Fine-scale genetic structure of the rice landrace population in Japan.

Rice cultivation was introduced into Japan 3000 years ago and has expanded across the country and encompasses a wide variety of environmental conditions. Here, we elucidated the differentiation of the genetic population structure of 1037 rice landraces across Japan. Using 4451 polymorphisms derived from genome-wide analysis of double-digest restriction-site-associated DNA analysis, population genomics including ADMIXTURE and principal component analysis was carried out. These landraces were classified into nine subpopulations based on geographical origin. Massive-scale genotyping and diversity analysis demonstrated that the differentiation of genetic population structure in rice landraces across Japan might consist of two phases, namely western to eastern and southern to northern phases. The differentiation of genetic population structure was detected only in landraces from three geographical regions, Hokuriku, Tohoku, and Hokkaido, as the southern to northern phase. Conversely, differentiation was not observed in landraces from six geographical regions, Kyushu, Shikoku, Chugoku, Kinki, Tokai, and Kanto, as the western to eastern phase. The genetic population structure may have facilitated the expansion of genetic diversity among local regions.

Artificial selection in the expansion of rice cultivation.

KEY MESSAGE: Gene distributions and population genomics suggest artificial selection of ghd7 osprr37, for extremely early heading date of rice, in the Tohoku region of Japan. The ranges of cultivated crops expanded into various environmental conditions around the world after their domestication. Hokkaido, Japan, lies at the northern limit of cultivation of rice, which originated in the tropics. Novel genotypes for extremely early heading date in Hokkaido are controlled by loss-of-function of both Grain number, plant height and heading date 7 (Ghd7) and Oryza sativa Pseudo-Response Regulator 37 (OsPRR37). We traced genotypes for extremely early heading date and analyzed the phylogeny of rice varieties grown historically in Japan. The mutations in Ghd7 and OsPRR37 had distinct local distributions. Population genomics revealed that varieties collected from the Tohoku region of northern Japan formed three clusters. Mutant alleles of Ghd7 and OsPRR37 appear to have allowed rice cultivation to spread into Hokkaido. Our results show that the mutations of two genes might be occurred in the process of artificial selection during early rice cultivation in the Tohoku region.

Translation of continuous artificial selection on phenotype into genotype during rice breeding programs.

Understanding genetic diversity among local populations is a primary goal of modern crop breeding programs. Here, we demonstrated the genetic relationships of rice varieties in Hokkaido, Japan, one of the northern limits of rice cultivation around the world. Furthermore, artificial selection during rice breeding programs has been characterized using genome sequences. We utilized 8,565 single nucleotide polymorphisms and insertion/deletion markers distributed across the genome in genotype-by-sequencing for genetic diversity analyses. Phylogenetics, genetic population structure, and principal component analysis showed that a total of 110 varieties were classified into four distinct clusters according to different populations geographically and historically. Furthermore, the genome sequences of 19 rice varieties along with historic representations in Hokkaido, nucleotide diversity and FST values in each cluster revealed that artificial selection of elite phenotypes focused on chromosomal regions. These results clearly demonstrated the history of the selections on agronomic traits as genome sequences among current rice varieties from Hokkaido.

Days to heading, controlled by the heading date genes, Hd1 and DTH8, limits rice yield-related traits in Hokkaido, Japan.

A key aspect of rice breeding programs is the optimization of days to heading (DTH) for maximizing grain productivity in cultivation areas. Here, the effects of genotypes for heading date on yield-related traits in rice (culm and panicle length (CL and PL), panicle number (PN), and total number of seeds) were investigated. Heading date 1 (Hd1) and Days to heading 8 (DTH8) are the main controllers of the variation in heading date in the rice population of Hokkaido, Japan. Thus, an F2 population (n = 192) derived from a cross between Kitaibuki (Hd1dth8) and Akage (hd1DTH8) was developed. Significant differences in DTH were found among all combinations. Each genotype for heading date showed variations in the yield-related traits without a significant difference. However, DTH exhibited high positive coefficient values (more than 0.709) with the yield-related traits except for PN, which had a negative coefficient value of -0.431. A later heading date resulted in a longer growth duration and a higher yield with a combination of longer PL and CL and lower PN. These results suggest that DTH limits the yield-related traits rather than the genotype for heading date.

