Search

Accurate diagnosis of the differentially aggressive fungi Leptosphaeria maculans ‘brassicae’ and L. biglobosa ‘brassicae’ causing blackleg disease in Brassica crops is crucial. Prior to the availability of fungal whole‐genome sequences, gene‐specific markers were designed on specific genes. But, the availability of the whole‐genome sequences of several isolates now offers a greater opportunity to develop more precise and novel markers. The draft genomes of L. maculans and L. biglobosa that are available in the repository were aligned, and species‐specific primers were designed from the variable sequences that encode for effector proteins. Three primer sets namely, Lm1, Lm2 and Lm5 and two primer sets namely Lb1’ and Lb3 specifically detected the isolates of target species in PCR assay. The primers Lm5 and Lb1’ were multiplexed for detection of L. maculans ‘brassicae’ and L. biglobosa ‘brassicae’, respectively, from a single PCR reaction. The markers were highly sensitive in amplifying target species from crude ‘pseudothecia and ascospore suspension’ without requiring DNA extraction. These markers, solitarily or in combination, could be used for precise, sensitive and rapid detection of L. maculans ‘brassicae’ and L. biglobosa ‘brassicae’ species and will be helpful for surveillance, management and transboundary quarantine of the devastating blackleg disease.

Blackleg is a devastating disease of cabbage (C genome), but so far, only a few resistant cabbage lines have been identified, and the resistance-related genes are yet to be identified. In contrast, numerous R loci have been mapped to the A or B genome. High ancestral synteny among Brassicaceae genomes suggest that homologous regions of R loci in the A or B genome may contain functional R gene(s) in the C genome. Here, we investigated the collinear region of a major Brassica napus R locus, LepR1′, in Brassica oleracea to identify genes of putative disease resistance class via comprehensive in silico analysis. The LepR1′ locus is collinear to a 17.29-Mbp region in chromosome C02 of Brassica oleracea and hosted a total of 1,392 genes, and 36 of these genes contained NBS, LRR, TIR, F-box and RLK domains. The expression of these 36 genes was profiled using qRT-PCR in the cotyledons of resistant (SCNU-03) and susceptible (SCNU-59) lines at 0, 6, 24, and 48 h after inoculation with Leptosphaeria maculans isolates 00–100s and 03–02s having the corresponding avirulent gene, AvrLep1. Among these genes, a cluster of 10 genes were differentially expressed. NBS-LRR gene Bo2g131620 had a 45-bp insertion/deletion (indel) mutation in the 3rd intron between resistant and susceptible lines. The higher expression in resistant lines against both isolates and the indel mutation in Bo2g131620 suggest that these genes may have roles in blackleg resistance in cabbage. Mapping and functional analysis will be necessary to validate such a role. Saturating the region with enough markers is recommended during mapping.

Plasmodiophora brassicae, an obligate biotrophic pathogen-causing clubroot disease, can seriously affect Brassica crops worldwide, especially Chinese cabbage. Understanding the transcriptome and metabolome profiling changes during the infection of P. brassicae will provide key insights in understanding the defense mechanism in Brassica crops. In this study, we estimated the phytohormones using targeted metabolome assays and transcriptomic changes using RNA sequencing (RNA-seq) in the roots of resistant (BrT24) and susceptible (Y510-9) plants at 0, 3, 9, and 20 days after inoculation (DAI) with P. brassicae. Differentially expressed genes (DEGs) in resistant vs. susceptible lines across different time points were identified. The weighted gene co-expression network analysis of the DEGs revealed six pathways including “Plant–pathogen interaction” and “Plant hormone signal transduction” and 15 hub genes including pathogenic type III effector avirulence factor gene (RIN4) and auxin-responsive protein (IAA16) to be involved in plants immune response. Inhibition of Indoleacetic acid, cytokinin, jasmonate acid, and salicylic acid contents and changes in related gene expression in R-line may play important roles in regulation of clubroot resistance (CR). Based on the combined metabolome profiling and hormone-related transcriptomic responses, we propose a general model of hormone-mediated defense mechanism. This study definitely enhances our current understanding and paves the way for improving CR in Brassica rapa.

