Widespread and transgenerational retrotransposon activation in inter- and intraspecies recombinant inbred populations of Lotus japonicus.

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.

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.

Comparative transcriptome analysis during tuberous stem formation in Kohlrabi (B. oleracea var. gongylodes) at early growth periods (seedling stages).

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.

Correction: EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

[This corrects the article DOI: 10.1371/journal.pgen.1007238.].

EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.

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.

The production and characterization of a BoFLC2 introgressed Brassica rapa by repeated backcrossing to an F1.

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 LORE1 insertion mutant resource.

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

IntroMap: a signal analysis based method for the detection of genomic introgressions.

BACKGROUND: Breeding programs often rely on marker-assisted tests or variant calling of next generation sequence (NGS) data to identify regions of genomic introgression arising from the hybridization of two plant species. In this paper we present IntroMap, a bioinformatics pipeline for the screening of candidate plants through the application of signal processing techniques to NGS data, using alignment to a reference genome sequence (annotation is not required) that shares homology with the recurrent parental cultivar, and without the need for de novo assembly of the read data or variant calling. RESULTS: We show the accurate identification of introgressed genomic regions using both in silico simulated genomes, and a hybridized cultivar data set using our pipeline. Additionally we show, through targeted marker-based assays, validation of the IntroMap predicted regions for the hybrid cultivar. CONCLUSIONS: This approach can be used to automate the screening of large populations, reducing the time and labor required, and can improve the accuracy of the detection of introgressed regions in comparison to a marker-based approach. In contrast to other approaches that generally rely upon a variant calling step, our method achieves accurate identification of introgressed regions without variant calling, relying solely upon alignment.

Loss of function mutations in the rice chromomethylase OsCMT3a cause a burst of transposition.

Methylation patterns of plants are unique as, in addition to the methylation at CG dinucleotides that occurs in mammals, methylation also occurs at non-CG sites. Genes are methylated at CG sites, but transposable elements (TEs) are methylated at both CG and non-CG sites. The role of non-CG methylation in transcriptional silencing of TEs is being extensively studied at this time, but only very rare transpositions have been reported when non-CG methylation machineries have been compromised. To understand the role of non-CG methylation in TE suppression and in plant development, we characterized rice mutants with changes in the chromomethylase gene, OsCMT3a. oscmt3a mutants exhibited a dramatic decrease in CHG methylation, changes in the expression of some genes and TEs, and pleiotropic developmental abnormalities. Genome resequencing identified eight TE families mobilized in oscmt3a during normal propagation. These TEs included tissue culture-activated copia retrotransposons Tos17 and Tos19 (Lullaby), a pericentromeric clustered high-copy-number non-autonomous gypsy retrotransposon Dasheng, two copia retrotransposons Osr4 and Osr13, a hAT-tip100 transposon DaiZ, a MITE transposon mPing, and a LINE element LINE1-6_OS. We confirmed the transposition of these TEs by polymerase chain reaction (PCR) and/or Southern blot analysis, and showed that transposition was dependent on the oscmt3a mutation. These results demonstrated that OsCMT3a-mediated non-CG DNA methylation plays a critical role in development and in the suppression of a wide spectrum of TEs. These in planta mobile TEs are important for studying the interaction between TEs and the host genome, and for rice functional genomics.

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.

Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1.

We established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population was composed of 2450 plant lines, from which a total of 4532 flanking sequence tags of LORE1 were recovered by pyrosequencing. The two-dimensional arrangement of the plant population, together with the use of multiple identifier sequences in the primers used to amplify the flanking regions, made it possible to trace insertions back to the original plant lines. The large-scale detection of new LORE1 insertion sites revealed a preference for genic regions, especially in exons of protein-coding genes, which is an interesting feature to consider in the interaction between host genomes and chromoviruses, to which LORE1 belongs, a class of retrotransposon widely distributed among plants. Forward screening of the symbiotic mutants from the population succeeded to identify five symbiotic mutants of known genes. These data suggest that LORE1 is robust as a genetic tool.

The SNARE protein SYP71 expressed in vascular tissues is involved in symbiotic nitrogen fixation in Lotus japonicus nodules.

Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor (SNARE) proteins are crucial for signal transduction and development in plants. Here, we investigate a Lotus japonicus symbiotic mutant defective in one of the SNARE proteins. When in symbiosis with rhizobia, the growth of the mutant was retarded compared with that of the wild-type plant. Although the mutant formed nodules, these exhibited lower nitrogen fixation activity than the wild type. The rhizobia were able to invade nodule cells, but enlarged symbiosomes were observed in the infected cells. The causal gene, designated LjSYP71 (for L. japonicus syntaxin of plants71), was identified by map-based cloning and shown to encode a Qc-SNARE protein homologous to Arabidopsis (Arabidopsis thaliana) SYP71. LjSYP71 was expressed ubiquitously in shoot, roots, and nodules, and transcripts were detected in the vascular tissues. In the mutant, no other visible defects in plant morphology were observed. Furthermore, in the presence of combined nitrogen, the mutant plant grew almost as well as the wild type. These results suggest that the vascular tissues expressing LjSYP71 play a pivotal role in symbiotic nitrogen fixation in L. japonicus nodules.

