Micro algae show a large potential to become a future food and fee d ingredient. They can be cultured at large scale and hence be a sustainable option to harvest oil rich wild marine species. with stable access and quality of essential fatty acids, not limited by nature (Ganuza et al., 2008) . Currently, culturing microalgae is not economically competitive to other fat and protein sources (Ruiz et al., 2016). Optimization of the culturing protocol is thereby necessary, including strain selection and optimization. For micro algae that do sexual reproduction, like the diatom Seminavis robusta , new strains can be generated through crossbreeding of existing strains and strain selection hence be made from a broader selection. If successful, this process can be repeated each generation to form a breeding program. The aim of this study was to document whether genetic variation between existing strains is inherited to the next generation after crossbreeding.
Materials and Methods
Eight strains of Seminavis robusta were used, four male and four female strains , coming from three different clades, derived from analysis of the rbcL gene . Each male was crossed with each female, to produce as many novel combinations as possible, but not all crosses were successful, producing 64 cross-bred cultures, from 11 full-sib families. The parental and offspring strains were grown in 2 replicates in a 12 days growth experiment where biomass development was measured through CY5 fluorescence data on day 0, 3, 6, 9 and 12 , using Cytation ™ 3 plate reader and imager from BioTek. Cell-length on day 0 was measured for 30 random cells from each culture. Based on visual inspection of the parental growth curve (Fig. 1) , three growth parameters were selected for analyses: growth from day 0 to day 6 (Gr06), growth from day 0 to day 9 (Gr09) and growth during the 3-day interval where this line had the highest growth (MaxGr ). The growth traits were analysed with a general linear model in R, fitting replicate, cell-length (as a proxy for culture age) and whether the crossing was performed within or between clades as fixed effects together with the fixed effects of the two parents.
The effects of parents were significant for all three growth parameters (p<0.01), demonstrating that genetic variation in growth between the strains exists and is passed on to the next generation when crossing the strains. Replicate and whether the cross was an inter-clade crossing was non-significant (p>0.05) for any of the studied traits. Cell length was significant for MaxGr (p<0.05), where cultures with longer cells, i.e. younger cultures, grew significantly better than cultures with shorter cells. By comparing r2 from models with and without parents fitted in the model, the effect of the two parents together could explain 0.25, 0.22 and 0.32 of the total variance in Gr06, Gr09 and MaxGr, respectively, indicating heritability spanning from 0.44 to 0.65.
Discussion and Conclusion
This study clearly showed a heritable variation in growth between the S. robusta strains which was herited from parents to offspring when crossing parents. Hence, selective breeding for growth is expected to be successful. Among the growth traits explored, MaxGr gave the highest heritability, estimated to 0.65. The parental growth curves (Fig. 1) showed that the strains differed in when they reached exponential growth as well as the growth rate in the exponential phase. The fact that MaxGr has a higher heritability than Gr06 and Gr09 indicates that the achieved maximum growth rate is controlled more by genetics and less by other random factors than the time when exponential growth is reached. MaxGr is also the only growth definition significantly affected by average cell-length in the culture. Gr06 and Gr09 seem more affected by other random variables, not controlled in this study, causing larger residual variation. This study is a proof of concept that growth in micro algae may have a significant and considerable genetic component which can be utilized for strain development and optimization. Crossing of existing strains can be used to generate variation to select among when the species of interest do sexual reproduction.
We acknowledge the funding support provided by the European Union’s Horizon 2020 research and innovation program under grant agreements No. 862658 for the NewTechAqua project, No. 730984 for the Assemble Plus project (SEBREDIFACEA, PID: 13290), and No. 824550 for the Forward project.
Ganuza, E., Benítez-Santana, T., Atalah, E., Vega-Orellana, O., Ganga, R., and Izquierdo, M.S. (2008). Crypthecodinium cohnii and Schizochytrium sp. as potential substitutes to fisheries-derived oils from seabream (Sparus aurata ) microdiets. Aquaculture 277(1), 109-116.
Ruiz, J., Olivieri , G., de Vree , J., Bosma , R., Willems, P., Reith, J.H., Eppink , M.H.M., Kleinegris , D.M.M., Wijffels, R.H., Barbosa, M.J. (2016). Towards industrial products from microalgae. Energy Environ. Sci., 9, 3036