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Objective Brain-dead donor lung transplantation frequently requires reduction in the size of oversized lung grafts for patients with a small chest cavity. We focused on the role of three-dimensional computed tomography (3D-CT) volumetry for recipients on downsizing oversized lung grafts. Methods We performed 53 brain-dead donor bilateral lung transplantations, including 15 lobar lung transplants (Lobar group) and 38 standard lung transplants with full-sized grafts (Full group), between December 2010 and December 2018. Recipient chest volume before transplantation was measured using 3D-CT volumetry, and donor lung volume was evaluated by predicted total lung capacity. Post-transplant outcomes and pulmonary function were retrospectively compared between the groups. Results The ratio of the recipient chest volume to the donor lung volume was significantly lower in the Lobar group (0.42 ± 0.15) than in the Full group (0.77 ± 0.30, P

Brassica rapa L. is an important vegetable and oilseed crop. We investigated the distribution of the histone mark tri-methylation of H3K27 (H3K27me3) in B. rapa and its role in the control of gene expression at two stages of development (2-day cotyledons and 14-day leaves) and among paralogs in the triplicated genome. H3K27me3 has a similar distribution in two inbred lines, while there was variation of H3K27me3 sites between tissues. Sites that are specific to 2-day cotyledons have increased transcriptional activity, and low levels of H3K27me3 in the gene body region. In 14-day leaves, levels of H3K27me3 were associated with decreased gene expression. In the triplicated genome, H3K27me3 is associated with paralogs that have tissue-specific expression. Even though B. rapa and Arabidopsis thaliana are not closely related within the Brassicaceae, there is conservation of H3K27me3-marked sites in the two species. Both B. rapa and A. thaliana require vernalization for floral initiation with FLC being the major controlling locus. In all four BrFLC paralogs, low-temperature treatment increases H3K27me3 at the proximal nucleation site reducing BrFLC expression. Following return to normal temperature growth conditions, H3K27me3 spreads along all four BrFLC paralogs providing stable repression of the gene.

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.

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.

The Brassica genus comprises of agro-economically important vegetables. Disease causes great yield loss of Brassica vegetables worldwide. Different traditional methods such as crop rotation and chemical control have limited effect on different diseases of Brassica vegetables and cannot completely eradicate the pathogens by these methods. Development of disease resistant cultivars is one of the most effective, ecofriendly, and cheapest measure to control Brassica diseases. With the development of genomics, molecular biology techniques, and biological methods, it is possible to discover and introduce resistance (R) genes to efficiently control the plant diseases caused by pathogens. Some R genes of major diseases such as Fusarium wilt and clubroot in Brassica vegetables have been already identified. Therefore, we will focus to review the Fusarium wilt and clubroot resistance in Brassica vegetables and the methodologies for identification, mapping, and pyramiding of R genes/quantitative trait loci (QTLs) to develop disease resistant cultivars. These techniques will be helpful for sustainable crop production and to maintain global food security and contribute to ensure protection of food supply in the Asian country as well as throughout the world.

KEY MESSAGE: Fusarium yellows resistant and susceptible lines in Brassica rapa showed different salicylic acid responses; the resistant line showed a similar response to previous reports, but the susceptible line differed. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease. Previous studies showed that genes related to salicylic acid (SA) response were more highly induced following Foc infection in Brassica rapa Fusarium yellows resistant lines than susceptible lines. However, SA-induced genes have not been identified at the whole genome level and it was unclear whether they were up-regulated by Foc inoculation. Transcriptome analysis with and without SA treatment in the B. rapa Fusarium yellows susceptible line 'Misugi' and the resistant line 'Nanane' was performed to obtain insights into the relationship between SA sensitivity/response and Fusarium yellows resistance. 'Nanane's up-regulated genes were related to SA response and down-regulated genes were related to jasmonic acid (JA) or ethylene (ET) response, but differentially expressed genes in 'Misugi' were not. This result suggests that Fusarium yellows resistant and susceptible lines have a different SA response and that an antagonistic transcription between SA and JA/ET responses was found only in a Fusarium yellows resistant line. SA-responsive genes were induced by Foc inoculation in Fusarium yellows resistant (RJKB-T23) and susceptible lines (RJKB-T24). By contrast, 39 SA-induced genes specific to RJKB-T23 might function in the defense response to Foc. In this study, SA-induced genes were identified at the whole genome level, and the possibility, the defense response to Foc observed in a resistant line could be mediated by SA-induced genes, is suggested. These results will be useful for future research concerning the SA importance in Foc or other diseases resistance in B. rapa.

