Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

Identification of a unique allele in the quantitative trait locus for crown root number in japonica rice from Japan using genome-wide association studies.

To explore the genetic resources that could be utilized to help improve root system architecture phenotypes in rice (Oryza sativa), we have conducted genome-wide association studies to investigate maximum root length and crown root number in 135 10-day-old Japanese rice accessions grown hydroponically. We identified a quantitative trait locus for crown root number at approximately 32.7 Mbp on chromosome 4 and designated it qNCR1 (quantitative trait locus for Number of Crown Root 1). A linkage disequilibrium map around qNCR1 suggested that three candidate genes are involved in crown root number: a cullin (LOC_Os04g55030), a gibberellin 20 oxidase 8 (LOC_Os04g55070), and a cyclic nucleotide-gated ion channel (LOC_Os04g55080). The combination of haplotypes for each gene was designated as a haploblock, and haploblocks 1, 2, and 3 were defined. Compared to haploblock 1, the accessions with haploblocks 2 and 3 had fewer crown roots; approximately 5% and 10% reductions in 10-day-old plants and 15% and 25% reductions in 42-day-old plants, respectively. A Japanese leading variety Koshihikari and its progenies harbored haploblock 3. Their crown root number could potentially be improved using haploblocks 1 and 2.

Validation of a quantitative trait locus for the white-core expression rate of grain on chromosome 6 in a brewing rice cultivar and development of DNA markers for marker-assisted selection.

Sake-brewing cultivars among varieties of Japanese rice (Oryza sativa L.) have traits adapted to the sake-brewing process, such as a high white-core expression rate (WCE). Our previous study detected putative quantitative trait loci (QTLs) associated with a high WCE derived from Yamadanishiki, a popular brewing rice cultivar. Because the occurrence of white-core grains depends on air temperature and the position of the grain on the panicle, phenotyping of WCE must consider these variable conditions. In this study, qWCE6, a QTL for the WCE on chromosome 6, was validated for the first time, and the phenotyping method examined for its suitability in fine-mapping. A clear tendency towards high WCE was observed in late-heading substituted lines which headed under low daily mean temperature at the experimental location. White-core grains were often expressed by the primary spikelets on the upper panicle, producing a high percentage of superior grains. The segregating population for qWCE6 in late heading revealed a distinct difference in WCE between the Koshihikari and Yamadanishiki homozygous alleles at qWCE6 as determined from that locality. Further, two insertion/deletion markers were developed for the marker-assisted selection of qWCE6. Our results will be useful for informing the breeding of sake-brewing rice cultivars.

Genetic and phenotypic assessment of sugar beet (Beta vulgaris L. subsp. vulgaris) elite inbred lines selected in Japan during the past 50 years.

Genetic diversity of Japanese sugar beet elite inbred line diversity (JSBDIV) set consisting of 63 lines was investigated using 33 cleaved amplified polymorphic sequence and 38 simple sequence repeat analyses. JSBDIV set was significantly subdivided into six (pedigree information), seven (Neighbor-Joining method) or 12 (population structure analysis) groups. The highest value of a pairwise population differentiation estimate, Φ PT value, among groups was yielded from population structure analysis with explained variation 32%. Some of the groups defined in this study exhibited close association with ancestral open-pollinated varieties (OPVs), suggesting that inter-OPV cross was rare during the establishment of JSBDIV set. On the other hand, low Φ PT values between some groups suggest that genetic backgrounds of ancestral OPVs had historically overlapped to some extent. Phenotypic traits showed significant differences both among and within groups. A nearly identical group was identified as the highest sugar content group irrespective of the grouping methods. Groups with Aphanomyces root rot resistance are associated with an OPV 'Tmm-1', suggesting it as a source of this trait. 'Tmm-1' is also associated with Cercospora leaf spot resistance, but an exceptional resistant line with no association of 'Tmm-1' supports a notion that different genetic resources exist for this trait.

QTL analysis of resistance to bacterial wilt caused by Ralstonia solanacearum in potato.