Plasmodiophora brassicae, an obligate biotrophic pathogen-causing clubroot disease, can seriously affect Brassica crops worldwide, especially Chinese cabbage. Understanding the transcriptome and metabolome profiling changes during the infection of P. brassicae will provide key insights in understanding the defense mechanism in Brassica crops. In this study, we estimated the phytohormones using targeted metabolome assays and transcriptomic changes using RNA sequencing (RNA-seq) in the roots of resistant (BrT24) and susceptible (Y510-9) plants at 0, 3, 9, and 20 days after inoculation (DAI) with P. brassicae. Differentially expressed genes (DEGs) in resistant vs. susceptible lines across different time points were identified. The weighted gene co-expression network analysis of the DEGs revealed six pathways including “Plant–pathogen interaction” and “Plant hormone signal transduction” and 15 hub genes including pathogenic type III effector avirulence factor gene (RIN4) and auxin-responsive protein (IAA16) to be involved in plants immune response. Inhibition of Indoleacetic acid, cytokinin, jasmonate acid, and salicylic acid contents and changes in related gene expression in R-line may play important roles in regulation of clubroot resistance (CR). Based on the combined metabolome profiling and hormone-related transcriptomic responses, we propose a general model of hormone-mediated defense mechanism. This study definitely enhances our current understanding and paves the way for improving CR in Brassica rapa.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is an economically devastating disease in melon. Molecular characterization of causal strains and identification of sources of resistance are necessary to control this important disease. Many strains of BFB were identified by partial sequencing and documented in GenBank. Recently, there was an outbreak of BFB strain NIHHS15-280 in melon fields (Goksung, Jeonnam, Korea) that severely hampered melon production in Korea. For this reason, we characterized NIHHS15-280 by partially sequencing the 16S rRNA gene using PCR-based markers. The partial sequence of the 16S rRNA gene of NIHHS15-280 was much more similar to A. citrulli strains associated with melon and watermelon hosts than other cucurbit hosts. A PCR-based assay identified the strain (NIHHS15-280) as belonging to A. citrulli group I, which predominantly infects non-watermelon cucurbits (virulent to melon). Thirty-five melon genotypes were screened against NIHHS15-280 in a detached-leaf inoculation assay. The lack of visible disease symptoms 11 days after inoculation in lines PI353814 and PI140471 indicated these to be highly resistant lines, whereas moderate resistance was observed in lines PI420145 and SCNU1154 and cultivars ME6 and ME5. Also, the most resistant lines were corroborated by a fruit inoculation assay. The characterized A. citrulli strain NIHHS15-280 (group I) and identified resistant lines will serve as a valuable resource for further genetic studies and improvement of BFB resistance in melon.

Clubroot, caused by the obligate biotrophic protist Plasmodiophora brassicae is a devastating disease of crucifers that causes substantial economic loss worldwide. The disease is characterized by the formation of galls in the root and hypocotyl of infected plants which restricts host vascular cambium development inhibiting efficient water and nutrient uptake by the plant. The pathogen- driven interference of hormonal homeostasis, particularly of cytokinin, in the root tissue is intricately linked with induction of hypertrophy and cell divisions leading to formation of galls. Levels of cytokinins and cell division generally increase at the onset of the disease which declines at the later stages of gall formation. The genes involved cytokinin biosynthesis such as cytokinin oxidase/ dehydrogenases and isopentenyl transferases shows differential expressions during clubroot infection and gall expansion in root tissues. Wider understanding of the roles of cytokinins and associated genes along the development of the disease will be helpful in unravelling plants defense mechanism against clubroot disease.

Recent advances in high-throughput genome sequencing technologies are now making the genetic dissection of the complex genome of cultivated strawberry easier. We sequenced Maehyang (short-day cultivar) × Albion (day-neutral cultivar) crossing populations using double digest restriction-associated DNA (ddRAD) sequencing technique that yielded 978,968 reads, 80.2% of which were aligned to strawberry genome allowing the identification of 13,181 high quality single nucleotide polymorphisms (SNPs). Total 3051 SNPs showed Mendelian segregation in F1, of which 1268 were successfully mapped to 46 linkage groups (LG) spanning a total of 2581.57 cM with an average interval genetic distance of 2.22 cM. The LGs were assigned to the 28 chromosomes of Fragaria × ananassa as determined by positioning the sequence tags on F. vesca genome. In addition, seven QTLs namely, qRU-5D, qRU-3D1, qRU-1D2, qRU-4D, qRU-4C, qRU-5C and qRU-2D2 were identified for runner production with LOD value ranging from 3.5–7.24 that explained 22–38% of phenotypic variation. The key candidate genes having putative roles in meristem differentiation for runnering and flowering within these QTL regions were identified. These will enhance our understanding of the vegetative vs sexual reproductive behavior in strawberry and will aid in setting breeding targets for developing perpetual flowering and profuse runnering cultivar.

