Genome-Wide Analysis of the WRKY Transcription Factor Family in Roses and Their Putative Role in Defence Signalling in the Rose–Blackspot Interaction

WRKY transcription factors are important players in plant regulatory networks, where they control and integrate various physiological processes and responses to biotic and abiotic stresses. Here, we analysed six rose genomes of 5 different species (Rosa chinensis, R. multiflora, R. roxburghii, R. sterilis, and R. rugosa) and extracted a set of 68 putative WRKY genes, extending a previously published set of 58 WRKY sequences based on the R. chinensis genome. Analysis of the promoter regions revealed numerous motifs related to induction by abiotic and, in some cases, biotic stressors. Transcriptomic data from leaves of two rose genotypes inoculated with the hemibiotrophic rose black spot fungus Diplocarpon rosae revealed the upregulation of 18 and downregulation of 9 of these WRKY genes after contact with the fungus. Notably, the resistant genotype exhibited the regulation of 25 of these genes (16 upregulated and 9 downregulated), while the susceptible genotype exhibited the regulation of 20 genes (15 upregulated and 5 downregulated). A detailed RT–qPCR analysis of RcWRKY37, an orthologue of AtWRKY75 and FaWRKY1, revealed induction patterns similar to those of the pathogenesis-related (PR) genes induced in salicylic acid (SA)-dependent defence pathways in black spot inoculation experiments. However, the overexpression of RcWRKY37 in rose petals did not induce the expression of any of the PR genes upon contact with black spot. However, wounding significantly induced the expression of RcWRKY37, while heat, cold, or drought did not have a significant effect. This study provides the first evidence for the role of RcWRKY37 in rose signalling cascades and highlights the differences between RcWRKY37 and AtWRKY75. These results improve our understanding of the regulatory function of WRKY transcription factors in plant responses to stress factors. Additionally, they provide foundational data for further studies

Robust markers associated with floral traits in roses are suitable for marker-assisted selection across gene pools

We investigated the potential of markers associated with floral traits for parental selection in a cut rose breeding program. We analysed six Kompetitive Allele Specific PCR (KASP) markers for three important floral traits, petal length, petal number and scent, derived from experiments in a garden rose population. The six markers were applied to genotype a collection of 384 parental genotypes used for commercial cut rose breeding. We phenotyped a selection of progeny derived from pairs of parents having either high or low dosages of (contrasting) marker alleles associated with these traits. Significant differences were found between the contrasting progeny groups for each of the traits, although parents with the optimal allele dosage combinations could not always be used for the crosses. This not only supports the robustness of these marker‒trait associations but also demonstrates their potential for commercial rose breeding. It also demonstrates the use of marker information generated in garden rose populations for cut rose breeding

Development of a multiplex amplicon-sequencing assay to detect low-frequency mutations in poinsettia (Euphorbia pulcherrima) breeding programmes

Poinsettia is an economically important ornamental potted plant in which certain bract colour variants are often obtained by mutation breeding. Previously, in poinsettia, we identified Bract1, a GST gene involved in the sequestration and transport of anthocyanins to the vacuole. This gene carries a short, highly mutable 4-bp repeat in its coding region. Loss of one repeat unit leads to a loss of function for Bract1, and in homozygous mutants, anthocyanin-based coloration is absent, resulting in white or cream-coloured bracts. Although mutation induction through ionizing radiation leads to a high frequency of mutations in Bract1, mutants are difficult to obtain from homozygous red genotypes. In this study, we used Bract1-specific amplicon sequencing as a tool to identify mutations in pools of tissues, which enabled the detection of mutations in dilutions of up to one mutant in 50 nonmutated samples. This approach enabled efficient screening of recalcitrant homozygous genotypes for mutated alleles and the reduction of the mutation load in the application of ionizing radiation in mutation breeding programmes

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication

Background: Black spot is one of the most severe and damaging diseases of garden roses. We present the draft genome sequence of its causative agent Diplocarpon rosae as a working tool to generate molecular markers and to analyze functional and structural characteristics of this fungus. Results: The isolate DortE4 was sequenced with 191x coverage of different read types which were assembled into 2457 scaffolds. By evidence supported genome annotation with the MAKER pipeline 14,004 gene models were predicted and transcriptomic data indicated that 88.5% of them are expressed during the early stages of infection. Analyses of k-mer distributions resulted in unexpectedly large genome size estimations between 72.5 and 91.4 Mb, which cannot be attributed to its repeat structure and content of transposable elements alone, factors explaining such differences in other fungal genomes. In contrast, different lines of evidences demonstrate that a huge proportion (approximately 80%) of genes are duplicated, which might indicate a whole genome duplication event. By PCR-RFLP analysis of six paralogous gene pairs of BUSCO orthologs, which are expected to be single copy genes, we could show experimentally that the duplication is not due to technical error and that not all isolates tested possess all of the paralogs. Conclusions: The presented genome sequence is still a fragmented draft but contains almost the complete gene space. Therefore, it provides a useful working tool to study the interaction of D. rosae with the host and the influence of a genome duplication outside of the model yeast in the background of a phytopathogen