Ralstonia solanacearum causes bacterial wilt, a soil-borne disease and one of the most important maladies of potato and other Solanaceae crops. We analyzed the resistance of a potato clone to bacterial wilt by quantitative trait locus (QTL) analysis. A resistant diploid potato clone 10-03-30 was crossed with a susceptible diploid clone F1-1 to generate a diploid, two-way pseudo-testcross F1 population comprised of 94 genotypes. Dense linkage maps, containing 4,139 single nucleotide polymorphism markers with an average distance of 0.6 and 0.3 cM between markers, were constructed for both parents. The resistance level was evaluated by in vitro inoculation test with R. solanacearum (phylotype I/biovar 4/race 1). Five QTLs (qBWR-1 to -5) were identified on potato chromosomes 1, 3, 7, 10, and 11, and they explained 9.3-18.4% of the phenotypic variance. The resistant parent had resistant alleles in qBWR-2, qBWR-3, and qBWR-4 and susceptible alleles in qBWR-1 and qBWR-5. Accumulation of the resistant alleles in all five QTLs increased the level of resistance compared with that of the resistant parent. This is the first study to identify novel QTLs for bacterial wilt resistance in potato by using genome-wide markers.

Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Identification of QTLs for rice grain size using a novel set of chromosomal segment substitution lines derived from Yamadanishiki in the genetic background of Koshihikari.

Grain size is important for brewing-rice cultivars, but the genetic basis for this trait is still unclear. This paper aims to identify QTLs for grain size using novel chromosomal segment substitution lines (CSSLs) harboring chromosomal segments from Yamadanishiki, an excellent sake-brewing rice, in the genetic background of Koshihikari, a cooking cultivar. We developed a set of 49 CSSLs. Grain length (GL), grain width (GWh), grain thickness (GT), 100-grain weight (GWt) and days to heading (DTH) were evaluated, and a CSSL-QTL analysis was conducted. Eighteen QTLs for grain size and DTH were identified. Seven (qGL11, qGWh5, qGWh10, qGWt6-2, qGWt10-2, qDTH3, and qDTH6) that were detected in F2 and recombinant inbred lines (RILs) from Koshihikari/Yamadanishiki were validated, suggesting that they are important for large grain size and heading date in Yamadanishiki. Additionally, QTL reanalysis for GWt showed that qGWt10-2 was only detected in early-flowering RILs, while qGWt5 (in the same region as qGWh5) was only detected in late-flowering RILs, suggesting that these QTLs show different responses to the environment. Our study revealed that grain size in the Yamadanishiki cultivar is determined by a complex genetic mechanism. These findings could be useful for the breeding of both cooking and brewing rice.

Genetic dissection of grain traits in Yamadanishiki, an excellent sake-brewing rice cultivar.

KEY MESSAGE: The grain traits of Yamadanishiki, an excellent sake-brewing rice cultivar in Japan, are governed by multiple QTLs, namely, a total of 42 QTLs including six major QTLs. Japanese rice wine (sake) is produced using brewing rice (Oryza sativa L.) that carries traits desirable for sake-brewing, such as a larger grain size and higher white-core expression rate (WCE) compared to cooking rice cultivars. However, the genetic basis for these traits in brewing rice cultivars is still unclear. We performed analyses of quantitative trait locus (QTL) of grain and days to heading over 3 years on populations derived from crosses between Koshihikari, a cooking rice, and Yamadanishiki, an excellent sake-brewing rice. A total of 42 QTLs were detected for the grain traits, and the Yamadanishiki alleles at 16 QTLs contributed to larger grain size. Two major QTLs essential for regulating both 100-grain weight (GWt) and grain width (GWh) were harbored in the same regions on chromosomes 5 and 10. An interaction was noted between the environment and the QTL associated with WCE on chromosome 6, which was detected in two of 3 years. In addition, two QTLs for WCE on chromosomes 3 and 10 overlapped with the QTLs for GWt and GWh, suggesting that QTLs associated with grain size also play an important role in the formation of white-core. Despite differences in the rate of grain growth in both Koshihikari and Yamadanishiki across 2 years, the WCE in Yamadanishiki remained consistent, thus demonstrating that the formation of white-core does not depend on grain filling speed. These data can be informative for programs involved in breeding better cooking and brewing rice cultivars.

Differences in glucose yield of residues from among varieties of rice, wheat, and sorghum after dilute acid pretreatment.