Derepression of the plant Chromovirus LORE1 induces germline transposition in regenerated plants.

Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposable elements in plants. Recent studies have identified chromoviruses to be a widely distributed lineage of Gypsy elements. These elements contain chromodomains in their integrases, which suggests a preference for insertion into heterochromatin. In turn, this preference might have contributed to the patterning of heterochromatin observed in host genomes. Despite their potential importance for our understanding of plant genome dynamics and evolution, the regulatory mechanisms governing the behavior of chromoviruses and their activities remain largely uncharacterized. Here, we report a detailed analysis of the spatio-temporal activity of a plant chromovirus in the endogenous host. We examined LORE1a, a member of the endogenous chromovirus LORE1 family from the model legume Lotus japonicus. We found that this chromovirus is stochastically de-repressed in plant populations regenerated from de-differentiated cells and that LORE1a transposes in the male germline. Bisulfite sequencing of the 5' LTR and its surrounding region suggests that tissue culture induces a loss of epigenetic silencing of LORE1a. Since LTR promoter activity is pollen specific, as shown by the analysis of transgenic plants containing an LTR::GUS fusion, we conclude that male germline-specific LORE1a transposition in pollen grains is controlled transcriptionally by its own cis-elements. New insertion sites of LORE1a copies were frequently found in genic regions and show no strong insertional preferences. These distinctive novel features of LORE1 indicate that this chromovirus has considerable potential for generating genetic and epigenetic diversity in the host plant population. Our results also define conditions for the use of LORE1a as a genetic tool.

Identification of a Male Sterile Candidate Gene in Lilium x formolongi and Transfer of the Gene to Easter Lily (L. longiflorum) via Hybridization.

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.

A chromosome-level genome sequence of Chrysanthemum seticuspe, a model species for hexaploid cultivated chrysanthemum.

Chrysanthemums are one of the most industrially important cut flowers worldwide. However, their segmental allopolyploidy and self-incompatibility have prevented the application of genetic analysis and modern breeding strategies. We thus developed a model strain, Gojo-0 (Chrysanthemum seticuspe), which is a diploid and self-compatible pure line. Here, we present the 3.05 Gb chromosome-level reference genome sequence, which covered 97% of the C. seticuspe genome. The genome contained more than 80% interspersed repeats, of which retrotransposons accounted for 72%. We identified recent segmental duplication and retrotransposon expansion in C. seticuspe, contributing to arelatively large genome size. Furthermore, we identified a retrotransposon family, SbdRT, which was enriched in gene-dense genome regions and had experienced a very recent transposition burst. We also demonstrated that the chromosome-level genome sequence facilitates positional cloning in C. seticuspe. The genome sequence obtained here can greatly contribute as a reference for chrysanthemum in front-line breeding including genome editing.

Extreme genetic signatures of local adaptation during Lotus japonicus colonization of Japan.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here, we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with colonization of Japan by Lotus japonicus over the past ~20,000 years. Based on patterns of genomic variation, we infer the details of the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We identify genomic regions with extreme genetic differentiation between northern and southern subpopulations and perform population structure-corrected association mapping of phenotypic traits measured in a common garden. Comparing the results of these analyses, we find that signatures of extreme subpopulation differentiation overlap strongly with phenotype association signals for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection during colonization and point to associated candidate genes.

Comparison of the genome structure of the self-incompatibility (S) locus in interspecific pairs of S haplotypes.

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, both of which are encoded in the S locus. The nucleotide sequence analyses of many SRK and SP11/SCR alleles have identified several interspecific pairs of S haplotypes having highly similar sequences between B. oleracea and B. rapa. These interspecific pairs of S haplotypes are considered to be derived from common ancestors and to have maintained the same recognition specificity after speciation. In this study, the genome structures of three interspecific pairs of S haplotypes were compared by sequencing SRK, SP11/SCR, and their flanking regions. Regions between SRK and SP11/SCR in B. oleracea were demonstrated to be much longer than those of B. rapa and several retrotransposon-like sequences were identified in the S locus in B. oleracea. Among the seven retrotransposon-like sequences, six sequences were found to belong to the ty3 gypsy group. The gag sequences of the retrotransposon-like sequences were phylogenetically different from each other. In Southern blot analysis using retrotransposon-like sequences as probes, the B. oleracea genome showed more signals than the B. rapa genome did. These findings suggest a role for the S locus and genome evolution in self-incompatible plant species.

Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress.

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 10 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.

Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L.

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ~95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.