Chinese cabbage (Brassica rapa L. var. pekinensis) is an economically important vegetable providing nutrients such as fiber, calcium and vitamins. Most cultivars of Chinese cabbage are F1 hybrids with vegetative heterosis, and harvesting of commercial F1 hybrid seeds makes use of self-incompatibility or cytoplasmic male sterility. Production of Chinese cabbage is always threatened by abiotic and biotic stress; climate change and increasing numbers of races and varieties of pathogens are also serious problems. The demand for abiotic or biotic resistant cultivars is growing year by year. An effective breeding method is desired, and marker-assisted selection (MAS) is a leading candidate. To apply MAS, identification of the causative gene or the locus linked to the causative gene controlling a trait for breeding is required. We review the recent research using molecular biology approaches and discuss how this information can apply to Chinese cabbage breeding.

Leaf spot of Brassica is an important bacterial seed borne disease caused by X. campestris pv. raphani (Xcr), one of the three pathovars of Xanthomonas campestris species which makes significant economic loss throughout the world. A rapid and reliable detection method will be helpful for early diagnosis of the disease. To do that, we designed Xcr specific sequence-characterized amplified region (SCAR) markers from the Xcr specific genomic fragments, identified by aligning the whole genome sequences of several closely related bacterial pathovars and other bacterial species. Our primer set Xcr_59 produced Xcr specific amplicon of 679 bp in PCR assays. This primer pair was also capable of detecting Xcr strains directly from the artificially infected leaf samples prepared by suspending the diseased leaf area in sterile water without extracting bacterial DNA. This rapid, sensitive and reliable detection technique will be useful for adopting any firsthand plant protection measures against the disease.

Background Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB. Results We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession. Conclusion We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.

Background Anthocyanins are plant secondary metabolites with key roles in attracting insect pollinators and protecting against biotic and abiotic stresses. They have potential health-promoting effects as part of the human diet. Anthocyanin biosynthesis has been elucidated in many species, enabling the development of anthocyanin-enriched fruits, vegetables, and grains; however, few studies have investigated Brassica napus anthocyanin biosynthesis. Results We developed a high-anthocyanin resynthesized B. napus line, Rs035, by crossing anthocyanin-rich B. rapa (A genome) and B. oleracea (C genome) lines, followed by chromosome doubling. We identified and characterized 73 and 58 anthocyanin biosynthesis genes in silico in the A and C genomes, respectively; these genes showed syntenic relationships with 41 genes in Arabidopsis thaliana and B. napus. Among the syntenic genes, twelve biosynthetic and six regulatory genes showed transgressively higher expression in Rs035, and eight structural genes and one regulatory gene showed additive expression. We identified three early-, four late-biosynthesis pathways, three transcriptional regulator genes, and one transporter as putative candidates enhancing anthocyanin accumulation in Rs035. Principal component analysis and Pearson’s correlation coefficients corroborated the contribution of these genes to anthocyanin accumulation. Conclusions Our study lays the foundation for producing high-anthocyanin B. napus cultivars. The resynthesized lines and the differentially expressed genes we have identified could be used to transfer the anthocyanin traits to other commercial rapeseed lines using molecular and conventional breeding. Electronic supplementary material The online version of this article (10.1186/s40709-018-0090-6) contains supplementary material, which is available to authorized users.

