OlCHR, encoding a chromatin remodeling factor, is a killer causing hybrid sterility between rice species Oryza sativa and O. longistaminata.

The genetic mechanisms of reproductive isolation have been widely investigated within Asian cultivated rice (Oryza sativa); however, relevant genes between diverged species have been in sighted rather less. Herein, a gene showing selfish behavior was discovered in hybrids between the distantly related rice species Oryza longistaminata and O. sativa. The selfish allele S13l in the S13 locus impaired male fertility, discriminately eliminating pollens containing the allele S13s from O. sativa in heterozygotes (S13s/S13l). Genetic analysis revealed that a gene encoding a chromatin-remodeling factor (CHR) is involved in this phenomenon and a variety of O. sativa owns the truncated gene OsCHR745, whereas its homologue OlCHR has a complete structure in O. longistaminata. CRISPR-Cas9-mediated loss of function mutants restored fertility in hybrids. African cultivated rice, which naturally lacks the OlCHR homologue, is compatible with both S13s and S13l carriers. These results suggest that OlCHR is a Killer gene, which leads to reproductive isolation.

The induced mutant allele flo4-303 confers floury characteristics on the japonica rice cultivar 'Hoshinoko'.

Rice flour is useful as a substitute for wheat flour, however, to obtain fine flour, millers need special milling facilities, which increase the cost of milling. To reduce the milling cost, we developed a floury mutant line by irradiating gamma-rays to dry seeds of the japonica cultivar 'Hoshinoyume'. The line was registered as a new cultivar, 'Hoshinoko'. Genetical analysis of the floury trait was conducted using an F2 population derived from a cross between 'Hoshinoko' and 'Corbetti' (a japonica rice cultivar with normal endosperm), which indicated the involvement of a single recessive gene located near the RM163 marker on the long arm of rice chromosome 5, flanking flo4 identified by Kang et al. (2005). Sequence analysis of flo4 showed a two-bp (CA) insertion in the eighth exon of in 'Hoshinoko' compared to that of 'Hoshinoyume', which led to a frameshift mutation. The CAPS-based genotype of flo4 gene completely correlated to the phenotype of endosperm in two populations. This CAPS marker could be helpful for rice breeders to develop new cultivars harboring floury endosperm of the flo4-303 gene.

Genetic region responsible for the differences of starch properties in two glutinous rice cultivars in Hokkaido, Japan.

Starch properties are major determinants of grain quality and food characteristics in rice (Oryza sativa L.). Control of starch properties will lead to the development of rice cultivars with desirable characteristics. We performed quantitative trait locus analysis and detected a putative region on chromosome 2 associated with phenotypic variation of starch properties in two glutinous rice varieties developed in the Hokkaido region of Japan: 'Kitayukimochi', which has a low pasting temperature and creates soft rice cakes, and 'Shirokumamochi', which has a high pasting temperature and creates hard rice cakes. Starch branching enzyme IIb (SbeIIb) was identified as a candidate gene within the region. Sequence analysis of SbeIIb in parental lines identified two single-nucleotide polymorphisms (SNPs) with non-synonymous mutations in the coding region of the 'Shirokumamochi' genotype (SbeIIbsr ). We genotyped over 100 rice cultivars, including 28 rice varieties in the Honshu region of Japan, using the CAPS marker, which was designed using one of the SNPs. However, SbeIIbsr was not found in rice cultivars in Honshu. Distribution analysis indicated that SbeIIbsr was introduced to the rice breeding population in Hokkaido from the American variety 'Cody' via the Hokkaido cultivar 'Kitaake'. As a result, SbeIIbsr was distributed only in progenies of 'Kitaake'.

A novel genotype DATTO5 developed using the five genes exhibits the fastest heading date designed in rice.