Genetic effect of a new allele for the flowering time locus Ghd7 in rice.

The optimization of flowering time is a key aspect in maximizing grain productivity in rice. Allelic variations in genes for flowering time are major drivers in the wide adaptability of cultivated rice around the world. Here, we identified a novel allele of flowering time gene Grain number, plant height and heading date 7 (Ghd7). Loss-of-function ghd7, Ghd7-0a, is important for extremely early flowering time for adaptability to cultivation in Hokkaido, Japan. However, the rice variety Sorachi lacks a key functional nucleotide polymorphism of Ghd7, which results in a loss of function of the gene. Based on the sequence of Ghd7 allele in Sorachi, we identified the insertion of a transposon-like sequence at an upstream site of Ghd7. Segregation analysis using an F2 population derived from the cross between Hoshinoyume and Sorachi demonstrated that the Ghd7 locus contributed to extremely early flowering time in Sorachi. This Ghd7 allele in Sorachi showed a weak function in terms of delay of flowering time, compared with loss-of-function allele, and a distinct distribution in northern Japan.

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.

Marker-assisted selection in rice breeding programs in Hokkaido.

Rice breeding programs in Hokkaido over the past 100 years have dramatically increased productivity and improved the eating quality of rice. Commercial varieties with high yield and good eating quality, such as Kirara 397, Hoshinoyume, and Nanatsuboshi, have been continuously registered since 1990. Furthermore, varieties with better eating quality using Wx1-1, which reduces amylose content to improve the taste of sticky rice, such as Oborozuki and Yumepirika, were registered in 2006 and 2008, respectively. However, to the best of our knowledge the genomic changes associated with these improvements have not been determined. Better understanding of the relationships between DNA sequences and agricultural traits could facilitate rice breeding programs in Hokkaido. Marker-assisted selection (MAS), which can select the plants with chromosomal regions tagged with DNA markers for desirable traits, is an advanced technology to manage genetic improvements. Here, we summarize the current states of MAS in rice breeding programs in Hokkaido before huge data sets of genome sequences using next-generation sequencing technology come into practical use in rice breeding programs.

Switching genetic effects of the flowering time gene Hd1 in LD conditions by Ghd7 and OsPRR37 in rice.

Flowering time control in plants is a major limiting factor on the range of species. Day length, perceived via the photoperiodic pathway, is a critical factor for the induction of flowering. The module of GIGANTEA (GI)-CONSTANS (CO)-FLOWERING LOCUS T in the long day (LD) plant Arabidopsis is conserved in diverse plant species including the short day (SD) plant rice, where this module comprises OsGI-Heading date 1 (Hd1)-Heading date 3a. Hd1, the rice ortholog of Arabidopsis CO, has dual functions in the regulation of flowering time, promoting flowering in SD conditions and delaying it in LD conditions. We herein show genetic interactions among three LD repressor genes: Hd1, Grain number, plant height and heading date 7 (Ghd7), and Oryza sativa Pseudo-Response Regulator37 (OsPRR37). Genetic analyses, including segregation analyses, evaluations of near isogenic lines, and transformation for flowering time demonstrated that Hd1 promoted flowering time in inductive SD and non-inductive LD conditions in genetic condition of loss-of-function Ghd7 and OsPRR37 (ghd7osprr37) in rice. Functional Ghd7 or OsPRR37 may switch the genetic effects of Hd1 from the promotion to the delay of flowering times in LD conditions.

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.

Phenotypic changes during 100-year rice breeding programs in Hokkaido.

