Putative R-genes Research Articles

De novo assembly and characterization of the draft genome of the cashew (Anacardium occidentale L.)

Cashew is the second most important tree nut crop in the global market. Cashew is a diploid and heterozygous species closely related to the mango and pistachio. Its improvement by conventional breeding is slow due to the long juvenile phase. Despite the economic importance, very little genomics/transcriptomics information is available for cashew. In this study, the Oxford nanopore reads and Illumina reads were used for de novo assembly of the cashew genome. The hybrid assembly yielded a 356.6 Mb genome corresponding to 85% of the estimated genome size (419 Mb). The BUSCO analysis showed 91.8% of genome completeness. Transcriptome mapping showed 92.75% transcripts aligned with the assembled genome. Gene predictions resulted in the identification of 31,263 genes coding for a total of 35,000 gene isoforms. About 46% (165 Mb) of the cashew genome comprised of repetitive sequences. Phylogenetic analyses of the cashew with nine species showed that it was closely related to Mangifera indica. Analysis of cashew genome revealed 3104 putative R-genes. The first draft assembly of the genome, transcriptome and R gene information generated in this study would be the foundation for understanding the molecular basis of economic traits and genomics-assisted breeding in cashew.

Discovery of Genome-Wide DNA Polymorphisms and Resistance-Relative Genes in Chaling Wild Rice (Oryza rufipogon Griff.) by Whole-Genome Sequencing

Chaling common wild rice (Chaling CWR, Oryza rufipogon Griff.) is a kind of germplasm resource which possesses abundant genetic diversity and plays a pivotal role in improving cultivated rice. In this research, we used next-generation sequencing technology to reveal the genome sequence of Chaling CWR and investigate its elite genes. The results showed that the average coverage was 93.99%. 1,743,368 single-nucleotide polymorphism (SNP) and 387,381 insertion and deletion (InDel) variants have been detected in Chaling CWR compared with the Nipponbare genome. The variant density was found to be highest in chromosome 10 and lowest in chromosome 5, with an average density of 467.08 SNPs/100 kb and 109.59 InDels/100 kb across the whole genome. 109,684 non-synonymous SNPs were identified in 14,315 genes, and 26,051 InDels were detected in the coding regions. We further classified these genes based on the protein domains that were involved in host defense mechanisms and in the analysis of 37 NBS-LRRs, 71 LRRs, 136 NB-ARCs, 119 PKs, 19 WRKY transcription factors, and 25 resistant protein genes. Among the putative R-genes, Pish, Pita, Pi25, Pi9, Pi21, Pid2, Pikm, and Xa21 were further identified, as exhibiting high rates of polymorphism. Results from this study provide a foundation for future basic research and marker-assisted breeding of rice resistance.

Characterization, identification and expression profiling of genome-wide R-genes in melon and their putative roles in bacterial fruit blotch resistance

BackgroundBacterial 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.ResultsWe 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.ConclusionWe 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.

In-silico identification and differential expressions of LepR4-syntenic disease resistance related domain containing genes against blackleg causal fungus Leptosphaeria maculans in Brassica oleracea

Blackleg, caused by Leptosphaeria maculans, is a devastating disease for which no resistant (R) gene is reported in cabbage (C-genome). All known R-genes for blackleg in Brassicaceae have been identified in A- or B-genome. However, identification of a few blackleg-resistant cabbage genotypes suggest that C-genome crops may also contain R-genes. Since, there is high ancestral synteny between these three genomes, we reasoned that mining the syntenic regions of known A- or B-genome R-genes may lead to the corresponding functional orthologues in C-genome. Here, we investigated the Brassica napus blackleg resistant-loci LepR4 and identified two syntenic blocks of 9.56 and 1 Mb in chromosomes C03 and C08, respectively, hosting a total of 1137 genes in C- genome. Among these, twelve genes have functional domains such NB-ARC, LRR, CC, TIR, MAP-kinase and S_TKc that have putatively roles in disease resistance. Higher expressions of leucine-rich repeat receptor-like kinase genes Bo3g110840 and Bo3g103150 against two L. maculans isolates in two resistant genotypes and of Serine/Threonine protein kinase genes Bo3g114980 and Bo3g130040 against both isolates in one resistant genotype and against one isolate in both resistant genotypes, respectively suggest their putative roles in conferring resistance to blackleg in cabbage. This approach is auxiliary to conventional mapping based approach and can be replicated for identifying all other A- or B-genome syntenic putative R-genes in C-genome which, upon validation by functional assays, will be helpful in enhancing our understanding and improving blackleg resistance in cabbage.

