**2. Material and methods**

## **2.1 Survey and collection of RWA at landscape level**

RWA samples were collected annually during the wheat growing season in South Africa from 2010 to 2019. All main wheat production areas within the known distribution of the RWA were sampled. The same areas were sampled each year and where possible the same fields (**Figures 1** and **2**). There are two main dryland wheat production areas in South Africa where RWA commonly occur, the Western Cape (winter rainfall area) (**Figure 1**) and the Free State (a summer rainfall area) (**Figure 2**), with irrigated wheat production areas in the Central and Western Free State and Northern Cape (**Figure 2**). Sampling sites were selected off primary or secondary roads that transected major wheat or barley production areas. Sites were 10-20 km apart with distances depending on the continuity of wheat fields. In the Western Cape an average of 32 fields were sampled (**Figure 1**) and in the Free State an average of 61 fields were sampled (**Figure 2**). Samples were collected from cultivated wheat, barley and oats as well as volunteer wheat, wild oats, rescue grass and false barley in road reserves and around cultivated fields. Infested leaves were placed in Petri dishes containing moist filter paper and stored in an icebox for transportation to the glasshouse. The number of aphids per plant, percentage plants infested, growth stage of the plants and damage on the plants were recorded. The geographical co-ordinates and elevation where the samples were collected were also captured on a GPS and all the information of each sample collected was entered into a database (Windows Office –Excel).

#### **2.2 Establishing clone colonies of collected RWA samples**

A single female aphid from each sample collected in the field was transferred to a wheat plant and caged (gauze size: 315micron) to produce a clone colony. RWA clone colonies are kept in glasshouse cubicles at night/day temperatures of 16 °C/22 °C and maintained on various wheat cultivars to avoid pre-adaptation to a specific cultivar until they multiplied sufficiently to be used for screening. Each clone colony is cultured for an average period of two to three months before screening.
