DECREASED IN DNA METHYLATION 1 (DDM1) is a chromatin remodeling factor that plays a major role in maintaining heterochromatin silencing and DNA methylation in plants. The soybean (Glycine max) genome, known for its large amount of pericentric heterochromatin, contains two DDM1 orthologs: GmDDM1a and GmDDM1b. In order to understand their functional roles in seed development, a process in which DNA methylation plays a significant role, we conducted mutant analysis. Single mutant and sesquimutant plants were healthy and produced seeds normally, but double mutants did not survive after germination. Methylome analysis of immature seeds detected an extensive decrease in methylation of CG and CHG sites in mutants. In fact, levels of CHG hypomethylation were correlated to the number of mutant alleles; however, severe CG methylation loss was only seen in double mutants. RNA-seq results showed that more than 3000 transposable elements (TEs) were activated transcriptionally in double mutants, which is consistent with the severe hypomethylation in their pericentromeric regions. Besides, we found 1388 protein-coding genes commonly increased in expression in all mutant genotypes ("Shared-up"), whereas 558 genes were only derepressed in double mutants ("Double-up"). These "Double-up" genes were largely found in heterochromatic regions, while "Shared-up" genes were mainly spread over the euchromatic chromosomal arms in which hypomethylation of mutants was substantially less than that of heterochromatin. These results explain the pivotal role of GmDDM1 in maintaining heterochromatin silencing and suggest the existence of a distinct GmDDM1-mediated euchromatin silencing mechanism during soybean seed development.

Common bean (Phaseolus vulgaris L.) is one of the most abundantly consumed legume crops as foods worldwide. In many African countries, this crop is an important staple food because of its rich nutrients. The Great Lakes region of Central Africa, which includes Rwanda, the nation with the highest per capita consumption of common beans worldwide, is known to be a center of common bean diversity in Africa. Increasing the amount of iron and zinc in common bean for biofortification has been a key breeding goal in Rwanda and other countries. In this study, using 192 accessions, including local landraces from Rwanda, breeding materials, released varieties, and others, we performed genome wide association studies (GWAS) to determine the loci governing those traits in addition to other agronomic traits. We identified a locus that was strongly associated with seed zinc concentration and candidate genes. The information might be a great help for marker-assisted breeding of this trait in common bean.

Tuberous stem is a key determinant of yield and quality in kohlrabi, and it is a prime target of artificial selection for breeding and domestication. To dissect the genetic basis of tuberization, a previously identified major QTL, REnBo2 on chromosome C03 was fine mapped to a 42.8-kb interval using the recombinant populations of three different backcross generations derived from the double haploid lines of kohlrabi and broccoli. Three genes namely Bol012972, Bol012973 and Bol012974 encoding a CBL-interacting protein kinase 6 (CIPK6), fatty acid desaturase 6 (FAD6) and WRKY transcription factor 32 (WRKY32), respectively, were annotated in the fine-mapped interval in Brassica oleracea reference genomes. FAD6 is unlikely to be the responsible gene due to its function as a fatty acid desaturase. Based on the gene structure and expression analyses of the remaining two candidates along with our previously conducted transcriptome and endogenous phytohormone profiles, Bol012972 (CIPK6) was identified as a promising candidate for REnBo2. Since the knockdown mutant of CIPK6 was known to exhibits swollen hypocotyls in Arabidopsis, we speculate that CIPK6 may regulate kohlrabi tuber development through regulating the auxin levels. The result will provide a foundation to elucidate the genetic mechanism for tuber formation in kohlrabi and other Brassica crops.

