On the cover

Preserved rice (Oryza sativa L.) seeds that had been stored as emergency food stock for about 100 years at the Furuhashi Kaikokan in Toyota City, Aichi Prefecture. By investigating such rice stock as a material, the genetic diversity of rice landraces in the Meiji era was evaluated. Clarifying changes in the genetic diversity of cultivated crops provides important insights in considering future conservation of genetic resources (Photo by Y.I. Sato, This issue, p. 347–354).

(C. Muto: Genetic Resources Center, National Agriculture and Food Research Organization (NARO))

Leaf chemistry traits are some of the key factors influencing tobacco quality, which can be significantly reduced by lower chemical components in cured leaf. To improve tobacco quality through breeding, genetic diversity analysis, population structure analysis, and genome-wide association studies were performed in a panel of 347 tobacco germplasms and the markers associated with five leaf chemistry traits, including total sugar (TS), reducing sugar (RS), total nitrogen (TN), nicotine (NIC), and total potassium (TP) contents were identified. Four groups were classified at a genetic distance of 0.316 by genetic diversity analysis based on coefficient parameter NEI72 using a program NTSYS-pc2.10e, whereas four well-differentiated subpopulations were postulated in the 347 tobacco accessions. A total of 47 target trait-associated SNPs was detected in at least three environments as well as the best linear unbiased predictions (BLUPs) across all environments, among which two, two, four, six, and one highly suggestive associated SNPs were repeatedly detected in all environments and BLUPs for TS, RS, TN, NIC, and TP, respectively. On the basis of the phenotypic effects of the alleles corresponding to suggestive associated SNPs, five tobacco accessions harboring favorable alleles with elite phenotypic performance in leaf chemistry traits were identified. The results could facilitate quality tobacco breeding for higher leaf chemistry trait contents through molecular marker-assisted approaches.

Novel mutant alleles related to isoflavone content are useful for breeding programs to improve the disease resistance and nutritional content of soybean. However, identification of mutant alleles from high-density mutant libraries is expensive and time-consuming because soybean has a large, complicated genome. Here, we identified the gene responsible for increased genistein-to-daidzein ratio in seed of the mutant line F333ES017D9. For this purpose, we used a time- and cost-effective approach based on selective genotyping of a small number of F₂ plants showing the mutant phenotype with nearest-neighboring-nucleotide substitution–high-resolution melting analysis markers, followed by alignment of short reads obtained by next-generation sequencing analysis with the identified locus. In the mutant line, GmCHR5 harbored a single-base deletion that caused a change in the substrate flow in the isoflavone biosynthetic pathway towards genistein. Mutated GmCHR5 was expressed at a lower level during seed development than wild-type GmCHR5. Ectopic overexpression of GmCHR5 increased the production of daidzein derivatives in both the wild-type and mutant plants. The present strategy will be useful for accelerating identification of mutant alleles responsible for traits of interest in agronomically important crops.

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.

While sweetpotato (Ipomoea batatas L.) improvement has generally been done by field-based selection, molecular genetic studies on traits of interest, i.e., molecular markers are needed for enhancing the breeding program of this world’s 7^th most important crop, as such markers facilitate marker-assisted selection. Here, we performed a combined approach of QTLs analyses and GWAS of storage root β-carotene content (BC), dry-matter (DM) and starch content (SC) using the genetic linkage maps constructed with 5,952 and 5,640 SNPs obtained from F₁ progenies between cultivars ‘J-Red’ and ‘Choshu’. BC was negatively correlated with DM (r = –0.45) and SC (r = –0.51), while DM was positively correlated with SC (r = 0.94). In both parental maps, a total of five, two and five QTL regions on linkage groups 7 and 8 were associated with BC, DM and SC, respectively. In GWAS of BC, one strong signal (P = 1.04 × 10^–9) was observed on linkage group 8, which co-located with one of the above QTL regions. The SNPs markers found here, particularly for β-carotene, would be useful base resources for future marker-assisted selection program with this trait.