Plant breeding programs in local regions may have genetic and phenotypic variations that are desirable and shape adaptability during the establishment of local populations. Despite the characterization of genetic population structures in various kinds of populations, the effects of variations in phenotype on agro-economical traits currently remain unclear. In the present study, we evaluated phenotypic changes in 26 agro-economical traits among the local population during rice breeding programs in Hokkaido. Wide variations were observed in all 26 agro-economical traits with continuous distributions. In order to elucidate improvements in these agro-economic traits during rice breeding programs in Hokkaido, values were compared between genetic population structures. Traits were classified into four patterns based on the timing of significant differences. Patterns A and B showed significant differences once and twice, respectively. Pattern C gradually showed significant differences. Pattern D showed no significant differences for the desired directions. Based on the changes in phenotype observed in the present study and the genetic population structure for the local population in Hokkaido, a model of the artificial selection for phenotypes in genetic diversity among the local population during plant breeding programs has been proposed.

[Serum ALP elevation early after treatment is a predictor for response in myeloma patients treated with bortezomib].

Several studies have shown the predictive value of elevated serum alkaline phosphatase (ALP) level in multiple myeloma (MM) patients treated with bortezomib (BTZ). We assessed the relationship between changes in ALP levels during treatment and response. Thirty patients treated with BTZ in our hospital were analyzed retrospectively. Of the patients analyzed, 12 were male, median age was 62 years (42-86), and 11 had a history of prior chemotherapy. Eighteen patients were treated with BTZ alone or in combination with dexamethasone, while the others were treated with a combination regimen employing an alkylating agent. Seven patients had undergone autologous stem cell transplantation following BTZ therapy. Ten of 28 patients showed ALP elevation of 25% or more from the baseline at 3 weeks, and 14 of the 28 had this finding at 6 weeks. Four of 5 patients who had achieved VGPR or more showed ALP elevation of 25% or more at 3 weeks, and all five had this finding by 6 weeks. No patient without ALP elevation achieved VGPR or a better response. ALP elevation exceeding 25% from the baseline by day 42 is significantly associated with a treatment response better than VGPR (p=0.019). In conclusion, ALP elevation during BTZ treatment is a valuable prognostic marker.

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.

Quantitative trait loci for rice blast resistance detected in a local rice breeding population by genome-wide association mapping.

Plant breeding programs aim to develop cultivars with high adaptability to the specific conditions in a local region. As a result, unique genes and gene combinations have been accumulated in local elite breeding populations during the long history of plant breeding. Genetic analyses on such genes and combinations may be useful for developing new cultivars with more-desirable agronomic traits. Here, we attempted to detect quantitative trait loci (QTL) for rice blast resistance (BR) using a local breeding rice population from Hokkaido, Japan. Using genotyping data on single nucleotide polymorphisms and simple sequence repeat markers distributed throughout the whole genomic region, we detected genetic regions associated with phenotypic variation in BR by a genome-wide association mapping study (GWAS). An additional association analysis using other breeding cultivars verified the effect and inheritance of the associated region. Furthermore, the existence of a gene for BR in the associated region was confirmed by QTL mapping. The results from these studies enabled us to estimate potential of the Hokkaido rice population as a gene pool for improving BR. The results of this study could be useful for developing novel cultivars with vigorous BR in rice breeding programs.

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.

Diversification of the plant-specific hybrid glycine-rich protein (HyGRP) genes in cereals.

Plant-specific hybrid proline- or glycine-rich proteins (HyP/GRPs) are involved in diverse gene functions including plant development and responses to biotic and abiotic stresses. The quantitative trait locus, qLTG3-1, enhances seed germination in rice under low-temperature conditions and encodes a member with a glycine-rich motif of the HyP/GRP family. The function of this gene may be related to the weakening of tissue covering the embryo during seed germination. In the present study, the diversification of the HyP/GRP gene family was elucidated in rice based on phylogenetic relationships and gene expression levels. At least 21 members of the HyP/GRP family have been identified in the rice genome and clustered in five regions on four chromosomes by tandem and chromosomal duplications. Of these, OsHyPRP05 (qLTG3-1) and its paralogous gene, OsHyPRP21, had a glycine-rich motif. Furthermore, orthologous genes with a glycine-rich motif and the HyP/GRP gene family were detected in four genome-sequenced monocots: 12 in barley, 10 in Brachypodium, 20 in maize, and 28 in sorghum, using a BLAST search of qLTG3-1 as the query. All members of the HyP/GRP family in these five species were classified into seven main groups, which were clustered together in these species. These results suggested that the HyP/GRP gene family was formed in the ancestral genome before the divergence of these species. The collinearity of chromosomal regions around qLTG3-1 and its orthologous genes were conserved among rice, Brachypodium, sorghum, and maize, indicating that qLTG3-1 and orthologous genes conserve gene function during seed germination.

