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Studies on Ribes plants, Cecidophyopsis mites and Blackcurrant Reversion virus for sustainable resistance breeding and cultivation of Ribes

 

 

Project Title: „Studies on Ribes plants, Cecidophyopsis mites and Blackcurrant Reversion virus for sustainable resistance breeding and cultivation of Ribes

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research” 

Project No.: 1.1.1.1/18/A/026

Period: 1 March 2019 – 28 February 2022

Project costs: 522 994,99 EUR

Principle Investigator: Ph.D. Inga Moročko-Bičevska (Institute of Horticulture)

Project partner: Dr. biol. Ina Baļķe (Latvian Biomedical Research and Study Centre)

 

Project summary:

The cultivation of plants of the genus Ribes is significantly affected by Cecidophyopsis ticks and Blackcurrant reversion virus (BRV). The breeding programs focus only on C. ribis and two resistance genes. Research shows that other species of Cecidophyopsis are also widespread, thus explaining the detected contradictions of host resistance.

The aim of the project is to explain the interaction of the Cecidophyopsis-BRV complex on Ribes plants: 1) by studying the concept and genetic diversity of the Cecidophyopsis species; 2) identifying the role of the Cecidophyopsis species in BRV transmission; 3) characterization of host defence to Cecidophyopsis and BRV using gene expression and transcriptome analyses by NGS; 4) by inventory and evaluation of local Ribes germplasm. The gained knowledge, evaluated local germplasm and virus-free in-vitro collection of local genotypes will serve the basis for sustainable resistance breeding and cultivation of Ribes promoting development of knowledge-based bioeconomy.

The implementation of the project will provide new knowledge for sustainable resistance breeding and growing of Ribes plants, at the same time increasing the number of scientific publications in internationally peer-reviewed journals and developing scientific capacity in agriculture and biotechnology. Performers are state research institutes that guarantee the public access of results (scientific publications, reports in scientific conferences) and the dissemination of knowledge to the industry (popular scientific publications, presentations in seminars, information in public databases). The project will support a continuation of started research on Cecidophyopsis mites and BRV, and initiated advanced studies in cooperation of both institutions in plant genetics, pathology, molecular biology and metagenomic analysis of environmental samples providing prerequisites for preparation of peer-reviewed publications.

Information published 01.03.2019.

 

Progress of the project:

1 March 2019 – 31 May 2019

Research

During the reporting period, five expeditions and samples for research in Latvia, Poland, Lithuania, and Finland were collected within Activity 1, in total collecting 134 samples of Ribes plants. Microscopy and testing of buds for the presence of mites were performed and samples were prepared for DNA isolation. The method for obtaining a DNA sample from a single mite individual was developed and 919 samples of DNA samples were prepared for the analysis. Work has been done to improve the specificity of amplification primers for the ITS region by developing and testing pairs of newly created primers. Practical testing of various types of microscopy for non-destructive analysis of mite specimens and species identification, and further isolation of DNA/RNA from one mite individual has been initiated. Investigation of information and available protocols for the selection of the most appropriate method for isolation of viral RNA from one mite individual has been started.

Within Activity 2. the updating of published information on Ribes plant resistance to Cecidophyopsis species and analysis of scientific literature was done. Information on Ribes plant samples analyzed in previous studies, genotyping results of Ce gene was collected and analyzed. The DNA collection of the existing Ribes plants, the samples available, the degree of their characterization, the quantitative and qualitative assessment of the DNA of the samples were evaluated. The collection of the existing Ribes plant material for DNA extraction has been evaluated.

An analysis of literature has been performed for the isolation of high quality total RNA from plant material with high polyphenol, polysaccharides and antioxidant content. Summarizations of used methods were performed. A protocol plan has been established for methods to be tested for total RNA isolation, for the suitable identification. Comparison of available NGS platforms available at BMC (Ion Torrent, Illumina and MGI) has been performed. Infestation trial for NGS research was established on potted plants in outdoor conditions for black currants, red currants and Ribes alpinum infested with three different mite species. Tests for infectious specimens for mite damage and samples for BRV diagnostics prior to infestation were performed to exclude possible contamination of plants before infestation. Experimental samples of Ribes plants from the infested plants for optimization of RNA extraction methodology, preparation for NGS analysis were collected and prepared.