Bio-refinery processes require use of the most suitable lignocellulosic biomass for enzymatic saccharification and microbial fermentation. Glucose yield from biomass solid fractions obtained after dilute sulfuric acid (1%) pretreatment (at 180 °C) was investigated using 14, 8, and 16 varieties of rice, wheat, and sorghum, respectively. Biomass solid fractions of each crop showed similar cellulose content. However, glucose yield after enzymatic hydrolysis (cellulase loading at 6.6 filter paper unit/g-biomass) was different among the varieties of each crop, indicating genotypic differences for rice, wheat, and sorghum. Nuclear magnetic resonance method revealed that the high residual level of lignin aromatic regions decreased glucose yield from solid fraction of sorghum.

Natural variation in the glucose content of dilute sulfuric acid-pretreated rice straw liquid hydrolysates: implications for bioethanol production.

Rice straw is a promising resource for bioethanol production. Because the glucose content of pretreatment liquid hydrolysates is highly correlated with ethanol yield, the selection of appropriate rice cultivars is essential. The glucose content in liquid hydrolysates of pretreated rice straws of 208 diverse cultivars was evaluated in natural field in 2013 and 2014 using a novel high-throughput system. The glucose content of the rice straw samples varied across cultivars and was affected by environmental factors such as temperature and solar radiation. Several high-quality cultivars exhibiting high glucose content in both years were identified. The results of this study can aid in development of novel rice cultivars suitable as both feedstocks for bioethanol production and cooking.

Exploring the areas of applicability of whole-genome prediction methods for Asian rice (Oryza sativa L.).

KEY MESSAGE: Our simulation results clarify the areas of applicability of nine prediction methods and suggest the factors that affect their accuracy at predicting empirical traits. Whole-genome prediction is used to predict genetic value from genome-wide markers. The choice of method is important for successful prediction. We compared nine methods using empirical data for eight phenological and morphological traits of Asian rice cultivars (Oryza sativa L.) and data simulated from real marker genotype data. The methods were genomic BLUP (GBLUP), reproducing kernel Hilbert spaces regression (RKHS), Lasso, elastic net, random forest (RForest), Bayesian lasso (Blasso), extended Bayesian lasso (EBlasso), weighted Bayesian shrinkage regression (wBSR), and the average of all methods (Ave). The objectives were to evaluate the predictive ability of these methods in a cultivar population, to characterize them by exploring the area of applicability of each method using simulation, and to investigate the causes of their different accuracies for empirical traits. GBLUP was the most accurate for one trait, RKHS and Ave for two, and RForest for three traits. In the simulation, Blasso, EBlasso, and Ave showed stable performance across the simulated scenarios, whereas the other methods, except wBSR, had specific areas of applicability; wBSR performed poorly in most scenarios. For each method, the accuracy ranking for the empirical traits was largely consistent with that in one of the simulated scenarios, suggesting that the simulation conditions reflected the factors that affected the method accuracy for the empirical results. This study will be useful for genomic prediction not only in Asian rice, but also in populations from other crops with relatively small training sets and strong linkage disequilibrium structures.

A unified mixed-model method for association mapping that accounts for multiple levels of relatedness.

As population structure can result in spurious associations, it has constrained the use of association studies in human and plant genetics. Association mapping, however, holds great promise if true signals of functional association can be separated from the vast number of false signals generated by population structure. We have developed a unified mixed-model approach to account for multiple levels of relatedness simultaneously as detected by random genetic markers. We applied this new approach to two samples: a family-based sample of 14 human families, for quantitative gene expression dissection, and a sample of 277 diverse maize inbred lines with complex familial relationships and population structure, for quantitative trait dissection. Our method demonstrates improved control of both type I and type II error rates over other methods. As this new method crosses the boundary between family-based and structured association samples, it provides a powerful complement to currently available methods for association mapping.

Artificial selection for a green revolution gene during japonica rice domestication.

The semidwarf phenotype has been extensively selected during modern crop breeding as an agronomically important trait. Introduction of the semidwarf gene, semi-dwarf1 (sd1), which encodes a gibberellin biosynthesis enzyme, made significant contributions to the "green revolution" in rice (Oryza sativa L.). Here we report that SD1 was involved not only in modern breeding including the green revolution, but also in early steps of rice domestication. We identified two SNPs in O. sativa subspecies (ssp.) japonica SD1 as functional nucleotide polymorphisms (FNPs) responsible for shorter culm length and low gibberellin biosynthetic activity. Genetic diversity analysis among O. sativa ssp. japonica and indica, along with their wild ancestor O. rufipogon Griff, revealed that these FNPs clearly differentiate the japonica landrace and O. rufipogon. We also found a dramatic reduction in nucleotide diversity around SD1 only in the japonica landrace, not in the indica landrace or O. rufipogon. These findings indicate that SD1 has been subjected to artificial selection in rice evolution and that the FNPs participated in japonica domestication, suggesting that ancient humans already used the green revolution gene.

