A genetic linkage map of black raspberry (Rubus occidentalis) and the
mapping of Ag (4) conferring resistance to the aphid Amphorophora
agathonica
Bushakra, J. M., Bryant, D. W., Dossett, M., Vining, K. J., VanBuren, R., Gilmore,
B. S., ... & Bassil, N. V. (2015). A genetic linkage map of black raspberry (Rubus
occidentalis) and the mapping of Ag 4 conferring resistance to the aphid
Amphorophora agathonica. Theoretical and Applied Genetics, 128(8), 1631-1646.
doi:10.1007/s00122-015-2541-x
10.1007/s00122-015-2541-x
Springer
Version of Record
http://cdss.library.oregonstate.edu/sa-termsofuse
Theor Appl Genet (2015) 128:1631–1646
DOI 10.1007/s00122-015-2541-x
ORIGINAL PAPER
A genetic linkage map of black raspberry (Rubus occidentalis)
and the mapping of Ag4 conferring resistance to the aphid
Amphorophora agathonica
Jill M. Bushakra1 · Douglas W. Bryant2 · Michael Dossett3 · Kelly J. Vining4 ·
Robert VanBuren2 · Barbara S. Gilmore1,5 · Jungmin Lee6 · Todd C. Mockler2,4 ·
Chad E. Finn7 · Nahla V. Bassil1 Received: 13 January 2015 / Accepted: 18 May 2015 / Published online: 3 June 2015
© The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract Key message We have constructed a densely popu‑
lated, saturated genetic linkage map of black raspberry
and successfully placed a locus for aphid resistance.
Abstract Black raspberry (Rubus occidentalis L.) is a
high-value crop in the Pacific Northwest of North America
with an international marketplace. Few genetic resources
are readily available and little improvement has been
achieved through breeding efforts to address production
challenges involved in growing this crop. Contributing to
Communicated by C. A. Hackett.
Electronic supplementary material The online version of this
article (doi:10.1007/s00122-015-2541-x) contains supplementary
material, which is available to authorized users.
* Jill M. Bushakra
jill.bushakra@ars.usda.gov
1
USDA-ARS National Clonal Germplasm Repository, 33447
Peoria Rd., Corvallis, OR 97333, USA
2
The Donald Danforth Plant Science Center, 975 N. Warson
Rd., St. Louis, MO 63132, USA
3
B.C. Blueberry Council (in Partnership with Agriculture
and Agri-Food Canada) - Pacific Agri-Food Research Centre,
6947 Hwy #7, P.O. Box 1000, Agassiz, BC V0M 1A0,
Canada
4
Center for Genome Research and Biocomputing, Oregon
State University, 3135 Agriculture and Life Science,
Corvallis, OR 97331, USA
5
USDA-ARS National Forage Seed and Cereal Research Unit,
3450 SW Campus Way, Corvallis, OR 97331, USA
6
USDA-ARS Horticultural Crops Research Unit Worksite,
29603 U of I Ln., Parma, ID 83660, USA
7
USDA-ARS Horticultural Crops Research Unit, 3420 NW
Orchard Ave., Corvallis, OR 97330, USA
its lack of improvement is low genetic diversity in elite cultivars and an untapped reservoir of genetic diversity from
wild germplasm. In the Pacific Northwest, where most production is centered, the current standard commercial cultivar is highly susceptible to the aphid Amphorophora agathonica Hottes, which is a vector for the Raspberry mosaic
virus complex. Infection with the virus complex leads to a
rapid decline in plant health resulting in field replacement
after only 3–4 growing seasons. Sources of aphid resistance have been identified in wild germplasm and are used
to develop mapping populations to study the inheritance of
these valuable traits. We have constructed a genetic linkage map using single-nucleotide polymorphism and transferable (primarily simple sequence repeat) markers for F1
population ORUS 4305 consisting of 115 progeny that segregate for aphid resistance. Our linkage map of seven linkage groups representing the seven haploid chromosomes
of black raspberry consists of 274 markers on the maternal map and 292 markers on the paternal map including a
morphological locus for aphid resistance. This is the first
linkage map of black raspberry and will aid in developing
markers for marker-assisted breeding, comparative mapping with other Rubus species, and enhancing the black
raspberry genome assembly.
Introduction
Genetic linkage map construction of rosaceous crops has
been used to understand genetics and as a precursor to
enabling molecular breeding for about 20 years. The earliest maps made during the 1990s were constructed mainly
by using isozymes, random amplification of polymorphic
DNA (RAPD), restriction fragment length polymorphism
(RFLP), and morphological markers (Chaparro et al. 1994;
13
1632
Foolad et al. 1995; Hemmat et al. 1994; Rajapakse et al.
1995; Stockinger et al. 1996; Viruel et al. 1995). Advancements in DNA technology in the 2000s led to the rapid
development of simple sequence repeat (SSR) markers
for de novo map construction (Castro et al. 2013; Celton
et al. 2009; Dirlewanger et al. 2004; Fernández-Fernández et al. 2008; Gisbert et al. 2009; Graham et al. 2004;
Hibrand-Saint Oyant et al. 2008; Olmstead et al. 2008) as
well as their incorporation into existing maps (Aranzana
et al. 2003; Dirlewanger et al. 2006; Etienne et al. 2002;
Liebhard et al. 2003; Paterson et al. 2013; Pierantoni et al.
2004; Silfverberg-Dilworth et al. 2006; Stafne et al. 2005;
Vilanova et al. 2008; Woodhead et al. 2008, 2010). Additional technological advances in high-throughput detection
of single-nucleotide polymorphic (SNP) loci using arrays,
or genotyping by sequencing (GBS), and the associated
improvement of data analysis have made SNP markers
increasingly useful for genetic map construction. Recently,
linkage maps for several members of the Rosaceae have
been constructed using SNP array technology (Antanaviciute et al. 2012; Clark et al. 2014; Frett et al. 2014;
Klagges et al. 2013; Montanari et al. 2013; Pirona et al.
2013; Yang et al. 2013).
The genus Rubus L. (Rosaceae, Rosoideae) has an
estimated 750 species distributed world-wide (Alice and
Campbell 1999; Thompson 1995). Of these, three are
of particular commercial importance, red raspberry (R.
idaeus L., subgenus Idaeobatus Focke), blackberry (Rubus
sp., subgenus Rubus L.), and black raspberry (subgenus
Idaeobatus). Genetic linkage maps have been constructed
for tetraploid blackberry (Castro et al. 2013), diploid red
raspberry (Sargent et al. 2007; Ward et al. 2013; Woodhead et al. 2010), and an interspecific cross between diploid red raspberry and diploid black raspberry (Bushakra
et al. 2012). While blackberry and red raspberry are highly
heterozygous, black raspberry, particularly R. occidentalis, is highly homozygous (Dossett et al. 2012b). Genetic
improvement of blackberry and red raspberry through
breeding has been a continual process for decades. For
example, from 1994 to 2014, the American Pomological Society’s Fruit and Nut Variety Registry Lists 38–47
(Clark and Finn 1999, 2002, 2006; Clark et al. 2008, 2012;
Daubeny 1997a, b, 1999, 2000, 2002, 2004, 2006, 2008;
Finn and Clark 2000, 2004, 2014; Finn et al. 2010; Moore
and Kempler 2010, 2012, 2014) records the release of 75
blackberry and hybrid berry and 189 red raspberry cultivars and only three black raspberry cultivars (‘Pequot’,
‘Niwot’, and ‘Explorer’). In addition, ‘Earlysweet,’ a selection derived from a purported cross between R. occidentalis
and the western black raspberry, R. leucodermis Dougl. ex
Torr. & Gray, was released in 1998 (Galletta et al. 1998).
Black raspberry figures prominently in the pedigrees of
many of the red raspberry cultivars released between
13
Theor Appl Genet (2015) 128:1631–1646
1994 and 2014. Difficulties in improving black raspberry
through breeding were first reported by Slate (1933) while
attempting to improve purple raspberries. Crossing with
other species was proposed as a way to increase genetic
diversity in cultivated black raspberry (Drain 1956; Hellman et al. 1982; Slate and Klein 1952), but has met with
limited success. Low genetic diversity was proposed by
Ourecky (1975) as the main reason for lack of development
of improved cultivars.
More recent interest in improving black raspberry has
been driven by research and commercial interest into its
bioactive compounds and their influence on human health,
specifically modulation of cancer cell proliferation, inflammation, cellular death, oxidation, etc. (Stoner et al. 2007).
Since the 1940s, Oregon has been the primary commercial
production region of black raspberry in North America.
