The appearance of brown rice grown under high temperature conditions is an important characteristic for improvement in Japanese rice breeding programs. We performed a QTL analysis of the appearance quality of brown rice using chromosome segment substitution lines of the indica cultivar ‘Habataki’ in the ‘Koshihikari’ genetic background. A line carrying a ‘Habataki’ segment on chromosome 7 showed a high percentage of perfect grains produced under high temperature conditions during the ripening period. To verify the role of this segment, and to narrow down the region containing the useful allele, substitution mapping was performed using multiple paired lines. As a result, the chromosomal location of a gene that we named Appearance quality of brown rice 1 (Apq1) was delimited to a 48-kb region. In addition, we developed an Apq1-near isogenic line (NIL) to evaluate the effect of Apq1 on various agronomic traits. Under high temperature conditions during the ripening period, the Apq1-NIL produced significantly higher percentages of perfect grains than ‘Koshihikari’. Other agronomic traits, including yield and palatability, were similar between the Apq1-NIL and ‘Koshihikari’. Therefore, the ‘Habataki’ allele of Apq1 will be useful in breeding programs aimed at improving the quality of grains ripened under high temperature conditions.

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Development of soybean cultivars with high seed yield is a major focus in soybean breeding programs. This study was conducted to identify genetic loci associated with seed yield-related traits in soybean and also to clarify consistency of the detected QTLs with QTLs found by previous researchers. A population of 135 F_2:3 lines was developed from a cross between a vegetable soybean line (MJ0004-6) and a landrace cultivar from Myanmar (R18500). They were evaluated in the experimental field of Kasetsart University, Kamphaeng Saen, Nakhon Pathom, Thailand in a randomized complete block design with two replications each in 2011 and 2012 growing seasons. The two parents exhibited contrasting characteristics for most of the traits that were mapped. Analysis of variance showed that the main effects of genotype and environment (year) were significant for all studied traits. Genotype by environment interaction was also highly significant for all the traits. The population was genotyped by 149 polymorphic SSR markers and the genetic map consisted of 129 SSR loci which converged into 38 linkage groups covering 1156 cM of soybean genome. There were 10 QTLs significantly associated with seed yield-related traits across two seasons with single QTLs explaining between 5.0% to 21.9% of the phenotypic variation. Three of these QTLs were detected in both years for days to flowering, days to maturity and 100 seed weight. Most of the detected QTLs in our research were consistent with earlier QTLs reported by previous researchers. However, four novel QTLs including SF1, SF2 and SF3 on linkage groups L and N for seed filling period and PN1 on linkage group D1b for pod number were identified in the present study.

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For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information.

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Lodging tolerance (LT) is an important trait for high yield and combine-harvesting efficiency in soybean [Glycine max (L.) Merr.]. Many previous studies have investigated quantitative trait loci (QTLs) for lodging score (LS) in soybean. Most of the investigated QTLs were located in the proximal region of maturity or growth habit loci. The aim of this study was to identify genetic factors for LT not associated with maturity or growth habit. QTL analysis was performed using a recombinant inbred line (RIL) population derived from a cross between ‘Toyoharuka’ (TH), a lodging-tolerant cultivar, and ‘Toyomusume’ (TM). The genotypes of TH and TM were estimated as both e1e2E3E4 and dt1. The average LS over 4 years was used for QTL analysis, identifying a major and stable QTL, qLS19-1, on chromosome 19. The LS of the near-isogenic line (NIL) with the TH allele at Sat_099, the nearest marker to qLS19-1, was significantly lower than the NIL with the TM allele at that position. The TH allele at Sat_099 rarely had a negative influence on seed yield or other agronomic traits in both NILs and the TM-backcrossed lines. Our results suggest that marker-assisted selection for qLS19-1 is effective for improving LT in breeding programs.

