RUBUS

Study Name: Late leaf rust and cane botrytis ratings of Rubus Experiment Type: Field FIELD Study Year: 1990

Curatorial selection of core accessions

Curatorial Additional Selections of Core Accessions.

Experiment Type: Field FIELD Study Year: 1986

Experiment Type: Field FIELD Study Year: 1987

Experiment Type: Field FIELD Study Year: 1988

Study Name: Standard descriptors for Rubus Experiment Type: Field FIELD

Flow cytometry performed on leaves with Vinca minor (perriwinkle) as the standard.

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).

Abstract. A lack of genetic diversity in cultivated black raspberry (Rubus occidentalis L.) germplasm has been widely recognized as a major factor limiting progress towards breeding improved cultivars. Despite this, little effort has been made since the early twentieth century to systematically collect and evaluate wild black raspberry for germplasm improvement. In recent years, there has been renewed interest in black raspberry breeding to replace existing cultivars that lack durability and disease resistance. We planted seedlings from 109 wild black raspberry populations, representing 24 US states and two Canadian provinces, in the field in replicated trial plots in Corvallis, Oregon (USA), to evaluate their performance. These populations showed wide variation in morphology, architecture, fruiting season, vigor, and apparent field tolerance to Verticillium wilt. For nearly every trait examined, wild black raspberry germplasm exhibited a range of variation beyond existing cultivars, and showed great potential for use in future breeding. While most populations were uniform phenotypically, segregation for fruit gloss and possible tolerance to Verticillium wilt was noted in a few, indicating the possibility of simple inheritance of these traits. A few populations with unusual morphology, such as spinelessness or flower abnormalities, were identified, as were populations that flowered on first year canes and produced fall fruit. Populations from the southern edge of the range appear to be particularly well adapted to western Oregon, with vigorous upright growth, strong canes, and some with a low incidence of Verticillium wilt. This germplasm will be of great value to breeders interested in developing improved black raspberry cultivars.

Plant materials and field maintenance
Collection of plant materials has been previously described (Dossett and Finn 2010; Dossett et al. 2012b). Briefly, during the summer of 2006, friends and colleagues living in eastern North America, within the native distribution of R. occidentalis, (USDA NRCS 2015) were solicited to send seed or fruit from wild plants in their area. Additional seed was obtained in 2007 through a similar request and from collecting trips across the southern and western edges of the native range (Hall et al. 2009; Hummer et al. 2008a, b). Through these efforts, seeds were obtained from more than 150 locations across the range, including 27 states and two Canadian provinces. Additional seed was obtained from all R. occidentalis seed accessions heldat the National Clonal Germplasm Repository (NCGR) in Corvallis, OR. Seeds were treated to promote germination as described by Dossett and Finn (2010). Sufficient seed germination was obtained from 109 wild populations for establishment of replicated field plantings. Due to differences in timing of seed acquisition, seedlings of 78 populations were planted in the field in September 2007, while 31 additional populations were planted in the field in May 2008 (Table 1). On each planting date, seedlings were planted in a randomized complete block design with four replications of at least three and no more than four plants, for a total of 12–16 plants from each popula-tion, depending on the total number of seedlings available. Four-plant plots of the cultivars Jewel, Mac Black, and Munger were also included in each replicate block for comparison. Extra seedlings and seedlings from seed lots that failed to produce at least 12 plants were planted as border rows of the field. Because of the difference in planting dates, popula-tions planted in the field in September 2007 and May 2008 were laid out separately with independent randomization and replication, but were directly adjacent to each other in the field.

Plants were planted 0.91 m apart in rows spaced 2.74 m apart at the USDA-ARS North Farm (Corval-lis, OR) and were trained to a three-wire trellis system with a lower wire at 0.50 m and two parallel wires hung 0.15 m apart at 0.91 m. Primocanes were trained between the parallel wires and primocane tips were pruned approximately 5–10 cm above the wires to induce branching in early June, just before fruit ripening. In the fall, primocane branches were pruned near the tips to help prevent them from rooting in the row. Floricanes from the previous fruiting season were removed during each dormant season. In late winter, new floricanes were pruned so that floricane branches were approximately 30 cm in length. Plants were fertilized, irrigated, and chemical weed controls applied per standard practices for commercially grown black raspberries in Oregon.

Collection of data and fruit samples
Evaluations of phenology and plant performance were performed in a similar manner as Dossett et al. (2008). In 2009 and 2010, dates of first bloom (first fully open flower) and fruit ripening (first fully colored fruit) were recorded for each plant. In addition, each plant was rated on a 1–9 scale for primocane vigor (1 = very poor vigor, 9 = extremely vigorous) in the spring of both years. Symptoms of Verticillium wilt (Verticillium dahliae Kleb.) were recorded in early fall 2008, after the first season of establishment, and again in the fall of 2009. Severity of symptoms was scored on a 0–6 scale (0 = no infection, 6 = all primocanes showing extensive discoloration and stunting). In the spring of 2009 and 2010, plants were scored on a 0–5 scale for the amount of cane death over the winter (0 = no floricane death or injury to cane tips, 5 = 80–100 % of floricanes dead). Samples of 25 randomly picked fruit were collected from each plant for weighing and evaluation. Fruit were scored for gloss on a 1–5 scale (1 = most pubescent, 5 = most glossy). In a few cases, 25 ripe fruit were not available from a given plant, so as many ripe fruit as could be collected were picked and weighed. Fruit were picked when they were fully colored and separated readily from the receptacle but before they were overripe. In 2009, plants were also rated on a 1–5 scale for primocane stiffness (1 = least stiff, 5 = most stiff) and lateral branching angle (1 = *75 –90 ,5= B15 ). Other morphological features were recorded in the field as they were noted. All traits were recorded in 2009 and 2010 for the seedlings planted in 2007. Due to the immaturity of plants established in 2008, observations of fruiting phenology, fruit characteristics, and winter floricane injury were recorded for the seedlings during 2010 only. A single seedling from each of ORUS 3815, ORUS 3827 and ORUS 4108 had morphological traits (e.g. canes with dense spines, poorly set reddish/purplish fruit, differences in leaflet shape) indicating they were probably the result of natural hybridization with wild red raspberry (R. strigosus Michx.). Data from these individuals were excluded from analysis. A few off-types of ‘Munger’ were noted, which ripened fruit approximately 7–10 days later than expected for this cultivar and which had a slightly different flower cluster shape (Dossett et al. 2012a). Data from these individuals were also excluded from analysis.