In 2014, 1650 acres were harvested that earned growers a
utilized production value of over US$16.8 million (Anonymous 2015). One hindrance to expanding production is
susceptibility of the predominant cultivar Munger to the
Raspberry mosaic virus complex vectored by the North
American large raspberry aphid, Amphorophora agathonica Hottes (Dossett and Finn 2010). Infection causes
rapid decline of plantings, often with field replacement
necessary after only three or four growing seasons (Halgren
et al. 2007). In contrast, under perennial production in open
fields for processed fruit, plantings of current cultivars of
red raspberry are typically kept in the field for 7–8 growing
seasons, and plantings of blackberry cultivars can last many
decades (C.E. Finn, personal communication). Selection
for cultivars with resistance to A. agathonica could significantly increase the longevity of the plants, reduce insecticide use, and therefore improve profitability for the grower
and quality of the environment.
A low level of genetic diversity in cultivated black raspberry has been found using molecular tools. Weber (2003),
using RAPD markers in 16 black raspberry cultivars, determined a level of similarity of 81 %. Two wild accessions
and five elite genotypes accounted for more than 50 % of
the similarity, while the remaining 11 cultivars shared 92 %
similarity compared to 70 % similarity among red raspberry cultivars found by Graham et al. (1994). In 2005,
Lewers and Weber used SSR markers from red raspberry
and strawberry to evaluate an F2 population of a red raspberry × black raspberry cross and found that the homozygosity of the black raspberry clone used was 80 % and
only 40 % in the red raspberry clone used. However, wild
populations of black raspberry show greater genetic diversity. For example, Nybom and Schaal (1990) sampled black
raspberry plants along a roadside in Missouri that were
then analyzed by RFLP. They found 17 informative fragments that identified 15 genotypes in the 22 samples collected. Dossett et al. (2012b) used SSR markers to examine
Theor Appl Genet (2015) 128:1631–1646
the genetic diversity among cultivars and wild germplasm.
They found that the diversity at 21 loci was much higher
among wild germplasm than in the elite cultivars, and that
six elite cultivars were identical at these 21 loci.
Genetic diversity in wild black raspberry germplasm as
detected by molecular tools (Dossett et al. 2012b; Nybom
and Schaal 1990) and through breeding experiments (Dossett et al. 2008) is currently untapped. To address this,
Dossett and Finn (2010) canvassed the native range of R.
occidentalis collecting seed, which was subsequently germinated and evaluated for multiple traits including aphid
resistance. From this study, three of 132 wild populations
were determined to segregate for resistance to A. agathonica. Two populations, ORUS 3817 collected from Maine,
and ORUS 3778 collected from Ontario, Canada, were
subsequently used to develop populations for genetic mapping and phenotypic analysis. F1 progeny of susceptible
cultivars Munger and Jewel crossed with individuals from
ORUS 3778 and ORUS 3817 were all resistant to aphids
under greenhouse conditions suggesting that the alleles for
resistance are dominant and that ORUS 3778 (Ag4) and
ORUS 3817 (Ag5) are homozygous for their respective
alleles. Dossett and Finn (2010) originally identified one
of the susceptible cultivars used in the crosses as ‘Black
Hawk’, however, subsequent fingerprinting work found it
to be ‘Jewel’ (Dossett et al. 2012a).
In this paper, we report the analysis of population ORUS
4305, an F1 black raspberry population, raised as one of
several populations to investigate genetic sources of resistance to the aphid A. agathonica with the intent of mapping
the aphid resistance allele Ag4. To quickly and efficiently
generate markers for mapping we have employed GBS following the protocol established by Elshire et al. (2011) with
modifications for Rubus (Ward et al. 2013), and anchored
the map with SSR markers from a variety of sources. We
have placed the phenotypic character of aphid resistance
on this linkage map which covers the seven Rubus linkage
groups (RLG) as defined by Bushakra et al. (2012).
Methods
Plant material
A full-sib (F1) family of 115 individuals was developed
from the controlled cross of ORUS 3021-2 (female, susceptible to aphids, postulated genotype ag4ag4) × ORUS
4153-1 (male, resistant to aphids, postulated genotype
Ag4ag4; Fig. 1). The source of this resistance is from ORUS
3778-1, an accession from wild seed collected in Ontario,
Canada (Dossett and Finn 2010). Progeny from this cross
were screened for aphid resistance as small seedlings in the
greenhouse as described by Dossett and Finn (2010) and
1633
‘Jewel’
ORUS 3021-2
NC84-10-3
ORUS 4305
‘Jewel’
ORUS 4153-1
Ag4 aphid resistance
ORUS 3778-1
Wild, Ontario, Can.,
Ag4 source
Fig. 1 Pedigree of mapping population ORUS 4305. Population
ORUS 4305 is derived from a wild-collected accession from Ontario,
Canada (ORUS 3778-1), that exhibited resistance to the North American large raspberry aphid that was crossed with aphid-susceptible
‘Jewel.’ One of the progeny from that cross, ORUS 4153-1 with proposed genotype Ag4ag4 representing the proposed gene conferring
resistance, was used as the male parent and crossed with aphid-susceptible ORUS 3021-2
followed the expected 1:1 segregation ratio (56 resistant, 59
susceptible, χ2 = 0.08, P = 0.78).
DNA extraction
Leaf samples were collected, bagged, kept cool, and transported to the laboratory. Leaf tissue aliquots of 30–50 mg
were placed in a cluster tube (Corning Life Sciences,
Tewksbury, MA, USA) containing a 4-mm stainless steel
bead (McGuire Bearing Company, Salem, OR, USA).
The samples were frozen in liquid nitrogen and stored at
−80 °C until extraction. Frozen tissue was homogenized
using the Retsch® MM301 Mixer Mill, (Retsch Inc., Hann,
Germany) at a frequency of 30 cycles/s using three 30 s
bursts. The E-Z 96® Plant DNA extraction kit (Omega BioTek, Norcross, GA, USA) was used as previously described
(Gilmore et al. 2011).
DNA quantification and quality
Genomic DNA was quantified using Quant-iT™ Picogreen® dsDNA Assay kit (Invitrogen, Eugene, OR, USA)
following manufacturer’s instructions modified to 100 μl
and compared against a λ standard DNA dilution series
with a Victor3V 1420 Multilabel Counter (PerkinElmer,
Downers Grove, IL, USA), followed by visualization on
1 % agarose gel in 1× TBE (Tris/Borate/EDTA) stained
13
1634
with ethidium bromide. Samples were stored at −20 °C
prior to use.
Marker sources
SSR primer pairs were selected from multiple sources
(Table 1). Markers derived from GBS were coded as S with
a number indicating the scaffold followed by an underscore
and a number indicating the physical SNP position on the
scaffold (i.e., S75_381030) (Bryant et al. 2014). Markers
developed from the sequencing of paired-end short reads
were coded with Ro (R. occidentalis) or Ri (R. idaeus) followed immediately by a number (i.e., Ro11481, Ri13528)
(Dossett et al. in press). All other markers are from published sources as indicated in Table 1. Ag4_AphidR is a
phenotypic marker for aphid resistance.
An additional 26 SSR and two high-resolution melting
(HRM) markers that mapped in multiple populations were
identified from the literature (Bushakra et al. 2012; Castillo
et al. 2010; Castro et al. 2013; Fernández-Fernández et al.
2013; Graham et al. 2004; Sargent et al. 2007) with the
intention of anchoring and orienting the linkage groups to
published maps (Table 2).
DNA amplification of SSR markers
DNA amplification was performed on a PTC-225 gradient thermal cycler (Bio-Rad, Hercules, CA, USA), a Dyad
Peltier thermal cycler (Bio-Rad, Hercules, CA, USA), an
Eppendorf Mastercycler (Eppendorf, Hamburg, Germany),
or a Nexus (Eppendorf, Hamburg, Germany). A fluorescent labeling polymerase chain reaction (PCR) protocol
(Schuelke 2000) was used for most SSR primer pairs. The
forward (F) primer of each pair was extended on the 5′-end
with an M13-TGTAAAACGACGGCCAGTAGC sequence
tag to which a universal M13-tagged fluorescent dye label
(WellRed D2, D3, D4; Integrated DNA Technologies, Inc.,
Coralville, IA, USA) annealed. The touch-down PCR protocol began with an initial denaturation for 3 min at 94 °C
followed by 10 cycles of 94 °C for 40 s, 65 °C (decreasing 1 °C every cycle) for 45 s, 72 °C for 45 s; 20 cycles
of 94 °C for 40 s, 52 °C for 45 s, 72 °C for 45 s; 10 cycles
of 94 °C for 40 s, 53 °C for 45 s, 72 °C for 45 s; followed
by a final extension of 72 °C for 30 min. Reactions were
performed in a final volume of 15 μl consisting of 6 ng
template DNA, 1× PCR buffer, 2 mM MgCl2, 200 μM
dNTP, 0.5 μM reverse primer, 0.12 μM M13-tagged forward primer, 0.5 μM WellRed labeled M13 primer (D2, D3
or D4), and 0.025 U GoTaq® Hot Start Polymerase (Promega Corporation, Madison, WI, USA). For a few SSR
primer pairs, the 5′-end of the F primer was fluorescently
labeled (WellRed D2, D3, or D4). The PCR protocol used
for labeled F-primers began with an initial denaturation for
13
Theor Appl Genet (2015) 128:1631–1646
3 min at 94 °C followed by 35 cycles of 94 °C for 40 s,
appropriate annealing temperature for 40 s, 72 °C for 30 s;
followed by a final extension of 72 °C for 30 min. The
reverse primer for Rub1C6 was PIG-tailed with 5′-GTTT-3′
(Brownstein et al. 1996) to minimize the occurrence of split
peaks and difficulties encountered in automated fragment
analysis following capillary electrophoresis.