Brassica rapa L., which includes Chinese cabbage, turnip, and pak choi, has more complex flowering time regulation than does Arabidopsis thaliana due to the presence of multiple paralogous flowering time genes. FLOWERING LOCUS C ( FLC ) is one of the key genes regulating the flowering time, and B. rapa has four FLC paralogs. BrFLC5 on the reference genome is deemed a pseudogene because of a mutation (from G to A) in the splice site of the third intron, but there are some accessions with a G nucleotide in the splice site. In this study, we genotyped 310 B. rapa accessions and found that 19 had homozygous and 81 had heterozygous putative functional BrFLC5 alleles. Accessions of turnip showed the highest proportion with a functional BrFLC5 allele. BrFLC5 acts as a floral repressor when overexpressed in A. thaliana . The BrFLC5 expression level varied in pre-vernalized plants, and this transcriptional variation was not associated with the G/A polymorphism in the third intron. Three accessions having a higher BrFLC5 expression in pre-vernalized plants had a 584-bp insertion in the promoter region. Many regions homologous to this 584-bp sequence are present in the B. rapa genome, and this 584-bp inserted region has tandem duplications of an AT-rich sequence in its central region. The possibility that a high expression of a functional BrFLC5 could contribute to producing premature bolting-resistant lines in B. rapa vegetables is discussed.

To unveil the mechanism of tuberous stem formation at early growth stages in kohlrabi, the comparative stem morphology, anatomy, and endogenous plant hormone profiles were studied at different days after germination (10 to 30 DAG). Stem anatomy revealed that the notable feature of the kohlrabi stem during the transition from primary to secondary growth was the formation of vascular bundle circles, with many phloems formed separately and arranged in a circular pattern along with the cambium layer, resulting in an expanded ring of primary and secondary phloem. In addition, medullary supplemental vascular bundles were differentiated in the central re-gion of pith and successively supplied small-sized meristematic parenchymatous cells which contribute to the tuber initiation in kohlrabi. Six phytohormones were quantified in the stems and leaves of both genotypes. The absence of Gibberellin A4 (GA4) in kohlrabi stem might be the cause of the stunted stem of kohlrabi. The total endogenous cytokinin in stem of kohlrabi was more than twice of that of broccoli, where trans-zeatin was the most abundant bioactive form. This can be explained by the meristematic nature of the cambium of kohlrabi stem. Profiles for Indole-3-acetic acid, Indole-3-acetylaspartic acid, and Abscisic acid of the stems and leaves of kohlrabi and broccoli were significantly different at several time points tested. Thus, the key features of kohlrabi during the initiation of tuberous stem were the formation of the phloem ring along with the cambium and the supplemental vascular bundles. This is the first paper showing the hormonal profile in the early seedling stage of kohlrabi. These findings provide useful information to elucidate the mechanism of tuberous stem formation of kohlrabi and other tuber crops.

シンテッポウユリ無花粉品種‘あきた清ひめ’とテッポウユリ品種の交配育種により，F2世代で無花粉の系統が得られることを明らかにした．この無花粉に関する遺伝子は潜性遺伝子であると推察される．F2世代における実際の無花粉株率は，種子の発芽率，抽苔率および開花率の影響を受け，変動する．育成された系統は，完全無花粉の性質に加えて，シンテッポウユリの生育旺盛な性質を有し，従来のテッポウユリ品種より小さな球根で切り花生産ができる新たな特性を有する．

Fusarium wilt is a significant disease in radish, but the genetic mechanisms controlling yellows resistance (YR) are not well understood. This study aimed to identify YR-QTLs and to fine-map one of them using F2:3 populations developed from resistant and susceptible radish parents. In this study, two high-density genetic maps each containing shared co-dominant markers and either female or male dominant markers that spanned 988.6 and 1127.5 cM with average marker densities of 1.40 and 1.53 cM, respectively, were generated using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) technology. We identified two YR-QTLs on chromosome R2 and R7, and designated the latter as ForRs1 as the major QTL. Fine mapping narrowed down the ForRs1 locus to a 195 kb region. Among the 16 predicted genes in the delimited region, 4 genes including two receptor-like protein and -kinase genes (RLP/RLK) were identified as prime candidates for ForRs1 based on the nucleotide sequence comparisons between the parents and their predicted functions. This study is the first to use a GRAS-Di for genetic map construction of cruciferous crops and fine map the YR-QTL on the R7 chromosome of radish. These findings will provide groundbreaking insights into radish YR breeding and understanding the genetics of YR mechanism.

