Molecular Breeding

, 38:83 | Cite as

Characterization of aphid resistance loci in black raspberry (Rubus occidentalis L.)

  • Jill M. Bushakra
  • Michael Dossett
  • Katherine A. Carter
  • Kelly J. Vining
  • Jana C. Lee
  • Douglas W. Bryant
  • Robert VanBuren
  • Jungmin Lee
  • Todd C. Mockler
  • Chad E. Finn
  • Nahla V. Bassil


Viruses vectored by the aphid Amphorophora agathonica cause decline in black raspberry plant health resulting in a shortened life and poor fruit quality of the infected plantings. New aphid resistant cultivars could increase the longevity of plantings providing growers and processors with consistent fruit production. Recent exploration of the native range of black raspberry identified three sources of aphid resistance: Ag4 from Ontario (ON), Canada, Ag5 from Maine (ME), and a third source from Michigan (MI) with no formal designation. The objectives of this study were to assess segregation of these three sources of aphid resistance in populations with single and combined sources and develop markers that can identify each source of resistance. A genetic linkage map constructed for ORUS 4305 placed the ON aphid resistance locus on Rubus linkage group (RLG) 6. Segregation ratios in populations with single and combined sources, and linkage mapping in two populations (ORUS 4304 and ORUS 4812) segregating for the Ag5 and MI sources, respectively, indicated that these three sources of resistance are each conferred by single dominant genes/alleles that are linked on RLG6. Confirmation of marker association in 16 validation populations identified four markers that could be used to predict resistance; however, none could distinguish between the ON and MI sources. These four markers may be useful for screening populations to enrich the field-planted progeny for aphid resistance. Fine mapping of the resistance loci is needed to develop functional markers at each of the resistance loci to enable pyramiding and durable aphid resistance.


Aphid resistance Linkage mapping Necrosis virus Virus vector Association analysis 



Thanks to Mary Peterson, Melissa Clark, Jessica Cesar, Victoria Skillman, Amanda Lake, Adam Cave, and Kathleen Knight at USDA-ARS Horticultural Crops Research Unit for care and maintenance of the aphid colonies and help with phenotyping and to summer interns Jamie Willard, Sabrina Teo, and Sarabeth Pearce-Smith for screening scaffold SSR primers.

Funding sources

The authors thank the Washington Red Raspberry Commission, the Oregon Raspberry and Blackberry Commission, the North American Raspberry and Blackberry Growers Association, the USDA-ARS, and the Northwest Center for Small Fruit Research for providing matching funds or other support. This work was funded by the USDA-National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative (SCRI) USDA-ARS CRIS 2072-21000-044-00D, 2072-21000-047-00D and 2072-21220-002-00D. Mention of trade names or commercial products in this publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the US Department of Agriculture.

Author contributions

JMB is the project coordinator who performed marker screening, designed primers, scored all markers, constructed the genetic linkage maps, phenotyped aphid resistance on population ORUS 4812 and additional aphid populations, and wrote the manuscript.

MD developed the mapping population, designed primers, performed the initial aphid screening, phenotyped aphid resistance in the mapping populations ORUS 4304 and ORUS 4305, and assisted with segregation interpretation and manuscript writing. MD is the primary drafter of the funded NIFA SCRI grant.

KAC extracted DNA, ran and prepared all markers for analysis, performed preliminary analysis, and provided technical assistance.

KJV performed the association analysis and assisted with bioinformatic analyses.

DWB developed a custom pipeline for bioinformatic analyses and performed GBS SNP calling.

DWB and RVB performed bioinformatic analyses, BLAST analyses, and linkage mapping.

JCL provided the aphid colonies and technical support.

JL is the PD (project director) on NIFA SCRI grant (project main funding) and contributed to manuscript writing.

TCM is the PI on NIFA SCRI grant (project main funding) and contributed computational resources and bioinformatics analysis.

CEF is the project director on NIFA SCRI grant (project main funding), helped assemble and phenotype the germplasm, develop the mapping population, and contributed to manuscript writing. CEF is the primary advisor for the phenotyping portion of the NIFA SCRI grant.