Initial evaluation of the local germplasm has been done, gathering data on past evaluation results of Ribes plant resistance to Cecidophyopsis mites in Institute of Horticulture collections and trials. Information on the phenotypic characterization of the existing Ribes genetic resources and statistical analysis of the data was performed. A manuscript of the publication "Phenotypical variability and diversity within the Ribes genetic resources collection of Latvia" was prepared and submitted for publication in Acta Horticulturae. Based on the current information on the phenotypic characterization of the Ribes genetic resources, an oral report was prepared on "XII International Rubus and Ribes Symposium: Innovative Rubus and Ribes Production for High Quality Berries in Changing Environments", 25-28 June 2019, Zurich. Inventory of local-origin Ribes genetic resource collections at LatHort field collections in Dobele and Pūre, and the data collected so far, was done to assess what additional observations and data records would be needed to select genotypes for inclusion in the national genetic resource conservation database and international databases.

The evaluation of the local genotypes of the Ribes available in the germplasm collections and collected in the earlier expeditions has been started. The evaluation was based on RIBESCO descriptors. A total of 23 gooseberries, 23 blackcurrants and 12 currant genotypes have been evaluated.

A list of genotypes needed for industrial research for in vitro propagation has been developed and the introduction of these plants in vitro has been started, as well as the introduction of the most valuable local genotypes in tissue culture for recovery and further preservation by creating a recovered, virus-free collection.

Information published 31.05.2019.

 

Progress of the project:

1 June 2019 – 31 August 2019

Within Activity 2., samples of three Ribes plants (blackcurrant, alpine currant, redcurrant) were collected and stored at various temperatures (+4°C, -20°C, -80°C) with and without RNAlater to determine the most effective sample storage conditions that allow the isolation of high quality total RNA for NGS analysis. Samples stored at + 4°C with and without RNAlater were used for RNA extraction with TRI Reagent, but after analysis of RNA samples, this method was found to be unsuitable for RNA extraction from blackcurrants, alpine currant, redcurrant. In parallel, three commercial RNA isolation kits were tested with introduced modifications according to available literature data by adding polyvinylpyrrolidone to the lysis buffer. A method of lysing matrix A, D, and E was tested for sample disintegration. It was concluded that lysis matrixes are suitable for disintegrating plant samples and two of the three kits used are compatible with this method. The introduced modification allows the isolation of RNA from blackcurrants, where the purity of RNA at 260/280 nm is 2.02 - 2.05. In addition, analysis of the quality and integrity of the isolated RNA samples on the Bioanalyzer has been started.

Information published 30.08.2019.

 

Progress of the project:

1 September 2019 – 30 November 2019

Within Activity No.1, Multiplex PCR amplification of mite DNA and PCR product preparation for fragment length analyses (FLA) with genetic analyser was continued. FLA was performed for 725 prepared mite DNA multiplex PCR samples on the ABI PRISM 3130xl genetic analyzer for species identification, and an automated analysis protocol for allele identification and selection was developed. Preliminary FLA data analyses and identification of mite species was performed in the collected plant material. Testing of non-destructive electron microscopy for the mite species identification and further isolation of nucleic acids from single mite individuals was continued and the optimal application protocols were elaborated and selected for further use. The testing of methods for viral nucleic acid isolation from mites and BRV PCR amplification was continued and the protocol for testing in the laboratory was established. Theoretical protocols for ITS/5.8S amplification and cloning was elaborated and their practical testing was started in the laboratory.

In Activity No.2, further development of methods for the Ce and P resistance gene identification in Ribes plants was performed on the Ribes plant material selected in earlier stages of the project, as well as involving additional Ribes samples. Modification of molecular marker reaction conditions was continued to ensure stable amplification of gene-specific fragments. In accordance with the project objectives, the collection of existing genetic information on the Ribes genetic resources was started, and the evaluation of existing genotyping data for preparation of paper manuscript has been undertaken. The obtained information will provide insight into the diversity and interrelation of genetic resources. To complement the existing genetic data, a review of the scientific literature and selection of molecular markers (cpSSR or chloroplast microsatellites) was done during the reporting period with aim to clarify the inter-specific structure of Ribes plant material, considering their complex origin, especially for blackcurrants, that can have the influence to resistance.

Total RNA isolation optimization experiments were continued from the previously harvested samples from genotypes of Ribes genus (black currant cultivar ‘Mara Eglite’, red currant cultivar ‘Kodu Suur Valge’ (‘Hele’) and genotype of Ribes alpinum, collected in the wild), as well as for the isolated RNA concentration, quality and integrity (RIN) analysis were performed. Comparing the concentration, quality and RIN of the isolated RNA in samples stored under different conditions, it was concluded that the RNA isolated from in RNAlater stored samples at +4°C and -20°C is higher in concentration and RIN, which is essential for the preparation of high quality NGS library. Conversely, samples stored at -80°C showed the opposite effect. Therefore, RNAlater can be used to store plant samples at higher temperatures. The optimal RNA isolation methods have been developed for Ribes plant samples by selecting the most appropriate lysis matrix, optimal PVP concentration and isolation kit for each species of the genus. Optimized RNA isolation methods will be used for RNA isolation from inoculation experiment samples for preparation of NGS libraries.

