Development of a breeding strategy for nitrogen use efficiency in spinach (Spinacia oleracea L.)

Spinach (<em>Spinacia oleracea </em>L.) is one of the most consumed leafy vegetables worldwide and it is considered to be highly nutritious. Spinach is a short-cycle leafy crop that has a high demand for nitrogen in order to rapidly come to a harvestable product that has the required dark green colour within a reasonable harvest window. In commercial production of spinach the recovery of N is poor, which may result in environmental pollution. To increase sustainability of both organic and conventional spinach cultivation there is a need to reduce the dependency on high levels of nitrogen. Growers therefore urgently need cultivars with a satisfactory yield under reduced N input conditions. Nitrogen use efficiency (NUE), defined as the ability to produce high biomass per unit N applied, is low in spinach. The present study aims to evaluate spinach genotypes for selectable traits under varying N supply and provide tools and knowledge to facilitate the development of varieties with good yield, quality and stability under low N input. To minimise environmental variation affecting the identification of traits related to NUE a screening method was developed using a hydroponics system. The genetic diversity for NUE related traits was first studied with 24 commercial cultivars under contrasting levels of N supply based on the Ingestad model with a steady-state N application. This demonstrated that the hydroponics screening strategy as a pre-screening tool enabled reliable detection of heritable variation among cultivars for NUE-related traits under optimal as well as suboptimal N input. Shoot dry weight and leaf area were preferred selectable traits for the detection of heritable differences contributing to NUE in spinach. The effect of N application strategy was examined in seven cultivars grown under hydroponics conditions with low and high N levels supplied either as a single bulk N application resembling N fertilization in field cultivation, or a steady-state N application according to Ingestad. The latter application strategy provided more stable and reproducible conditions for determination of genetic differences in NUE under low N conditions for a short-cycle leafy vegetable crop. Several tools for molecular genetic evaluation of NUE in spinach were provided as well, including a SNP marker set for marker-assisted breeding, a genetic mapping population with a corresponding genetic map, and the identification of two major QTL regions contributing to growth under low N conditions. With these tools, an efficient strategy for breeding for NUE efficiency in spinach would include screening under controlled conditions at high and low N using leaf area, biomass and root to shoot ratio as selectable traits, and QTL identification of genetic factors that can be targeted and combined using marker-assisted selection. An in depth genotype by environment interaction analysis using six field trials showed that environmental factors like temperature, soil, and management strongly influence nitrogen availability in the soil in a short cycle crop like spinach. This severely complicates selection and breeding for NUE of spinach under field conditions, and emphasizes the importance of performing trials under better controllable conditions for genetic dissection of NUE and discovery of genetic factors contributing to NUE. It also underscores the importance of validating these findings in various field trials. Multi-environment field trials with different levels of N fertilization will then allow selection of cultivars that combine stable performance under various low input growing conditions with high yields under more favorable conditions.