Quantitative trait locus (QTL) analysis and fine-mapping for Fusarium oxysporum disease resistance in Raphanus sativus using GRAS-Di technology.

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.

Role of DNA methylation in hybrid vigor in Arabidopsis thaliana.

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.

Allele specific DNA marker for fusarium resistance gene FocBo1 in Brassica oleracea.

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.

Genome-Wide Analysis of Parent-of-Origin Allelic Expression in Endosperms of Brassicaceae Species, Brassica rapa.

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.

Molecular and cellular characteristics of hybrid vigour in a commercial hybrid of Chinese cabbage.

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.

The importance of reproductive barriers and the effect of allopolyploidization on crop breeding.

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.

Map-based cloning of a candidate gene conferring Fusarium yellows resistance in Brassica oleracea.

KEY MESSAGE: We identified the candidate gene conferring yellow wilt resistance (YR) in B. oleracea . This work will facilitate YR breeding programs for B. oleracea and its closely related species. Yellow wilt disease is one of the most serious diseases of cabbage worldwide. Type A resistance to the disease is controlled by a single dominant gene that is used in cabbage breeding. Our previous QTL study identified the FocBo1 locus controlling type A resistance. In this study, the FocBo1 locus was fine-mapped by using 139 recombinant F2 plants derived from resistant cabbage (AnjuP01) and susceptible broccoli (GCP04) DH lines. As a result, we successfully delimited the location of FocBo1 within 1.00 cM between markers, BoInd 2 and BoInd 11. Analysis of BAC and cosmid sequences corresponding to the FocBo1 locus identified an orthologous gene of Bra012688 that was recently identified as an candidate gene that confers yellows resistance in Chinese cabbage. The candidate gene-specific DNA markers and phenotypes in F1 cabbage cultivars and their selfed F2 populations showed a perfect correlation. Our identification of the candidate gene for FocBo1 will assist introduction of fusarium resistance into B. oleracea cultivars and contribute further understanding of interaction between Brassica plants and fusarium.

Identification of candidate genes for Fusarium yellows resistance in Chinese cabbage by differential expression analysis.

Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans is an important disease of Brassica worldwide. To identify a resistance (R) gene against Fusarium yellows in Chinese cabbage (Brassica rapa var. pekinensis), we analyzed differential expression at the whole genome level between resistant and susceptible inbred lines using RNA sequencing. Four hundred and eighteen genes were significantly differentially expressed, and these were enriched for genes involved in response to stress or stimulus. Seven dominant DNA markers at putative R-genes were identified. Presence and absence of the sequence of the putative R-genes, Bra012688 and Bra012689, correlated with the resistance of six inbred lines and susceptibility of four inbred lines, respectively. In F(2) populations derived from crosses between resistant and susceptible inbred lines, presence of Bra012688 and Bra012689 cosegregated with resistance, suggesting that Bra012688 and Bra012689 are good candidates for fusarium yellows resistance in Chinese cabbage.

Production of high yield short duration Brassica napus by interspecific hybridization between B. oleracea and B. rapa.

Brassica napus is a leading oilseed crop throughout many parts of the world. It is well adapted to long day photoperiods, however, it does not adapt well to short day subtropical regions. Short duration B. napus plants were resynthesized through ovary culture from interspecific crosses in which B. rapa cultivars were reciprocally crossed with B. oleracea. From five different combinations, 17 hybrid plants were obtained in both directions. By self-pollinating the F1 hybrids or introgressing them with cultivated B. napus, resynthesized (RS) F3 and semi-resynthesized (SRS) F2 generations were produced, respectively. In field trial in Bangladesh, the RS B. napus plants demonstrated variation in days to first flowering ranging from 29 to 73 days; some of which were similar to cultivated short duration B. napus, but not cultivated short duration B. rapa. The RS and SRS B. napus lines produced 2-4.6 and 1.6-3.7 times higher yields, respectively, as compared to cultivated short duration B. napus. Our developed RS lines may be useful for rapeseed breeding not only for subtropical regions, but also for areas such as Canada and Europe where spring rapeseed production can suffer from late spring frosts. Yield and earliness in RS lines are discussed.

Molecular mechanisms of epigenetic variation in plants.

Natural variation is defined as the phenotypic variation caused by spontaneous mutations. In general, mutations are associated with changes of nucleotide sequence, and many mutations in genes that can cause changes in plant development have been identified. Epigenetic change, which does not involve alteration to the nucleotide sequence, can also cause changes in gene activity by changing the structure of chromatin through DNA methylation or histone modifications. Now there is evidence based on induced or spontaneous mutants that epigenetic changes can cause altering plant phenotypes. Epigenetic changes have occurred frequently in plants, and some are heritable or metastable causing variation in epigenetic status within or between species. Therefore, heritable epigenetic variation as well as genetic variation has the potential to drive natural variation.

