Evaluation location: Oregon, United States
ABSTRACT. The large raspberry aphid (Amphorophora agathonica Hottes) is an important vector of viruses in Rubus L. across North America. Although breeding for aphid resistance has long been recognized as an important tool for protecting red raspberries (Rubus idaeus L.) from viral infection, this is the first report of resistance to A. agathonica in black raspberry (Rubus occidentalis L.). Seedlings from 132 wild populations of black raspberries, representing the species’ native range, were screened for resistance to A. agathonica. Strong resistance was found in three of these populations, one from Ontario (PI 653326, ORUS 3778), one from Maine (PI 653347, ORUS 3817), and one from Michigan (No PI number, ORUS 4109). Resistance to the large raspberry aphid in ORUS 3778 and ORUS 3817 is dominant and appears to be conferred by different genes. We propose that the genes for resistance in ORUS 3778 and ORUS 3817 be designated Ag4 and Ag5, respectively. Resistance to A. agathonica in ORUS 4109 also appears to be controlled by a dominant allele at a single locus, but cannot be differentiated from Ag4 at this time.
Curator's note: PI 659143 = ORUS 4109-1 = was selected from this study.
PLANT MATERIALS AND APHIDS. During Summer 2006, friends and colleagues living in eastern North America, within the native distribution of R. occidentalis, were solicited to send seed or fruit from wild plants in their area. Additional seed was obtained in 2007 through a similar request and collecting trips across the southern and western edges of the native range (Hall et al., 2009). Through these efforts, seeds were obtained from more than 150 locations across the range, including 27 states and two Canadian provinces. From some locations, seed from multiple maternal clones were sent as separate samples and were kept separate from each other after arrival. From other locations, the seeds represented bulk fruit samples of many individuals (Table 1). Upon arrival, seeds were extracted from the fruit, dried, and stored in a cool dry place until scarification. Seed was also obtained from eight populations held at the National Clonal Germplasm Repository in Corvallis, OR. Seeds were scarified in concentrated sulfuric acid for 45 min in an ice bath, rinsed with sodium bicarbonate solution (Church and Dwight, Princeton, NJ), soaked for 6 d ina 3 g_L–1 calciumhypochlorite solution (Sigma-Aldrich, St. Louis) with an excess of calcium hydroxide (Mallinckrodt, Phillipsburg, NJ), and then planted onmoist peat and held at 4 _C for 8 weeks. Seedling trays were then placed on the mist bench with bottom heat at 24 _C for germination. After the emergence of their first true leaf, seedlings were planted in 72-cell trays (cell dimensions 4 · 4 · 6 cm), and then placed in the greenhouse under a 16-h daylength. When seedlings were _10 to 12 cm tall, they were repotted individually and then subjected to screening for aphid resistance in the greenhouse under similar conditions to those described above. Seedlings were watered regularly and given a liquid fertilizer (20N–8.7P–16.6K; Scotts, Marysville, OH) once per week throughout this period to promote fast growth and tender tissue attractive to aphids.
Alate and apterous A. agathonica were collected from a commercial red raspberry field and were raised in screened cages on the susceptible ‘Meeker’ red raspberry. Adult aphids from this colony were placed in petri dishes with a detached leaf and moist filter paper to isolate nymphs as they were born. Nymphs were removed from the petri dishes at 12-h intervals for 5 d and were transferred to virus-free plants of the susceptible black raspberry ‘Munger’ in a separate cage to ensure that aphids used for screening seedlings did not carry viruses from the field. This colony was maintained in a screened cage in the greenhouse under a 16-h daylength for the duration of the project. All aphids used in resistance screening came from this colony.