Capillary electrophoresis of SSR markers
Success of the PCR was confirmed by 2 % agarose gel electrophoresis. Up to six fragments were pooled based on dye
and predicted fragment size and separated on a Beckman
CEQ 8000 capillary genetic analyzer (Beckman Coulter,
Fullerton, CA, USA). Separation was followed by analysis
of allele size and marker visualization using the fragment
analysis module of the CEQ 8000 software.
High‑resolution melting
The HRM technique (Wittwer et al. 2003) was used to
amplify markers from Bushakra et al. (2012). Reactions
were performed on PTC-225 gradient thermal cycler
(Bio-Rad, Hercules, CA, USA), followed by HRM on the
LightScanner® System (BioFire Defense, Salt Lake City,
UT, USA). Reactions were performed in a final volume
of 10 μl consisting of 6 ng DNA, 1× LightScanner Master Mix, 1 μM each forward and reverse primer. Each well
was topped with one drop of mineral oil. The PCR amplification protocol was 94 °C for 30 s, followed by 30 s at
the appropriate annealing temperature (57 or 58 °C) and
extension at 72 °C for 30 s for 40 cycles. Following a final
melting step at 95 °C for 30 s, the samples were cooled to
4 °C until HRM analysis. Amplicon melting occurred on
the LightScanner where samples were heated to 98 °C over
a period of 8 min with default settings. Analysis was performed using the LightScanner® Instrument & Analysis
Software small amplicon genotyping module.
GBS library construction and sequencing
GBS libraries were constructed following Ward et al.
(2013) and Elshire et al. (2011). Briefly, 100 ng of genomic
DNA per sample were digested with 4 U of ApeKI (New
England Biolabs, Ipswich, MA, USA) and then ligated with
T4 ligase to 1.8 ng of combined common and unique barcode adapters (Elshire et al. 2011). Annealed and quantitated unique barcode and common adapters were provided
by the Buckler Lab for Maize Genetics and Diversity, Cornell University (Ithaca, NY, USA) and Clemson University
(Clemson, SC, USA) (Supplementary Table 1).
The GBS libraries were submitted to the Oregon State
University Center for Genome Research and Biocomputing
1
5
2
2
2
2
4
6
6
7
6
4
3
Ri18886_SSR
Ri20047_SSR
Ri20466_SSR
Ri3758_SSR
Ri5037_SSR
Ro10488_SSR
Ro1079_SSR
Ro11481_SSR
Ro12112_SSR
Ro14925_SSR
Ro15590_SSR
Ro16697_SSR
Ro1682_SSR
7
Ri10139_SSR
7
5
Ro_CBEa0011M11_SSR
Ri16959_SSR
4
Ro_CBEa0010N20_SSR
2
6
Rub1C6_SSR
Ri14075_SSR
6
Rh_MEa0006bG05_SSR
7
4
RiM017_SSR
Ri13528_SSR
3
RhM003_SSR_RLG3
3
4
Ro_CBEa0009J05_SSR
Ri12319_SSR
4
Ro_CBEa0004G23_SSR
2
1
Ro_CBEa0003K17_SSR
3
1
Ro_CBEa0002P01b_SSR
Ri11086_SSR
2
Ro_CBEa0001L24_SSR
Ri11795_SSR
NA
NA
5
Ru_EE284382_HRM_RLG5
119, 125
152, 160
179, 211
117
157, 167
156
223, 225
119
336, 339
392, 406
369
376, 393
308, 317
283, 286
351, 355
460, 463
304
299, 313
268, 300
295
243, 245
114, 118
244, 260
294, 303
121
160
201, 205
114, 117
140
156, 158
223
121, 131
336, 339
400, 406
369, 374
376, 405
314, 317
286
357, 359
463
292, 304
299, 313
268, 302
295, 314
241, 243
118
262, 268
294
212
236, 238
227, 236
212, 214
292, 294
507, 509
307, 309
228
292
509
307
226, 228
277, 279
NA
NA
7
Ri_1B16_HRM_RLG7
275, 277
NA
4153-1 allele
sizes
NA
RLG 3021-2 allele
sizes
ERubLR_SQ07-3_C07_HRM_ 7
RLG7
Locus
M13-AAAATGGAGACTAGATCCAGCG
M13-AGGGTGCGTGTCAGAAGTAAGT
M13-CACGAGTAACACTCCCAAATGA
M13-GTTTTGCCTACGGACTTGAATC
M13-GGTTTTCTGGGAAAAACAGAAG
M13-CCCTGTTTGATCTATTCAATCCC
M13-CCCAAAGGACAGAAGTATGGAC
M13-AAAATGTGATTGAGCCGACG
M13-ACAAATTCCAGTCAGTCCATGC
M13-CTCTGCTACAACCCAACGAGTC
M13-GAGTCTGATATCAAGCAGCCCT
M13-ATCCAACCCTTCATTCTCTGTT
M13-AAAATTCTGATTGGGCCGAC
M13-GTCTCGGCCGAATAATAAACAA
M13-GGGCATGAACCACATAAAGG
M13-GGGGGCTTTACATCATCATT
(AT)6
(AT)8–(TA)6
(GA)6
(TGT)7
(TC)8
M13-AGGAGCGATGTTATAGGCATGT
M13-CCAGTGAGTGAGCCTTGAGATA
M13-GGAGCAAGAAGCCTTGAAGATA
M13-AGCTGGTCGAAGAAGGTTTATG
M13-TACTCCCAAAAACCCAGAATTG
TAGAGGGAGAAAAAGGGAGTGC
ATTTGGAAGGAATACGGAACCT
GTTGCCTCTGGATTGCTTTTAT
AACTTTCTCCCGTTCTCCTAGC
GTCTGAGCAGAAATGGGAAATC
CTGTTTCCAGCAAACCTAACCT
GGCAGAGATTTGAGGTTTCTGA
GCTGATAGTGGGGTTTGGATAA
TCTTGGAATTGGGGTTATTCTG
TCTATCTCTCCGTTGTGGATTT
CGCGTTTTCACTGTTCACTTTA
GAGGAGCAGCTTGTCGAGAT
CGGGTCTTACAGGCAAGTGATA
GGGAAAACTGAAGAACACGAAG
CCAGACGCATTAAATCTGTCAC
GGCAATTTGGAGATTTCTTGCT
AAAGGTAGAAGTGGAGGACTCA
GCGAAGACGAGGAAGATGAAT
ACAACACGAAGAACACGAGAGA
CACGAAGAACAACGAGAGAAAA
TCCATTTCCAAAACACATTGA
TTCGTAGTCTTGCCCTTGCT
Dossett et al., in press
Dossett et al. (2012b)
GTTTAGGTAAGCAATGGGAAAGCTC Dossett and Finn
(2010)
Castro et al. (2013)
Castillo et al. (2010)
GTTCAGGCCAGTCAATGTCA
CATTGTGCTTATGATGGTTTCG
D4-TCTGCCTCTGCATTTTACACAG
AGCAGAATCGGTTCTTACAAGC
M13-GAAACAGGTGGAAAGAAACCTG
Unpublished
Bushakra et al. (2012)
Source
M13-GAAGCAGCAGCAAGACCTTT
TTTGCTCGTCGTACTCATCG
M13-CCATCTCCAATTCAGTTCTTCC
GTTCCCTTTGCTTTGAGACG
CTTACGCTTCTTGGCTCCAC
GCGCTTGAGCATCAAATGTA
GACGTCTCCATTGGGAAGAA
AGGTGAGGTGGGAGATGATG
AAGAAGAAGGGTGGGTTTCA
CCGTGGTGGTTGTTATGTTG
Reverse primer sequence
M13-CCAAGTCCAACCACTCACAC
M13-GACGCGGTGAGATTTTGATT
M13-GGAAAGAAAACCCTAGCCGA
(AG)6–(TA)6–(AT)5 M13-AAGATAAGAGGAGAAGTGCGGA
(TA)6
(TC)9–(TC)14
(GAA)6
(AT)7
(GA)10
(CT)9
(ATG)9
(TC)6
(TC)8
(TC)10
(TCA)7
(GAA)8
(TC)9
(TC)8
(AG)14
(GA)9
(CT)15
(AAG)8
(TG)6
(TG)10
(CA)4–(GA)3
(AG)8
(TC)6
M13-CCCTCCCTCTCTCCAGTTTC
M13-TAAAGAAAGGGGTTGTTGCG
(CCA)4–(TA)7
(TC)4–(CT)7
ACGGAGGATGACAGAGAACC
CTTGGGCAGCTTTAGCCTTT
GGATCAAGGAGTGAGGATGG
Forward primer sequence
NA
NA
NA
Repeat motif
