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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.




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".