During current period the summarizing of knowledge and scientific information on Ribes/Cecidophyopsis/BRV complex interaction and previous observation results in Ribes collections of Institute of Horticulture was performed and the preparation of review article was started. Data collection and description using RIBESCO descriptors of Ribes genotypes and inventory of present genetic resources in field collections in Dobele and Pure was continued, to select more valuable genotypes for including in national and international genetic resource databases. Data analysis for the results obtained in season 2019 has begun. Work was continued on the development of evaluation methods. Selected Ribes genotypes for further industrial research and valuable local accessions for creating of virus and pest free core collection were propagated and maintained in vitro.

The project progress and goals were communicated to the society during the European Science Night, where at the Institute of Horticulture (LatHort), Plant Pathology and Entomology laboratories scientists in a popular science format presented poster and provided practical demonstrations about research on Cecidophyopsis bud mites, blackcurrant reversion virus and Ribes plants. Information about activities was posted on the LatHort Facebook site.

Information published 29.11.2019.

 

Progress of the project:

1 December 2019 - 29 February 2020

Within Activity No.1, application protocols for amplification and cloning of the ITS/5.8S region of mites were developed, the cloning and preparation of samples for sequencing has been done and first samples sent for sequencing to the project partner BMC. The studies on Cecidophyopsis morphology using non-destructive electron microscopy has been started and isolation of nucleic acids from single mites performed. The testing of BRV in single mites after electron microscopy was started.

In Activity No.2, further development of methods for the Ce and P resistance gene identification in Ribes plants was performed on the Ribes plant material selected in earlier stages of the project, as well as involving additional Ribes samples. The material for cloning and sequencing of the Ce gene specific amplification fragment was prepared, cloned and sent for sequencing to the project partner BMC. Work was continued on the analysis of existing Ribes genetic resources genotyping information for preparation of a publication manuscript. Adaptation of the methodology was started on the set of chloroplast molecular markers (cpSSR or chloroplast microsatellites) selected in the previous reporting period, to select the most suitable markers for explaining of the inter-specific structure of Ribes plant material. The isolation of total RNA from mite-infested and control samples of blackcurrant (cultivar ‘Mara Eglite’) sampled in May and August and stored at -80°C has been initiated by BMC following the previously optimized RNA isolation protocol. The work has been undertaken on the preparation of NGS libraries for sequencing on MGISeq2000.

The evaluation of genotypes according to RIBESCO descriptors in Ribes germplasm collections of LatHort in field collections was continued to select the most valuable genotypes for including in the National Genetic Resources Conservation Database and international databases. The evaluation data of the previous season were summarized. The assessment of damages by gall mites in the Ribes LatHort field collections in Dobele and Pūre was done and development of evaluation methods was continued. In vitro propagation and maintenance of the most valuable local Ribes genotypes and selected genotypes for industrial research was continued in Plant Tissue Laboratory.

The work was continued to collect information and writing an article on resistance of Ribes to Cecidophyopsis and BRV, and their interactions. Interested parties were informed about the progress of the project and the achieved results in the practical conference "Sustainable Agriculture", where scientists from the Institute of Horticulture presented a poster and submitted a manuscript for publication in the conference proceedings.

Information published 28.02.2020.

 

Progress of the project:

1 March 2020 – 31 May 2020

Within Activity No.1, the amplification and cloning of the ITS/5.8S region of mites were continued, and the next samples sent for sequencing to the project partner BMC where sequencing was performed by the Sanger method. Additional blackcurrant samples were received from UK, mite DNA isolated and samples prepared for the species detection by FLA. FLA was performed by the project partner on the new mite multiplex PCR samples on the genetic analyzer ABI PRISM 3130xl for species determination using the previously developed automated analysis protocol. The testing of BRV in single mites after electron microscopy was continued.

Within Activity No.2 the collecting of information and writing an article on resistance of Ribes to Cecidophyopsis and BRV, and their interactions was continued. The analysis of existing Ribes genetic resources genotyping information for preparation of a publication manuscript was continued by performing additional literature and data analysis. Adaptation of the methodology of application of chloroplast molecular markers (cpSSR) selected in the previous stages of the project has been performed; the set of the most informative markers has been selected to explain the structure of interspecific crossings of Ribes plant material. DNA extraction was performed using the existing Ribes sample collection. In order to ensure the representation of Ribes species, additional samples were collected in the LatHort collection and their DNA extraction and quality assessment was performed. PCR of resistance Ce genes specific amplification fragments was performed for further cloning and sequencing, and analysis of the obtained sequences was started. In BMC the sequencing of cloned amplification fragment of Ce genes samples transferred by the Leading partner was performed by the Sanger method.