Table 1 Transferable locus primer sequences used to construct the genetic linkage maps for black raspberry F1 population ORUS 4305
Theor Appl Genet (2015) 128:1631–1646
1635
13
3
2
1
6
4
4
5
4
5
5
3
1
6
7
4
5
6
6
4
1
3
6
2
5
1
1
4
4
2
6
6
4
3
5
Ro17045_SSR
Ro17803_SSR
13
Ro18036_SSR
Ro19042_SSR
Ro20267_SSR
Ro2173_SSR
Ro2432_SSR
Ro2579_SSR
Ro2827_SSR
Ro3003_SSR
Ro3017_SSR
Ro3237_SSR
Ro3981_SSR
Ro4104_SSR
Ro4261_SSR
Ro4345_SSR
Ro4532_SSR
Ro5263_SSR
Ro5378_SSR
Ro6594_SSR
Ro7270_SSR
Ro8167_SSR
Ro8486_SSR
Ro9206_SSR
Ro9324_SSR
Ro942_SSR
Rubus110a_SSR_RLG4
Rubus116a_SSR_RLG4
Rubus126b_SSR_RLG2
Rubus16a_SSR_RLG6
Rubus223a_SSR
Rubus26a_SSR
Rubus270a_SSR_RLG3
Rubus275a_SSR_RLG5
218, 224
157, 171
164, 166
222, 224
171
170, 172
137, 143
184, 186
165
139, 141
182, 184
139, 165
158
203, 205
187, 207
158, 162
161, 181
164, 201
126, 137
172
96
177
171
191, 207
201
210, 213
114
204, 219
181, 183
115
133, 135
161
145, 152
137, 141
179, 181
114
203, 221
153, 159
196, 201
119
139, 141
164, 167
4153-1 allele
sizes
155, 161
156, 164
135, 139
172, 178
94, 96
168, 171
171, 177
207
201, 203
210, 213
108, 114
204, 219
181, 185
115, 117
131, 135
159, 173
145, 199
133, 141
181
114, 116
199, 240
159
201
104, 119
139, 147
164
RLG 3021-2 allele
sizes
Locus
Table 1 continued
M13-CCTGCATTTTTCTGTATTTTGG
M13-TGTTGTACGTGTTGGGCTTT
(CT)31–(CA)22
(AT)8(GT)11
(AG)27
(GA)10
(CT)11–(CA)29
GATCCTGGAAAGCGATGAAA
CCCACCTCCATTACCAACTC
CATTTCATCCAAATGCAACC
D3-GCATCAGCCATTGAATTTCC
M13-CACAACCAGTCCCGAGAAAT
TTAAGGCGTCGTGGATAAGG
GGGTGTTTGCCAGTTTCAGT
TCAGTTTTCTTCCCACGGTTA
M13-AACACCGGCTTCTAAGGTCT
(AT)4–(TA)8–(AT)10 M13-TCTCTTGCATGTTGAGATTCTATT
M13-CCAACCCAAAAACCTTCAAC
GTTGTGGCATGGCCTTTTAT
M13-AAACAAAGGATAAAGTGGGAAGG TGTCAGTTGGAGGGAGAACA
CAAATTCGACACCACCTATCAG
GCAATCACACATTAAAAGGTCC
CAAGATTGTCACGTACTCGGAA
AAGGAATGAAATAGGGACGGTT
GAAGGAATGCAAAACCAGAAAG
TGGTCTTCCATAACCCTTCAGT
CTGTAATACTAGGCTCCACCGC
TCAGCTGAGTTTTTGCAGAGAT
TTTGTTTGCCTTAGAGTCCTCC
TCGATGATCATATCATTCCACC
AAAGAAATAGGGAAAGGGGGAG
TCTCATTCCAGATGGGTTATCA
ATGATATGACGGCTGAGATCAA
AAATGTCCTTCCTGATGATTGG
ATTGGCAGGCTTTCCTTACATA
GCACAAGTAGCACAACTCAACA
CTTCCCATAGCAACTCTATCCC
GAGCGCAGAAGCAGACTTATCT
ATTATAGAAATTGGGCGCACTC
CTTCTCAAGAACACGGCGAT
GGTGTATTTTAATGCGGTCACA
GTTGGATTTCATGGAAAGTGTC
TGGGTTTCAAAGGTCAATCTCT
CTGTGGATTCAGACGAAGATGA
(CT)12–(T)10
M13-AATCGTCGCCTGCAATATTTAC
M13-CCTACTTTCAAAGCCCATTTTG
M13-ACAGTTCCTACAAAGGATCGGA
M13-TCGCGCTGATAGTGTTTCATAC
M13-CAATTGCACATAACCCATCATC
M13-CTCAGGAAACCGTCATACTTCC
M13-TTTGAGAGGACGAATGTCGTTA
M13-TCTTCACACATGTCCACTGGTT
M13-AACCTTTTGCGTTTGATACTCC
M13-AGTTCATCAATTTGAGGGATGG
M13-TTACAGCAATTGAAGGATGAGC
M13-AATAGCATGGAATCCACTCACC
M13-AAAGCTTCCTCATTTTGTGAGC
M13-GATCTCTGATTCCCGCATTATT
M13-AACCCAAAGCTTTCCTTCTTGT
M13-CAACCGCTTTAATGAAGTGTGA
M13-ACGTTGATCATAGCCTCCAAAT
M13-GCGTCTGCTTTCTTCTCAGTCT
M13-TTTTATATGCTTGTCCCACACG
M13-CGGATGAATTTAAGAAAGCTGG
M13-TATTGGGAGTGAAAGAGCCCTA
M13-GAACCAAAGCTTTTGATTGGTC
M13-GGGTATATTCCAAAACCCCAAT
M13-CTTCTTGGGACGAAAAACAAAC
GTTCAGAATGCAGTTGAAACCA
ACTTTTGCATCTGCTTCATCTG
M13-TCCAACATTGGTGACAGTTTTC
M13-GCCCGATAGATTAAAAGGGAAA
Reverse primer sequence
Forward primer sequence
(TC)8
(GAA)7
(AG)7
(AT)8
(CT)9
(TC)6
(GAA)8
(TTC)9
(TA)8
(GA)6
(TTG)6
(TC)7
(TTC)9
(TA)7
(TG)7–(TG)7
(TA)7
(GA)7
(GA)6–(GA)7
(CT)6
(CA)10
(TC)8
(TTA)8
(TGA)8
(GA)6
(CCT)8
(TA)10
(TGA)8
Repeat motif
Graham et al. (2004)
Source
1636
Theor Appl Genet (2015) 128:1631–1646
ACGAATTGATTTCATCAACC
Locus names prefaced with Ro were derived from black raspberry (Rubus occidentalis L.), those prefaced with Ri and Rubus were derived from red raspberry (R. idaeus L.), those prefaced with
Rh were derived from blackberry (Rubus sp.). Names in italics designate those markers that were used to anchor the linkage groups and were selected because they map in multiple Rubus linkage maps. Two markers (Rub1C6 and Rubus270a) were designed with a fluorescent tag on their forward primer. Each entry includes the linkage group to which the locus mapped, the allele size
in population ORUS 4305, the repeat motif, the forward and reverse primer sequence, and source. The sequence of the M13 tag is 5′-TGTAAAACGACGGCCAGTAGC
CCTCCGAAGAAAAACAGCAG
M13-CTTCTTTCCAACCGATTTC
(AGC)7
M13-TTTGTCTCCGTCTTTTTGCC
(TC)15
260, 266
260
6
ERubLRSQ_07-4_D05_SSR_
RLG6
254, 256
1
Rh_MEa0013cF08_SSR
254, 266
GTAAGGCCACCCCATTGAG
M13-CCCTCCATCTCCACCATAAA
(GA)38
248, 250
2
Rh_MEa0013bG01_SSR
248
GAAATGCTTGAAGCCGAGAG
Woodhead et al.
(2008)
Lewers et al. (2008)
TTCTGCTCATCTTTGGGGTC
M13-TGGTGGTTCACCGTTCACTA
(CT)15
M13-CCCCAAACTCCAAAATCTCA
(CT)17
148, 154
Rh_MEa0007aB01_SSR_RLG5 5
148, 152
268, 274
2
Rh_MEa0002cA01_SSR
268, 272
Reverse primer sequence
Forward primer sequence
Repeat motif
4153-1 allele
sizes
RLG 3021-2 allele
sizes
Locus
Table 1 continued
Source
Theor Appl Genet (2015) 128:1631–1646
1637
core facilities (Corvallis, OR, USA) for quantitation using a
Qubit® fluorometer (Invitrogen, Carlsbad, CA, USA). The
size distribution of the library was confirmed by checking
1000 pg of DNA with the Bioanalyzer 2100 HS-DNA chip
(Agilent Technologies, Santa Clara, CA, USA). Libraries were diluted to 10 nM based on Qubit® readings and
quantitative PCR (qPCR) was used to quantify the diluted
libraries. For each pooled library, 15.5 pM were loaded
for single-end Illumina® sequencing of 101 cycles with
the HiSeq™ 2000 (Illumina, Inc.) and analyzed using the
Version 3 cluster generation and sequencing kits (Illumina,
Inc.).
