Investigation of regulatory regions of Scots pine disease resistance genes

European Regional Development Fund Operational Programme "Growth and Employment" Specific Aid Objective 1.1.1 "To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure" Activity 1.1.1.2 "Post-doctoral Research Aid" agreement No. 1.1.1.2/VIAA/3/19/510

Postdoctoral researcher Vilnis Šķipars.
Scientific consultant Dainis Ruņģis.

Despite the importance of P. sylvestris, the amount of genomic information of this species is limited. A full genome reference sequence has not been published. However, the amount of available information is increasing as Scots pine transcriptome studies have been published by several groups and there is some whole genome sequence data available. Also, whole genome sequences and transcriptome studies have been published for other conifer species (e.g. Pinus taeda and Picea abies). This supports the novelty and credibility of the proposed project, as, while there is a lack of genomic studies in P. sylvestris, genomic resources and analytical approaches are available from other conifer species.

The molecular basis of phenotypic differentiation and adaptive traits is not only based on protein coding DNA sequence polymorphisms, leading to changes in protein structure, but also in gene regulation, including epigenetic effects. Epigenetic mechanisms include methylation of gene promoter regions, and gene expression levels can also be influenced by gene copy number variations.

Scientific goal is to clarify structure, variability and epigenetic status of resistance linked gene 5’ flanking sequences, characterise epigenetic responses to inoculation, gaining insights in the regulation of these genes, and creation of screening markers for selection of Scots pine individuals for further studies.

Study impact: considering the economic and ecological importance of P. sylvestris in Northern Europe and the existence of current Scots pine breeding programs, the results can have a positive impact in ensuring sustainability of forest ecosystems and wood production in face of increasing damage by root rot and other pathogens.

Project implementation:

1. Sample material selection and preparation.
2. Oligonucleotide selection and technology approbation.
3. Investigation of gene flanking region structure.
4. Investigation of DNA methylation status and dynamics and the impact on gene expression.
5. Development of screening markers and protocol.
6. Public engagement and communication.

Actualities

1. 1.01.2020. The project started on January 1, 2020.
2. 24.01.2020. Lecture in 42nd National Biology Olympiad.
3. In January–February 2020, the selection of phenotypically different individuals (in terms of terpene composition and resistance to pathogens) was performed, evaluating the data of LSFRI "Silava" forest selection and tree plant adaptation department, the objects in which the selected individuals have survived were selected in the database of long-term research objects.
4. In February–March 2020, selected field surveys, sampling for genetic analysis and genetic testing of individuals using SSR markers were performed to confirm the identity of the individuals referred to in point 3 in the plantations.
5. In March–April 2020, 25 grafts were collected from 5 individuals (125 grafts in total), grafting was performed by JSC Latvia’s State Forests Seeds and Seedlings department specialists.
6. In April 2020, a procurement list for the next steps of the project was compiled and the estimated reagent costs were clarified, and an analysis of previously obtained common pine transcriptome data was performed to improve primer selection in the next step of the study.
7. In April–June 2020, the design of oligonucleotide sequences was performed, changes in the experimental design were made; due to the mobility difficulties caused by COVID-19, the possibility to perform all stages of oligonucleotide approbation in LSFRI "Silava" is planned. Due to both changes in the experimental design and the use of another sequencer for approbation of oligonucleotides, changes were made in the prepared reagent procurement. In order to make these changes, both scientific and technical literature was analyzed and reagent and sequencer manufacturers were consulted. Condition of the grafted clones was also examined.
8. On September 25, 2020 Vilnis Šķipars presented the work in the Molecular genetics lab (video). It is part of a series of TV stories dedicated to young people to encourage them to choose science as a career.
9. On October 1, 2020, during a science cafe-type online event from the series Online with a scientist Vilnis Šķipars presented the research on forest tree genetics in Latvia and neighbouring countries as well as about this research. The event was organised by Association of Latvian Young Scientists (video).
10. 12.2020. Journal "Baltijas Koks" December issue the local forest industry-related audience is informed about the aims and significance of this study (article in Latvian).
11. 26–27/01.2021. The research was presented at the popular scientific conference "Knowledge-Based Forest Industry".
12. The results of the study will be presented at the international conference Forest Genetics Student & PostDoc Symposium 2021 on May 19–20, 2021. This conference is co-hosted by Western Forest Genetics Association (WFGA) and Canadian Forest Genetics Association (CFGA/ACGF).
13. The current results of the study have been presented at the international conference Forest Genetics Student & PostDoc Symposium 2021" on May 19–20, 2021.
14. The first results obtained by massive parallel sequencing have been analyzed. Although they confirm that the newly developed method can give the expected results, its effectiveness without further optimization is insufficient, so it was decided to use another method to achieve the research goals, modifying it to use the already invested resources efficiently.
15. In order to promote mutual communication of local scientists and public understanding of science, an informal science café-format event "Zinātnes pusstops" (Science pint) organized by the Latvian Association of Young Scientists was attended on 13 September. 10 persons participated in the event, one was from Japan. Participants represented economics, molecular genetics, microbiology, chemistry, bio-medicine, archeology and other branches of science. Unfortunately, non-scientists did not participate this time.
16. We have acquired the Oxford Nanopore Technologies sequencer MinION, which is special in that it allows us to read the nucleotide sequences of long DNA fragments without interruption. This has specific advantages in data analysis.
17. The use of the Oxford Nanopore Technologies sequencer MinION has been successfully launched. We will be happy to introduce you to this technology, the cost of experiments and other issues.
18. Results obtained until now will be presented in the 20th IUFRO Tree Biotech and the 2nd FTMB (Forest Tree Molecular Biology and Biotechnology) Conference (July 7–9, Harbin, China). Remote participation.
19. The results of the research obtained until now will be presented at the conference "7th IUFRO International Workshop on the Genetics of Tree-Parasite Interactions in Forestry", which will be held in Pontevedra, Spain, September 12–16, 2022. In-person participation intended.
20. During the Forestry Afternoon (a popular science event) in LSFRI "Silava" on September 30, students and adults, were introduced with the course and significance of the research. Thank you to the audience for attending the lecture!

Primer sets for screening of regulatory regions of Scots pine disease resistance-linked genes