Securing adaptation of timothy cultivars under climate change and during seed multiplication using genomics and big-data approaches

Timothy is the most important forage grass grown in Norway, especially in the northern parts of the country. Climate change leads to greater variation in temperature and precipitation, and this will affect both plant production in the growing season and winter survival. Production of commercial seeds of northern Norwegian timothy cultivars is in southern Norway because the climate in the north is not good enough for safe seed production. The aim of the project was to investigate whether frost and ice-encasement tolerance, as well as genetic structure, changes during generations of propagation of northern timothy cultivars in the south. Frost and ice-encasement tolerance were tested in three seed batches, pre-basic, basic and certified seed of the northern adapted cultivars 'Engmo' and 'Noreng'. Frost tolerance is reduced in the cultivars when we compare plants from pre-basic with plants from today's commercial seed lots that have been propagated in the south. No significant differences between seed lots or cultivars were found for tolerance to ice-encasement, maybe because plants are not exposed to ice cover during propagation in southern Norway. Genetic changes were detected in the cultivars over time, with greatest change in Noreng, a synthetic cultivar with a narrower genetic background than Engmo. The results show that seed production in south-east Norway can reduce the winter hardiness of northern Norwegian cultivars and indicate that the current system of propagation does not preserve the cultivars' characteristics well enough. Three generations of the northern timothy cultivars Engmo and Nordeng were bred in each of three locations in Norway, in the north (Tromsø), in central Norway (Steinkjer) and in the south (Landvik). These locations represent the traditional areas for propagation of pre-basic, basic and commercial seed of northern timothy cultivars. The aim was to investigate whether propagation in southern parts of the country reduces the overwintering ability. Both cultivars, to different degrees, lose frost tolerance and ice-encasement tolerance with repeated propagation in the south. Genotyping with SNP markers shows that there are clear changes over generations, especially in the 1st seed generation. Surviving plants from field trials at four locations in northern Norway with the cultivars 'Engmo', 'Noreng', 'Grindstad' and 'Snorri' were collected and tested for frost tolerance and ice-encasement tolerance. Frost tolerance of surviving plants was worse than that of plants from the seed lots that were sown in the field. This age effect was also evident in gene expression under frost and ice cover; plants from the original seed lots were clearly different from surviving plants from the field. Engmo and Snorri were the most tolerant to frost and ice cover. The results showed that although surviving plants had lower frost tolerance than plants from the sown seed lots, many stress-responsive genes were up-regulated in them. We found significant upregulation of genes related to ethylene response, glycolysis and stress-related proteins under ice cover, and gene expression during ice cover, hypoxia (flooding) and frost is partially overlapping. Field survivors and plants from original seed lots, as well as northern and southern cultivars differed in gene expressions. These differences may be due to selection in the field, plant age and/or genetic background. A genotype from the cultivar 'Liljeros' (Graminor) has been sequenced. The genome sequence is of high quality and has been used both in the gene expression studies (RNA sequencing) and in the population genetic studies (genetic markers). We are collaborating with a group at the Swedish University of Agricultural Sciences (SLU) that is sequencing the parent species of timothy (wild timothy and alpine timothy) on joint publication of the timothy genome. Data from the official variety testing (VCU) of fodder crops (18,477 observations for the period 1988-2017 covering 166 combinations of location and year of establishment and 119 different cultivars) was used to find a statistical prediction model for yield based on agroclimatic variables. The number of degree days (GDD) in July and the number of days with rain (>1 mm) in June–July provided the best prediction. The prediction model was combined with several climate ensembles (RCP4.5) to predict yields for the decades 2050–2059 and 2090–2099 at 8 locations in Norway. The predictions show a significantly reduced yield in South-East Norway, while it will increase in Northern Norway by the middle of the century due to increased temperatures and changing rainfall patterns. The project has been presented in the media and trade press, and at market days. A closing seminar with participants from the reference group and the agricultural consultancy in Northern Norway represented in addition to all the project participants was arranged at NIBIO Tromsø, Holt in December 2023.

Kompetansebygging: 1) Prosjektet har bygd opp kompetanse hos partnerne på gjennomføring og analyse av frost- og isdekketoleranse. 2) Kompetanse på genekspresjonsanalyser (RNA sekvensering) av responser på frost- og isdekketoleranse er utviklet. En NMBU-stipendiat har utført sitt doktorgradsarbeid på dette. Studiene av genekspresjon under isdekke er unike. 3) Økt kompetanse innen bioinformatikk for analyse og sammenstilling av genomsekvenseringsdata for et komplisert polyploid timoteigenom. 4) Kompetanse på bruk av data fra langvarige serier av feltforsøk for å modellere avling basert på agro-klimatiske variabler er utviklet ved samarbeid med Norsk regnesentral. Betydning for partnere og næring 5) Resultatene viser at frøproduksjonen i Sørøst-Norge kan redusere vinterherdigheten hos nordnorske sorter, og tyder på at dagens system for oppformering ikke bevarer sortenes egenskaper godt nok. 6) Resultatene vil få betydning for virksomheten for bedriftspartnerne i prosjktet, dvs. frøfirmaene Felleskjøpet og Stand Unikorn, og Graminor som utvikler timoteisorter. 7) Resultatene har bekreftet utsagn fra bøndene i Nord Norge om at timoteisorten Noreng, som erstattet Engmo i frøblandingene, har dårligere overvintring enn Engmo. Samfunnsmessig betydning: 8) Økt varighet av eng ved at sortene har god overvintringsevne har stor betydning for landbruket. Det gir økt bærekraft ved reduserte utslipp, mindre bearbeiding av jorda, reduserte kostnader og økt lønnsomhet for bonden.

Grassland based forage production is the cornerstone of agriculture in Northern Norway. It is important for the economy and it is expected to use local feed resources for the production of milk and meat. Achieving this goal requires higher and more stable yields of high quality forage over years. Timothy is the most important forage species in Norway, especially in the north. The predicted climate change will lead to higher and more variable precipitation patterns. These changes are expected to be more rapid in the north. Development of timothy cultivars that are adapted is crucial for a sustainable forage production in the north. Breeding of new cultivars takes long time with traditional methods – up to 20 years. By using machine-learning methods and genomic selection, it is possible to combine climate and soil data with phenotype and genomic data to predict genomic breeding values. Production of commercial seeds of timothy cultivars bred for Northern Norway is challenging since the production is located in the South Eastern part of Norway. This can lead to risks of genetic shifts in the cultivars and thus change in key traits that are important in the north, e.g. winter survival. Studies of genetic shifts during seed multiplication are limited. In this project, we will study winter survival, estimated as freezing and ice-encasement tolerance in different seed generations of northern timothy cultivars and breeding populations. This information will be combined with molecular marker data for identification of genome regions in timothy associated in genetic regulation of these traits. In addition, we will study potential genetic shifts in seed generations of different age of the northern timothy cultivars ‘Engmo and ‘Noreng’. Historical phenotypic data from multi-location-year yield trials will be combined with climate, soil, and genomic data using advanced machine learning models to develop genomic prediction models for developing of adapted cultivars in the north.