NVB is the project director on NIFA SCRI grant (project main funding), helped develop and test molecular markers, and contributed to manuscript writing. NVB is the primary advisor for the genomics portion of the NIFA SCRI grant.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11032_2018_839_MOESM1_ESM.docx (2 mb)
ESM 1 (DOCX 2070 kb)
11032_2018_839_MOESM2_ESM.docx (59 kb)
ESM 2 (DOCX 59 kb)


  1. Aliu O, Chung KC (2012) Assessing strength of evidence in diagnostic tests. Plast Reconstr Surg 129:989e–998e. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bassil, NV, Gilmore, Hummer KE, Dossett M, Mockler T, Filichkin S, Peterson M, Finn CE, Lee J, Fernandez G, Perkins-Veazie P, Weber C, Agunga R, Rhodes E, Scheerens JC, Lewers K, Graham J, Fernández-Fernández F, and Yun SJ (2014) Genetic and developing genomic resources in black raspberry. Acta Hortic (ISHS) 1048:19–24Google Scholar
  3. Bassil NV, Davis TM, Zhang H, Ficklin S, Mittmann M, Webster T, Mahoney L, Wood D, Alperin ES, Rosyara UR, Koehorst-vanc Putten H, Monfort A, Sargent DJ, Amaya I, Denoyes B, Bianco L, van Dijk T, Pirani A, Iezzoni A, Main D, Peace C, Yang Y, Whitaker V, Verma S, Bellon L, Brew F, Herrera R, van de Weg E (2015) Development and preliminary evaluation of a 90K Axiom® SNP array for the allo-octoploid cultivated strawberry Fragaria ×ananassa. BMC Genomics 16:30. CrossRefGoogle Scholar
  4. Birch ANE, Jones AT, Fenton B, Malloch G, Geoghegan I, Gordon SC, Hillier J, Begg G (2002) Resistance-breaking raspberry aphid biotypes: constraints to sustainable control through plant breeding. Acta Hortic (ISHS) 585:315–317CrossRefGoogle Scholar
  5. Boissot N, Thomas S, Chovelon V, Lecoq H (2016) NBS-LRR-mediated resistance triggered by aphids: viruses do not adapt; aphids adapt via different mechanisms. BMC Plant Biol 16:1–12. CrossRefGoogle Scholar
  6. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics btu170Google Scholar
  7. Bradish CM (2016) Studies of linkage mapping, trait heritability, anstern black and red raspberry. North Carolina State University. PhD Thesis at
  8. Bus VGM, Chagné D, Bassett HCM, Bowatte D, Calenge F, Celton JM, Durel CE, Malone MT, Patocchi A, Ranatunga AC, Rikkerink EHA, Tustin DS, Zhou J, Gardiner SE (2008) Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.). Tree Genet Genomes 4:223–236. CrossRefGoogle Scholar
  9. Bus V et al (2010) Genome mapping of an apple scab, a powdery mildew and a woolly apple aphid resistance gene from open-pollinated Mildew Immune Selection. Tree Genet Genomes 6:477–487CrossRefGoogle Scholar
  10. Bushakra JM, Stephens MJ, Atmadjaja AN, Lewers KS, Symonds VV, Udall JA, Chagné D, Buck EJ, Gardiner SE (2012) Construction of black (Rubus occidentalis) and red (R. idaeus) raspberry linkage maps and their comparison to the genomes of strawberry, apple, and peach. Theor Appl Genet 125:311–327. CrossRefPubMedGoogle Scholar
  11. Bushakra JM, Bryant DW, Dossett M, Vining KJ, VanBuren R, Gilmore BS, Lee J, Mockler TC, Finn CE, Bassil NV (2015) A genetic linkage map of black raspberry (Rubus occidentalis) and the mapping of Ag 4 conferring resistance to the aphid Amphorophora agathonica. Theor Appl Genet 128:1631–1646. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88:57–63. CrossRefPubMedGoogle Scholar
  13. Cevik V, King G (2002) High-resolution genetic analysis of the Sd-1 aphid resistance locus in Malus spp. Theor Appl Genet 105:346–354. CrossRefPubMedGoogle Scholar
  14. Daubeny HA (1966) Inheritance of immunity in the red raspberry to the North American strain of the aphid Amphorophora rubi Kltb. Proc Natl Acad Sci 88:346–351Google Scholar
  15. Daubeny HA, Stary D (1982) Identification of resistance to Amphorophora agathonica in the native North American red raspberry. J Amer Soc Hortic Sci 107:593–597Google Scholar
  16. Dossett M, Finn CE (2008) Variation and inheritance of vegetative characteristics and reproductive traits in black raspberry (Rubus occidentalis L.). Acta Hortic (ISHS) 777:147–152CrossRefGoogle Scholar
  17. Dossett M, Finn CE (2010) Identification of resistance to the large raspberry aphid in black raspberry. J Amer Soc Hortic Sci 135:438–444Google Scholar