Population structure in Japanese rice population.

It is essential to elucidate genetic diversity and relationships among even related individuals and populations for plant breeding and genetic analysis. Since Japanese rice breeding has improved agronomic traits such as yield and eating quality, modern Japanese rice cultivars originated from narrow genetic resource and closely related. To resolve the population structure and genetic diversity in Japanese rice population, we used a total of 706 alleles detected by 134 simple sequence repeat markers in a total of 114 cultivars composed of 94 improved varieties and 20 landraces, which are representative and important for Japanese rice breeding. The landraces exhibit greater gene diversity than improved lines, suggesting that landraces can provide additional genetic diversity for future breeding. Model-based Bayesian clustering analysis revealed six subgroups and admixture situation in the cultivars, showing good agreement with pedigree information. This method could be superior to phylogenetic method in classifying a related population. The leading Japanese rice cultivar, Koshihikari is unique due to the specific genome constitution. We defined Japanese rice diverse sets that capture the maximum number of alleles for given sample sizes. These sets are useful for a variety of genetic application in Japanese rice cultivars.

Development of INDEL markers to discriminate all genome types rapidly in the genus Oryza.

The wild Oryza species are rich in genetic diversity and are good resources for modern breeding of rice varieties. The reliable ex situ conservation of various genetic resources supports both basic and applied rice research. For this purpose, we developed PCR-based and co-dominant insertion/deletion (INDEL) markers which enable the discrimination of the genome types or species in the genus Oryza. First, 12,107 INDEL candidate sequences were found in the BAC end sequences for 12 Oryza species available in public databases. Next, we designed PCR primers for INDEL-flanking sequences to match the characteristics of each INDEL, based on an assessment of their likelihood to give rise to a single or few PCR products in all 102 wild accessions, covering most Oryza genome types. Then, we selected 22 INDEL markers to discriminate all genome types in the genus Oryza. A phylogenetic tree of 102 wild accessions and two cultivars according to amplicon polymorphisms for the 22 INDEL markers corresponded well to those in previous studies, indicating that the INDEL markers developed in this study were a useful tool to improve the reliability of identification of wild Oryza species in the germplasm stocks.

Identification of Novel Quantitative Trait Loci for Culm Thickness of Rice Derived from Strong-Culm Landrace in Japan, Omachi.

Increasing the lodging resistance of rice through genetic improvement has been an important target in breeding. To further enhance the lodging resistance of high-yielding rice varieties amidst climate change, it is necessary to not only shorten culms but strengthen them as well. A landrace rice variety, Omachi, which was established more than 100 years ago, has the largest culm diameter and bending moment at breaking in the basal internodes among 135 temperate japonica accessions. Using unused alleles in such a landrace is an effective way to strengthen the culm. In this study, we performed quantitative trait locus (QTL) analysis to identify the genetic factors of culm strength of Omachi using recombinant inbred lines (RILs) derived from a cross between Omachi and Koshihikari, a standard variety in Japan. We identified three QTLs for the culm diameter of the 5th internode on chromosomes 3 (qCD3) and 7 (qCD7-1, qCD7-2). Among them, qCD7-2 was verified by QTL analysis using the F2 population derived from a cross between one of the RILs and Koshihikari. RNA-seq analysis of shoot apex raised 10 candidate genes underlying the region of qCD7-2. The increase in culm strength by accumulating Omachi alleles of qCD3, qCD7-1 and qCD7-2 was 25.0% in 2020. These QTLs for culm diameter pleiotropically increased spikelet number per panicle but did not affect days to heading or culm length. These results suggest that the Omachi alleles of qCD3, qCD7-1 and qCD7-2 are useful for breeding to increase lodging resistance and yield.

High-Density Linkage Maps from Japanese Rice japonica Recombinant Inbred Lines Using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di).

