. Here, we assess the relationship between transmission ratio distortion (TRD) of marker loci and parental divergence in intraspecific rice (Oryza sativa L.) crosses. We produced 10 diverse F(2) populations by crossing a temperate japonica reference accession with each of 10 donor accessions belonging to subpopulations different from the reference accession, and then genotyped the F(2) populations using molecular markers distributed across the entire genome. Significant TRDs (α = 0.05) were detected in 9 of the 10 F(2) populations. TRD regions on chromosomes 3 and 6 were common to several populations; in contrast, other TRD regions were unique to a single population, indicating the diversification of genomic location of TRDs among the populations. The level of TRD (estimated from the overall number and magnitude of TRDs) was significantly correlated with the genetic distance between the donor accessions and the reference accession. Our results suggest that in intraspecific rice crosses, parental divergence may result in diversification of the TRD pattern, followed by an increase in the level of TRD.