  18. Dossett M, Kempler C (2012) Biotypic diversity and resistance to the raspberry aphid Amphorophora agathonica in Pacific Northwestern North America. J Amer Soc Hortic Sci 137:445–451Google Scholar
  19. Dossett M, Kempler C (2016) Breeding raspberries for aphid resistance in British Columbia: progress and challenges. In: Acta Hortic (ISHS). Internatl Soc Hortic Sci (ISHS), Leuven, Belgium, pp 115–120.
  20. Dossett M, Lee J, Finn CE (2010) Variation in anthocyanins and total phenolics of black raspberry populations. J Funct Foods 2:292–297. CrossRefGoogle Scholar
  21. Dossett M, Bassil NV, Lewers KS, Finn CE (2012) Genetic diversity in wild and cultivated black raspberry (Rubus occidentalis L.) evaluated by simple sequence repeat markers. Genet Resour Crop Evol 59:1849–1865. CrossRefGoogle Scholar
  22. Evans KM, Govan CL, Fernández-Fernández F (2008) A new gene for resistance to Dysaphis pyri in pear and identification of flanking microsatellite markers. Genome 51:1026–1031. CrossRefPubMedGoogle Scholar
  23. Fernández-Fernández F, Antanaviciute L, Knight VH, Dunwell JM, Battey NH, Sargent DJ (2013) Genetics of resistance to Amphorophora idaei in red raspberry. Acta Hortic (ISHS) 976:501–508CrossRefGoogle Scholar
  24. Gilmore BS, Bassil NV, Hummer KE (2011) DNA extraction protocols from dormant buds of twelve woody plant genera. J Amer Pomol Soc 65:201–207Google Scholar
  25. Halgren A, Tzanetakis IE, Martin RR (2007) Identification, characterization, and detection of Black raspberry necrosis virus. Phytopathology 97:44–50. CrossRefPubMedGoogle Scholar
  26. Haskell G (1960) Biometrical characters and selection in cultivated raspberry. Euphytica 9:17–34Google Scholar
  27. Jung S, Jesudurai C, Staton M, du Z, Ficklin S, Cho I, Abbott A, Tomkins J, Main D (2004) GDR (Genome Database for Rosaceae): integrated web resources for Rosaceae genomics and genetics research. BMC Bioinformatics 5:130CrossRefPubMedPubMedCentralGoogle Scholar
  28. Knight RL, Keep E, Briggs JB (1959) Genetics of resistance to Amphorophora rubi (Kalt.) in the raspberry. I. The gene A 1 from Baumforth A. J Genet 56:261–280CrossRefGoogle Scholar
  29. Knight JR, Briggs JB, Keep E (1960) Genetics of resistance to Amphorophora rubi (Kalt.) in the raspberry. II. The genes A 2 - A 7 from the American variety, Chief. Genet Res, Cambridge 1:319–331CrossRefGoogle Scholar
  30. Lambert P, Pascal T (2011) Mapping Rm2 gene conferring resistance to the green peach aphid (Myzus persicae Sulzer) in the peach cultivar “Rubira®”. Tree Genet Genomes 7:1057–1068. CrossRefGoogle Scholar
  31. Lee J (2016) Rosaceae products: anthocyanin quality and comparisons between dietary supplements and foods. NFS J 4:1–8. CrossRefGoogle Scholar
  32. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lightle D, Dossett M, Ebert T, Finn CE, Martin RR, Lee JC (2015) Effects of three novel resistant black raspberry selections on Amphorophora agathonica feeding behavior and performance. Arthropod Plant Interact 9:487–496. CrossRefGoogle Scholar
  34. Mace TA, King SA, Ameen Z, Elnaggar O, Young G, Riedl KM, Schwartz SJ, Clinton SK, Knobloch TJ, Weghorst CM, Lesinski GB (2014) Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling. Cancer Immunol Immunother 63:889–900. CrossRefPubMedPubMedCentralGoogle Scholar
  35. MacFarlane SA, Tzanetakis IE, Halgren AB, Martin RR (2017) Raspberry mosaic virus. In: Martin RR, Ellis MA, Williamson B, Williams RN (eds) Compendium of raspberry and blackberry diseases and pests, 2nd edn. APS Press, St. Paul, pp 75–78Google Scholar
  36. Martin RR (2002) Virus diseases of Rubus and strategies for their control. Acta Hortic (ISHS) 585:265–270CrossRefGoogle Scholar
  37. Martin RR, MacFarlane S, Sabanadzovic S, Quito D, Poudel B, Tzanetakis IE (2013) Viruses and virus diseases of Rubus. Plant Dis 97:168–182. CrossRefGoogle Scholar
  38. McMenemy LS, Mitchell C, Johnson SN (2009) Biology of the European large raspberry aphid (Amphorophora idaei): its role in virus transmission and resistance breakdown in red raspberry. Agric For Entomol 11:61–71. CrossRefGoogle Scholar
  39. Montrose DC, Horelik NA, Madigan JP, Stoner GD, Wang LS, Bruno RS, Park HJ, Giardina C, Rosenberg DW (2011) Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis. Carcinogenesis 32:343–350. CrossRefPubMedGoogle Scholar