Low-temperature tolerance during the germination and bud stages is an important characteristic of direct-seeded rice (DSR). Recombinant inbred lines (RILs) derived from indica rice H335, which is highly tolerant to low temperature, and indica rice CHA-1, which is sensitive to low temperature, were used to identify quantitative trait loci (QTLs) associated with low-temperature tolerance during the germination and bud stages. a total of 11 QTLs were detected based on a high-density genetic map; among these, six QTLs explained 5.13–9.42% of the total phenotypic variation explained (PVE) during the germination stage, and five QTLs explained 4.17–6.42% of the total PVE during the bud stage. All QTLs were distributed on chromosome 9, and all favourable alleles originated from H335. The physical position of each QTL was determined, and 11 QTLs were combined into five genetic loci; three of these loci are involved during the germination stage (loci 1, 2, and 3), and three are involved during the bud stage (loci 3, 4, and 5). Loci 2, 4 and 5 were repeatedly detected in the wet season (WS) and dry season (DS). Notably, loci 3 was detected during both the germination and bud stages. These loci are good candidates for future studies of gene function and could serve as highly valuable genetic factors for improving cold tolerance during the germination and bud stages of rice.

Rice (Oryza sativa L.) has been considered one of the most important crops in Ethiopia. Landraces and improved accessions in Ethiopia were characterized on the basis of polymorphism data for SSR markers, and classified into two groups: I and II. Cluster I was further divided into two sub-clusters, Ia and Ib. Cluster Ia corresponded to Japonica-like type, Cluster Ib to Japonica type, and Cluster II to Indica type with some Indica-like type. Many landraces and improved varieties belonged to Cluster Ia. Superior landraces were included in Cluster Ib. Further categorization based on blast resistance demonstrated three groups: Clusters A, B1, and B2. Cluster A comprised accessions with relatively high resistance, whereas Clusters B1 and B2 included susceptible accessions. Most of the improved varieties were found in Cluster A. Superior landraces, X-Jigna classified into Ib or DNA type tended to be susceptible in Cluster B2 for blast resistance. These results demonstrated that traditional landraces preferred by farmers should be improved for disease resistance using blast-resistant varieties. In order to avoid hybrid sterility occurring in cross-hybridizing breeding between Indica and Japonica types, desirable parental accessions can be chosen within the same DNA cluster. The clustering information among accessions may be useful in breeding schemes for selection of counterparts in cross-breeding programs.

Tobacco bushy top disease (TBTD) is a viral disease of tobacco (Nicotiana tabacum L.) caused by mixed infection of Tobacco bushy top virus or Ethiopian tobacco bushy top virus and a helper virus. Despite its damage to tobacco, practical genetic resources for disease resistance have not been found. Here, we report that a mutation of tobacco eIF(iso)4E genes (eIF(iso)4E-S and eIF(iso)4E-T), which encode eukaryotic translation initiation factors, confers resistance (reduced susceptibility) to TBTD caused by a virus from Malawi (designated as tobacco bushy top virus Malawi isolate, TBTV-MW). RNAi lines in which eIF(iso)4E genes were silenced showed reduced susceptibility to TBTV-MW. We also tested chemically-induced single (eIF(iso)4E-S or eIF(iso)4E-T) and double (eIF(iso)4E-S and eIF(iso)4E-T) nonsense mutants for resistance to TBTV-MW. Suppression of eIF(iso)4E-S showed reduced susceptibility, and the resistance of the double mutant tended to be even stronger. eIF(iso)4E mutants also showed reduced susceptibility to TBTV-MW transmitted by aphids. To the best of our knowledge, the eIF(iso)4E-S mutant is the first genetic resource for TBTD resistance breeding in tobacco.

Wild plants with a wide distribution, including those exposed to a wide variety of environmental conditions, may have variations in key functional traits relevant for agricultural applications. The East Asian wild radish (Raphanus sativus var. raphanistroides) is an appropriate model plant because it is widely distributed and has outstanding sink capacity as well as two cultivars within the species. Multiple common garden trials with 14 populations and three testing sites were conducted across the Japanese archipelago to quantify variations in yield and allocation. Significant inter-population variations and interaction effects with testing sites were detected for the root and shoot mass and the root mass fraction (RMF). While the rank order of the population changed drastically among sites and the variance components of genetic effects were small in yield traits (2.4%–4.7%), RMF displayed a large genetic variance (23.2%) and was consistently higher in the northern populations at all sites. Analyses revealed that the mean temperature of growing season of the seed origin was the most prominent factor explaining variation in RMF, irrespective of the sites. We concluded that the trait of resource allocation had a temperature-related cline and plants in cooler climates could invest more resources into their roots.

