Kiyoaki Kato

Lipoxygenase (LOX) is a multifunctional enzyme involved in flavor-associated metabolite production, plant development, and stress responses. However, the LOX gene family in adzuki bean (Vigna angularis) has not been systematically characterized. We aimed to identify and characterize in the LOX gene family in adzuki bean and investigate their potential roles in seed LOX activity. Genome-wide identification of LOX genes was performed, followed by phylogenetic, structural, and duplication analyses. Gene expression patterns were analyzed using RNA-seq and validated by qRT-PCR. LOX activity in seeds was evaluated using a colorimetric assay. We identified 24 LOX genes, including 4 9-LOX, 13 type I 13-LOX, and 7 type II 13-LOX members. Gene expansion was associated with segmental and tandem duplication. Expression analysis revealed tissue-specific patterns, with VaLOX8 and VaLOX11 highly expressed in seeds. LOX2 and LOX3 activities were detected in mature seeds, whereas LOX1 activity was absent across 32 adzuki cultivars. Based on phylogenetic, sequence alignment, and synteny analyses, VaLOX8 and VaLOX11 were found to be orthologous to GmLOX3 and GmLOX2, respectively, suggesting roles in LOX3 and LOX2 activities. This study provides foundational insights into the LOX gene family in adzuki bean.

In this study, we mapped quantitative trait loci (QTLs) associated with heading date and grain quality traits in a novel set of 44 chromosome segment substitution lines (CSSLs) derived from closely related rice (Oryza sativa L. ssp. japonica) cultivars ‘Yukihikari’ (good grain quality) and ‘Joiku462’ (superior eating and high grain appearance qualities). Days to heading (DTH), apparent amylose content (AAC), protein content (PC), thousand brown-grain weight (TBGW), brown grain length (BGL), brown grain width (BGWI), brown grain thickness (BGT), and the contents of 12 mineral elements (S, P, Mg, Ca, K, Mo, Cu, Zn, Mn, Fe, Sr, and Si) in polished rice were evaluated in 44 CSSLs grown in two different environments. We identified 78 QTLs, including 8, 7, 8, 8, 19, 10, and 10 for DTH, AAC, PC, TBGW, BGL, BGWI, and BGT, respectively, and 2, 1, 3, and 2 for S, Mo, Cu, and Zn contents, respectively. Several QTLs were observed in the same region, forming 17 clusters on chromosomes 1–10. These QTLs can facilitate gene isolation and breeding to develop rice cultivars with optimum heading time and improved grain quality.

Hybrid breakdown is a post-zygotic reproductive isolation that hinders genetic exchange between species or populations in both animals and plants. Two complementary recessive genes, temperature sensitive hybrid breakdown1 (thb1) and thb2, cause hybrid breakdown in rice (Oryza sativa). The present study delimited the THB1 locus to a 9.1-kb sequence, containing a single gene encoding a putative transmembrane protein with unknown functions. Haplotype analysis of THB1 in the two core collections of 119 accessions revealed that these accessions were divided into 22 haplotypes. A test cross with thb2 carrier showed that haplotype2 (H2) was assigned to thb1 and was restricted to temperate japonica. A nonsynonymous nucleotide polymorphism (SNP) specific to H2 was identified as a causal mutation in thb1. A test cross with thb1 carrier indicated that six accessions, including temperate japonica, tropical japonica, and indica, carried thb2. These results suggest that thb1 has recently evolved in temperate japonica, whereas thb2 arose in an ancient japonica and introgressed into the present three subgroups. Furthermore, we developed a derived cleaved amplified polymorphic sequence (dCAPS) marker to detect causal SNP in THB1. Our findings provide new insights into reproductive isolation and may benefit rice breeding.

Increasing the epicotyl length (ECL) of adzuki bean cultivars enhances the suitability for mechanical weeding during the vegetative stages and harvesting at pod maturity. To explore the genetic control of ECL, and to identify molecular markers that could facilitate breeding for increased ECL, recombinant inbred lines (RILs) were developed from a cross between Toiku161 (long epicotyls) and Chihayahime (ordinary length epicotyls). In this study, four quantitative trait loci (QTLs) were identified for ECL by QTL-seq analysis, one each on chromosomes 2, 7, 10 and 11. Insertion and deletion (InDel)-based mapping also detected QTLs on chromosomes 7, qECL7.1, and 10, qECL10.1. Substitution mapping using InDel, cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), and single nucleotide polymorphism (SNP) markers narrowed the chromosomal location of qECL7.1 to a 418 kb region flanked by DNA markers TC99_10,211,134 bp and TC102_10,628,880 bp. A total of 35 genes were predicted within the qECL7.1 region. The ECL QTLs and molecular markers identified here will contribute towards marker-assisted selection of desirable long ECL genotypes that allow for increased mechanization and more efficient adzuki bean production.