The libraries were sequenced in three lanes at three
different times. The first sequencing lane included 95
samples (91 progeny, and two replicated samples per parent). The second sequencing run included 88 samples (26
black raspberry including parents, grandparents, standards and progeny, and 62 unrelated strawberry samples).
The third sequencing run included 64 samples (ORUS
3021-2 repeated 4 times, ORUS 4153-1 repeated 5 times
and 55 progeny). Over all three runs, the parents ORUS
3021-2 and ORUS 4153-1 were sequenced at least twice in
each lane. Forty-four progeny were sequenced more than
once due to low initial quality and numbers of reads per
individual.
GBS SNP calling
Version 3.0 of the TASSEL GBS discovery software pipeline (Li et al. 2009) was used to call SNP loci using a
repeat-masked version of the genome sequence. Three GBS
runs representing 112 individuals as described above were
analyzed simultaneously. Data were initially subjected to
sequence and nucleotide read quality control using Trimmomatic (Bolger et al. 2014) (http://www.usadellab.org/
cms/?page=trimmomatic) and were then analyzed with
TASSEL.
Genetic linkage map construction
All loci were converted into segregation codes for JoinMap® v. 4.1 (Van Ooijen 2006). Loci were then organized
into parental sets and subjected to the maximum likelihood (ML) mapping algorithm. Independence Likelihood
of Odds (LOD) threshold of 5 was used for establishing
the linkage groups (LG). All other settings were default.
Five progeny (ORUS 4305-38, 39, 41, 59, and 65) were
excluded based on incongruous SNP data occurring from
30 (ORUS 4305-39) to 90 (ORUS 4305-65) times. GBS
data were not available for ORUS 4305-7, 19, 45, 54, 58,
75, 95, 97, 103, and 110 due to poor DNA quality. The
consensus map of seven linkage groups was generated by
combining the parental linkage maps of ORUS 3021-2 and
13
1638
Theor Appl Genet (2015) 128:1631–1646
Table 2 Summary of loci mapped in F1 population ORUS 4305
Genotyping by sequencing (GBS)
Total number of GBS SNP identified over three sequencing
runs
Number of monomorphic or ambiguous loci
Number of loci heterozygous in both parents
Number of loci heterozygous in ORUS 3021-2
Number of loci heterozygous in ORUS 4153-1
Total scaffolds represented
Scaffolds mapping to multiple RLG
Total number of GBS SNP mapped
Simple sequence repeat (SSR)
Total number of loci screened
Number of monomorphic or ambiguous loci
Number of loci that failed
Number of loci that are heterozygous in both parents
Number of loci heterozygous in ORUS 3021-2
Number of loci heterozygous in ORUS 4153-1
Number of loci mapped
High-resolution melting (HRM)
Total number of loci screened
Number of monomorphic or ambiguous loci
Number of loci that failed
Number of loci heterozygous in ORUS 3021-2
Number of loci heterozygous in ORUS 4153-1
Number of loci mapped
Anchor loci (26 SSR + 2 HRM)
Not mapped
Mapped
7911
3472
921
318
326
356
13
399
552
235
118
138
29
32
70
80
69
0
7
4
3
28
16
12
Genotyping by sequencing (GBS) single nucleotide polymorphic
(SNP) loci were generated by DNA digestion and subsequent highthroughput sequencing. Data were analyzed for mapping using the
TASSEL computer software provided through the Buckler Lab for
Maize Genetics and Diversity. Simple sequence repeat (SSR) and
high-resolution melting (HRM) loci were derived from a number of
sources
ORUS 4153-1 using the regression algorithm of the mapping software JoinMap v. 4.1. Linkage map visualization
was accomplished with MapChart 2.2 (Voorrips 2002).
The quality of genotype calls and of each map were
evaluated with a graphical genotyping approach in Microsoft Excel (Redmond, WA, USA) as previously described
(Bassil et al. 2015; Young and Tanksley 1989).
Results
Transferable markers
In total, 552 SSR markers from new and published sources
were evaluated for the amplification of polymorphic PCR
13
products in the parents and one progeny. Of these, 118
failed to amplify, 235 were homozygous in both parents
or gave ambiguous results, 138 were heterozygous in both
parents, 29 were heterozygous in ORUS 3021-2, and 32
were heterozygous in ORUS 4153-1 (Table 2).
A total of 30 primer pairs (SSR and HRM) for 28 anchor
loci were assessed for the production of a polymorphic
PCR product in the parents and six progeny of population ORUS 4305. Twelve of these loci were successfully
mapped (Table 2).
Eighty HRM primer pairs (Bushakra et al. 2012) were
evaluated for the amplification of polymorphic PCR products on the parents and 14 progeny. Of these 80 HRM
primer pairs, 57 were monomorphic, 12 were unclear or
had poor amplification, and 11 were evaluated in the full
population. Three of these HRM markers were mapped successfully, two in ORUS 3021-2 and one in ORUS 4153-1
(Table 2). Out of 660 transferable markers evaluated, a total
of 72 (11 %) were successfully mapped. BLAST analysis
(Altschul et al. 1990) of the forward and reverse primer and
nucleotide sequences (when available), allowed scaffold
assignment of most mapped transferable markers (Supplementary Table 2).
GBS SNP markers
The first sequencing run of 95 samples generated 596 K
sequence clusters/mm2 (optimal density is 750–850 K clusters/mm2; MyIllumina Support Bulletin); the second and
third sequencing runs were within the optimum range at
825 and 752 K clusters/mm2, respectively. These cluster
densities provided raw reads ranging from approximately
165 million to 310 million. Over the three sequencing runs,
112 progeny and the two parents were sequenced to generate an average number of reads per individual of 3,105,333,
with 20,317,182 (5.8 %) of reads unaligned. Default TASSEL filtering parameters using the parent information identified 57,238 SNP positions. Further filtering of the SNP
data to remove those loci with more than 10 % missing data
resulted in a data set of 7911 SNP loci, of which 3472 were
monomorphic or ambiguous, 921 were heterozygous in
both parents, 318 were heterozygous in ORUS 3021-2, and
326 were heterozygous in ORUS 4153-1 (Table 2).
Linkage mapping
Of the five progeny excluded based on incongruous SNP
data, ORUS 4305-65 showed obvious phenotypic differences from the rest of the population and may be the result
of a pollen contamination; however, the other four progeny were not phenotypically different from the rest of the
population. A total of 100 progeny were used to construct
the seven linkage groups for the parental linkage maps, the
Theor Appl Genet (2015) 128:1631–1646
characteristics of which are summarized in Table 3. To construct the linkage map for ORUS 3021-2, five GBS-generated SNP markers were removed for skewed segregation
ratios, four were removed for creating double recombination events within a distance of 10 cM or less, and one was
removed due to unsuccessful linkage phase determination.
For ORUS 3021-2 (Supplementary Fig. 1) the resulting 274
markers comprising the seven LGs spanned 779.4 centiMorgans (cM) with an average distance of 2.9 cM between
markers. RLG7 had the greatest number of markers (56),
and was also the longest (134.5 cM) with an average distance of 2.4 cM between markers. RLG2 was the shortest
at 84.1 cM, with an average distance of 2.8 cM between the
30 markers, and two gaps of 11.4 and 11.9 cM. The largest
gap for the map of ORUS 3021-2 was 22.2 cM on RLG6.
Of the 222 GBS SNP markers used for map construction,
200 (90 %) segregated as expected, either 1:1 or 1:2:1; two
loci (1 %) varied from expected at a significance level of
0.01, 11 loci (5 %) varied from expected at a significance
level of 0.05, and nine loci (4 %) varied from expected at a
significance level of 0.1.
To construct the linkage map for ORUS 4153-1, 18 GBSgenerated SNP markers were removed for skewed segregation ratios, 14 were removed for creating double recombination events within a distance of 10 cM or less, and one
SSR marker was removed due to unsuccessful linkage
phase determination. For ORUS 4153-1 (Supplementary
Fig. 2) the resulting 292 markers comprising the seven
LGs spanned 892.1 cM with an average distance of 3.2 cM
between markers. RLG7 had the greatest number of markers
(64) and was also the longest (151.4 cM) with an average
distance of 2.4 cM between markers, and three gaps greater
than 10 cM, the largest of which was 12.2 cM; RLG1 was
the shortest at 101.7 cM with 23 markers, an average distance of 4.4 cM between markers, and three gaps greater
than 10 cM, the largest of which was 14.8 cM. The largest
gap for the map of ORUS 4153-1 was 14.8 cM at the end
of RLG1. Of the 249 GBS SNP markers used for map construction, 230 (92 %) segregated as expected, either 1:1 or
1:2:1; a single locus (0.4 %) varied from expected at a significance level of 0.01, nine loci (4 %) varied from expected
at a significance level of 0.05, and nine loci (4 %) varied
from expected at a significance level of 0.1.