Fragaria × ananassa, a strawberry evolved from hybridization between F. virginiana and F. chiloensis, is a globally cultivated and consumed fruit crop valued for its flavor and nutritional value. Flavor and quality of fruits are determined by factors such as sugars and organic acids present during fruit development. These characteristics are highly subjective in different genotypes and affected by various environmental factors. In this study, we analyzed contents of major sugar compounds including fructose, glucose and sucrose by HPLC analysis in four cultivars namely, Maehyang, Seolhyang, Festival and Sweet Charlie. We identified 55 genes related to fructose, glucose, sucrose and soluble sugar regulation whose expression were analyzed in four cultivars at three developmental stages of the fruit namely, green, white and ripened stages. Expression of these genes across these progressive fruit developmental stages varied among cultivars. Among the 55 genes, genes FaFru3, FaSuc11 and FaGlu8 revealed differential patterns of expression along developmental stages of the fruit in high and low sugar-containing genotypes, respectively and may be putative candidates for sugar content in strawberries. Expression of genes are discussed with regard to corresponding sugar content in these genotypes. Further analysis and application of these genes may be valuable in developing high sugar containing cultivars via marker-assisted breeding.

Clubroot is a devastating disease of Brassicaceae caused by the biotrophic protist Plasmodiophora brassicae. The progression of clubroot disease is modulated by the glucosinolate (GSL) profile of the host plant. GSL is hydrolysed by the enzyme myrosinase upon cell disruption and gives rise to metabolites like isothiocyanate, nitriles, thiocyanates, epithionitriles and oxazolidines. Some of these metabolites play important roles in the plant’s defence mechanism. We identified 13 Myrosinase (Myro) and 28 Myrosinase-Binding Protein-like (MBP) genes from Brassica oleracea L. using a comparative genomics approach and characterised them through in silico analyses. We compared the expression patterns of these genes in a clubroot-susceptible line and a resistant line following inoculation with P. brassicae. Two BolMyro and 12 BolMBP genes were highly expressed in the susceptible line, whereas only one BolMyro and five BolMBP genes were highly expressed in the resistant line. Principal component analysis confirmed that specific GSL profiles and gene expression were modulated due to pathogen infection. Plants with higher levels of neoglucobrassicin, glucobrassicin and methooxyglucobrassicin produced disease symptoms and formed galls, whereas, plants with higher levels of sinigrin, hydroxyglucobrassicin and progoitrin produced less symptoms with almost no galls. Our results provide insights into the roles of Myro and MBP genes in GSL hydrolysis during P. brassicae infection, which will help for developing clubroot resistant cabbage lines.

Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F2 individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F1 plants were resistant to Acidovorax citrulli strain KACC18782, and F2 plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP A2035T in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F2 and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.

Anthocyanins are the resultant end-point metabolites of phenylapropanoid/flavonoid (F/P) pathway which is regulated at transcriptional level via a series of structural genes. Identifying the key genes and their potential interactions can provide us with the clue for novel points of intervention for improvement of the trait in strawberry. We profiled the expressions of putative regulatory and biosynthetic genes of cultivated strawberry in three developmental and characteristically colored stages of fruits of contrastingly anthocyanin rich cultivars: Tokun, Maehyang and Soelhyang. Besides FaMYB10, a well-characterized positive regulator, FaMYB5, FabHLH3 and FabHLH3-delta might also act as potential positive regulators, while FaMYB11, FaMYB9, FabHLH33 and FaWD44-1 as potential negative regulators of anthocyanin biosynthesis in these high-anthocyanin cultivars. Among the early BGs, Fa4CL7, FaF3H, FaCHI1, FaCHI3, and FaCHS, and among the late BGs, FaDFR4-3, FaLDOX, and FaUFGT2 showed significantly higher expression in ripe fruits of high anthocyanin cultivars Maehyang and Soelhyang. Multivariate analysis revealed the association of these genes with total anthocyanins. Increasingly higher expressions of the key genes along the pathway indicates the progressive intensification of pathway flux leading to final higher accumulation of anthocyanins. Identification of these key genetic determinants of anthocyanin regulation and biosynthesis in Korean cultivars will be helpful in designing crop improvement programs.