  40. Ourecky DK (1975) Brambles. In: Janick J, Moore JN (eds) Advances in fruit breeding. Purdue University Press, West Lafayette, pp 98–129Google Scholar
  41. Pagliarani G, Dapena E, Miñarro M, Denancé C, Lespinasse Y, Rat-Morris E, Troggio M, Durel CE, Tartarini S (2016) Fine mapping of the rosy apple aphid resistance locus Dp-fl on linkage group 8 of the apple cultivar ‘Florina’. Tree Genet Genomes 12:1–12. CrossRefGoogle Scholar
  42. Preacher KJ (2001) Calculation for the chi-square test: an interactive calculation tool for chi-square tests of goodness of fit and independence.
  43. Rodrigo KA, Rawal Y, Renner RJ, Schwartz SJ, Tian Q, Larsen PE, Mallery SR (2006) Suppression of the tumorigenic phenotype in human oral squamous cell carcinoma cells by an ethanol extract derived from freeze-dried black raspberries. Nutr Cancer 54:58–68. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Rossi M, Goggin FL, Milligan SB, Kaloshian I, Ullman DE, Williamson VM (1998) The nematode resistance gene Mi of tomato confers resistance against the potato aphid. Proc Natl Acad Sci 95:9750–9754. CrossRefPubMedGoogle Scholar
  45. Salinas NR, Zurn JD, Mathey M, Mookerjee S, Denoyes B, Perrotte J, Potier A, Finn CE, Hancock JF, Stewart P, Bassil NV (2017) Validation of molecular markers associated with perpetual flowering in octoploid Fragaria germplasm. Mol Breed 37:70. CrossRefGoogle Scholar
  46. Sargent D, Fernández-Fernández F, Rys A, Knight V, Simpson D, Tobutt K (2007) Mapping of A 1 conferring resistance to the aphid Amphorophora idaei and dw (dwarfing habit) in red raspberry (Rubus idaeus L.) using AFLP and microsatellite markers. BMC Plant Biol 7:15CrossRefPubMedPubMedCentralGoogle Scholar
  47. Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233–234CrossRefPubMedGoogle Scholar
  48. Takahashi Y, Nagata T (1992) parB: an auxin-regulated gene encoding glutathione S-transferase. Proc Natl Acad Sci 89:56–59CrossRefPubMedGoogle Scholar
  49. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40:e115–e115. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Van Ooijen JW (2006) JoinMap® 4, Software for the calculation of genetic linkage maps in experimental populations. Kyazma B.V, WageningenGoogle Scholar
  51. VanBuren R, Bryant D, Bushakra JM, Vining KJ, Edger PP, Rowley ER, Priest HD, Michael TP, Lyons E, Filichkin SA, Dossett M, Finn CE, Bassil NV, Mockler TC (2016) The genome of black raspberry (Rubus occidentalis). Plant J 87:535–547. CrossRefPubMedGoogle Scholar
  52. Weber CA (2003) Genetic diversity in black raspberry detected by RAPD markers. HortSci 38:269–272Google Scholar
  53. Winter JD (1929) A preliminary account of the raspberry aphids. U Minnesota Agricult Exp Station Tech Bull 61:1–30Google Scholar
  54. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCgreen. Clin Chem 49:853–860. CrossRefPubMedGoogle Scholar
  55. Young ND, Tanksley SD (1989) Restriction fragment length polymorphism maps and the concept of graphical genotypes. Theor Appl Genet 77:95–101. CrossRefPubMedGoogle Scholar
  56. Züst T, Agrawal AA (2016) Mechanisms and evolution of plant resistance to aphids. Nat Plants 2:15206. CrossRefPubMedGoogle Scholar

Copyright information

© This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2018

Authors and Affiliations

  • Jill M. Bushakra
    • 1
  • Michael Dossett
    • 2
  • Katherine A. Carter
    • 1
  • Kelly J. Vining
    • 3
  • Jana C. Lee
    • 4
  • Douglas W. Bryant
    • 5
  • Robert VanBuren
    • 5
    • 6
  • Jungmin Lee
    • 7
  • Todd C. Mockler
    • 5
  • Chad E. Finn
    • 4
  • Nahla V. Bassil
    • 1
  1. 1.USDA-ARS National Clonal Germplasm RepositoryCorvallisUSA
  2. 2.BC Berry Cultivar Development Inc. (in partnership with Agriculture and Agri-Food Canada)Pacific Agri-Food Research CentreAgassizCanada
  3. 3.Department of Horticulture, Agriculture and Life ScienceOregon State UniversityCorvallisUSA
  4. 4.USDA-ARS Horticultural Crops Research UnitCorvallisUSA
  5. 5.The Donald Danforth Plant Science CenterSt. LouisUSA
  6. 6.Department of HorticultureMichigan State UniversityEast LansingUSA
  7. 7.USDA-ARS Horticultural Crops Research Unit worksiteParmaUSA

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