The genus Brassica contains important vegetable crops, which serve as a source of oil seed, condiments, and forages. However, their production is hampered by various diseases such as clubroot and Fusarium wilt, especially in Brassica vegetables. Soil-borne diseases are difficult to manage by traditional methods. Host resistance is an important tool for minimizing disease and many types of resistance (R) genes have been identified. More than 20 major clubroot (CR) disease-related loci have been identified in Brassica vegetables and several CR-resistant genes have been isolated by map-based cloning. Fusarium wilt resistant genes in Brassica vegetables have also been isolated. These isolated R genes encode the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rice-repeat (TIR-NBS-LRR) protein. DNA markers that are linked with disease resistance allele have been successfully applied to improve disease resistance through marker-assisted selection (MAS). In this review, we focused on the recent status of identifying clubroot and Fusarium wilt R genes and the feasibility of using MAS for developing disease resistance cultivars in Brassica vegetables.

Heterosis or hybrid vigour is a phenomenon in which hybrid progeny exhibit superior yield and biomass to parental lines and has been used to breed F1 hybrid cultivars in many crops. A similar level of heterosis in all F1 individuals is expected as they are genetically identical. However, we found variation in rosette size in individual F1 plants from a cross between C24 and Columbia-0 accessions of Arabidopsis thaliana. Big-sized F1 plants had 26.1% larger leaf area in the first and second leaves than medium-sized F1 plants at 14 days after sowing in spite of the identical genetic background. We identified differentially expressed genes between big- and medium-sized F1 plants by microarray, genes involved in the category of stress response were overrepresented. We made transgenic plants overexpressing 21 genes, which were differentially expressed between the two size classes, and some lines had increased plant size at 14 or 21 days after sowing but not at all time points during development. Change of expression levels in stress-responsive genes among individual F1 plants could generate the variation in plant size of individual F1 plants in A. thaliana.

DNA methylation is an epigenetic gene regulatory mechanism that plays an essential role in gene expression, transposon silencing, genome imprinting and plant development. We investigated the influence of DNA methylation on gene expression in Brassica rapa L., to understand whether epigenetic differences exist between inbred lines. Genome-wide DNA methylation was analysed by methylated DNA immunoprecipitation sequencing (MeDIP-seq) of 14-day-old first and second leaves from two inbred lines of Chinese cabbage, one susceptible and one resistant to fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans. MACS (model-based analysis for ChIP-seq) identified DNA methylation peaks in genic regions including 2 kb upstream, exon, intron and 2 kb downstream. More than 65% of genes showed similar patterns of DNA methylation in the genic regions in the two inbred lines. DNA methylation states of the two inbred lines were compared with their transcriptome. Genes having DNA methylation in the intron and in the 200 bp upstream and downstream regions were associated with a lower expression level in both lines. A small number of genes showed a negative correlation between differences in DNA methylation levels and differences in transcriptional levels in the two inbred lines, suggesting that DNA methylation in these genes results in transcriptional suppression.

Heterosis or hybrid vigor is a phenomenon where hybrid progeny have superior performance compared to their parental inbred lines. This is important in the use of F1 hybrid cultivars in many crops and vegetables. However, the molecular mechanism of heterosis is not clearly understood. Gene interactions between the two genomes such as dominance, overdominance, and epistasis have been suggested to explain the increased biomass and yield. Genetic analyses of F1 hybrids in maize, rice, and canola have defined a large number of quantitative trait loci, which may contribute to heterosis. Recent molecular analyses of transcriptomes together with reference to the epigenome of the parents and hybrids have begun to uncover new facts about the generation of heterosis. These include the identification of gene expression changes in hybrids, which may be important for heterosis, the role of epigenetic processes in heterosis, and the development of stable high yielding lines.

Flowering time is an important agronomic trait for Brassica rapa crops, and previous breeding work in Brassica has successfully transmitted other important agronomic traits from donor species. However, there has been no previous attempts to produce hybrids replacing the original Brassica FLC alleles with alien FLC alleles. In this paper, we introduce the creation of a chromosome substitution line (CSSL) containing a homozygous introgression of Flowering Locus C from Brassica oleracea (BoFLC2) into a B. rapa genomic background, and characterize the CSSL line with respect to the parental cultivars. The preferential transmission of alien chromosome inheritance and the pattern of transmission observed during the production of the CSSLs are also discussed.

The genome-wide characterization of single nucleotide polymorphism (SNP) between cultivars or between inbred lines contributes to the creation of genetic markers that are important for plant breeding. Functional markers derived from polymorphisms within genes that affect phenotypic variation are especially valuable in plant breeding. Here, we report on the genome re-sequencing and analysis of the two parental inbred lines of the commercial F1 hybrid Chinese cabbage cultivar "W77". Through the genome-wide identification and classification of the SNPs and indels present in each parental line, we identified about 1,500 putative non-functional genes in each parent. We designed cleaved amplified polymorphic sequence (CAPS) markers using specific mutations found at Eco RI restriction sites in the parental lines and confirmed their Mendelian segregation by constructing a linkage map using 96 F2 plants derived from the F1 hybrid cultivar, "W77". Our results and data will be a useful genomic resource for future studies of gene function and metagenomic studies in Chinese cabbage.

Epigenetic regulation, covalent modification of DNA and changes in histone proteins are closely linked to plant development and stress response through flexibly altering the chromatin structure to regulate gene expression. In this review, we will illustrate the importance of epigenetic influences by discussing three agriculturally important traits of Brassicaceae. (1) Vernalization, an acceleration of flowering by prolonged cold exposure regulated through epigenetic silencing of a central floral repressor, FLOWERING LOCUS C. This is associated with cold-dependent repressive histone mark accumulation, which confers competency of consequence vegetative-to-reproductive phase transition. (2) Hybrid vigor, in which an F1 hybrid shows superior performance to the parental lines. Combination of distinct epigenomes with different DNA methylation states between parental lines is important for increase in growth rate in a hybrid progeny. This is independent of siRNA-directed DNA methylation but dependent on the chromatin remodeler DDM1. (3) Self-incompatibility, a reproductive mating system to prevent self-fertilization. This is controlled by the S-locus consisting of SP11 and SRK which are responsible for self/non-self recognition. Because self-incompatibility in Brassicaceae is sporophytically controlled, there are dominance relationships between S haplotypes in the stigma and pollen. The dominance relationships in the pollen rely on de novo DNA methylation at the promoter region of a recessive allele, which is triggered by siRNA production from a flanking region of a dominant allele.

Resistant and susceptible lines in Brassica rapa have different immune responses against Fusarium oxysporum inoculation. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease of Brassicaceae; however, the mechanism of how host plants respond to Foc is still unknown. By comparing with and without Foc inoculation in both resistant and susceptible lines of Chinese cabbage (Brassica rapa var. pekinensis), we identified differentially expressed genes (DEGs) between the bulked inoculated (6, 12, 24, and 72 h after inoculation (HAI)) and non-inoculated samples. Most of the DEGs were up-regulated by Foc inoculation. Quantitative real-time RT-PCR showed that most up-regulated genes increased their expression levels from 24 HAI. An independent transcriptome analysis at 24 and 72 HAI was performed in resistant and susceptible lines. GO analysis using up-regulated genes at 24 HAI indicated that Foc inoculation activated systemic acquired resistance (SAR) in resistant lines and tryptophan biosynthetic process and responses to chitin and ethylene in susceptible lines. By contrast, GO analysis using up-regulated genes at 72 HAI showed the overrepresentation of some categories for the defense response in susceptible lines but not in the resistant lines. We also compared DEGs between B. rapa and Arabidopsis thaliana after F. oxysporum inoculation at the same time point, and identified genes related to defense response that were up-regulated in the resistant lines of Chinese cabbage and A. thaliana. Particular genes that changed expression levels overlapped between the two species, suggesting that they are candidates for genes involved in the resistance mechanisms against F. oxysporum.

As climatic changes occur over the coming decades, our scientific understanding of plant responses to environmental cues will become an increasingly important consideration in the breeding of agricultural crops. This review provides a summary of the literature regarding vernalization research in Brassicaceae, covering both the historical origins of vernalization research and current understanding of the molecular mechanisms behind the regulatory pathways involved in vernalization and subsequent inflorescence. We discuss the evolutionarily conserved biology between the model organism Arabidopsis thaliana and the Brassica genus of crop cultivars and contrast the differences between the genera to illustrate the importance of Brassica-specific research into vernalization.

Covalent modifications of histone proteins act as epigenetic regulators of gene expression. We report the distribution of two active histone marks (H3K4me3 and H3K36me3) in 14-day leaves in two lines of Brassica rapa L. by chromatin immunoprecipitation sequencing. Both lines were enriched with H3K4me3 and H3K36me3 marks at the transcription start site, and the transcription level of a gene was associated with the level of H3K4me3 and H3K36me3. H3K4me3- and H3K36me3-marked genes showed low tissue-specific gene expression, and genes with both H3K4me3 and H3K36me3 had a high level of expression and were constitutively expressed. Bivalent active and repressive histone modifications such as H3K4me3 and H3K27me3 marks or antagonistic coexistence of H3K36me3 and H3K27me3 marks were observed in some genes. Expression may be susceptible to changes by abiotic and biotic stresses in genes having both H3K4me3 and H3K27me3 marks. We showed that the presence of H3K36me3 marks was associated with different gene expression levels or tissue specificity between paralogous paired genes, suggesting that H3K36me3 might be involved in subfunctionalization of the subgenomes.

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 F2 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 F2 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.

Objectives The aim of the study was to evaluate the feasibility of Ramelteon for the prevention of delirium after lung cancer surgery in elderly patients. Methods Medical records of patients over 70 years old, who underwent anatomical pulmonary resection for lung cancer at our institution from January 2013 to December 2015, were reviewed. Patients treated in 2013 and 2014 were used as a control group. Ramelteon was administered daily for 7 days after surgery. The incidence of delirium was determined based on the Intensive Care Delirium Screening Checklist (ICDSC). Scores of ≥4 and 1-3 points were used for the diagnoses of delirium and a pre-delirious state, respectively. Results There were 24 patients in the Ramelteon group and 58 patients in the control group. ICDSC scores of ≥4 points were found for no patients in the Ramelteon group and 5 (9%) in the control group, whereas 21 (88%) and 49 (85%) patients, in the respective groups, had ICDSC scores of 0 points. The average incidence of events, associated with delirium, showed a trend of being lower in the Ramelteon group (0.25 ± 0.74 vs 1.58 ± 4.93, P = 0.061), and all events in the Ramelteon group occurred on the day of surgery. Thus, only one day was required for complete recovery from delirium in the Ramelteon group, whereas 8 days were needed in the control group. The peak delirious state occurred after 5 days in the control group. Conclusions Ramelteon is likely to reduce the incidence and intensity of delirium after surgery for lung cancer in elderly patients.

Chinese cabbage ( Brassica rapa L. var. pekinensis ) is an important vegetable in Asia. Most Japanese commercial cultivars of Chinese cabbage use an F 1 hybrid seed production system because of the high yielding cultivars produced. An efficient method for predicting hybrid performance in the parental generations is desired, and genetic distance between parental lines might be a good indicator of the level of hybrid vigor in a cross. Information concerning the genetic relationships among parental candidate inbred lines is useful for variety protection. The number of DNA markers available that can be used to assess the purity of inbred lines is limited in B. rapa . The aim of this study is to use DNA markers to assess the genetic distance between inbred lines to examine early developmental and yield heterosis so as to develop methods for selecting the best parental lines for the production of hybrids. We screened highly polymorphic SSR and CAPS markers to assess the genetic uniformity of inbred lines and characterize their genetic relationship. We examined the early size and yield heterosis in 32 F 1 hybrids of Chinese cabbage. There was a moderate correlation in mid-parent heterosis between leaf size at 21 days after sowing and harvested biomass but not in best-parent heterosis. In contrast there was no correlation between genetic distance and mid-parent or best-parent heterosis, indicating that genetic distance does not predict the heterosis phenotype.

Chinese cabbage (Brassica rapa L. var. pekinensis) is an important vegetable in Asia, and most Japanese commercial cultivars of Chinese cabbage use an F1 hybrid seed production system. Self-incompatibility is successfully used for the production of F1 hybrid seeds in B. rapa vegetables to avoid contamination by non-hybrid seeds, and the strength of self-incompatibility is important for harvesting a highly pure F1 seeds. Prediction of agronomically important traits such as disease resistance based on DNA markers is useful. In this dataset, we identified the S haplotypes by DNA markers and evaluated the strength of self-incompatibility in Chinese cabbage inbred lines. The data described the predicted disease resistance to Fusarium yellows or clubroot in 22 Chinese cabbage inbred lines using gene associated or gene linked DNA markers. Keywords: Brassica rapa, F1 hybrid cultivar, Marker assisted selection, Fusarium yellows, Clubroot disease, Self-incompatibility

Inter-specific hybrids are a useful source for increasing genetic diversity. Some reproductive barriers before and/or after fertilization prevent production of hybrid plants by inter-specific crossing. Therefore, techniques to overcome the reproductive barrier have been developed, and have contributed to hybridization breeding. In recent studies, identification of molecules involved in plant reproduction has been studied to understand the mechanisms of reproductive barriers. Revealing the molecular mechanisms of reproductive barriers may allow us to overcome reproductive barriers in inter-specific crossing, and to efficiently produce inter-specific hybrids in cross-combinations that cannot be produced through artificial techniques. Inter-specific hybrid plants can potentially serve as an elite material for plant breeding, produced through the merging of genomes of parental species by allopolyploidization. Allopolyploidization provides some benefits, such as heterosis, increased genetic diversity and phenotypic variability, which are caused by dynamic changes of the genome and epigenome. Understanding of allopolyploidization mechanisms is important for practical utilization of inter-specific hybrids as a breeding material. This review discusses the importance of reproductive barriers and the effect of allopolyploidization in crop breeding programs.

While some cases of nocardial pneumonia develop secondary empyema, tension pyopneumothorax is a very rare and lethal complication. A 74-year-old man who exhibited thrombocytopenia during steroid therapy for autoimmune hepatitis, presented to our department with a nocardial tension pyopneumothorax. He underwent a left lower lobectomy after chest drainage, and was discharged without any complication other than reoperation to remove a postoperative hematoma.

Uniparental gene expression, observed in both animals and plants, is termed genomic imprinting. Genomic imprinting is a well-known epigenetic phenomenon regulated through epigenetic modifications such as DNA methylation and histone modifications. Recent genome-wide studies of endosperm transcription have revealed the rapid change of imprinted genes between species, suggesting the flexibility of this phenomenon. Although the functional significance and evolutionary trends of imprinted genes are still obscure, it can be clarified by inter-species comparisons. In this study, we analyzed the pattern of genomic imprinting in Brassica rapa, a species related to Arabidopsis thaliana. Compared with the ancient karyotype of A. thaliana and B. rapa, B. rapa has a triplicated genome. Many imprinted genes, beyond the estimated number previously reported in other species, were observed. Several imprinted genes have been conserved among species in Brassicaceae. We also observed rapid molecular evolution of imprinted genes compared to non-imprinted genes in B. rapa. Especially, imprinted gene overlapping between species showed more rapid molecular evolution and preferential expression in endosperms. It may imply that a small number of imprinted genes have retained functional roles among diverged species and have been the target of natural selection.

© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. Epigenetic gene regulation is crucial to plant life and can involve dynamic interactions between various histone modifications, DNA methylation, and small RNAs. Detailed analysis of epigenome information is anticipated to reveal how the DNA sequence of the genome is translated into the plant’s phenotype. The aim of this study was to map the DNA methylation state at the whole genome level and to clarify the relationship between DNA methylation and transcription, small RNA expression, and histone H3 lysine 9 di-methylation (H3K9me2) in Brassica rapa. We performed whole genome bisulfite sequencing, small RNA sequencing, and chromatin immunoprecipitation sequencing using H3K9me2 antibody in a Chinese cabbage inbred line, RJKB-T24, and examined the impact of epigenetic states on transcription. Cytosine methylation in DNA was analysed in different sequence contexts (CG, CHG, and CHH) (where H could be A, C, or T) and position (promoter, exon, intron, terminator, interspersed repeat regions), and the H3K9me2 and 24 nucleotide small interfering RNAs (24nt-siRNA) were overlaid onto the B. rapa reference genome. The epigenome was compared with that of Arabidopsis thaliana and the relationship between the position of DNA methylation and gene expression, and the involvement of 24nt siRNAs and H3K9me2 are discussed.

Heterosis or hybrid vigor is a phenomenon in which a hybrid progeny exhibits superior performance compared to their parental inbred lines. F1 hybrid seed production system is used for many crops and vegetables because of the high yield due to hybrid vigor. It has been over 100 years since the discovery of the hybrid vigor phenomenon; however, the molecular mechanism of hybrid vigor is still a mystery. Understanding the mechanism of hybrid vigor will allow developing more efficient breeding systems and contribute to food security. At this time, hybrid vigor research has mainly used genetic approaches providing a number of findings in many plant species. High throughput sequencing will make genetic analysis more efficient, and large-scale quantitative trait analysis or genome wide association analysis has been performed in some plant species. However recent works have revealed that epigenetic regulations also contribute to hybrid vigor. In this chapter, we will discuss the most recent findings suggesting that epigenetic regulation may play important roles in hybrid vigor.

Cabbage (Brassica oleracea L. var. capitata) is an important vegetable worldwide. Most Japanese commercial cultivars of cabbage use an F1 hybrid seed production system. The purity of F1 hybrid seeds is important and the assessment of purity based on DNA markers can be highly accurate. In addition, selection of agronomically important traits such as disease resistance based on DNA markers is useful for breeding of cabbage. The aim of this study is to demonstrate the effectiveness of DNA marker-assisted selection in cabbage. In this study we distinguished the parental S haplotypes in 35 F1 hybrid cultivars by combining several linked DNA markers. Thirty-one highly polymorphic simple sequence repeats (SSR) markers were screened from 175 reported SSR markers, which are useful for assessment of the purity of F1 hybrid seeds. We examined the relationship between the DNA marker based genotype and the phenotype by an inoculation test of clubroot disease. A co-dominant PCR–RFLP marker was developed for selection of Fusarium yellows resistance and the genotypes using this marker were consistent with inoculation test in all tested samples.

We report the case of a 64-year-old woman with recurrent mediastinal dedifferentiated liposarcoma. She had undergone surgical resection twice. Subsequently, on regular follow-up visits, chest computed tomography demonstrated a third local recurrence of the tumor with invasion into adjacent intrathoracic organs. Adhesion of the heart to the anterior chest wall through a pericardial defect that had been inadvertently created at the second operation was suspected. We decided to use a thoracoabdominal approach with four-dimensional computed tomography (4DCT) for preoperative guidance. The patient had also undergone MRI; however, the result was equivocal regarding adhesion of intrathoracic organs. The use of 4DCT enabled successful en bloc resection of the tumor. Results of the pathology examination showed complete resection of a dedifferentiated liposarcoma. The patient's postoperative course was uneventful, and she was discharged on postoperative day 19.

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.

Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis.

Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4(th) lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.

Fusarium oxysporum f. sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change >=2 fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and ten of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.

The tuberous stem of kohlrabi is an important quantitative trait, which affects its yield and quality. Genetic control of this trait has not yet been unveiled. To identify the QTLs controlling stem swelling of kohlrabi, a BC1 population of 92 plants was developed from a cross of broccoli DH line GCP04 and kohlrabi var. Seine. A wide range of variation in tuberous stem diameter was observed among the mapping populations. We constructed a genetic map of nine linkage groups (LGs) with different types of markers, spanning a total length of 913.5 cM with an average marker distance of 7.55 cM. Four significant QTLs for radial enlargement of kohlrabi stem, namely, REnBo1, REnBo2, REnBo3, and REnBo4 were detected on C02, C03, C05, and C09, respectively, and accounted for the phenotypic variation of 59% for the stem diameter and 55% for the qualitative grading of tuberous stem in classes. Then, we confirmed the stability of identified QTLs using BC1S1 populations derived from the BC1 plants having heterozygous alleles at the target QTL and homozygous kohlrabi alleles at the remaining QTLs. REnBo1and REnBo2 using 128 plants of BC168S1 and 94 plants of BC143S1, respectively, and REnBo3 and REnBo4 using 152 plants of BC157S1 were detected at the same positions as the respective QTLs of the BC1 population. Confirmation of QTLs in two successive generations indicates that the QTLs are persistent. The QTLs obtained in this study could be useful in marker-assisted selection of kohlrabi breeding, and to understand the genetic mechanisms of stem swelling and storage organ development in kohlrabi and other Brassica species.

Background Heterosis or hybrid vigour is a phenomenon in which hybrid progeny exhibit superior performance compared to their parental inbred lines. Most commercial Chinese cabbage cultivars are F1 hybrids and their level of hybrid vigour is of critical importance and is a key selection criterion in the breeding system. Results We have characterized the heterotic phenotype of one F1 hybrid cultivar of Chinese cabbage and its parental lines from early- to late-developmental stages of the plants. Hybrid cotyledons are larger than those of the parents at 4 days after sowing and biomass in the hybrid, determined by the fresh weight of leaves, is greater than that of the larger parent line by approximately 20 % at 14 days after sowing. The final yield of the hybrid harvested at 63 days after sowing is 25 % greater than the yield of the better parent. The larger leaves of the hybrid are a consequence of increased cell size and number of the photosynthetic palisade mesophyll cells and other leaf cells. The accumulation of plant hormones in the F1 was within the range of the parental levels at both 2 and 10 days after sowing. Two days after sowing, the expression levels of chloroplast-targeted genes in the cotyledon cells were upregulated in the F1 hybrid relative to their mid parent values. Shutdown of chlorophyll biosynthesis in the cotyledon by norflurazon prevented the increased leaf area in the F1 hybrid. Conclusions In the cotyledons of F1 hybrids, chloroplast-targeted genes were upregulated at 2 days after sowing. The increased activity levels of this group of genes suggested that their differential transcription levels could be important for establishing early heterosis but the increased transcription levels were transient. Inhibition of the photosynthetic process in the cotyledon reduced heterosis in later seedling stages. These observations suggest early developmental events in the germinating seedling of the hybrid may be important for later developmental vigour and yield advantage. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0734-3) contains supplementary material, which is available to authorized users.

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed., Author Summary Genomic imprinting, mono-allelic gene expression depending on the parent-of-origin, is an epigenetic process known in mammals and flowering plants. A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. The FWA promoter is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is necessary. Here we show that the direct repeat structure is highly diverse in species within the genus Arabidopsis. Unexpectedly, we found that the direct repeat structure is dispensable for the epigenetic silencing and methylation of the FWA promoter. Rather, the SINE-related promoter sequence is sufficient for these features. Frequent independent formation of the tandem repeats suggests that they may be a consequence of the epigenetically controlled system.