Transferable markers for the parental maps ranged
from a low of three markers on ORUS 3021-2 RLG7 and
4153-1 RLG6 to a high of 12 on ORUS 4153-1 RLG4. A
total of 72 transferable markers were mapped in this population. BLAST analysis of the transferable markers against
the draft genome assembly allowed scaffold assignment
for 65 of 72 markers (90 %) so that 356 scaffolds were
represented.
The phenotypic marker for aphid resistance, Ag4_
AphidR, was located on RLG6 of the aphid-resistant parent
1639
ORUS 4153-1 and maps to the same location as S99_32802
(Fig. 2).
The seven consensus RLGs (Table 3; Fig. 3) assembled
from merging the parental maps consisted of 438 markers spanning 546.4 cM with an average distance between
markers of 1.3 cM. Consensus RLG6 was the longest
(90.2 cM) with an average distance between the 69 markers of 1.3 cM, and one gap of 10.4 cM. Consensus RLG7
had the most markers (77) that spanned 81.0 cM with an
average distance of 1.1 cM between markers. RLG2 was
the shortest at 70.8 cM with an average distance between
the 59 markers of 1.2 cM. The 12 anchor markers identified from the literature (Table 1; Supplementary Figs. 1,
2, markers in italics) allowed the positive identification
of consensus RLG 2-7, with the last, RLG1, identified by
default.
Thirteen of the 356 represented scaffolds (3.6 %) map
to more than one linkage group (Table 4); 33 of the loci are
SNPs and five are SSRs. Four scaffolds (S10, S26, S134,
and S142) are represented by SNP loci on more than two
linkage groups. Four scaffolds (S14, S71, S78, and S279)
are represented by at least one SNP and a single SSR locus
on more than one linkage group.
Discussion
We present the first linkage map constructed from a pure
black raspberry cross. The first attempt at genetic linkage mapping using SSR markers on an F2 generation of
a black raspberry × red raspberry cross identified high
homozygosity as well as severe segregation distortion and
did not result in a linkage map (Lewers and Weber 2005).
The linkage map constructed using non-anonymous
DNA sequences for black raspberry selection 96395S1
comprises 29 markers spaced on average at intervals of
10 cM over six LG spanning 306 cM (Bushakra et al.
2012). The first published red raspberry map of ‘Glen
Moy’ × ‘Latham’ consisted of 273 markers derived from
amplified fragment length polymorphic and genomic-SSR
markers and spanned 789 cM over nine LG (Graham et al.
2004). Over the next 6 years as more markers were developed and added, the improved ‘Glen Moy’ × ‘Latham’
map reported by Woodhead et al. (2010) consisted of 228
markers over seven LG spanning 840.3 cM with transferable markers present on each LG. Paterson et al. (2013)
subsequently added gene-based markers to the linkage
map constructed by Woodhead et al. (2010) by mining
Rubus transcriptome and EST databases for candidate
genes in the fruit volatiles pathway. The efficiency of
marker generation used here is a vast improvement over
previous marker development techniques in Rubus. The
saturated consensus linkage map presented here spans
13
779.4 2.9
52
274
Total
Each Rubus linkage group (RLG) details the number of loci mapped, the number of loci that are transferable (either simple sequence repeat or high-resolution melting), the length of the linkage
group in centiMorgans (cM), the average distance between each locus in cM, and the number of gaps larger than 10 cM
292
42
892.1 3.2
438
70
546.4 1.3
1
1
90.2 1.3
81.0 1.1
12
7
3
3
98.4 3.1
134.5 2.4
RLG1
RLG2
RLG3
RLG4
RLG5
RLG6 32
RLG7 56
10
3
1
0
46
64
3
6
149.4 3.2
151.4 2.4
69
77
2.0
1.2
1.1
1.2
1.2
77.5
70.8
73.2
74.6
79.1
9
11
7
14
10
3
1
1
1
2
3.4
2.8
2.6
3.0
3.0
99.5
84.1
113.6
115.3
134.0
13
29
30
44
38
45
8
8
5
8
10
2
2
1
1
1
23
40
33
43
43
6
7
4
12
4
101.7
115.9
102.9
143.0
127.8
4.4
2.9
3.1
3.3
3.0
39
59
67
63
64
Average
distance
between
loci in cM
Number of cM
transferable loci
Locus
number
Gaps over
10 cM
Average
distance
between
loci in cM
Number of cM
transferable loci
Average
distance
between
loci in cM
Locus
number
Number of cM
transferable
loci
Gaps over
10 cM
Locus
number
Consensus Map
ORUS 4153-1 (male parent)
ORUS 3021-2 (female parent)
Table 3 Summary of genetic linkage maps for female parent ORUS 3021-2, male parent ORUS 4153-1, and the consensus map for F1 population ORUS 4305
0
0
0
0
0
Theor Appl Genet (2015) 128:1631–1646
Gaps over
10 cM
1640
ORUS 4153-1
RLG6
0.0
1.0
12.2
15.8
19.5
22.5
24.6
25.6
28.7
29.7
30.7
39.5
40.7
46.8
51.1
55.1
59.3
S47_428315
S83_31915
S487_120066
S144_116431
S24_348547
S671_62748
S64_165666
S28_107186
S87_64177
S252_232489
S114_40714
S380_62888
S316_186017
S431_37187
S842_37425
S41_43599
S371_49180
S376_162362
Ro11481_S328
S215_218295
114.3
121.7
SQ07-4_D05_RLG6_S14
S818_21981
S477_117436
S1797_556
S447_61626
S394_124946
S977_26034
S99_104800
S122_74787
S187_259241
S140_40577
S196_140013
S684_63660
Ag4_AphidR
S99_32802
S525_75530
S275_97574
S474_45156
S79_351594
S65_461926
130.9
132.9
S33_584984
S457_17312
142.0
145.8
S214_34656
Ro4532_S13
S13_940743
S63_573175
69.9
70.2
74.6
77.4
78.9
84.4
87.5
91.8
93.9
105.1
108.5
110.5
111.4
112.4
149.4
Fig. 2 Rubus linkage group (RLG) 6 for black raspberry mapping population parent ORUS 4153-1. The morphological locus for
Ag4 aphid resistance against the North American large raspberry
aphid is shown in blue bold font. The linkage map is constructed of
single-nucleotide polymorphic (SNP) loci generated by genotyping
by sequencing (GBS) (prefaced with S) and simple sequence repeat
(SSR) loci from various Rubus sources (prefaced with Ro, Ri, Rh,
Ru, Rub, and SQ). Transferable loci are indicated in bold font; anchor
loci for comparisons with other Rubus linkage maps are indicated in
bold italic font (color figure online)
546.2 cM and is composed of 374 GBS-generated SNP
markers and 68 transferable markers with an average of
1.3 cM between markers. The transferable markers are
distributed among the LG and can be used for alignment
to other Rubus maps. The scaffold assignment allows for
Theor Appl Genet (2015) 128:1631–1646
Consensus_RLG1
0.0
0.1
1.1
7.7
8.9
10.0
S20_736564
S70_150228
S335_26380
S20_127876
S42_237147
Ro_CBE2P01b_S71
15.2
Ri18886_S9
18.0
S134_217734
Consensus_RLG2
0.0
0.9
1.0
S2_650888
Ri3758
S2_739570
4.5
5.3
6.2
S71_420905
S7_379506
S7_295570
15.3
15.7
16.9
19.8
25.2
25.5
28.3
26.2
33.3
S3_792848
S75_381030
36.6
S15_274316
38.3
S81_168310
43.3
43.8
45.0
46.1
S97_272432
S154_190648
S234_258231
Ro942_S234
32.2
32.4
33.2
34.4
35.2
35.8
41.4
43.5
43.8
43.9
44.8
45.6
46.3
46.4
47.4
48.5
54.0
58.4
60.0
62.1
62.7
64.7
65.5
68.8
69.7
70.3
70.4
71.2
71.3
71.7
72.6
73.6
75.6
77.5
S88_93014
S10_617269
S804_55106
S565_20930
Rh_MEa13cF08_S82
Ro_CBEa3K17_S565
S143_181231
S648_18748
Ro9324_S289
S929_43718
Ro6594_S674
Ro18036_S322
S14_952525
S414_7874
S426_20245
S142_215410
S798_31088
S999_22487
S721_59214
Ro3237_S199
S921_19756
S278_218184
S472_89825
1641
48.8
49.0
50.5
51.1
52.4
53.4
53.5
54.6
55.9
57.1
58.4
59.3
60.3
62.0
63.4
64.6
70.4
70.7
70.8
Ri3758_S2
S4_1405440
S2_372319
S17_207928
S4_725978
S4_142584
S62_439154
S14_877960
S14_44081
S468_92150