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Hybrid incompatibility plays an important role in establishment of post-zygotic reproductive isolation. To unveil genetic basis of hybrid incompatibilities between diverged species of genus Oryza AA genome species, we conducted genetic dissection of hybrid sterility loci, S22(t), which had been identified in backcross progeny derived from Oryza sativa ssp. japonica (recurrent parent) and South American wild rice O. glumaepatula near the end of the short arm of chromosome 2. The S22(t) region was found to be composed of two loci, designated S22A and S22B, that independently induce F₁ pollen sterility. Pollen grains containing either of the sterile alleles (S22A-glum^s or S22B-glum^s) were sterile if produced on a heterozygous plant. No transmission of the S22A-glum^s allele via pollen was observed, whereas a low frequency of transmission of S22B-glum^s was observed. Cytological analysis showed that the sterile pollen grains caused by S22A could reach the bicellular or tricellular stage, and the nearly-sterile pollen grains caused by S22B could reach the tricellular stage. Our genetic analysis showed repulsion linkage effect is possible to induce strong reproductive barrier by high pollen sterility based on recombination value and transmission ratio of hybrid sterility gene to the progeny was influenced by frequency of competitors on fertilization.

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Assessing the level of genetic diversity within a germplasm collection contributes to evaluating the potential for its utilization as a gene pool to improve the performance of cultivars. In this study, 45 high-quality simple sequence repeat (SSR) markers were screened and used to estimate the genetic base of a world-wide collection of 248 rapeseed (Brassica napus) inbred lines. For the whole collection, the genetic diversity of A genome was higher than that of C genome. The genetic diversity of C genome for the semi-winter type was the lowest among the different germplasm types. Because B. oleracea is usually used to broaden the genetic diversity of C genome in rapeseed, we evaluated the potential of 25 wild B. oleracea lines. More allelic variations and a higher genetic diversity were observed in B. oleracea than in rapeseed. One B. oleracea line and one oilseed B. rapa line were used to generate a resynthesized Brassica napus line, which was then crossed with six semi-winter rapeseed cultivars to produce 7 F₁ hybrids. Not only the allele introgression but also mutations were observed in the hybrids, resulting in significant improvement of the genetic base.

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Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line (‘Touhoku 129’) and a sensitive leading cultivar (‘Tachinagaha’) during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, ‘Touhoku 129’ genotypes exhibited more GSD insensitivity than ‘Tachinagaha’ genotypes. The lower incidence of GSD for ‘Touhoku129’ was attributable primarily to these three QTLs because RILs harboring a ‘Touhoku 129’ genotype at the three QTLs exhibited a GSD incidence similar to that of ‘Touhoku 129.’ Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.

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In a screening of about 500 lines of Tartary buckwheat, we identified lines that contained no detectable rutinosidase isozymes using an in-gel detection assay. We confirmed that seeds of these individuals had only a trace level of in-vitro rutinosidase activity. To investigate the heritability of the trace-rutinosidase characteristic, we analyzed the progeny of crosses between rutinosidase trace-lines, ‘f3g-162’, and the ‘Hokkai T8’. The F₂ progeny clearly divided into two groups: those with rutinosidase activity under 1.5 nkat/g seed (trace-rutinosidase) and those with activity over 400 nkat/g seed (normal rutinosidase). The segregation pattern of this trait in F₂ progeny exhibited 1 : 3 ratio (trace-rutinosidase : normal rutinosidase), suggesting that the trace-rutinosidase trait is conferred by a single recessive gene; rutinosidase-trace A (rutA). In addition, sensory panelists evaluated the bitterness of flour from trace-rutinosidase individuals and did not detect bitterness, whereas flour from normal rutinosidase individuals was found to have strong bitterness. Although at least three bitter compounds have been reported in Tartary buckwheat seeds, our present findings indicate that rutin hydrolysis is the major contributing factor to bitterness. In addition, the trace-rutinosidase line identified here, ‘f3g-162’, is a promising material for generating a non-bitter Tartary buckwheat variety.

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Here, we developed a new Tartary buckwheat cultivar ‘Manten-Kirari’, whose flour contains only trace amounts of rutinosidase and lacked bitterness. The trace-rutinosidase breeding line ‘f3g-162’ (seed parent), which was obtained from a Nepalese genetic resource, was crossed with ‘Hokkai T8’ (pollen parent), the leading variety in Japan, to improve its agronomic characteristics. The obtained progeny were subjected to performance test. ‘Manten-Kirari’ had no detectable rutinosidase isozymes in an in-gel detection assay and only 1/266 of the rutinosidase activity of ‘Hokkai T8’. Dough prepared from ‘Manten-Kirari’ flour contained almost no hydrolyzed rutin, even 6 h after the addition of water, whereas the rutin in ‘Hokkai T8’ dough was completely hydrolyzed within 10 min. In a sensory evaluation of the flour from the two varieties, nearly all panelists detected strong bitterness in ‘Hokkai T8’, whereas no panelists reported bitterness in ‘Manten-Kirari’. This is the first report to describe the breeding of a Tartary buckwheat cultivar with reduced rutin hydrolysis and no bitterness in the prepared flour. Notably, the agronomic characteristics of ‘Manten-Kirari’ were similar to those of ‘Hokkai T8’, which is the leading variety in Japan. Based on these characteristics, ‘Manten-Kirari’ is a promising for preparing non-bitter, rutin-rich foods.

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Using an F₁ population from a cross between Japanese pear (Pyrus pyrifolia Nakai) cultivars ‘Akiakari’ and ‘Taihaku’, we performed quantitative trait locus (QTL) analysis of seven fruit traits (harvest time, fruit skin color, flesh firmness, fruit weight, acid content, total soluble solids content, and preharvest fruit drop). The constructed simple sequence repeat-based genetic linkage map of ‘Akiakari’ consisted of 208 loci and spanned 799 cM; that of ‘Taihaku’ consisted of 275 loci and spanned 1039 cM. Out of significant QTLs, two QTLs for harvest time, one for fruit skin color, and one for flesh firmness were stably detected in two successive years. The QTLs for harvest time were located at the bottom of linkage group (LG) Tai3 (nearest marker: BGA35) and at the top of LG Tai15 (nearest markers: PPACS2 and MEST050), in good accordance with results of genome-wide association study. The PPACS2 gene, a member of the ACC synthase gene family, may control harvest time, preharvest fruit drop, and fruit storage potential. One major QTL associated with fruit skin color was identified at the top of LG 8. QTLs identified in this study would be useful for marker-assisted selection in Japanese pear breeding programs.

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Boiled seed hardness is an important factor in the processing of soybean food products such as nimame and natto. Little information is available on the genetic basis for boiled seed hardness, despite the wide variation in this trait. DNA markers linked to the gene controlling this trait should be useful in soybean breeding programs because of the difficulty of its evaluation. In this report, quantitative trait locus (QTL) analysis was performed to reveal the genetic factors associated with boiled seed hardness using a recombinant inbred line population developed from a cross between two Japanese cultivars, ‘Natto-shoryu’ and ‘Hyoukei-kuro 3’, which differ largely in boiled seed hardness, which in ‘Natto-shoryu’ is about twice that of ‘Hyoukei-kuro 3’. Two significantly stable QTLs, qHbs3-1 and qHbs6-1, were identified on chromosomes 3 and 6, for which the ‘Hyoukei-kuro 3’ alleles contribute to decrease boiled seed hardness for both QTLs. qHbs3-1 also showed significant effects in progeny of a residual heterozygous line and in a different segregating population. Given its substantial effect on boiled seed hardness, SSR markers closely linked to qHbs3-1, such as BARCSOYSSR_03_0165 and BARCSOYSSR_03_0185, could be useful for marker-assisted selection in soybean breeding.

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Soybean (Glycine max (L.) Merr.) oil typically contains 8% α-linolenic acid that is highly unstable and easily oxidized. This property is undesirable in many food and industrial applications. Genetic strategies for reducing α-linolenic acid content would enhance the commercial value. However, genetic resources for low α-linolenic acid content are limited among natural soybean variations. Microsomal omega-3-fatty acid desaturase (FAD3) is responsible for the synthesis of α-linolenic acid in the polyunsaturated fatty acid pathway. There are four FAD3 homologs (Glyma02g39230, Glyma11g27190, Glyma14g37350 and Glyma18g06950) in the soybean genome. While non-functional alleles have been reported for Glyma02g39230 (GmFAD3-1a) and Glyma14g37350 (GmFAD3-1b), little variation is seen in Glyma18g06950 (GmFAD3-2a). We isolated seven mutant GmFAD3-2a alleles, each containing a single-nucleotide substitution, from 39,100 independent mutant lines by using targeting induced local lesions in genomes (TILLING). Analysis of GmFAD3-2a transcripts and enzyme activities revealed that one missense mutant, ‘P1-A9’, contains a non-functional allele of GmFAD3-2a. By combining three non-functional alleles (GmFAD3-1a, GmFAD3-1b, and GmFAD3-2a), we generated soybean lines containing <2% α-linolenic acid in their seeds. The reverse-genetics-based development of novel mutant alleles in the fatty acid metabolic pathway will allow the improvement of soybean with better oil quality through conventional breeding.

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Two hundred ninety-six Asian barley (Hordeum vulgare L.) accessions were assessed to detect QTLs underlying salt tolerance by association analysis using a 384 single nucleotide polymorphism (SNP) marker system. The experiment was laid out at the seedling stage in a hydroponic solution under control and 250 mM NaCl solution with three replications of four plants each. Salt tolerance was assessed by leaf injury score (LIS) and salt tolerance indices (STIs) of the number of leaves (NL), shoot length (SL), root length (RL), shoot dry weight (SDW) and root dry weight (RDW). LIS was scored from 1 to 5 according to the severity of necrosis and chlorosis observed on leaves. There was a wide variation in salt tolerance among Asian barley accessions. LIS and STI (SDW) were the most suitable traits for screening salt tolerance. Association was estimated between markers and traits to detect QTLs for LIS and STI (SDW). Seven significant QTLs were located on chromosomes 1H (2 QTLs), 2H (2 QTLs), 3H (1 QTL), 4H (1 QTL) and 5H (1 QTL). Five QTLs were associated with LIS and 2 QTLs with STI (SDW). Two QTLs associated with LIS were newly identified on chromosomes 3H and 4H.

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Environmental sustainability concerns make improving yield under lower N input a desirable breeding goal. To evaluate genetic variation and heterosis for low-N tolerance breeding, 28 F1 hybrids from a diallel scheme, along with their eight parental lines, were tested for agronomic traits including kernel number per ear (KNE) and grain yield per plant (GY), in replicated plots over two years under low-nitrogen (LN, without nitrogen application) and normal-nitrogen (NN, 220 kg N ha⁻¹) conditions. Taken together the heritability in this and our previous studies, the correlation with grain yield, and the sensitivity to the stress for target trait selection, KNE was a good secondary target trait for LN selection in maize breeding. KNE also showed much higher mid-parent heterosis than hundred-kernel weight under both nitrogen levels, particularly under LN, indicating that KNE contributed the majority of GY heterosis, particularly under LN. Therefore, KNE can be used as a positive target trait for hybrid performance prediction in LN tolerance breeding. Our results also suggest that breeding hybrids for LN tolerance largely relies on phenotypic evaluation of hybrids under LN condition and yield under LN might be improved more by selection for KNE than by direct selection for GY per se.

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The wild ancestral form of barley, Hordeum vulgare ssp. spontaneum, is a valuable source for gene enrichment of cultivated barley. The purpose of this work was to study the area of distribution as well as the extent and structure of genetic variation of wild barley populations grown in Kazakhstan. It was found that distribution of wild barley populations in Kazakhstan is restricted to the most southern province. A genome wide single nucleotide polymorphism (SNP) analysis was performed in order to study the level of the genetic diversity in 96 accessions representing 14 wild barley populations from Kazakhstan and 25 accessions from the Middle East which is the center of diversity of this subspecies. The oligonucleotide pooled assay was used to genotype 384 SNPs distributed throughout the genome. In total 233 polymorphic SNPs were selected for further statistical analysis. The level of genetic diversity of wild barley populations from Kazakhstan was predictably narrower (He = 0.19 ± 0.01) in comparison with wild barley samples from the Middle East (He = 0.29 ± 0.01). The results suggested that H. vulgare ssp. spontaneum populations in Kazakhstan probably represent a recent spread of a limited number of plants from the primary distribution area and might be well adapted to winter low temperature.