APHID RESISTANCE SCREENING. Between June and Aug. 2007, 5415 seedlings were screened for resistance to A. agathonica in the greenhouse. An additional 1140 wild seedlingswere screened for resistance between Feb. and Apr. 2008. We began screening 72 seedlings from each seed lot for resistance, but quickly determined that seed lots could be adequately sampled for resistance with only 36 seedlings. Thus, in 27 seed lots, 72 seedlings were screened for resistance to A. agathonica, and in 151 seed lots, up to 36 seedlings were screened. Some seed lots experienced poor germination; in cases where fewer than 36 seedlings were available, all seedlings were screened for resistance. In those populations where seed from different maternal clones at a location were sent as separate samples, 36 seedlings from each were screened for resistance for a total of up to 216 seedlings from some locations. The methods for screening plants for resistance to A. agathonica were similar to those previously used by others screening for resistance to aphids in red raspberry (Daubeny and Stary, 1982; Knight et al., 1959). Three apterous adult aphids were placed on the youngest leaves of each seedling and were allowed to remain undisturbed for 1week. After 1week, each seedling was examined for evidence of colonization. Plants with more than 10 aphids on them were scored as susceptible and were placed back on the bench. Plants with fewer than 10 aphids were marked and the total number of adult and nymph aphids was recorded before plants were placed back on the bench. Plants with fewer than three aphids had the appropriate number of adult aphids added to them to bring the number up to three. Four days after this initial inspection, this procedure was repeated. Plants assumed to be susceptible during the first inspection were reexamined to confirmsusceptibility,while the procedure for plants with fewer than 10 aphids remained the same. This was repeated at 4-d intervals three more times for a total of five challenges. In this manner, plants that continually exhibited a non-preference reaction by supporting fewer than 10 aphids were identified under
conditions of extreme aphid feeding pressure.
In Apr. 2008, a colony of Amphorophora rubitoxica Knowlton was started from aphids collected in the field, and was maintained on ‘Munger’ using the same procedures as outlined above. Singlepotted plants of ‘Munger’, ORUS 3778–1, ORUS 3817–1, and ORUS 4109–1 were simultaneously placed in the colony and were monitored over a 2-week period for colonization. This was repeated three times over the course of the summer to determine whether resistance to A. agathonica coincided with resistance to A. rubitoxica. Seedlings from the cross ORUS 4158–2 · ORUS 3021–2 were also screened for resistance to A. rubitoxica using the same procedures described above for A. agathonica. F1 POPULATIONS. Two aphid-resistant seedlings from ORUS 3778 (Simcoe, ON, Canada) and ORUS 3817 (Gardiner, ME) were selected for crossing the following year: ORUS 3778–1, ORUS 3778–2, ORUS 3817–1, and ORUS 3817–2 (a four-digit ORUS number followed by a dash and another number indicates a selection from within that cross). These plants were moved to large pots and were grown in the greenhouse until September, when they were moved outside. In Jan. 2008, these four plants were brought back into the greenhouse, along with potted plants of the susceptible cultivars Munger and Black Hawk. These plants were allowed to break dormancy and flower, at which time crosses between the resistant and susceptible genotypes were performed (Table 2). Fruit and seeds from these crosses were treated as described above and, after 2 weeks drying, were subjected
to the same germination procedures. A subset of seedlings from each cross was screened in the greenhouse for resistance to A. agathonica using the procedures described above. This procedure was repeated in Jan. 2009 with the single resistant seedling from Bath,MI (ORUS 4109–1), and an aphid susceptible
selection, ORUS 3021–1.
BC1 POPULATIONS. Five seedlings from each of four F1 populations ORUS 4153, ORUS 4155, ORUS 4157, and ORUS 4158 were moved to large pots in Sept. 2008. The plants were fertilized weekly with a liquid fertilizer (20N–8.7P–16.6K; Scott’s, Marysville, OH) and kept in a warm greenhouse under a 16-h daylength until 21 Nov. 2008 to promote growth and maturity. These seedlings were then treated to induce flower bud initiation and dormancy before being brought back into the greenhouse on 1 Apr. 2009 to begin flowering. In early May, crosses were performed between these plants and field-grown plants of ORUS 3021–2 and ‘Jewel’. Seeds and seedlings from these crosses were treated in the same manner as above and in Dec. 2009, seedlings were screened for aphid resistance as already described.
STATISTICS. Chi-square tests to determine goodness-of-fit and probability of observed segregation ratios were performed using SAS (version 9.1; SAS Institute, Cary, NC).