Climate change-induced events are causing salinization of many rice-growing areas, requiring the identification of new sources of genetic variation for salt tolerance in plant genetic resources since commonly grown cultivars are sensitive to salt. To identify the level of salt tolerance across a wide range of genotypes, we used a multivariate screening method using multiple growth and physiological traits simultaneously. For this purpose, four indica, two japonica and two wild rice genotypes were grown hydroponically under 40 and 80 mM NaCl stresses; fourteen different growth, qualitative and physiological traits, e.g. plant height, biomass, root and shoot elongation rates, and tissue ion accumulation, were recorded. In general, indica varieties performed better than both japonica and wild species. Our approach identified the existence of qualitatively different mechanisms of salt tolerance across the genotypes. For example, Pokkali, a salt-tolerant indica variety, displayed both ‘Na exclusion’ and ‘ion balance’ mechanisms, whereas PSBRc50 and IR58 showed only ‘Na exclusion’, and the Japonica genotypes Banikat and Nipponbare showed only ‘ion balance’. The results demonstrated that the tolerance is dependent on the level of stress and that this varies between genotypes; Nipponbare is moderately tolerant to 40 mM NaCl but not to 80 mM. We also suggest that the use of multivariate analyses can simplify the complex salinity tolerance picture and can effectively reveal the salinity tolerant genotype from a wide range of germplasm. The results reported here identify different physiological mechanism of tolerance across the genotypes and provide a sound basis for future studies examining their underlying molecular mechanisms.

Aims: The purple leaf blotch (PLB) disease, for which there is no released resistant variety in Bangladesh, causes loss in production of onion in every year. The mutants of a released variety, BARI Piaz - 2 has shown to possess resistance against PLB whi ch is yet to be characterized at molecular level. An easy and simple molecular detection technique of the gene responsible for this disease will be of great use in future. The present study was thus carried out to molecularly characterize four mutant lines of onion using Simple Sequence Repeat ( SSR) marker to detect the presence or absence of PLB gene conferring resistance against purple blotch disease in onion. P lace and Duration of Study: The research was carried out during the period from March, 2013 to

The inheritance and causal loci for resistance to blackleg, a devastating disease of Brassicaceous crops, are yet to be known in cabbage (Brassica oleracea L.). Here, we report the pattern of inheritance and linked molecular marker for this trait. A segregating BC1 population consisting of 253 plants was raised from resistant and susceptible parents, L29 (♀) and L16 (♂), respectively. Cotyledon resistance bioassay of BC1 population, measured based on a scale of 0&ndash, 9 at 12 days after inoculation with Leptosphaeria maculans isolate 03&ndash, 02 s, revealed the segregation of resistance and ratio, indicative of dominant monogenic control of the trait. Investigation of potential polymorphism in the previously identified differentially expressed genes within the collinear region of &lsquo, B. napus blackleg resistant loci Rlm1&prime, in B. oleracea identified two insertion/deletion (InDel) mutations in the intron and numerous single nucleotide polymorphisms (SNPs) throughout the LRR-RLK gene Bol040029, of which six SNPs in the first exon caused the loss of two LRR domains in the susceptible line. An InDel marker, BLR-C-InDel based on the InDel mutations, and a high resolution melting (HRM) marker, BLR-C-2808 based on the SNP C2808T in the second exon were developed, which predicated the resistance status of the BC1 population with 80.24%, and of 24 commercial inbred lines with 100% detection accuracy. This is the first report of inheritance and molecular markers linked with blackleg resistance in cabbage. This study will enhance our understanding of the trait, and will be helpful in marker assisted breeding aiming at developing resistant cabbage varieties.

Acidovorax citrulli (A. citrulli) strains cause bacterial fruit blotch (BFB) in cucurbit crops and affect melon significantly. Numerous strains of the bacterium have been isolated from melon hosts globally. Strains that are aggressively virulent towards melon and diagnostic markers for detecting such strains are yet to be identified. Using a cross-inoculation assay, we demonstrated that two Korean strains of A. citrulli, NIHHS15-280 and KACC18782, are highly virulent towards melon but avirulent/mildly virulent to the other cucurbit crops. The whole genomes of three A. citrulli strains isolated from melon and three from watermelon were aligned, allowing the design of three primer sets (AcM13, AcM380, and AcM797) that are specific to melon host strains, from three pathogenesis-related genes. These primers successfully detected the target strain NIHHS15-280 in polymerase chain reaction (PCR) assays from a very low concentration of bacterial gDNA. They were also effective in detecting the target strains from artificially infected leaf, fruit, and seed washing suspensions, without requiring the extraction of bacterial DNA. This is the first report of PCR-based markers that offer reliable, sensitive, and rapid detection of strains of A. citrulli causing BFB in melon. These markers may also be useful in early disease detection in the field samples, in seed health tests, and for international quarantine purposes.