Statistics
Following visual inspection of residual plots and Levene’s test to confirm assumptions of normality and homoscedasticity, the GLM and CORR procedures in random mix from these seed lots SAS (version 9.1; SAS Institute, Cary, NC) were used for analysis of variance for all traits as well as correlations (Pearson) between ratings of Verticillium wilt symptoms, winter cane injury, vigor, branching angle, and cane stiffness.

Abstract
Because of its intense anthocyanin pigments, black raspberry (Rubus occidentalis L.) has a long history of use as a natural colorant and dye. Recent studies showing black raspberries to be a rich source of anthocyanins and other dietary phytochemicals have led to renewed interest in breeding better adapted cultivars that meet the demands of these markets. Anthocyanin content is a critical indicator of fruit quality for fresh and processed markets. Previous studies characterizing black raspberry anthocyanins have focused on existing cultivars comprising a narrow genetic base; however, progress in breeding new cultivars with better adaptability and disease resistance will rely on the use of new germplasm sources. Using high performance liquid chromatography/diode array detector/ion trap mass spectrometer, we examined anthocyanin content and profiles in the juice of fruit from black raspberry seedlings representing 78 wild populations from across the species’ native range over a two year period. Anthocyanin profiles were similar to those previously reported, however total anthocyanin content varied widely. Total anthocyanins in individual clones ranged from 39 to 996 mg/100 ml (expressed as cyanidin-3-glucoside) and averaged slightly higher in 2010 than in 2009. Black raspberry cultivars fell in the middle of this range, with individual wild clones ranging from less than one fourth to nearly three times the anthocyanin concentration of the industry standard, ‘Munger’. Genetic diversity for anthocyanin content is present in recently collected wild black raspberry germplasm and should be carefully evaluated when using this material for breeding improved cultivars.

Materials and Methods
During the summer of 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. Through this effort, seeds were obtained from more than 110 locations across the range, including 18 US states and two Canadian provinces. Upon arrival in the lab, seeds were extracted from the fruit, dried, and stored in a cool dry place until being treated to promote germination as described by Dossett and Finn (2010).
In September of 2007, seedlings were planted in the field in a randomized complete block design with four replications representing 78 wild populations at the USDA-ARS North Farm (Corvallis, OR, USA). Wild populations were represented by four plants per replication as were plants of ‘Jewel’, ‘Mac Black’, and ‘Munger’ for comparison. Plants were spaced 0.9 m apart and trained to a three-wire trellis system with a lower wire at 0.5 m and two parallel wires hung 0.15 m apart at 0.9 m. In early June, primocane tips were pruned at 1 m to induce branching. In late winter, floricanes from the previous fruiting season were removed and branches on new floricanes were pruned to approximately 30 cm in length.
In 2009, two seedlings from each plot were randomly selected and approximately 100 g of fruit from each were harvested for further evaluation. Initial analysis of this data (not shown) indicated that variation between plots was generally greater than variation from within a plot. This, combined with disease pressure limiting the availability of fruit in many plots, led to the decision to harvest a bulk sample of approximately 100 g of fruit from each plot for analysis in 2010.
Collection, handling, and extraction of fruit samples was otherwise performed as described by Dossett et al. (2008). Anthocyanin profiles were determined by HPLC/diode array detector/ion trap XCT mass spectrometer (HPLC/DAD/ESI-MS/MS) on an Agilent 1100 series system (Agilent Technologies, Santa Clara, CA, USA). The guard and analytical columns, mobile phase composition, and the gradient program used for HPLC analysis are described by Lee and Finn (2007), and the protocol for identifying and quantifying anthocyanins was described by Dossett et al. (2008, 2010, 2011). Total anthocyanins were determined by summing the amounts of the individual anthocyanins detected.

The type and amount of anthocyanins in raspberries, and other small fruits, has recently received increased attention. Black raspberry (Rubus occidentalis L.), in particular, has long been recognized as a rich source of anthocyanins and has been the focus of many recent studies examining their potential health benefits. In this study, we characterized a novel anthocyanin profile found in seedlings of two wild black raspberry populations collected from South Dakota, USA. Seedlings from these populations lack pigments glycosylated with rutinoside in their fruit, have elevated levels of cyanidin-3-sambubioside, and contain a small but significant amount of pelargonidin-3-glucoside, a pigment reported only once previously in black raspberry. Affected fruit also have lower than typical total anthocyanins (77.5–134.4 mg 100 mL−1). Based on the available evidence, we believe the plants have a mutation in the gene encoding anthocyanidin-3-glycoside rhamnosyltransferase (3RT), providing a unique opportunity to identify and study one of the major genes in the anthocyanin pathway and its effect on fruit anthocyanins and color.

Study Name: Determination of TSS, SS, pH and TA in small fruits at NCGR Study Year: 1988

Cuttings or tip layers

Ethnobotanical knowledge gathered by Pricilla Russell Kari from Athabascan Elders. Published by US National Park Service in 1995

chromosome count of buds or root tips at NCGR-Corvallis by Dr. Maxine Thompson