The optimization of heading date is a key aspect for maximizing grain productivity in cereal crops including rice. The combinations of genes for heading date, a quantitative trait, are a major driver in the wide adaptability of cultivated rice worldwide. Here, we identified a novel QTL, qDTH3 (quantitative trait locus for days-to-heading on chromosome 3), for early flowering time in the F2 population derived from a cross between Hoshinoyume (HS) and Daichinohoshi (DH) among local rice populations with extremely early heading date. The DH allele at qDTH3, qDTH3DH , headed 2.7 days earlier than the HS allele at qDTH3, qDTH3HS . We sought to design a genotype for earlier heading date by pyramiding of five heading date genes. We designated this aggregate of the five genes as DATTO5. Plants with DATTO5 were selected from the F2 population derived from a cross between DH and HShd5, which is a near-isogenic line carrying a loss-of-function of days to heading 8 in a genetic background of HS. Plants with DATTO5 exhibited earlier heading date but reduced fitness, including shorter culm and panicle length and fewer seeds compared with HS, as a representative local rice variety with extremely early heading date.

Introgression of the chromosomal region with the Pi-cd locus from Oryza meridionalis into O. sativa L. during rice domestication.

KEY MESSAGE: The genotype of the Pi-cd locus found in blast-resistant rice variety Kitakurin, which is a cultivated rice from Japan belonging to Oryza sativa japonica, is identical to that of its wild relative O. meridionalis. Crop domestication from wild relatives to cultivated species has encompassed significant phenotypic changes. However, little is known about the genetic changes involved in domestication. Here, we surveyed the origin of the Pi-cd locus across Oryza species with AA genomes by comparison with the genome sequences of Hoshinoyume (HS), which does not carry the Pi-cd blast resistance gene, and Kitakurin (KK), which carries the Pi-cd blast resistance gene. We found that variety-specific transposons were enriched at the Pi-cd locus. The genotype of the Pi-cd locus characterized by transposons in HS and KK was specific to each Oryza species with the AA genome. The Kitaake (KT) genotype at the Pi-cd locus found in KK was identical only to that of O. meridionalis and distributed only in subgroups of japonica in the World Rice Collection and tropical japonica in the Japanese Rice Collection, whereas it was not present in O. rufipogon accessions. The distinct distributions of genotypes of the Pi-cd locus clearly demonstrated that the Pi-cd locus was introgressed from O. meridionalis into O. sativa, specific to tropical japonica.

Establishment of adaptability to the northern-limit of rice production.

The domestication of cultivated crops from their wild relatives narrowed down their genetic diversity in a bottleneck effect. Subsequently, the cultivation areas of crops have expanded all over the world into various environmental conditions from the original area along with human migration after domestication. Here, we demonstrated the genetic changes in the adaptation of rice to Hokkaido (41°2-45°3N latitude), Japan, from the tropics of their origin in Asian cultivated rice, Oryza sativa L. Although cultivated rice originated from the tropics, Hokkaido is one of the northern-limits of rice cultivation worldwide. Population genomics focusing on the local populations showed the varieties had genetically distinct classes with limited genetic diversity. In addition, some varieties in the class carried unique genotypes for flowering time, exhibiting extremely early flowering time. Certain mutations in unique genotypes can split off the varieties that are able to grow in Hokkaido. Furthermore, the changes in the genotype for flowering time during rice cultivation in Hokkaido demonstrated novel combinations of genes for flowering time owing to the intensive artificial selection on natural variation and rice breeding programs to achieve stable rice production in Hokkaido.

Colocalization of QTLs for hull-cracked rice and grain size in elite rice varieties in Japan.

The control of insects that consume cereal grains is important for the production and storage of grains. Hull-cracked rice, which has splits in the hull, becomes more susceptible to insects both in the paddy field and during storage. The development of varieties with a low frequency of hull-cracked rice is the most economical and effective strategy to avoid insect damage and the environmental risks from agricultural chemical entering rice grains. In this study, we identified that QTLs for the frequency of hull-cracked rice and for grain width are located on the same chromosome using recombinant inbred lines derived from a cross between the elite rice varieties in Hokkaido, Japan, which are from the same pedigree and are genetically closely related. These QTLs were detected close to different molecular markers, which were separated by 1,101,675 bp, on chromosome 5 in the reference Nipponbare genome. In addition, low coefficient values of the phenotype were found between hull-cracked rice and grain size. These results suggested that the ratio of hull-cracked rice is independent of grain size. Using these QTLs, new varieties with low hull-cracked rice could be developed regardless of grain size.