Tuberous stem of kohlrabi is an important agronomic trait, however, the molecular basis of tuberization is poorly understood. To elucidate the tuberization mechanism, we conducted a comparative transcriptomic analysis between kohlrabi and broccoli at 10 and 20 days after germination (DAG) as tuberous stem initiated between these time points. A total of 5580 and 2866 differentially expressed transcripts (DETs) were identified between genotypes (kohlrabi vs. broccoli) and growth stages (10 DAG vs. 20 DAG), respectively, and most of the DETs were down-regulated in kohlrabi. Gene ontology (GO) and KEGG pathway enrichment analyses showed that the DETs between genotypes are involved in cell wall loosening and expansion, cell cycle and division, carbohydrate metabolism, hormone transport, hormone signal transduction and in several transcription factors. The DETs identified in those categories may directly/indirectly relate to the initiation and development of tuberous stem in kohlrabi. In addition, the expression pattern of the hormone synthesis related DETs coincided with the endogenous IAA, IAAsp, GA, ABA, and tZ profiles in kohlrabi and broccoli seedlings, that were revealed in our phytohormone analysis. This is the first report on comparative transcriptome analysis for tuberous stem formation in kohlrabi at early growth periods. The resulting data could provide significant insights into the molecular mechanism underlying tuberous stem development in kohlrabi as well as in other tuberous organ forming crops.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes, including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a 'shock' that stimulates genome reorganization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained unclear. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the RILs investigated. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as an unexpectedly common event in plant reproduction.

Pollen-free varieties are advantageous in promoting cut-flower production. In this study, we identified a candidate mutation which is responsible for pollen sterility in a strain of Lilium × formolongi, which was originally identified as a naturally occurred male-sterile plant in a seedling population. The pollen sterility occurred due to the degradation of pollen mother cells (PMCs) before meiotic cell division. Genetic analysis suggested that the male-sterile phenotype is attributed to one recessive locus. Transcriptome comparison between anthers of sterile and fertile plants in a segregated population identified a transcript that was expressed only in pollen-fertile plants, which is homologous to TDF1 (DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1) in Arabidopsis, a gene encoding a transcription factor AtMYB35 that is known as a key regulator of pollen development. Since tdf1 mutant shows male sterility, we assumed that the absence transcript of the TDF1-like gene, named as LflTDF1, is the reason for pollen sterility observed in the mutant. A 30 kbp-long nanopore sequence read containing LflTDF1 was obtained from a pollen-fertile accession. PCR analyses using primers designed from the sequence suggested that at least a 30kbp-long region containing LflTDF1 was deleted or replaced by unknown sequence in the pollen-sterile mutant. Since the cross between L. × formolongi and Easter lily (L. longiflorum) is compatible, we successfully introgressed the male-sterile allele, designated as lfltdf1, to Easter lily. To our knowledge, this is the first report of molecular identification of a pollen-sterile candidate gene in lily. The identification and marker development of LflTDF1 gene will assist pollen-free lily breeding of Easter lilies and other lilies.

In vegetables of Brassica rapa L., Fusarium oxysporum f. sp. rapae (For) or F. oxysporum f. sp. conglutinans (Foc) cause Fusarium yellows. A resistance gene against Foc (FocBr1) has been identified, and deletion of this gene results in susceptibility (focbr1-1). In contrast, a resistance gene against For has not been identified. Inoculation tests showed that lines resistant to Foc were also resistant to For, and lines susceptible to Foc were susceptible to For. However, prediction of disease resistance by a dominant DNA marker on FocBr1 (Bra012688m) was not associated with disease resistance of For in some komatsuna lines using an inoculation test. QTL-seq using four F-2 populations derived from For susceptible and resistant lines showed one causative locus on chromosome A03, which covers FocBr1. Comparison of the amino acid sequence of FocBr1 between susceptible and resistant alleles (FocBr1 and FocBo1) showed that six amino acid differences were specific to susceptible lines. The presence and absence of FocBr1 is consistent with For resistance in F-2 populations. These results indicate that FocBr1 is essential for For resistance, and changed amino acid sequences result in susceptibility to For. This susceptible allele is termed focbr1-2, and a new DNA marker (focbr1-2m) for detection of the focbr1-2 allele was developed.