The genetic dissection of agronomically important traits in closely related Japanese rice cultivars is still in its infancy mainly because of the narrow genetic diversity within japonica rice cultivars. In an attempt to unveil potential polymorphism between closely related Japanese rice cultivars, we used a next-generation-sequencing-based genotyping method: genotyping by random amplicon sequencing-direct (GRAS-Di) to develop genetic linkage maps. In this study, four recombinant inbred line (RIL) populations and their parents were used. A final RIL number of 190 for RIL71, 96 for RIL98, 95 for RIL16, and 94 for RIL91 derived from crosses between a common leading Japanese rice cultivar Koshihikari and Yamadanishiki, Taichung 65, Fujisaka 5, and Futaba, respectively, and the parent plants were subjected to GRAS-Di library construction and sequencing. Approximately 438.7 Mbp, 440 Mbp, 403.1 Mbp, and 392 Mbp called bases covering 97.5%, 97.3%, 98.3%, and 96.1%, respectively, of the estimated rice genome sequence at average depth of 1× were generated. Analysis of genotypic data identified 1050, 1285, 1708, and 1704 markers for each of the above RIL populations, respectively. Markers generated by GRAS-Di were organized into linkage maps and compared with those generated by GoldenGate SNP assay of the same RIL populations; the average genetic distance between markers showed a clear decrease in the four RIL populations when we integrated markers of both linkage maps. Genetic studies using these markers successfully localized five QTLs associated with heading date on chromosomes 3, 6, and 7 and which previously were identified as Hd1, Hd2, Hd6, Hd16, and Hd17. Therefore, GRAS-Di technology provided a low cost and efficient genotyping to overcome the narrow genetic diversity in closely related Japanese rice cultivars and enabled us to generate a high density linkage map in this germplasm.

Integrated genome-wide differentiation and association analyses identify causal genes underlying breeding-selected grain quality traits in japonica rice.

Improving grain quality is a primary objective in contemporary rice breeding. Japanese modern rice breeding has developed two different types of rice, eating and sake-brewing rice, with different grain characteristics, indicating the selection of variant gene alleles during the breeding process. Given the critical importance of promptly and efficiently identifying genes selected in past breeding for future molecular breeding, we conducted genome scans for divergence, genome-wide association studies, and map-based cloning. Consequently, we successfully identified two genes, OsMnS and OsWOX9D, both contributing to rice grain traits. OsMnS encodes a mannan synthase that increases the white core frequency in the endosperm, a desirable trait for sake brewing but decreases the grain appearance quality. OsWOX9D encodes a grass-specific homeobox-containing transcription factor, which enhances grain width for better sake brewing. Furthermore, haplotype analysis revealed that their defective alleles were selected in East Asia, but not Europe, during modern improvement. In addition, our analyses indicate that a reduction in grain mannan content during African rice domestication may also be caused a defective OsMnS allele due to breeding selection. This study not only reveals the delicate balance between grain appearance quality and nutrition in rice but also provides a new strategy for isolating causal genes underlying complex traits, based on the concept of "breeding-assisted genomics" in plants.

Diversity and population structure of black soybean landraces originating from Tanba and neighboring regions.

Black soybean landraces that had been cultivated in Tanba region and the neighboring regions and conserved black soybean landraces, including those from other regions in Japan, were used in this study. The polymorphisms of 78 SSR markers in nuclear DNA and 6 SSRs in chloroplast DNA were analyzed in the black soybean landrace populations. The result of phylogenic analysis revealed that the black soybeans can be classified into six clades. The landraces originating from Tanba region were classed into first and second clades, and two chloroplast genotypes were found in the population of black soybeans from the Tanba region. Genotype A chloroplast was predominantly identified in major populations of the Tanba, while genotype B was widely distributed in the black soybean population. Population structure analysis in the Japanese black soybean accessions inferred there are six groups. The black soybean landrace from the Tanba region was classified into three groups, mainly corresponding to the distance-based phylogenic results. The two groups were probably derived from different ancestors with Type A and B chloroplast genomes, respectively, whereas the other group showed both types of chloroplast genome. The admixture situations suggested that the landraces in the main group have been widely cultivated in Tanba region, while the landraces that belong to other groups were cultivated in localized area. Several phenotypes were compared among genotype groups, dividing into two sub-groups: founder sub-group and admixed sub-group. Phenotypic differences were observed between founder landraces in group 1 and group 3. On the other hand, landraces in admixture landraces in group 1 and group 2 segregated for several traits, while founder landraces in group 1 were stabled for each trait. These observations suggest that gene flow events have occurred between different founder landraces.

Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene.

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1-1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.