QTL pyramiding for improving of cold tolerance at fertilization stage in rice.

Vigorous cold tolerance at the fertilization stage (CTF) is a very important characteristic for stable rice production in cold temperature conditions. Because CTF is a quantitatively inherited trait, pyramiding quantitative trait loci (QTLs) using marker-assisted selection (MAS) is effective for improving CTF levels in rice breeding programs. We previously identified three QTLs controlling CTF, qCTF7, qCTF8 and qCTF12, using backcrossed inbred lines derived from a cross between rice cultivar Eikei88223 (vigorous CTF) and Suisei (very weak CTF). However, pyramiding of these QTLs for the application of MAS in practical rice breeding programs have not yet been elucidated. In this study, we examined the effect of pyramiding QTLs for improvement of CTF level using eight possible genotype classes from the 152 F3 population derived from a cross between Eikei88223 and Suisei. Increasing of CTF levels in combinations between qCTF7 and qCTF12 and between qCTF8 and qCTF12 were detected. Furthermore, we compared the haplotype pattern around the QTLs for CTF among the rice cultivars from Hokkaido. These results are useful for improvement of new cultivars with high CTF levels using MAS and identification of genetic resources with the novel QTL(s) for CTF.

Historical changes in population structure during rice breeding programs in the northern limits of rice cultivation.

KEY MESSAGE: The rice local population was clearly differentiated into six groups over the 100-year history of rice breeding programs in the northern limit of rice cultivation over the world. Genetic improvements in plant breeding programs in local regions have led to the development of new cultivars with specific agronomic traits under environmental conditions and generated the unique genetic structures of local populations. Understanding historical changes in genome structures and phenotypic characteristics within local populations may be useful for identifying profitable genes and/or genetic resources and the creation of new gene combinations in plant breeding programs. In the present study, historical changes were elucidated in genome structures and phenotypic characteristics during 100-year rice breeding programs in Hokkaido, the northern limit of rice cultivation in the world. We selected 63 rice cultivars to represent the historical diversity of this local population from landraces to the current breeding lines. The results of the phylogenetic analysis demonstrated that these cultivars clearly differentiated into six groups over the history of rice breeding programs. Significant differences among these groups were detected in five of the seven traits, indicating that the differentiation of the Hokkaido rice population into these groups was correlated with these phenotypic changes. These results demonstrated that breeding practices in Hokkaido have created new genetic structures for adaptability to specific environmental conditions and breeding objectives. They also provide a new strategy for rice breeding programs in which such unique genes in local populations in the world can explore the genetic potentials of the local populations.

Genetical and morphological characterization of cold tolerance at fertilization stage in rice.

Cold temperature during the reproductive phase leads to seed sterility, which reduces yield and decreases the grain quality of rice. The fertilization stage, ranging from pollen maturation to the completion of fertilization, is sensitive to unsuitable temperature. Improving cold tolerance at the fertilization stage (CTF) is an important objective of rice breeding program in cold temperature areas. In this study, we characterized fertilization behavior under cold temperature to define the phenotype of CTF and identified quantitative trait loci (QTLs) for CTF. A wide variation in CTF levels has been identified among local cultivars in Hokkaido, which is one of the most northern regions for rice cultivation in the world. Clear varietal differences in pollen germination, and pollen tube elongation due to cold temperature have been observed. These differences may confer a degree of CTF among this population. We conducted QTL analysis for CTF using 120 backcrossed inbred lines derived from a cross between Eikei88223 (vigorous CTF) and Suisei (very weak CTF). Three QTLs for CTF were identified. A clear effect by QTL, qCTF7, for increasing the level of CTF was validated using advanced progeny. These results will facilitate marker-assist selection for desirable QTLs for CTF in rice breeding program.