The genus Brassica includes oil crops, vegetables, condiments, fodder crops, and ornamental plants. Brassica species underwent a whole genome triplication event after speciation between ancestral species of Brassica and closely related genera including Arabidopsis thaliana . Diploid species such as Brassica rapa and Brassica oleracea have three copies of genes orthologous to each A. thaliana gene, although deletion in one or two of the three homologs has occurred in some genes. The floral transition is one of the crucial events in a plant’s life history, and time of flowering is an important agricultural trait. There is a variation in flowering time within species of the genus Brassica , and this variation is largely dependent on a difference in vernalization requirements. In Brassica , like in A. thaliana , the key gene of vernalization is FLOWERING LOCUS C ( FLC ). In Brassica species, the vernalization response including the repression of FLC expression by cold treatment and the enrichment of the repressive histone modification tri-methylated histone H3 lysine 27 (H3K27me3) at the FLC locus is similar to A. thaliana . B. rapa and B. oleracea each have four paralogs of FLC , and the allotetraploid species, Brassica napus , has nine paralogs. The increased number of paralogs makes the role of FLC in vernalization more complicated; in a single plant, paralogs vary in the expression level of FLC before and after vernalization. There is also variation in FLC expression levels between accessions. In this review, we focus on the regulatory circuits of the vernalization response of FLC expression in the genus Brassica .

Chinese cabbage (Brassica rapa L. var. pekinensis) is an economically and agriculturally significant leafy vegetable that is extensively cultivated throughout the world. Vernalization is exposure to prolonged cold that alters gene expression and accelerates a transition from the vegetative to reproductive phase. Premature bolting caused by exposure to cold inhibits head production and reduces the yield of Chinese cabbage, and developing a late bolting line is important for breeding. Therefore, it is critical to identify the genes showing differential expression patterns during cold treatment in Chinese cabbage. However, there are few studies on the transcriptome profiling of different durations of cold treatments in Chinese cabbage. Here, we analyzed the gene expression profiles in a Chinese cabbage inbred line, RJKB-T24, given different durations of cold treatments using RNA sequencing. Differentially expressed genes (DEGs) between non-vernalized and vernalized samples tended to be downregulated, and some genes involved in the flowering pathway (including BrFLC and BrMAF genes) were downregulated following cold treatment. Functional enrichment analysis indicated that some DEGs were involved in the stress response and hormone signaling pathways. For genes involved in the FRI-containing complex, a known activator of FLC in Arabidopsis thaliana, only BrFRL1 showed changes in expression. In contrast to A. thaliana, BrVIP and BrVRN genes showed different expression patterns between paralogs during cold treatment, suggesting that Chinese cabbage's flowering pathway is somehow different and more complex than in A. thaliana. These outcomes provide significant insights into the genetic control of bolting and flowering that occur during the vernalization of Chinese cabbage.

Vernalization, a floral transition stimulated by the prolonged exposure to cold temperatures, is an undesirable feature in the breeding of leafy vegetables that impairs productivity by promoting bolting. Cabbage is categorized as a green plant vernalization type; only older plants possess vernalization competence. Despite being of agricultural importance in the breeding of bolting resistance, the molecular mechanism of agedependent acquisition of vernalization competence is poorly understood. In Arabidopsis, FLOWERING LOCUS C (FLC) is a key vernalization response gene that functions as a floral repressor. Its expression decreases upon cold exposure, leading to floral induction. In this study, we characterized three FLC orthologs in cabbage (BoFLC1, BoFLC2, and BoFLC3). Predicted amino acid sequences of the three BoFLC cDNAs were highly conserved with the MADS-box domain. The overexpression of the three BoFLCs in Arabidopsis displayed late flowering phenotypes, indicating the functional conservation of these paralogs as floral repressors in cabbage. The expression analyses before and after constant cold treatment given at different plant ages showed that the three BoFLCs displayed distinct responses to prolonged cold exposure. Regardless of plant age, BoFLC1 declined less markedly, whereas BoFLC2 and BoFLC3 were repressed by sufficient long cold treatment. In both younger and older plants, the expression of the ortholog of floral integrator gene, FLOWERING LOCUS T (FT), was induced by cold treatment, probably due to a reduction in the repressive effect of BoFLCs, suggesting that the fate of age-dependent vernalization is determined independently or downstream of BoFT induction. Our results provide new insights into the mechanism of green plant vernalization in Brassica vegetables.