Adzuki bean is an important legume crop originating in temperate regions, with photoperiod in sensitivity being a key factor in its latitudinal adaptation. The Flowering Date1 (FD1) gene has a large effect on the photoperiodic response of flowering time, but the molecular basis for the effect of this locus is undetermined. The present study delimited the FD1 locus to a 17.1 kb sequence, containing a single gene, an E1 ortholog (VaE1). A comparison between Vigna angularis ‘Shumari’ (photoperiod insensitive) and ‘Acc2265’ (photoperiod sensitive) identified 29 insertions/deletions and 178 SNPs upstream of VaE1 in the FD1 locus. VaE1 expression in ‘Acc2265’ was greater under long-day than short-day conditions, whereas VaE1 expression in ‘Shumari’ was lower regardless of day length. These findings suggested that responsible gene of FD1 is a VaE1, which acts as a floral repressor by being upregulated in response to long-day conditions. The inability to upregulate VaE1 under long-day conditions was linked to its ability to flower under these conditions. These results provide greater understanding of the molecular control of a flowering date and clues enabling the breeding of adzuki bean at higher latitudes.

Hybrid breakdown (HB) is an important type of post-zygotic reproductive barrier that inhibits hybrid production during the process of cross-breeding. A novel low temperature-dependent HB was identified in a chromosomal segment substitution line (CSSL) library derived from a cross of two rice (Oryza sativa L. japonica) cultivars, Yukihikari and Kirara397. A set of weakness symptoms in a target CSSL was observed at 23°C, but was rescued at 27°C and/or 30°C. Genetic analysis of HB using an F2:3 population of a cross between a target CSSL and Kirara397 found that a recessive temperature sensitive hybrid breakdown1 (thb1) gene from Yukihikari caused HB in the genetic background of Kirara397. Molecular mapping showed that thb1 was located within a 199-kb fragment on chromosome 6. A genetic study of F2 populations of reciprocal crosses between Yukihikari and Kirara397 confirmed that this HB was induced by the interaction of two recessive genes. These results provide important clues to further dissect the mechanism of generation of a novel temperature sensitive HB in rice intrasubspecific crosses and suggest that these linked markers will useful in rice breeding.

Grain yield-related traits and grain quality-related traits are important for rice cultivars. The quantitative trait loci (QTLs) involved in controlling the natural variation in these traits among closely related cultivars are still unclear. The present study describes the development of a novel chromosome segment substitution line (CSSL) population derived from a cross between the temperate japonica cultivars Yukihikari and Kirara397, which are grown in Hokkaido, the northernmost limit for rice cultivation. Days to heading, culm length, panicle length, panicle number, brown grain weight per plant, thousand brown grain weight, brown grain length, brown grain width, brown grain thickness, apparent amylose content, and protein content were evaluated. Panicle length, brown grain length and amylose content differed significantly in the parental cultivars. Thirty-five significant changes in the evaluated traits were identified in the CSSLs. A total of 28 QTLs were located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 12. These findings could be useful for breeding rice cultivars in the northernmost limit for rice cultivation.

Epicotyl length (ECL) of adzuki bean (Vigna angularis) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F2 population derived from a cross between the breeding line ‘Tokei1121’ (T1121, long epicotyls) and the cultivar ‘Erimo167’ (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F5 residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation.

Quantitative trait loci (QTLs) associated with eating quality, grain appearance quality and yield-related traits were mapped in recombinant inbred lines (RILs) derived from closely related rice (Oryza sativa L. subsp. japonica) cultivars, Yukihikari (good eating quality) and Joiku462 (superior eating quality and high grain appearance quality). Apparent amylose content (AAC), protein content (PC), brown grain length (BGL), brown grain width (BGWI), brown grain thickness (BGT), brown grain weight per plant (BGW) and nine yield-related traits were evaluated in 133 RILs grown in four different environments in Hokkaido, near the northernmost limit for rice paddy cultivation. Using 178 molecular markers, a total of 72 QTLs were detected, including three for AAC, eight for PC, two for BGL, four for BGWI, seven for BGT, and six for BGW, on chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 11 and 12. Fifteen intervals were found to harbor multiple QTLs affecting these different traits, with most of these QTL clusters located on chromosomes 4, 6, 8, 9 and 12. These QTL findings should facilitate gene isolation and breeding application for improvement of eating quality, grain appearance quality and yield of rice cultivars.

The induction of flowering under long‐day conditions is an important adaptation by short‐day plants, such as adzuki beans (Vigna angularis), to high‐latitude environments. This study clarified the genetic control underlying the long‐day insensitivity of adzuki bean cultivar ‘Shumari’. ‘Shumari’ was found to be insensitive to a 16‐h day, whereas landrace Acc2265 was highly sensitive. When grown under natural long‐day conditions at Obihiro (42°9′N), Acc2265 initiated flowering at least 80 days after ‘Shumari’. When 86 recombinant inbred lines (RILs) derived from crosses between ‘Shumari’ and Acc2265 were grown under these conditions, their flowering dates ranged from the middle of July to the end of October. The distinct bimodal distribution in the RIL population was due to a single major gene, designated Flowering Date1 (FD1). Molecular mapping showed that FD1 was located between the SSR markers Az02‐37M3 and Az02‐40M9, at distances of 6 and 10.4 cM, respectively, on linkage group 2. RILs carrying FD1^S lacked long‐day sensitivity, whereas RILs carrying FD1^A were sensitive to long‐day conditions, confirming that FD1 controls long‐day sensitivity.