Work continued on the preparation of NGS libraries of blackcurrant (cultivar ‘Mara Eglite’) mite-infested and control samples for sequencing on MGISeq2000, as well as the isolation of total RNA from Ribes alpinum and red currant (cultivar ‘Kodu Suur Valge’ (‘Hele’)) for mite-infested and control samples sampled in May and August and stored at -80°C has been initiated by BMC following the previously optimized RNA isolation protocols. The work has been undertaken on the preparation of NGS libraries for sequencing.

Primers based on the literature search were selected and new primers developed for amplification of COX1, TEF 1-a, HSP70 gene fragments and sequencing to develop mite detection method.

The evaluation of Ribes genetic resources field collections was continued according to RIBESCO descriptors. The second year data set on plant phenological and vegetative development, frost resistance was collected. The collections in Pūre and Dobele were assessed for the prevalence and symptoms of BRV. Samples were collected from the red and white currants for the determination of BRV to develop a disease evaluation methodology. In the tissue culture laboratory, 9 Ribes genotypes from different species were propagated and rooted in the substrate, which will be used for resistance studies, and 9 blackcurrant and 4 gooseberry genotypes for a creating of virus and pest free core collection, which will be tested for healthiness. In parallel, samples of all genotypes are maintained and preserved in vitro.

Information published 29.05.2020

 

Progress of the project

1 June 2020 – 31 August 2020 

Within Activity No.1, the amplification and cloning of the ITS/5.8S region of mites was done for the samples from UK and after electron microscopy. The prepared samples were sent for sequencing to the project partner BMC. Sequencing of the ITS/5.8S region of mites transferred by the Leading Partner was performed by the Sanger method. Fragment Length analysis (FLA) was performed on new mite multiplex PCR samples on the genetic analyzer ABI PRISM 3130xl for the species determination, using the previously developed automated analysis protocol. The research on mite morphology by an electron microscopy and testing of BRV in single mites after the electron microscopy and plant material was continued.

Within Activity No.2 the writing an article on resistance of Ribes to Cecidophyopsis and BRV, and their interactions was continued.

The analysis of the sequences obtained from resistance Ce gene-specific amplification fragments was continued. A draft of the publication on the results of SSR marker genotyping of Ribes genetic resources has been prepared, and experts from different project activities have been involved to start the improvements of draft publication. The chloroplast molecular markers (cpSSR) adapted in the previous stages of the project were used to start the genotyping of Ribes plant material in order to explain the possible species structure of interspecific crosses. The cpSSR genotyping was performed on 126 samples of blackcurrants (including samples of Ribes species used in blackcurrant breeding), as well as 13 samples of red currants, 3 gooseberries, 8 Ribes alpinum and 10 other Ribes species. At the BMC, length analysis for 23 chloroplast molecular markers, selected in the previous stages of the project, of FLA samples (3812 samples) transferred by the Leading Partner were started on the genetic analyzer ABI PRISM 3130xl, and development of automated analysis protocols.

NGS libraries were prepared of blackcurrant (cultivar ‘Mara Eglite’) mite-infested and control samples for sequencing on MGISeq2000. Work continued on the preparation of NGS libraries of mite-infested and control samples of Ribes alpinum and red currant (cultivar ‘Kodu Suur Valge’ (‘Hele’)) harvested in May and stored at -80°C. The evaluating of experimental infestation of Ribes plants with several bud mite species was undertaken. A new infestation experiment was set up using several species of Ribes in vitro propagated plants and mites collected from various host plants.

Protocols for PCR amplification of COX1, TEF 1-α, and HSP70 genes were established and practical testing in the laboratory was started using the newly designed primers.

The evaluation of Ribes genetic resources field collections and genotype describing according to RIBESCO descriptors was continued. The second year data set on genotype yield, berry quality, pest and disease resistance was collected and phenotypic berry characterization and photography was performed. In the Tissue Culture Laboratory, the propagation and maintenance in vitro of plants required for industrial research, which was started in 2019, was continued, as well as the in vitro propagation of another 16 genotypes required for research was started. The maintenance of 10 blackcurrant and 5 gooseberry genotypes in vitro for the creation of virus and pest free genetic resource core collection was continued and the samples were collected and tested. Additionally, 6 currants and 1 gooseberry genotype of Latvian origin were introduced in vitro.

Information published 31.08.2020.



Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".