The superwoman1-cleistogamy2 mutant is a novel resource for gene containment in rice.

Outcrossing between cultivated plants and their related wild species may result in the loss of favourable agricultural traits in the progeny or escape of transgenes in the environment. Outcrossing can be physically prevented by using cleistogamous (i.e. closed-flower) plants. In rice, flower opening is dependent on the mechanical action of fleshy organs called lodicules, which are generally regarded as the grass petal equivalents. Lodicule identity and development are specified by the action of protein complexes involving the SPW1 and OsMADS2 transcription factors. In the superwoman1-cleistogamy1 (spw1-cls1) mutant, SPW1 is impaired for heterodimerization with OsMADS2 and consequently spw1-cls1 shows thin, ineffective lodicules. However, low temperatures help stabilise the mutated SPW1/OsMADS2 heterodimer and lodicule development is restored when spw1-cls1 is grown in a cold environment, resulting in the loss of the cleistogamous phenotype. To identify a novel, temperature-stable cleistogamous allele of SPW1, targeted and random mutations were introduced into the SPW1 sequence and their effects over SPW1/OsMADS2 dimer formation were assessed in yeast two-hybrid experiments. In parallel, a novel cleistogamous allele of SPW1 called spw1-cls2 was isolated from a forward genetic screen. In spw1-cls2, a mutation leading to a change of an amino acid involved in DNA binding by the transcription factor was identified. Fertility of spw1-cls2 is somewhat decreased under low temperatures but unlike for spw1-cls1, the cleistogamous phenotype is maintained, making the line a safer and valuable genetic resource for gene containment.

Genetic shift in local rice populations during rice breeding programs in the northern limit of rice cultivation in the world.

The rapid accumulation of pre-existing mutations may play major roles in the establishment and shaping of adaptability for local regions in current rice breeding programs. The cultivated rice, Oryza sativa L., which originated from tropical regions, is now grown worldwide due to the concerted efforts of breeding programs. However, the process of establishing local populations and their origins remain unclear. In the present study, we characterized DNA polymorphisms in the rice variety KITAAKE from Hokkaido, one of the northern limits of rice cultivation in the world. Indel polymorphisms were attributed to transposable element-like insertions, tandem duplications, and non-TE deletions as the original mutation events in the NIPPONBARE and KITAAKE genomes. The allele frequencies of the KITAAKE alleles markedly shifted to the current variety types among the local population from Hokkaido in the last two decades. The KITAAKE alleles widely distributed throughout wild rice and cultivated rice over the world. These have accumulated in the local population from Hokkaido via Japanese landraces as the ancestral population of Hokkaido. These results strongly suggested that combinations of pre-existing mutations played a role in the establishment of adaptability. This approach using the re-sequencing of local varieties in unique environmental conditions will be useful as a genetic resource in plant breeding programs in local regions.

Genome-wide association mapping focusing on a rice population derived from rice breeding programs in a region.

Plant breeding programs in local regions may generate genetic variations that are desirable to local populations and shape adaptability during the establishment of local populations. To elucidate genetic bases for this process, we proposed a new approach for identifying the genetic bases for the traits improved during rice breeding programs; association mapping focusing on a local population. In the present study, we performed association mapping focusing on a local rice population, consisting of 63 varieties, in Hokkaido, the northernmost region of Japan and one of the northern limits of rice cultivation worldwide. Six and seventeen QTLs were identified for heading date and low temperature germinability, respectively. Of these, 13 were novel QTLs in this population and 10 corresponded to the QTLs previously reported based on QTL mapping. The identification of QTLs for traits in local populations including elite varieties may lead to a better understanding of the genetic bases of elite traits. This is of direct relevance for plant breeding programs in local regions.