Ro10488_S420
S32_375704
S124_714
S22_493758
Ri14075_S36
S295_217369
S418_91496
S77_522635
S294_233966
S152_94166
S615_66181
S77_150939
S294_240516
S269_65757
S112_10927
S217_52295
Ro_CBEa1L24_S382
S456_103577
S332_25238
Ri11086
Rh_MEa2cA01_S755
Ro8486_S330
S134_83955
S419_74798
S134_74067
S241_119603
S727_30152
Rh_MEa13bG01_S324
Ro17803_S176
S176_295101
S348_11401
S650_32526
S458_3294
S714_73912
S590_9650
S449_31741
S169_39974
S149_322226
S353_156014
S23_120199
S10_66813
S53_419253
Rub126b_RLG2_S622
Consensus_RLG3
0.0
0.9
2.4
4.3
6.4
8.7
10.2
10.3
10.7
12.4
12.5
13.4
13.5
13.9
18.1
18.8
19.8
21.3
23.1
24.6
24.9
25.7
27.8
28.4
28.6
31.5
31.6
32.4
32.5
32.7
33.3
34.7
36.1
41.6
41.8
42.2
44.2
45.9
46.3
50.0
50.1
51.6
54.1
56.5
57.5
58.4
58.5
59.3
59.5
59.8
60.6
63.3
65.0
67.0
68.0
68.2
69.7
70.3
70.7
72.0
73.2
S107_124243
S46_579890
S29_168757
S73_33754
S46_702636
S139_144731
S142_7482
S95_41560
Ro3017_S334
S139_162862
S75_81406
S29_647815
Ro7270_S959
S6_272056
S177_269209
S107_118411
S113_308895
S40_488292
S27_903196
S113_305041
S111_349602
S80_303146
Ro1682_S226
S239_140073
S233_65965
S45_71463
S202_203089
S226_285133
S80_151911
S355_30318
S279_96154
S194_27933
S409_72552
S318_155707
S409_72630
S86_272676
S408_156553
S115_211947
S52_429297
S261_153798
S231_29430
S35_190714
RhM003_RLG3
S168_55970
S380_138595
S76_223569
S167_271810
S25_886295
S167_305583
S175_120669
S201_50091
S156_263195
S209_270566
S156_147409
S209_265706
S118_113626
S21_897073
S21_213189
S31_314682
S26_4209
S314_179125
Ro17045_S26
S26_335827
Ri12319_S72
Rub270a_RLG3_S72
S119_192278
S68_477340
Consensus_RLG4
0.0
0.9
3.1
3.5
4.2
5.4
Rub116a_RLG4_S462
S1_2397101
Ro16697_S101
RiM017_RLG4_S101
S101_278550
S462_77258
7.7
S1_1883174
10.8
S1_42487
13.8
13.9
14.3
14.7
S1_8641
S153_124032
Rub26a_RLG4_S8
Ro4261_S1
22.8
25.3
28.2
32.0
33.5
33.9
35.9
36.4
37.8
Ro_CBEa9J05_S8
S391_173965
S37_479829
S440_123121
S288_52689
Ro_CBEa4G23_S54
S357_177604
S357_177550
S485_41160
S327_60426
S30_582093
S258_212619
Ro2579_S228
Ro20267_S228
Ro2173_S108
S174_269589
S109_307463
S1023_16069
S364_80657
S537_5881
S116_118019
S502_126385
S537_5882
S630_40278
S314_148478
S529_27629
S10_1249211
S850_14502
S161_355312
S469_61140
S235_147689
S494_12849
S1174_19186
S653_21318
Ro_CBEa10N20_S103
Ro5378_S400
S103_169793
S398_55678
S494_12860
S225_138869
S454_132548
S454_55758
S253_22396
Rub110a_RLG4_S100
S612_66375
S723_48232
S120_78654
S120_199903
S147_375410
Ro1079_S120
S509_84964
39.2
40.0
41.1
44.4
45.5
51.6
53.4
53.6
53.7
54.0
55.0
55.3
55.7
57.2
57.5
57.7
58.3
60.0
61.2
62.2
63.2
63.9
64.0
64.2
65.2
65.3
66.2
69.3
69.9
70.3
71.2
71.6
73.8
74.6
Fig. 3 Consensus linkage map for population ORUS 4305. Each of
the linkage groups consists of single-nucleotide polymorphic (SNP)
loci generated by genotyping by sequencing (GBS) (prefaced with S)
and simple sequence repeat (SSR) loci from various Rubus sources
(prefaced with Ro, Ri, Rh, Ru, Rub, and SQ). Transferable loci are
indicated in bold font; anchor loci for comparisons with other Rubus
linkage maps are indicated in bold italic font. The morphological
locus for Ag4 aphid resistance against the North American large raspberry aphid is shown in blue bold font (color figure online)
future fine mapping, QTL analysis, and improved black
raspberry genome assembly.
The reduced-representation sequencing accomplished
with GBS has generally been used in crop plants with high
levels of heterozygosity. For example, Poland et al. (2012)
were able to map 20,000 and 34,000 GBS-generated SNP
loci in wheat and barley reference linkage maps, respectively;
Lu et al. (2013) performed GBS in tetraploid switchgrass
(Panicum virgatum) and were able to map 88,217 SNP loci;
Truong et al. (2012) used GBS to generate SNP in Arabidopsis thaliana and lettuce (Lactuca sativa) and were able
to map 1200 and 1113 SNP loci, respectively; Russell
et al. (2014) mapped 790 SNP loci in blackcurrant (Ribes
nigrum). This is the first use of GBS on black raspberry, a
13
1642
Theor Appl Genet (2015) 128:1631–1646
Consensus_RLG5
0.0
S26_210580
2.0
3.1
Ro2827_S12
S12_409515
64.6
66.4
67.4
69.2
69.7
RuEE284382_RLG5_S178
S11_566199
S11_701791
Ro_CBEa11M11_S5
S5_481465
S5_672048
S386_162892
S247_263810
Ro9206_S138
S247_264439
S138_172691
Rh_MEa7aB01_RLG5_S96
S96_10539
S222_4415
S190_165898
S134_333655
S85_209336
S146_178483
S78_386742
S134_395808
S51_580471
S240_274593
S90_260135
S38_226913
S90_16944
Ri20047_S90
Ro4345_S1079
S39_50026
S259_46181
S427_121604
S220_216318
S259_100913
S220_216348
S733_27643
S344_12492
S127_265724
Rub275_RLG5
S530_106983
S1127_16040
S411_144987
S1313_1723
S276_219889
S395_59407
Ro3003_S1163
S654_42261
S776_33747
S536_107187
S323_22819
S117_462560
S505_61312
S102_135460
S961_14723
Ro2432
S402_61441
S171_213638
S93_6530
S1029_12169
74.6
75.9
76.3
S59_190044
S387_8229
S548_89207
79.1
S370_45404
6.8
7.5
7.6
12.0
14.7
15.6
17.8
18.5
20.0
20.7
20.8
22.8
24.0
24.9
25.9
26.9
27.3
28.2
29.2
29.8
30.5
31.5
32.6
33.7
33.9
36.0
41.2
42.9
44.2
44.5
44.8
45.0
45.9
47.0
48.8
50.5
51.5
52.1
53.2
53.6
54.3
54.8
55.4
56.0
56.8
57.3
58.2
58.6
63.6
Consensus_RLG6
0.0
1.2
11.6
12.0
13.5
15.8
17.7
18.7
18.8
19.5
22.6
24.9
25.8
26.0
28.0
28.9
29.1
29.2
30.0
30.2
30.6
Rub16a_RLG6_S57
S487_120066
S47_293993
S144_116431
S155_368623
Ro15590_S48
Ro3981_S78
S24_348547
S671_62748
S64_165666
S28_107186
S28_64542
S87_253649
Ro8167_S28
S252_232489
S87_64177
Ro12112_S114
S114_40714
S380_62888
38.4
39.0
40.2
40.4
40.8
S215_218295
S316_186017
S842_37425
S41_43599
S371_49180
Ro11481_S328
S376_162362
S477_117436
SQ07-4_D05_RLG6_S14
S818_21981
S447_61626
S1797_556
S977_26034
S394_124946
S122_74787
S140_40577
S99_104800
S187_259241
S99_32802
Ag4_AphidR
S780_47083
S256_68947
S266_83737
Ro5263_S266
S525_75530
S474_45156
S275_97574
S79_351594
S238_33526
S492_56949
S1158_2328
S122_230445
Rub1C6_S99
S474_19898
S260_4228
S165_315500
S136_346904
S331_120820
S33_584984
S49_534988
S457_17312
Rub223a_S179
S214_34663
Rh_MEa6bG05_S13
S214_34656
Ro4532_S13
S13_940743
S63_573175
41.1
41.5
42.0
42.8
43.3
43.7
44.5
45.2
46.7
47.4
48.6
50.0
50.1
50.3
50.4
50.8
51.2
51.8
53.8
56.5
58.1
60.9
61.9
64.0
65.0
65.1
67.7
68.2
69.9
70.4
74.9
76.4
79.9
80.7
81.7
85.6
86.5
90.2
Fig. 3 continued
13
S47_428315
S83_31915
Consensus_RLG7
0.0
S1089_19238
6.3
6.8
Ri13528
S44_599959
11.4
Ro14925_S279
12.7
Ro4104_S421
17.2
18.3
19.5
20.8
22.0
22.3
22.6
23.0
24.8
25.5
25.9
26.1
26.9
27.1
27.2
27.3
27.6
27.7
27.8
28.0
28.2
28.4
28.6
28.8
29.0
29.2
30.2
30.4
31.2
32.2
32.4
32.6
33.5
34.0
34.9
37.1
37.2
37.4
38.4
39.4
40.1
40.4
41.0
41.6
42.3
42.5
42.7
43.4
44.4
44.6
47.5
48.8
49.4
50.0
51.8
52.9
53.6
60.2
64.2
65.7
68.7
S151_134836
S151_34946
RiCHEa1B16_RLG7_S574
S325_109080
S121_410272
S219_198144
S219_194825
S249_52424
S604_66577
S547_52503
S1034_26022
S277_211346
S681_58391
S142_7508
S781_3964
S399_120712 S609_54570
S161_75403 S500_53440
S608_26380 S799_55672
S1001_19490
S595_84360
S496_44208
S212_266279 S1104_1113
S500_45749
S495_85717 S756_65801
S475_16395
S337_42274
S337_126476
S244_154037 S471_114555
S651_61319
S464_41282
S341_24920
Ri10139_S566
S577_10160
S780_9754
S1391_6231
Ri16959
S287_112188