Cadmium (Cd) is as an extremely toxic metal that can contaminate agricultural soils. To reduce the risk of Cd intake in food cereals, the development of cultivars with low grain Cd concentration (GCC) is an effective countermeasure. We analyzed quantitative trait loci (QTLs) for GCC in a doubled haploid (DH) common wheat (Triticum aestivum L.) population derived from ‘Chugoku 165’ (low GCC) × ‘Chukei 10-22’ (high GCC). We found novel loci for low GCC on the short arm of chromosome 4B and on the long arm of chromosome 6B. These QTLs accounted for 9.4%–25.4% (4B) and 9.0%–17.8% (6B) of the phenotypic variance in the DH population. An association analysis with 43 cultivars identified 3 loci at these QTLs: QCdc.4B-kita, QCdc.6B-kita1, and QCdc.6B-kita2. In contrast to durum wheat and barley, no QTL was detected on the chromosomes of homeologous group 5 for heavy metal P1B-type ATPase 3. These results will contribute to marker-assisted selection for low GCC in breeding of common wheat.

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.

Preserved rice (Oryza sativa L.) seeds stored for nearly a century as an emergency food stocks from the Mikawa area were investigated for their genetic diversity. Morphologically, the seeds appeared to be typical Japonica. One chloroplast INDEL petN-trnC, two nuclear INDELs Acp1 and Cat1, and three SNP markers in Starch synthase IIa were amplified to characterize the molecular profile. The efficiency of amplification varied among the markers. Most of preserved seeds were classified as Japonica, but some were identified as Indica. The heterozygous genotypes detected suggested a high frequency of outcrossing at that time. On the other hand, 21 SSR markers showed quite a high degree of amplification efficiency. Principal coordinate analysis and STRUCTURE analysis based on the SSR polymorphisms proved that the preserved seeds contained alleles that were not detected among current landraces and breeding varieties, and there were the expected three subpopulations among 96 preserved seeds. These results indicated that these preserved seeds from Mikawa area in Meiji era had high genetic diversity and consisted of some subpopulations including Indica landraces with typical Japonica seed shape. These lines were considered to have been lost from current genetic resources.

Alloplasmic plants exhibit various phenotypic changes such as cytoplasmic male sterility (CMS). We have been attempting to produce an alloplasmic Brassica rapa CMS line (2n = 20) carrying Diplotaxis tenuifolia cytoplasm (cyt-Dt) for several years, but a single extra chromosome always remained in all lines produced. We confirmed a D. tenuifolia-specific band in the alloplasmic line carrying D. tenuifolia cytoplasm by RAPD analysis, indicating that the additional chromosome was derived from D. tenuifolia. Here, we observed the phenotypic characteristics of the alloplasmic B. rapa monosomic addition line, named (cyt-Dt) B. rapa MAL, and investigated why a single extra chromosome is required in its genetic background for viability. When the (cyt-Dt) B. rapa MALs were crossed with pollen of several B. rapa lines, approximately 50% of the ovules attracted pollen tubes, and all the progeny had the additional chromosome. These results suggested that only the female gametes with n = 11 rather than n = 10 were fertilized and developed into mature seeds, and that cytoplasmic female sterility was overcome by nuclear restorer gene(s) derived from the cytoplasmic donor species.

A citrus cultivar identification system using CAPS marker has been developed on nursery trees, but this needs to be extended to include various product types, such as imported fruits and processed products. Here, we developed a new cultivar identification system using TaqMan-MGB SNP genotyping assay. Eight probe and primer sets were designed to amplify PCR fragments <100 bp to enable the genotyping of fresh and processed fruits in which predicted that insufficient quantities of DNA and residual impurities in the DNA extracts. The TaqMan-MGB SNP genotyping assay was stable and reproducible, and were confirmed to apply various sample sources, including leaves, fresh fruit, juice, canned fruit, and dry fruit. They could provide at least a single differentiating SNP to discriminate any paired combination among 48 citrus cultivars. Minimal marker subsets to identify the target cultivar were listed for each of 18 registered cultivars with valid patent. The allelic SNP genotypes of 48 citrus cultivars, which cover more than 98% of all citrus fruit shipment produced in Japan, is valuable for the referencing information in the DNA-based identification for fresh and processed fruits. This identification system will help protect registered cultivars and facilitate food fraud inspections.

