The importance for frost tolerance in white lupin: phenotypic and genotypic tools for its improvement

White lupin (Lupinus albus) is a cool-season grain legume of high potential interest for European agriculture. It may play an important role for producing novel high-protein foods and reducing our dependency on international markets for high-protein feedstuff, while enhancing soil nitrogen availability and biodiversity. The interest in this crop is driven by its high seed protein content (36–40 percent) compared to other cool-season grain legumes. Greater frost tolerance can expand autumn sowing of the crop northwards, to take advantage of milder winters and to escape the increasing risk of terminal drought. In warmer regions, sudden frosts following mild-temperature periods may produce high winter mortality of insufficiently acclimated plants. The field-based selection for frost tolerance is challenged by the wide and increasing year-to-year climatic variability, which emphasise the interest of selection under controlled conditions to ensure applicability, efficiency, and replicability. CREA’s Research Centre for Animal Productions and Aquaculture in Lodi (Italy) set up an easy-to-build phenotyping platform for frost tolerance assessment consisting of a walk-in growth chamber capable of freezing temperatures and within the project Diversilience, CREA performed a methodological study aimed to identify optimal platform-based selection procedures, and a large-scale evaluation study aimed to explore the genetic architecture of the frost tolerance trait and to explore opportunities for molecular marker-based selection. 

Methodological study

The study included 11 genotypes broadly representing low, medium, and high classes of frost tolerance according to previous field-based experiments in northern Italy. After an acclimation period of 15 days at 4 °C, seedlings of these genotypes were subjected to four freezing temperature treatments, i.e., -7, -9, -11, and -13 °C, in an experiment with four replications and 10 plants per experimental unit. Frost tolerance was assessed for each genotype in each treatment using two criteria: plant mortality (percent), and a visual aerial biomass injury score (on a scale from 1 to 10) based on the amount of necrosis and mortality after recovery and regrowth. We estimated the Lethal Temperature 50 (LT₅₀), representing the temperature at which 50 percent mortality occurs. LT₅₀ averaged -11.0 °C, which also corresponded to the freezing treatment that maximised the genotype variation, with mortality ranging from 0.26 to 0.88. Mortality following this treatment was highly correlated with biomass injury score (r = 0.97) and LT₅₀ (r = 0.94) of the genotypes, indicating a strong consistency among these indicators of frost tolerance. Importantly, the consistency of genotype mortality across platform and field conditions was high, supporting the use of the platform for a reliable assessment of genotype frost tolerance.

Large-scale evaluation

We evaluated 144 white lupin inbred lines derived from 16 crosses between 4 sweet-seed cultivars and 4 elite international landraces, in an experiment with four replications. The lines were tested at the freezing temperature of -11 °C (which showed the highest screening value in the methodological experiment). The broad-sense heritability of lupin frost tolerance according to plant mortality values was high (H² = 0.82). A preliminary genome-wide association study (GWAS) identified a significant SNP on chromosome 11. Several other peaks were present although they did not reach the significance threshold, suggesting a polygenic control of the trait. Therefore, we performed a genomic prediction study encompassing three prediction models (rrBLUP, Bayesian LASSO, and Bayes B), which exhibited prediction accuracy values (i.e., correlations between observed and genomically-predicted genotype values in cross validations) close to 0.70. Such values could justify a genome-based prediction of frost tolerance for large numbers of genotypes, to restrict the number of genotypes undergoing a phenotypic selection.

Conclusion

The high consistency for genotype mortality across platform and field conditions encourages the use of the platform for genotype selection as well as for molecular and eco-physiological studies. Genomic selection may contribute to frost tolerance improvement according to preliminary results for breeding lines, which will be verified by a subsequent study focusing on landrace germplasm.