Genomic analysis of powdery mildew resistance in a hop (Humulus lupulus L.) bi-parental population segregating for \u201cR6-locus\u201d

Genetic response in hop to fungal pathogen infection has been evaluated at the chromosomal level through QTL analyses but very little information exists on the expression of genes during infection periods. Raw GBS reads and phenotypic data from a previously published QTL analysis along with a newly assembled PacBio-derived hop reference genome were used to re-evaluate resistance to races v4/v6 of powdery mildew (PM; Podosphaera humuli). QTL analyses revealed two tightly linked regions of association on a single linkage group. The three SNP markers most tightly linked to PM resistance (found on contig 000559F) were observed downstream from a putative R-gene locus for powdery mildew resistance. This 230 kb region contained a series of seven putative R-genes surrounded by seven putative peroxidase-3 genes downstream and seven putative glucan endo-1,3-beta-glucosidase upstream and an expressed F-box domain protein. RNAseq data showed all putative R-genes along with all putative glucan endo-1,3-beta-glucosidase genes were expressed under diseased conditions, while none of the peroxidase genes were expressed. The second region contained three SNPs found on contig 002916F next to two putative R-genes. RNAseq data showed complex expression of exons contained in putative isoforms of R-genes. This preliminary information will prove valuable information for development of precise markers located either within or next to genes responsible for race v4/v6 PM resistance.

Disease Resistance Genetics and Genomics in Octoploid Strawberry

Octoploid strawberry (Fragaria ×ananassa) is a valuable specialty crop, but profitable production and availability are threatened by many pathogens. Efforts to identify and introgress useful disease resistance genes (R-genes) in breeding programs are complicated by strawberry’s complex octoploid genome. Recently-developed resources in strawberry, including a complete octoploid reference genome and high-resolution octoploid genotyping, enable new analyses in strawberry disease resistance genetics. This study characterizes the complete R-gene collection in the genomes of commercial octoploid strawberry and two diploid ancestral relatives, and introduces several new technological and data resources for strawberry disease resistance research. These include octoploid R-gene transcription profiling, dN/dS analysis, expression quantitative trait loci (eQTL) analysis and RenSeq analysis in cultivars. Octoploid fruit eQTL were identified for 76 putative R-genes. R-genes from the ancestral diploids Fragaria vesca and Fragaria iinumae were compared, revealing differential inheritance and retention of various octoploid R-gene subtypes. The mode and magnitude of natural selection of individual F. ×ananassa R-genes was also determined via dN/dS analysis. R-gene sequencing using enriched libraries (RenSeq) has been used recently for R-gene discovery in many crops, however this technique somewhat relies upon a priori knowledge of desired sequences. An octoploid strawberry capture-probe panel, derived from the results of this study, is validated in a RenSeq experiment and is presented for community use. These results give unprecedented insight into crop disease resistance genetics, and represent an advance toward exploiting variation for strawberry cultivar improvement.

Identification of expressed R-genes associated with leaf spot diseases in cultivated peanut

Peanut (Arachis hypogaea L.) is an important food and oilseed crop worldwide. Yield and quality can be significantly reduced by foliar fungal diseases, such as early and late leaf spot diseases. Acceptable levels of leaf spot resistance in cultivated peanut have been elusive due to environmental interactions and the proper combination of QTLs in any particular peanut genotype. Resistance gene analogs, as potential resistance (R)-genes, have unique roles in the recognition and activation of disease resistance responses. Novel R-genes can be identified by searches for conserved domains such as nucleotide binding site, leucine rich repeat, receptor like kinase, and receptor like protein from expressed genes or through genomic sequences. Expressed R-genes represent necessary plant signals in a disease response. The goals of this research are to identify expressed R-genes from cultivated peanuts that are naturally infected by early and late spot pathogens, compare these to the closest diploid progenitors, and evaluate specific gene expression in cultivated peanuts. Putative peanut R-genes (381) were available from a public database (NCBI). Primers were designed and PCR products were sequenced. A total of 214 sequences were produced which matched to proteins with the corresponding R-gene motifs. These R-genes were mapped to the genome sequences of Arachis duranensis and Arachis ipaensis, which are the closest diploid progenitors for tetraploid cultivated peanut, A. hypogaea. Identification and association of specific gene-expression will elucidate potential disease resistance mechanism in peanut and may facilitate the selection of breeding lines with high levels of leaf spot resistance.

A novel workflow correlating RNA-seq data to Phythophthora infestans resistance levels in wild Solanum species and potato clones.