Multiple mechanisms and challenges for the application of allopolyploidy in plants.

An allopolyploid is an individual having two or more complete sets of chromosomes derived from different species. Generation of allopolyploids might be rare because of the need to overcome limitations such as co-existing populations of parental lines, overcoming hybrid incompatibility, gametic non-reduction, and the requirement for chromosome doubling. However, allopolyploids are widely observed among plant species, so allopolyploids have succeeded in overcoming these limitations and may have a selective advantage. As techniques for making allopolyploids are developed, we can compare transcription, genome organization, and epigenetic modifications between synthesized allopolyploids and their direct parental lines or between several generations of allopolyploids. It has been suggested that divergence of transcription caused either genetically or epigenetically, which can contribute to plant phenotype, is important for the adaptation of allopolyploids.

Genome wide gene expression in artificially synthesized amphidiploids of Arabidopsis.

The merging of two different genomes occurs during the formation of amphidiploids, and the merged regulatory networks have the potential to generate a new gene expression pattern. We examined the genome-wide gene expression of two newly synthesized amphidiploids between Arabidopsis thaliana and the related species Arabidopsis lyrata subsp. lyrata and Arabidopsis halleri subsp. gemmifera. 1,137 (4.7%) and 1,316 (5.4%) of probesets showed differential gene expression in A. thaliana-A. halleri and A. thaliana-A. lyrata hybrids respectively, compared to the mid parent value and of these, 489 were in common. Genes that differed in expression between the parental lines tended to have an expression level in both hybrids differing from the mid parent value. In contrast to protein coding genes, there is little differential expression of transposons. Genes in the categories of chloroplast-targeted and response to stress were overrepresented in the non-additively expressed genes in both amphidiploids. As these genes have the potential to contribute directly to the plant phenotype, we suggest that rapid changes of gene expression in amphidiploids might be important for producing greater biomass.

One-pot synthesis of cyclophane-type macrocycles using manganese(III)-mediated oxidative radical cyclization.

Cyclophane-type macrocyclic compounds from 21 to 56 members having two fused dihydrofuran rings were synthesized by the manganese(III)-mediated oxidation of terminal dienes with bis(3-oxobutanoate)s containing aromatics. The reaction detail, characterization and reaction pathways are described.

In Arabidopsis thaliana Heterosis Level Varies among Individuals in an F1 Hybrid Population.

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.

Divergence of RNA editing among Arabidopsis species.

RNA editing altered the RNA sequence by replacing the C nucleotide to U in the organellar genomes of plants. RNA editing status sometimes differed among distant species. The pattern of conservation and variation of RNA editing status made it possible to evaluate evolutionary mechanisms impacting functional aspects of RNA editing. In this study, divergence of RNA editing in the chloroplast genome among Arabidopsis species was analyzed to determine 9 losses and 1 gain in RNA editing. All changes in A. thaliana lineage resulted from changes to the chloroplast genome sequence, whereas changes in the A. lyrata / halleri lineage were possibly due to exclusive changes in the nuclear editing factors. One loss of RNA editing in A. lyrata was caused by a deficiency in the PPR gene OTP80. The changes in RNA editing occurred approximately every two million years and were not observed at functionally important sites. These results highlight the conserved nature of RNA editing status suggesting the importance of RNA editing during evolution.

The complete chloroplast genome of Sisymbrium irio.

The complete chloroplast genome of Sisymbrium irio was determined. The length of the complete chloroplast genome is 154,001 bp. The whole chloroplast genome consists of 83,891 bp long single copy (LSC) and 17,630 bp small single copy (SSC) regions, separated by a pair of 26,240 bp inverted repeat (IR) regions. The S. irio chloroplast genome encodes 112 annotated known unique genes including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic position of S. irio is sister to Brassiceae and Thlaspideae.

Gene duplication, silencing and expression alteration govern the molecular evolution of PRC2 genes in plants.

PRC2 genes were analyzed for their number of gene duplications, dN/dS ratios and expression patterns among Brassicaceae and Gramineae species. Although both amino acid sequences and copy number of the PRC2 genes were generally well conserved in both Brassicaceae and Gramineae species, we observed that some rapidly evolving genes experienced duplications and expression pattern changes. After multiple duplication events, all but one or two of the duplicated copies tend to be silenced. Silenced copies were reactivated in the endosperm and showed ectopic expression in developing seeds. The results indicated that rapid evolution of some PRC2 genes is initially caused by a relaxation of selective constraint following the gene duplication events. Several loci could become maternally expressed imprinted genes and acquired functional roles in the endosperm.

Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in Brassica rapa L.

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.

Genetic characterization of inbred lines of Chinese cabbage by DNA markers; towards the application of DNA markers to breeding of F1 hybrid cultivars.

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.

Draft sequences of the radish (Raphanus sativus L.) genome.

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥ 300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.