Wheat yellow mosaic (WYM) is a soilborne disease caused by Wheat yellow mosaic virus (WYMV). Symptoms include yellow mosaic coloring of leaves, stunting, and growth inhibition. Severe infection may result in yield loss. WYM is one of the most serious diseases affecting wheat production in East Asia. The most effective control is through breeding resistant cultivars. A winter wheat cultivar, ‘OW104’, shows little to no symptoms in heavily WYMV-infested fields in Hokkaido, Japan. Here we detected Qym4, a QTL accounting for 45%–57% of WYMV resistance, in the vicinity of the markers Xcfd49, Xbarc183, and Xgpw4357 on wheat chromosome arm 6DS. F₃ progenies with ‘OW104’ allele at Qym4 showed significantly higher resistance than those with ‘Hokushin’ homozygote or heterozygote. We developed ‘Hokushin’ near-isogenic lines by backcrossing with ‘Hokushin’ as the recurrent parent and ‘OW104’ as the resistance donor. All the WYMV-resistant BC₅F₁/BC₄F₁ plants carried ‘OW104’ allele only at Xcfd49. Our results suggest that the introduction of Qym4 confers resistance to WYMV in winter wheat.

To clarify the genetic mechanisms of fertility restoration in sorghum F₁ hybrids produced in Japan (‘Ryokuryu’, ‘Hazuki’, ‘Haretaka’, ‘Natsuibuki’, ‘Hanaaoba’, ‘Akidachi’ and ‘Kazetachi’), we analyzed QTLs for fertility restoration using seven F₂ populations derived from those hybrids. By QTL mapping with a series of SSR markers, we detected three major QTLs for fertility restoration. These data and the results of haplotype analysis of known fertility restorer (Rf) genes showed that qRf5, corresponding to the Rf5 locus, was the most widely used Rf gene for fertility restoration of sorghum F₁ hybrids among the lines tested. Other major Rf genes detected were qRf8, corresponding to Rf1, and qRf2, corresponding to Rf2. QTLs for grain weight also corresponded to these Rf loci. A minor QTL, qRf3, may also affect restoration of fertility. Our data show that three major Rfs—Rf1, Rf2, and Rf5—were used in F₁ hybrid sorghum production in Japan. This knowledge can be used to improve the efficiency of the F₁ sorghum breeding program.

Powdery mildew (PM), caused by Erysiphe cruciferarum, is an epidemic of oil rapeseed (Brassica napus L.) growing worldwide, but PM resistant germplasm is rare in this species. We screened 102 accessions of B. napus and other cruciferous species and found an Ethiopian mustard (Brassica carinata) cultivar ‘White flower’ immune to PM in both the field and greenhouse. Outcrossing in the female parent ‘White flower’ was promoted by using a chemical gametocide tribenuron-methyl, to obtain hybrid seeds of distant hybridization with an elite B. napus cultivar ‘Zhongshuang11’. Three true F₁ hybrids with B. carinata cytoplasm were obtained without using embryo rescue, which showed complete male sterility and light yellow petals. The hybrid plants and the progenies derived from backcrossing were validated using morphological traits, seed quality, and molecular markers. Five lines in the BC₁F₃ generation, named ‘W7-1’, ‘W7-4’, ‘W7-6’, ‘W8-1’, and ‘W8-3’, and one BC₂F₂ line ‘W3PS-1’, whose young leaf was yellow green, were identified to be resistant or moderately resistant to PM. The seed quality and some morphological traits of these lines resembled the parent ‘Zhongshuang11’, indicating that the resistance gene(s) has been preliminarily introduced into B. napus.

Polymorphism information generated by next-generation sequencing (NGS) technologies has enabled applications of genome-wide markers assisted breeding. However, handling such large-scale data remains a challenge for experimental researchers and breeders, calling for the urgent development of a flexible and straightforward analysis tool for NGS data. We developed “IonBreeders” as bioinformatics plugins that implement general analysis steps from genotyping to genomic prediction. IonBreeders comprises three plugins, “ABH”, “IMPUTATION”, and “GENOMIC PREDICTION”, for format conversion of genotyping data, preprocessing and imputation of genotyping data, and genomic prediction, respectively. “ABH” converts genotyping data derived from NGS into the ABH format, which is acceptable for our further plugins and with other breeding software tools, R/qtl, MapMaker, and AntMap. “IMPUTATION” filters out non-informative markers and imputes missing marker genotypes. In “GENOMIC PREDICTION”, users can use four statistical methods based on their target trait, quantitative trait locus effect, and number of markers, and construct a prediction model for genomic selection. IonBreeders is operated in Torrent Suite, but can also handle genotype data in standard formats, e.g., Variant Call Format (VCF), by format conversion using free software or our provided scripts.

To elucidate the resistance mechanisms of the rice (Oryza sativa L.) cultivar ‘Milyang 44’ against rice stink bugs, we compared the number of stylet sheaths, husk perforations, and feeding marks on the surface of the grains caused by Leptocorisa chinensis and Cletus punctiger on Milyang 44 and the control cultivar, i.e., ‘Aichinokaori SBL’. We also examined the cross-sectional structure of the rice husks. We found that the number of stylet sheaths per panicle was higher in Milyang 44 than in Aichinokaori SBL for both rice stink bug species, except in one test involving C. punctiger. However, Milyang 44 had significantly less damage per number of stylet sheaths than Aichinokaori SBL, resulting in a lower percentage rates of pecky rice grains in Milyang 44. Interestingly, there was no difference in the percentage rates of pecky rice between the two cultivars after removing one third of the husks. Histological analysis showed that the sclerenchymatous cell wall containing lignin of husk was thicker in Milyang 44 than in Aichinokaori SBL, suggesting that the husk of Milyang 44 plays an important role in its resistance to these two rice stink bug species.

Resistant starch (RS) is beneficial for human health, and especially for diabetics. Considering the high cost and low productivity of the Jiangtangdao 1 rice variety with high RS content, breeding high RS rice varieties exhibiting high productivity is essential. A molecular marker-assisted selection strategy was applied to increase RS content in a three-line hybrid rice variety. The functional rice variety Jiangtangdao 1, which contains sbe3-rs (on chr2) that controls the RS content, was used as the high RS content donor parent. Subsequently, male sterile maintainer and restorer lines containing homozygous sbe3-rs were bred using molecular marker-assisted selection combined with traditional breeding methods. The male sterile line was crossed with the restorer lines to identify the optimal hybrid combination with a high RS content. We obtained four combinations for which the yields were >50% higher than those of the control Jiangtangdao 1. In addition, there was no significant difference in the RS content between the combinations and Jiangtangdao 1. The hybrid rice plants with high RS content exhibited favorable agronomic traits and therefore have broad prospects for commercial application.

Simple sequence repeat (SSR) markers provide a reliable tool for the identification of accessions and the construction of genetic linkage maps because of their co-dominant inheritance. In the present study, we developed new SSR markers with next-generation sequencing using the Roche 454 GS FLX+ platform. Five hundred SSR primer sets were tested for the genetic identification of pineapple, including 100 each for the di-, tri-, tetra-, penta-, and hexa-nucleotide motif SSRs. In total, 160 SSR markers successfully amplified fragments and exhibited polymorphism among accessions. The SSR markers revealed the number of alleles per locus (ranging from 2 to 13), the expected heterozygosity (ranging from 0.041 to 0.823), and the observed heterozygosity (ranging from 0 to 0.875). A total of 117 SSR markers with tri- or greater nucleotide motifs were shown to be effective at facilitating accurate genotyping. Using the SSR markers, 25 accessions were distinguished genetically, with the exception of accessions ‘MD-2’ and ‘Yonekura’. The developed SSR markers could facilitate the establishment of efficient and accurate genetic identification systems and the construction of genetic linkage maps in the future.