Comparative transcriptomics between species can provide valuable understanding of plant-pathogen interactions. Here, we focus on wild Solanum species and potato clones with varying degree of resistance against Phytophthora infestans, which causes the devastating late blight disease in potato. The transcriptomes of three wild Solanum species native to Southern Sweden, Solanum dulcamara, Solanum nigrum, and Solanum physalifolium were compared to three potato clones, Desiree (cv.), SW93-1015 and Sarpo Mira. Desiree and S. physalifolium are susceptible to P. infestans whereas the other four have different degrees of resistance. By building transcript families based on de novo assembled RNA-seq across species and clones and correlating these to resistance phenotypes, we created a novel workflow to identify families with expanded or depleted number of transcripts in relation to the P. infestans resistance level. Analysis was facilitated by inferring functional annotations based on the family structure and semantic clustering. More transcript families were expanded in the resistant clones and species and the enriched functions of these were associated to expected gene ontology (GO) terms for resistance mechanisms such as hypersensitive response, host programmed cell death and endopeptidase activity. However, a number of unexpected functions and transcripts were also identified, for example transmembrane transport and protein acylation expanded in the susceptible group and a cluster of Zinc knuckle family proteins expanded in the resistant group. Over 400 expressed putative resistance (R-)genes were identified and resistant clones Sarpo Mira and SW93-1015 had ca 25% more expressed putative R-genes than susceptible cultivar Desiree. However, no differences in numbers of susceptibility (S-)gene homologs were seen between species and clones. In addition, we identified P. infestans transcripts including effectors in the early stages of P. infestans-Solanum interactions.

Transcriptome of Erysiphe necator-infected Vitis pseudoreticulata leaves provides insight into grapevine resistance to powdery mildew.

Powdery mildew (PM), which is caused by the pathogen Erysiphe necator (Schw.) Burr., is the single most damaging disease of cultivated grapes (Vitis vinifera) worldwide. However, little is known about the transcriptional response of grapes to infection with PM. RNA-seq analysis was used for deep sequencing of the leaf transcriptome to study PM resistance in Chinese wild grapes (V. pseudoreticulata Baihe 35-1) to better understand the interaction between host and pathogen. Greater than 100 million (M) 90-nt cDNA reads were sequenced from a cDNA library derived from PM-infected leaves. Among the sequences obtained, 6541 genes were differentially expressed (DEG) and were annotated with Gene Ontology terms and by pathway enrichment. The significant categories that were identified included the following: defense, salicylic acid (SA) and jasmonic acid (JA) responses; systemic acquired resistance (SAR); hypersensitive response; plant–pathogen interaction; flavonoid biosynthesis; and plant hormone signal transduction. Various putative secretory proteins were identified, indicating potential defense responses to PM infection. In all, 318 putative R-genes and 183 putative secreted proteins were identified, including the defense-related R-genes BAK1, MRH1 and MLO3 and the defense-related secreted proteins GLP and PR5. The expression patterns of 16 genes were further illuminated by RT-qPCR. The present study identified several candidate genes and pathways that may contribute to PM resistance in grapes and illustrated that RNA-seq is a powerful tool for studying gene expression. The RT-qPCR results reveal that effective resistance responses of grapes to PM include enhancement of JA and SAR responses and accumulation of phytoalexins.

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.

PRGdb 2.0: towards a community-based database model for the analysis of R-genes in plants

The Plant Resistance Genes database (PRGdb; http://prgdb.org) is a comprehensive resource on resistance genes (R-genes), a major class of genes in plant genomes that convey disease resistance against pathogens. Initiated in 2009, the database has grown more than 6-fold to recently include annotation derived from recent plant genome sequencing projects. Release 2.0 currently hosts useful biological information on a set of 112 known and 104 310 putative R-genes present in 233 plant species and conferring resistance to 122 different pathogens. Moreover, the website has been completely redesigned with the implementation of Semantic MediaWiki technologies, which makes our repository freely accessed and easily edited by any scientists. To this purpose, we encourage plant biologist experts to join our annotation effort and share their knowledge on resistance-gene biology with the rest of the scientific community.

PRGdb: a bioinformatics platform for plant resistance gene analysis

PRGdb is a web accessible open-source (http://www.prgdb.org) database that represents the first bioinformatic resource providing a comprehensive overview of resistance genes (R-genes) in plants. PRGdb holds more than 16 000 known and putative R-genes belonging to 192 plant species challenged by 115 different pathogens and linked with useful biological information. The complete database includes a set of 73 manually curated reference R-genes, 6308 putative R-genes collected from NCBI and 10463 computationally predicted putative R-genes. Thanks to a user-friendly interface, data can be examined using different query tools. A home-made prediction pipeline called Disease Resistance Analysis and Gene Orthology (DRAGO), based on reference R-gene sequence data, was developed to search for plant resistance genes in public datasets such as Unigene and Genbank. New putative R-gene classes containing unknown domain combinations were discovered and characterized. The development of the PRG platform represents an important starting point to conduct various experimental tasks. The inferred cross-link between genomic and phenotypic information allows access to a large body of information to find answers to several biological questions. The database structure also permits easy integration with other data types and opens up prospects for future implementations.