Isolation, molecular characterization, and mapping of four rose MLO orthologs

Powdery mildew is a major disease of economic importance in cut and pot roses. As an alternative to conventional resistance breeding strategies utilizing single-dominant genes or QTLs, mildew resistance locus o (MLO)-based resistance might offer some advantages. In dicots such as Arabidopsis, pea, and tomato, loss-of-function mutations in MLO genes confer high levels of broad-spectrum resistance. Here, we report the isolation and characterization of four MLO homologs from a large rose EST collection isolated from leaves. These genes are phylogenetically closely related to other dicot MLO genes that are involved in plant powdery mildew interactions. Therefore, they are candidates for MLO genes involved in rose powdery mildew interactions. Two of the four isolated genes contain all of the sequence signatures considered to be diagnostic for MLO genes. We mapped all four genes to three linkage groups and conducted the first analysis of alternative alleles. This information is discussed in regards to a reverse genetics approach aimed at the selection of rose plants that are homozygous for loss-of-function in one or more MLO genes.BMBF/0315542ANatural Science Foundation of China/No31160403Natural Science Foundation of Yunnan/2011FB12

Inheritance genetics of the trait vector competence in Frankliniella occidentalis (Western flower thrips) in the transmission of Tomato spotted wilt virus

The complexity of tospovirus–vector–host plant interaction is linked to a range of factors influencing vector's efficacy in virus transmission, leading to high variability in the transmission efficiency within vector populations. Main shortcomings of most studies are the missing information on the intrinsic potential of individual insects to serve as efficient vectors, both at phenotypic and at genotypic levels. Moreover, detailed analysis of vector competence heredity and monitoring the splitting of both genotypes and phenotypes in filial generations has not been reported. In this study, using the model system Frankliniella occidentalis and Tomato spotted wilt virus, we evaluated the inheritance and stability of the trait vector competence in a population through basic crossings of individually characterized partners, as well as virgin reproduction. We hypothesized that the trait is heritable in F. occidentalis and is controlled by a recessive allele. From the results, 83% and 94% of competent and noncompetent males respectively, inherited their status from their mothers. The trait was only expressed when females were homozygous for the corresponding allele. Furthermore, the allele frequencies were different between males and females, and the competent allele had the highest frequency in the population. These suggest that the trait vector competence is inherited in single recessive gene in F. occidentalis, for which the phenotype is determined by the haplodiploid mechanism. These findings are fundamental for our understanding of the temporal and spatial variability within vector populations with respect to the trait vector competence and at the same time offer an essential basis for further molecular studies.DFG/207/37-

The type of ploidy of chrysanthemum is not black or white: a comparison of a molecular approach to published cytological methods

Polyploidy is a widespread phenomenon among higher plants and a major factor shaping the structure and evolution of plant genomes. The important ornamental chrysanthemum (Chrysanthemum indicum hybrid) possesses a hexaploid genome with 54 chromosomes and was classified based on its evolutionary origin and cytological methods as an allopolyploid. However, it is questionable whether cytological methods are sufficient to determine the type of ploidy, and there are more informative methods available based on molecular marker analyses. Therefore, we collected segregation data for 406 dominant molecular marker alleles [327 amplified fragment length polymorphism (AFLPs), 65 single-strand conformation polymorphism (SSCPs) and 14 microsatellites (EST-SSRs)] in a biparental F1 population of 160 individuals. We analyzed these data for the characteristics that differ between allopolyploids and autopolyploids, including the segregation ratio of each marker, the ratio of single-dose (SD) to multi-dose (MD) markers, the ratio of SD markers in coupling to those in repulsion and the banding patterns of the SSRs. Whereas the analysis of the segregation ratio of each polymorphic marker indicated disomic (13 markers) as well as hexasomic (eight markers) inheritance, the ratio of SD markers in coupling to those in repulsion was 1:0, which is characteristic of autopolyploids. The observed ratio of SD to MD markers was 0.67:0.33 which is significantly different to the expected segregation for auto- and allohexaploids. Furthermore, the three EST-SSR alleles were inherited in all possible combinations and were not independent of each other, as expected for fixed heterozygosity in allopolyploids. Combining our results with published cytological data indicates that cultivated chrysanthemums should be classified as segmental allohexaploids.BMEL