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The yellowing strain of Soybean dwarf virus (SbDV-YS) causes yellowing and yield loss in common bean (Phaseolus vulgaris). The most effective control is achieved through breeding for resistance. An indeterminate climbing cultivar with a white seed coat, ‘Oofuku’, is resistant to SbDV-YS in inoculation tests. We crossed ‘Oofuku’ with an elite cultivar, ‘Taisho-Kintoki’, which is SbDV-YS-susceptible, determinate dwarf with a red-purple seed coat, and performed amplified-fragment-length polymorphism analysis of F₃ lines. From nucleotide sequences of the resistant-specific fragments and their flanking regions, we developed five DNA markers, of which DV86, DV386, and DV398 were closely linked to Sdvy-1, a resistance gene. Using the markers, we developed ‘Toiku-B79’ and ‘Toiku-B80’, the near-isogenic lines (NILs) incorporating Sdvy-1 in the background of ‘Taisho-Kintoki’. The NILs had similar growth habit, maturity date and seed shape to those of ‘Taisho-Kintoki’. The quality of boiled beans was also similar, except that the NILs had more seed coat cracking than ‘Taisho-Kintoki’. The NILs showed no SbDV-YS infection in inoculation tests. We suggest that Sdvy-1 is a useful source of SbDV-YS resistance in common bean.

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Black soybeans have been used as a food source and also in traditional medicine because their seed coats contain natural phenolic compounds such as proanthocyanidin and anthocyanin. The objective of this research is to reveal the genetic variation in the phenolic compound contents (PCCs) of seed coats in 227 black soybean cultivars, most of which were Japanese landraces and cultivars. Total phenolics were extracted from seed coats using an acidic acetone reagent and the proanthocyanidin content, monomeric anthocyanin content, total flavonoids content, total phenolics content, and radical scavenging activity were measured. The cultivars showed wide genetic variation in PCCs. Each of the contents was highly correlated with one another, and was closely associated with radical scavenging activity. PCCs were also moderately associated by flowering date but not associated by seed weight. Cultivars with purple flowers had a tendency to produce higher PCCs compared with cultivars with white flowers, suggesting that the W1 locus for flower color can affect phenolic compound composition and content. Our results suggest that developing black soybean cultivars with high functional phenolic compounds activity is feasible.

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Chlorosis level is a useful parameter to assess imidazolinone resistance in sunflower (Helianthus annuus L.). The aim of this study was to quantify chlorosis through two different methods in sunflower plantlets treated with imazapyr. The genotypes used in this study were two inbred lines reported to be different in their resistance to imidazolinones. Chlorosis was evaluated by spectrophotometrical quantification of photosynthetic leaf pigments and by a bioinformatics-based color analysis. A protocol for pigment extraction was presented which improved pigment stability. Chlorophyll amount decreased significantly when both genotypes were treated with 10 μM of imazapyr. Leaf color was characterized using Tomato Analyzer^® color test software. A significant positive correlation between color reduction and chlorophyll concentration was found. It suggests that leaf color measurement could be an accurate method to estimate chlorosis and infer chlorophyll levels in sunflower plants. These results highlight a strong relationship between imidazolinone-induced chlorosis and variations in leaf color and in chlorophyll concentration. Both methods are quantitative, rapid, simple, and reproducible. Thus, they could be useful tools for phenotyping and screening large number of plants when breeding for imidazolinone resistance in this species.

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A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5′-T/TAA-3′ in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.

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‘Niigata S3’ is a new strawberry (Fragaria × ananassa Duch.) cultivar that is early flowering and possesses high soluble solid content and good coloration. It was selected from a cross between Kei812 (seed parent) and ‘Asuka-Ruby’ (pollen parent). The first harvest date of ‘Niigata S3’ was December 27, 34 days earlier than ‘Echigohime’ and 9 days earlier than ‘Asuka-Ruby’ (means of 2007 and 2008). The marketable yield of ‘Niigata S3’ was 85% of ‘Echigohime’, 107% of ‘Asuka-Ruby’, while the early yield was 145% of ‘Echigohime’, 85% of ‘Asuka-Ruby’ (based on 2007 and 2008 means). The shape of the fruit is long conical, and its skin color medium-red. The fruit skin hardness of ‘Niigata S3’ was 31.5 g/mm², which was harder than ‘Echigohime’, and its average soluble solid content was 11.4%, which was higher than the values for ‘Echigohime’ and ‘Asuka-Ruby’ (2008). Furthermore, ‘Niigata S3’ did not bear apical overripe fruit. This new cultivar is adaptable to the climatic conditions of Niigata, as well as other regions that experience low winter temperatures and insolation.

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