S473_16767
S329_82033
S563_15061
S540_84061
S1212_14404 S384_49752
S472_89891
S61_295208
S705_64391
S115_45212 S110_263544
S61_40795
S865_3480
SQ07-3_C07_RLG7_S148
S110_362170
S200_237792
S232_134490
S251_17084
S200_242671
S272_158581
S94_313488
S173_278143
S272_71197
S43_656815
S631_78691
S129_153488
S98_274616
S60_604050
81.0
S34_759158
27.4
Theor Appl Genet (2015) 128:1631–1646
1643
Table 4 Summary of the genomic scaffolds with loci on more than
one Rubus linkage group (RLG)
Scaffold
RLG
Parent
SNP, SSR
S10
S14
S14
S21
S26
S71
S78
S115
S134
1, 2, 4
1, 2
2, 6
3, 7
3, 5, 7
1, 2
5, 6
3, 7
2, 5
4153-1
3021-2
4153-1
3021-2
3021-2
3021-2
3021-2
3021-2
3021-2
3, 0
2, 0
1, 1
2, 0
3, 0
1, 1
1, 1
2, 0
2, 0
S134
S142
S142
S161
S161
S279
S380
S380
1, 2, 5
1, 3
3, 7
7
4
3, 7
3
6
4153-1
3021-2
4153-1
3021-2
4153-1
4153-1
3021-2
4153-1
3, 0
2, 0
2, 0
1, 0
1, 0
1, 1
1, 0
1, 0
S472
1, 7
3021-2
2, 0
Each entry details the linkage group and the parental map on which
the loci are found and the type of locus, either single-nucleotide polymorphic (SNP) or simple sequence repeat (SSR)
crop of relatively low genetic diversity. Even with an average
number of reads per individual of 3,105,333 over the three
sequencing runs, only 1545 SNP loci were found that met
criteria for linkage mapping and of those 399 were mapped
successfully (Table 2). While this is a sufficient number of
markers to develop a well-populated map, it, along with the
low mapping success rate of transferable markers, illustrates
the low level of heterozygosity found in black raspberry. In
contrast, the linkage maps constructed of GBS-derived SNP
and SSR markers for red raspberry parents ‘Heritage’ and
‘Tulameen’ comprise 4521 markers spaced on average at
intervals of 0.1 cM over seven LG spanning 462.7 cM and
2391 markers spaced on average at intervals of 0.1 cM spanning 376.6 cM, respectively (Ward et al. 2013). While digestion by a more frequent restriction enzyme cutter for GBS
may be a way to increase the number of SNP loci identified, this does not guarantee mapping success as segregation
within the population is essential for linkage mapping.
Up to 97 % of the mapped scaffolds were placed on a
single linkage group indicating high quality assembly of
the draft genome. The 13 scaffolds that map to multiple
LGs will need to be investigated further to assess whether
these inconsistencies represent errors in the genome assembly; however, initial observations could indicate regions of
high chromosome homology or possible regions of genome
duplication especially between RLG3 and RLG7.
The placement of the aphid-resistance morphological
marker representing gene Ag4 on RLG6 corresponds to
the red raspberry genomic region found by Sargent et al.
(2007) for A1. The only other aphid-resistance gene in
Rubus that has been mapped is A10, which was found to be
located on red raspberry RLG4 (Fernández-Fernández et al.
2013). A1 originated from the old red raspberry ‘Baumforth’s A’ and confers race-specific resistance to three
biotypes of the European large raspberry aphid, Amphorophora idaei Börner (biotypes 1, 3 and the A10-breaking;
McMenemy et al. 2009), but is ineffective against the
North American species A. agathonica. Ag4_AphidR maps
to the same position as SNP S99_32802, providing us with
a clearly defined region on which to focus our future finemapping efforts and comparative mapping to red raspberry.
This linkage map region is associated with many quantitative trait loci (QTL) having to do with resistance to aphids
(Sargent et al. 2007), and fungal (Graham et al. 2006) and
fungal-like (Graham et al. 2011) pathogens in red raspberry
and we hope to use our linkage map to better understand
the underlying reasons for these associations.
Conclusions
We present here the first genetic linkage map of black
raspberry comprised of GBS-generated SNP and transferable markers. The presence of SSR and HRM markers
selected from the literature, along with the other transferable markers allowed us to positively identify all RLG as
per Bushakra et al. (2012), and provide an opportunity to
align all existing Rubus linkage maps. These maps will
serve as a framework for anchoring scaffold sequences in
the black raspberry draft genome sequence. Comparative
mapping using the common markers and the draft genome
sequence will be useful for aligning QTL among different
species of Rubus. Future studies on the different sources of
aphid resistance, including construction of densely populated linkage maps and cloning of loci associated with
aphid resistance, will provide information on the loci and
will result in the development of markers that can be used
for marker-assisted breeding for aphid resistance in black
raspberry.
Author contribution statement JMB Project Coordinator performed marker screening, selected anchor markers,
ran and scored all markers, constructed the genetic linkage
map, and wrote the manuscript. DWB developed a custom pipeline for bioinformatic analyses, and performed
GBS SNP calling. MD developed the mapping population, short-read Ro and Ri primers, performed the initial
marker screening, and phenotyped aphid-resistance in the
mapping population. KJV and RVB assisted in GBS SNP
13
1644
calling and other bioinformatic analyses, BLAST analyses,
and linkage mapping. BSG assisted in developing and performed the initial marker screening. JL PI on NIFA SCRI
grant (project main funding) and contributed to manuscript
writing. TCM PI on NIFA SCRI grant (project main funding) and contributed computational resources and bioinformatics analysis. CEF PI on NIFA SCRI grant (project
main funding), helped assemble and phenotype the germplasm, develop the mapping population, and contributed to
manuscript writing. Primary advisor for the phenotyping
portion of the NIFA SCRI grant. NVB PI on NIFA SCRI
grant (project main funding), helped analyze short-read
sequencing results, develop and test molecular markers,
and contributed to manuscript writing. Primary advisor for
the genomics portion of the NIFA SCRI grant.
Acknowledgments The authors thank Dr. Jana Lee (USDA-ARS
HCRU, Corvallis, OR, USA) for her help with rearing aphids, Dr.
Eric van de Weg (University of Wageningen, The Netherlands) for
his assistance with graphical genotyping and consultation, The Barley
Lab at Oregon State University for access to JoinMap, Drs. Kim Lewers (USDA-ARS BARC, Beltsville, MD, USA) and Christopher Saski
(Clemson University, Clemson, SC, USA) for access to unpublished
SSR marker data, and Drs. Kim Lewers, James Olmstead (University of Florida, Gainesville, FL, USA) and two anonymous reviewers for their helpful suggestions. 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) USDAARS CRIS 2072-21000-044-00D, 2072-21000-047-00D and 207221220-002-00D. 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
U.S. Department of Agriculture.
Conflict of interest The authors declare that they have no conflict
of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit
to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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