Evolution of the Rdr1 TNL-cluster in roses and other Rosaceous species

Background: The resistance of plants to pathogens relies on two lines of defense: a basal defense response and a pathogen-specific system, in which resistance (R) genes induce defense reactions after detection of pathogen-associated molecular patterns (PAMPS). In the specific system, a so-called arms race has developed in which the emergence of new races of a pathogen leads to the diversification of plant resistance genes to counteract the pathogens' effect. The mechanism of resistance gene diversification has been elucidated well for short-lived annual species, but data are mostly lacking for long-lived perennial and clonally propagated plants, such as roses. We analyzed the rose black spot resistance gene, Rdr1, in five members of the Rosaceae: Rosa multiflora, Rosa rugosa, Fragaria vesca (strawberry), Malus x domestica (apple) and Prunus persica (peach), and we present the deduced possible mechanism of R-gene diversification.Results: We sequenced a 340.4-kb region from R. rugosa orthologous to the Rdr1 locus in R. multiflora. Apart from some deletions and rearrangements, the two loci display a high degree of synteny. Additionally, less pronounced synteny is found with an orthologous locus in strawberry but is absent in peach and apple, where genes from the Rdr1 locus are distributed on two different chromosomes. An analysis of 20 TIR-NBS-LRR (TNL) genes obtained from R. rugosa and R. multiflora revealed illegitimate recombination, gene conversion, unequal crossing over, indels, point mutations and transposable elements as mechanisms of diversification.A phylogenetic analysis of 53 complete TNL genes from the five Rosaceae species revealed that with the exception of some genes from apple and peach, most of the genes occur in species-specific clusters, indicating that recent TNL gene diversification began prior to the split of Rosa from Fragaria in the Rosoideae and peach from apple in the Spiraeoideae and continued after the split in individual species. Sequence similarity of up to 99% is obtained between two R. multiflora TNL paralogs, indicating a very recent duplication.Conclusions: The mechanisms by which TNL genes from perennial Rosaceae diversify are mainly similar to those from annual plant species. However, most TNL genes appear to be of recent origin, likely due to recent duplications, supporting the hypothesis that TNL genes in woody perennials are generally younger than those from annuals. This recent origin might facilitate the development of new resistance specificities, compensating for longer generation times in woody perennials.DFG/DE 511/4-1DFG/DE 511/4-

Genetic analysis of callus formation in a diversity panel of 96 rose genotypes

In a diversity panel of 96 rose genotypes, variation in the capacity to form calluses on leaf explants in vitro was investigated, and a genome-wide association study (GWAS) was performed to identify genetic factors associated with callus formation. Calluses were induced from wounded in vitro leaflets on two media differing in their plant growth regulator composition. Significant differences between genotypes were observed in callus size on the first callus-inducing medium (CIM1, containing 10.7 µM naphthylene acetic acid) using a 0–4 scale, as well as on a second callus-inducing medium (CIM2, containing 4.5 µM dichlorophenoxyacetic acid and 2 µM 6-(γ,γ-dimethylallylaminopurine)) with callus size scales of 0.82–4. GWAS utilizing the WagRhSNP 68K SNP array for callus size induced on either CIM1 or CIM2 enabled the identification of 26 and 13 significantly associated SNPs, respectively. Among these SNPs, we found the SNPs Rh12GR_12098_1092Q (uncharacterized gene) and RhMCRND_2903_1233Q in a gene encoding a pentatricopeptide repeat-containing protein were associated with callus size on CIM1, with large effects being observed between alleles. Two SNPs, RhK5_5473_763P (S-formylglutathione hydrolase) and Rh12GR_37799_568Q (polyglutamine binding protein, WW domain binding protein), were associated with callus size on CIM2 with large effect sizes. The markers associated with callus size on CIM1 form a large cluster on chromosome 3 and minor clusters on other chromosomes and provide the first preliminary indications of candidate genes responsible for the observed phenotypic variation

P Starvation in Roses Leads to Strongly Genotype-Dependent Induction of P-Transporter Genes during Black Spot Leaf Disease

Phosphorous starvation in plants has been reported to have contrasting effects on the interaction with pathogens in different plant pathogen systems and plant species. Both increases and decreases in susceptibility have been observed in numerous reports. Here, we analysed black spot infection and the leaf expression of two plant phosphate transporters and one defence marker gene in roses after phosphorous starvation. We varied three factors: phosphate starvation versus full supply of phosphorous, black spot infection vs. mock inoculation, and different susceptible and resistant progeny of a biparental rose population. Black spot susceptibility or resistance was not significantly changed upon phosphate starvation in either compatible or incompatible interactions. The expression of phosphate transporters was strongly induced upon starvation, but in some genotypes, expression was altered by black spot interaction as well. The marker for pathogenic interactions was exclusively induced by interaction with black spot, but the expression was altered by a combination of phosphate starvation and interaction with the fungus in some genotypes. In summary, phosphate starvation has clear effects on the gene expression of phosphate transporters in rose leaves, and the interaction with a hemibiotrophic leaf pathogen is strongly genotype dependent