Brassica rapa L. is an important agricultural crop that requires a period of prolonged cold for flowering. This process is known as vernalization. Studies have shown that long noncoding RNAs (lncRNAs) play important roles in abiotic stress responses and several cold-responsive noncoding RNAs have been suggested to be involved in vernalization. We examined the transcriptome of the Chinese cabbage inbred line ( B. rapa L. var. pekinensis ) RJKB-T24, and identified 1,444 long intergenic noncoding RNAs (lincRNAs), 551 natural antisense transcripts (NATs), and 93 intronic noncoding RNAs (incRNAs); 549 of the 2,088 lncRNAs significantly altered their expression in response to four weeks of cold treatment. Most differentially expressed lncRNAs did not lead to a change of expression levels in mRNAs covering or near lncRNAs, suggesting that the transcriptional responses to four weeks of cold treatment in lncRNA and mRNA are independent. However, some differentially expressed mRNAs had NATs with expression altered in the same direction. These genes were categorized as having an abiotic stress response, suggesting that the paired-expression may play a role in the transcriptional response to vernalization or cold treatment. We also identified short-term cold treatment induced NATs in BrFLC and BrMAF genes, which are involved in vernalization. The lncRNAs we identified differed from those reported in Arabidopsis thaliana , suggesting the role of lncRNAs in vernalization differ between these two species.

The fusarium yellows resistance (YR) gene FocBo1 was previously identified and the DNA markers were developed to assist the breeding of YR cultivars in Brassica oleracea. However, the further analysis revealed discrepancies between the phenotypes and the genotypes predicted by those DNA markers in cabbage commercial cultivars. Since this discrepancy seemed to be due to unknown susceptible alleles of focbo1, we sequenced the gene in 19 accessions to determine the sequence variations between alleles and found that there were two resistant FocBo1 alleles and six susceptible alleles in the investigated population. The newly designed PCR markers detected three mutations in the susceptible alleles that generate premature termination codons. These were shown to accurately distinguish resistant and susceptible alleles in more than 200 accessions of B. oleracea inbred lines and cultivars. The study revealed that the locus is represented by 37.2% resistant and 62.8% susceptible alleles within seventy-eight commercial cultivars. Structural analysis of the gene revealed that a part of the allelic variation comes from intragenic recombination between alleles. Our results enable a more precise prediction of the phenotype by marker assisted selection, promoting the production of YR cultivars in B. oleracea.

Brassica rapa is an important oilseed crop species next to B. napus in Brassicaceae. However, genetic improvement of B. rapa oilseeds has not been intensively conducted compared to B. napus , which resulted in the limited number of low erucic acid (LEA) cultivars, which is an essential trait for edible oil materials. Candle and Tobin are LEA B. rapa cultivars bred in Canada that are closely related to each other; however, the causal mutation has not been identified. This study was initially aimed to investigate whether the alleles of BrFAE1 , the gene encoding a key enzyme for erucic acid synthesis, in Candle and Tobin have mutations impairing their functions. An insertion of the long terminal repeat (LTR) retrotransposon, designated as BRACOPIA , was identified in the 5′ end of the coding region of the gene in both Candle and Tobin. BRACOPIA disrupted the transcription in developing seeds, resulting in a loss of function allele designated as brfae1re . Next, we found that Span, the world’s first LEA B. rapa cultivar developed in Canada, has brfae1re , suggesting that the BRACOPIA insertion is the founder mutation of Canadian LEA B. rapa cultivars. Finally, we investigated the distribution of BRACOPIA family retrotransposons in the Brassica genus, and found that they are present in both the A and C genomes, but the activity has been kept rather modest. Since brfae1re is easily distinguishable from the wild-type allele by PCR, the identification of this mutation could enhance LEA breeding in B. rapa .

There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower.